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Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analys is.

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ANTIMICROBIAL ACTIVITY OF BIOACTIVE COMPOUNDS ISOLATED FROM MARINE BACTERIA ASSOCIATED WITH SPONGE Jaspis sp. AND THEIR GENETICS ANALYSIS EFFENDI GRADUATE SCHOOL BOGOR AGRICULTURAL UNIVERSITY BOGOR 2012 DECLARATION I hereby declare that this thesis entitled “Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. an d Their Genetics Analysis” is the result of my own work through the guidance from my academic supervisors and has not been submitted in any form for another de gree at any other university. Sources of information de rived or quoted from published and unpublished works of other authors is mentioned in the text and listed in the list of References at the end o f this thesis. Bogor, August 2012 Effendi G351100021 ABSTRACT EFFENDI. Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analys is. Under direction of ARIS TRI WAHYUDI and MUNTI YUHANA. Antimicrobial compounds of three marine bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 which associated with sponge Jaspis sp. were extracted using ethyl acetate solvent. Each of bacterial crude extract showed different activity against non-pathogenic microbes i.e. Bacillus subtilis, Escherichia coli, and pathogenic microbes i.e. Staphylococcus aureus, enteropathogenic E. coli K1-1 (EPEC K1-1), Pseudomonas aeruginosa, Candida albicans and C. tropicalis. Bacterial crude extract of isolate SAB E-41 demonstrated the best antimicrobial activity against non-pathoge nic and pathogenic microbes. Active fractions of each bacterial crude extract were detected using bioautography method. Fractionation and purification of antimicrobial compound for bacterial crude extract from isolate SAB E-41 was carried out using silica gelcolumn chromatography and preparative thin layer chromatography (PTLC) technique. Analysis of 16S rDNA for those three isolates showed that they were included in the genus of Bacillus. Sequence analysis of cloned DNA fragments encoding ketosynthase (KS) do main showed that they had homology with PKS type I of B. amyloliquefaciens LL3 for SAB E-41 and putative polyketide synthase pksL of B. amyloliquefaciens subsp. plantarum CAU-B946 for SAB E-57. The adenylation (A) domain of SAB E-31 showed its homology with bacitracin synthetase I of B. pumilus ATCC 7061, whereas for SAB E-41 and SAB E-57 showed their homology to sur factin synthetase B of B. amyloliquefaciens subsp. plantarum CAU-B946 and surfactin synthetase A of B. amyloliquefaciens subsp. plantarum CAU-B946, respectively. Keywords : Antimicrobial compound, fractionation, cloning, 16S rDNA, KS and A domain, Jaspis sp. SUMMARY EFFENDI. Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analys is. Under direction of ARIS TRI WAHYUDI and MUNTI YUHANA. The increase of global resistance of the pathogenic microbes against various antibiotics becomes a serious concern in public health. Many efforts are conducted in order to solve this problem, such as finding the new bioactive compounds from marine bacteria which associated with marine sponges. Three marine bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 that associated with sponge Jaspis sp. showed their capability in producing antimicrobial compounds. In this study, extraction of antimicrobial compounds from these bacteria was conducted using ethyl acetate solvent. Each of bacterial crude extract displayed significant activity against B. subtilis, E. coli as non-pathogenic microbes and S. aureus, P. aeruginosa, enterop athogenic E. coli K1-1, C. albicans and C. tropicalis as pathogenic microbes. Bacterial crude extract of isolate SAB E-41 demonstrated better activity than bacterial crude extracts of isolates SAB E-31 and SAB E-57. Analysis of constituent component for each bacterial crude extract was conducted using thin layer chromatography (TLC). Each of bacterial crude extract was spotted onto silica gel plate and eluted with n-butanol-ethyl acetate solvent mixture (3:7). Six spots/fractions were successfully detected by viewing under UV light at 254 nm and 366 nm wave- length. Active spots/fractions from each bacterial crude extract were detected using bioautography method. Four spots from each bacterial crude extract showed antimicrobial activity against P. aeruginosa and two spots showed antimicrobial activity against S. aureus. Fractionation of bacterial crude extract from isolate SAB E-41 was carried out using silica gel-column chromatography. Two hundred and five fractions were successfully collected from fraction collector and combined into thirty compos ite fractions based on the same chromatogram by using TLC ana lysis. The antimicrobial activity of thirty compos ite fractions was tested to S. aureus, P. aeruginosa, enteropathogenic E. coli K1-1, C. albicans and C. tropicalis. Fifteen of the m named by BA-1, BA-2, BA-3, BA-4, BA-5, BA-6, BA-7, BA-8, BA-11, BA12, BA-13, BA-14, BA-15, BA-17 and BA-18 have different antimicrobial activity against S. aureus, P. aeruginosa, EPEC K1-1 and C. albicans. Fraction BA-2 that was eluted by chloroform- methanol (90% -10%) solvent system showed antifungal activity against C. albicans while fraction BA-13 that was eluted with chloroformmethanol (50% -50%) solvent system showed the highest inhibition against S. aureus followed by fraction BA-17. The diameter of inhibition zone that formed by these two active fractions were about 12 mm and 14 mm. Fraction BA-2 and BA-4 that was eluted with chloroform- methanol (90%-10%) showed the best activity against enteropathogenic E. coli K1-1. The diameter of inhibition zone that formed by these two active fractions were about 10 mm. Purification of fifteen compos ite fractions was conducted using preparative thin layer chromatography (PTLC) technique. Four active fractions with the Rf values of 0.87; 0.50; 0.41 and 0.12 were successfully collected from fraction BA- 13 and displayed significant activity against S. aureus and EPEC K1-1. Fractions BA-17 and BA-18 carried two kinds of active compounds with the Rf values of 0.93; 0.12 for the BA-17 and 0.93; 0.25 for the BA-18. Both of these active compounds were successfully collected and showed antimicrobial activity against S. aureus. Morphological and molecular identification of those three isolates were carried out in order to identify these bacteria. Sequences analysis of 16S rDNA showed that three isolates were included in the genus of Bacillus. Isolate SAB E-31 had 98% of homology level with B. pumilus strain KD3 while isolate SAB E-41 had 98 % of homology level with B. amyloliquefaciens strain zy2 and isolate SAB E-57 had 97% of homology level with B. subtilis strain YRL02. PCR amplification of ketosynthase (KS) domain of PKS and adenylation (A) domain of NRPS were successfully amplified and sub-cloned into T-Vector pMD20. All isolates coded as SAB E-31, SAB E-41 and SAB E-57 possessed A domain and only two isolates coded as SAB E-41 and SAB E-57 possessed KS domain. DNA fragment encoding KS domain was in a size of 700 bp while for A domain was in a size of 1000 bp. Sequences analysis of DNA fragment encoding KS domain using BlastX program indicated that isolate SAB E-41 showed a similarity level of 97% with type I PKS from Bacillus amyloliquefaciens LL3 and isolate SAB E-57 showed a similarity level of 98% with putative polyketide synthase pksL from B. amyloliquefaciens subsp. plantarum CAU-B946 whereas for A domain indicated that isolate SAB E-31 showed a similarity level of 81% with bacitracin synthetase 1 from B. pumilus ATCC 7061. Isolate SAB E-41 s howed a similarity level of 80% with sur factin synthetase B from B. amyloliquefaciens subsp. plantarum CAUB946 and isolate SAB E-57 showed a similarity level of 81% with surfactin synthetase A from the same strain of Bacillus, CAU-B946. Keywords : Antimicrobial compound, fractionation, cloning, 16S rDNA, KS and A domain, Jaspis sp. Copyright © 2012, by Bogor Agricultural University Copyright are protected by law 1. It is prohibited to cite all or part of this thesis without referring to and mentioning the source a. Citation only permitted for the sake of education, research, scientific writing, report writing, critical writing or reviewing scientific problem. b. Citation doesn’t inflict the name and honor of Bogor Agricultural University 2. It is prohibited to republish and reproduce all or part of this thesis without the written permission from Bogor Agricultural University ANTIMICROBIAL ACTIVITY OF BIOACTIVE COMPOUNDS ISOLATED FROM MARINE BACTERIA ASSOCIATED WITH SPONGE Jaspis sp. AND THEIR GENETICS ANALYSIS EFFENDI A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Microbiology Study Program GRADUATE SCHOOL BOGOR AGRICULTURAL UNIVERSITY BOGOR 2012 External Examiner of the Thesis Examination Committee: Dr. Ir. Widanarni, M.Si Title Name Student ID : Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analysis : Effendi : G351100021 Approved Advisory Committee Dr. Aris Tri Wahyudi, M.Si Chairman Dr. Munti Yuhana, M.Si Committee Member Agreed Coordinator of Microbiology Mayor Dr. Ir. Gayuh Rahayu Date of Examination: August 15th , 2012 Dean of Graduate Schoo l Dr. Ir. Dahrul Syah, M.Sc. Agr. Date of Graduation: ACKNOWLEDGEMENTS First and foremost, I would like to thanks God for the health, grace and gifts so this thesis can be completed on time. The topic for this thesis was “Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analysis. I would like to say gratefully thanks to Dr. Aris Tri Wahyudi, M.Si as the main supervisor who gave me the opportunity to work on this truly exciting research project and Dr Munti Yuhana, M.Si as the committee member. I wish to express my sincere thanks and gratitude for their guidance, great support, encouragement, patience and scientific knowledge that were given to me. Thank you very much also to Dr. Ir Ence Darmo Jaya Supena, M.S as the head of Department of Biology and Dr. Ir Widanarni, M.Si as the examination committee that would like to give their suggestion for the improvement of this thesis. Gratefully thanks also to Prof. Masafumi Yohda and Prof. Masafumi Odaka which gave the permission for me and all of the facility during the research in the laboratory of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology, Japan. Gratefully thanks also to Prof. Wuled Lenggoro which supported me during I followed the Short Stay/Shor t Visit (SSSV) program in Japan. Thank you very much to Neng Risma Liasari and all of the stude nts in Yohda Lab. and Lenggoro Lab. that would like to share about their scientific knowledge and helped me during the research in Japa n. Thank you also to head of laboratory, all of the staff and stude nts in the labo rator y of Microb iology and Biopharmaca Research Center, IPB, Bogor for their research’s permission and helpful effort. Finally, I would like to say gratefully thanks to my parents and brothers that already supported me during I study at Bogor Agricultural University. “This work is dedicated to my lovely parents and my best brothers”. Bogor, August 2012 Effendi BIOGRAPHY The author was born in Medan on August 21st , 1987 as the third son from Mr. Tan Po Tiam (R.I.P) and Mrs. Ong Siu Ing. In 2005, the author completed his senior high school at Yayasan Perguruan Tinggi Sutomo 2, Medan and in the same year, the author was accepted as an undergraduate student at University of Sumatera Utara, especially in Biology Study Program. During the study, the author was active at Microb iology Study Club (MSC) organization that held by Department of Biology. Beside of that, the author also worked as a Lab. Assistant in the laboratory of Microbiology and the laboratory of Plant Structure and Develop ment. In 2009, the author finished the unde rgraduate schoo l and got the Bachelor of Science degree. After finished the study, the author worked as a private teacher for one year. In 2010, the author was accepted as a Graduate student at Bogor Agricultural University, especially in Microb iology Study Program. During the study, the author worked at PT. Mitra Pelajar as an instructor of Biology for 4 months. In 2011, the author was accepted as a participant in the Short Stay/Short Visit (SSSV) program for 3 months which was held by Tokyo University of Agriculture and Technology (TUAT), Naka-cho, Koganei, Tokyo, Japan. On June 6th – 8th, 2012, part of this thesis with the title “Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp.” has been presented in the International Seminar on Advances in Molecular Genetics and Biotechnology for Public Education hosted by Atma Jaya Catholic University of Indo nesia. TABLE OF CONTENT Page TABLE OF CONTENT................................................................................... i LIST OF TABLES ...........................................................................................ii LIST OF FIGURES ........................................................................................iii LIST OF APPENDIXES ................................................................................iv INTRODUCTION Background ............................................................................................... 1 Aims and Scope of the Study .................................................................... 2 LITERATURES Marine Natural Products ........................................................................... 3 Marine Spo nge-Associated Bacteria ......................................................... 4 Bioactive Compounds ............................................................................... 6 Polyketide Synthase and Nonribosomal Peptide Synthetase .................... 8 MATERIALS AND METHODS Duration and P lace of Study ................................................................... 11 Materials.................................................................................................. 11 Extraction of Antimicrobial Compounds ................................................ 11 Antimicrobial Activity Test .................................................................... 12 Detection of Antimicrobial Compounds ................................................. 13 Fractionation of Bacterial Crude Extract ................................................ 13 Purification of Antimicrobial Compounds.............................................. 14 Morphological and Molecular Identification .......................................... 14 DNA Extraction ...................................................................................... 14 PCR Amplification of KS and A Domain............................................... 15 Cloning of DNA Fragments Encoding KS and A Domain ..................... 16 Sequencing a nd Bioinformatics Analysis of KS and A Domain ............ 16 RESULTS Antimicrobial Activity of Bacterial Crude Extracts ............................... 17 Thin Layer Chromatography and Bioautography ................................... 18 Fractionation of Bacterial Crude Extract from Isolate SAB E-41 .......... 20 Purification of Antimicrobial Compounds using Preparative TLC ........ 22 Morphological and Molecular Identification Based on 16S rDNA Analysis................................................................................................... 24 Amplification of DNA Fragments Encoding KS and A Domain ........... 26 Cloning a nd Bioinformatics Analysis ..................................................... 26 DISCUSSION ................................................................................................. 33 CONCLUSION AND SUGGESTION ......................................................... 45 REFERENCES............................................................................................... 47 APPENDIXES ................................................................................................ 53 LIST OF TABLES Page 1 Bacterial isolates identification from marine sponges ................................. 5 2 Bioactive compounds produced by marine sponges .................................... 7 3 Diameter average of inhibition zone (mm) from three bacterial crude extracts (100 mg/ml) produced by sponge-associated bacteria........ 17 4 Variation of Rf values from three bacterial crude extracts eluted with different solvent systems ................................................................... 18 5 Active spots/fractions of three bacterial crude extracts detected using bioautography method ....................................................... 20 6 Antimicrobial activity of thirty composite fractions collected from silica gel-column chromatography..................................... 21 7 Rf values of active compounds from fifteen composite fractions ............. 23 8 Similarity of 16S rDNA sequences from isolates SAB E-31 SAB E-41 and SAB E-57 compared with GenBank Database .................. 25 9 Bioinformatics sequences analysis of DNA fragments encoding KS domain using BlastX Program ............................................. 27 10 Bioinformatics sequences analysis of DNA fragments encoding A domain using BlastX Program ............................................... 28 LIST OF FIGURES Page 1 Organization of the multimodular PKS gene cluster in metagenomic libraries from D. dissolute ..................................................... 9 2 Two A domains ins ide the biosynthetic gene cluster of Onnamide B ................................................................................................. 9 3 Flowchart of procedural steps used in this study ....................................... 12 4 Antimicrobial activity of three bacterial crude extracts isolates SAB E-31, SAB E-41 and SAB E-57 using agar diffusion method .......... 18 5 Profile of each bacterial crude extract on silica gel plate merck 60 F 254 eluted with n-butanol and ethyl acetate mixture (3:7).................. 19 6 Antimicrobial activity of active spots/fractions using bioautography method ................................................................................ 19 7 Antimicrobial activity of thirty compo site fractions .................................. 22 8 Profile of active compounds from fraction BA-13, BA-17 and BA-18 o n silica gel plate merck 60 F 254 .................................................... 24 9 Antimicrobial activity of fifteen composite fractions purified using PTLC Technique .............................................................................. 24 10 Gram staining o f three bacterial isolates coded as: A). SAB E-31; B). SAB E-41 and C). SAB E-57............................................................... 24 11 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57 with the reference strains ba sed o n 16S rDNA sequences......................... 25 12 Agarose gel electrophoresis of DNA fragments encoding KS domain and A Domain ......................................................................... 26 13 Restriction of recombinant plasmid digested with BamHI + XbaI A). pMD20-KS Domain and B). pMD20-A Domain ................................ 27 14 Alignment of amino acid sequences of KS domain from isolates SAB E-41 and SAB E-57 w ith the reference strains in GenBank Database using ClustalW program............................................................. 29 15 Phylogenetic tree of isolates SAB E-41 and SAB E-57 with the reference strains based on amino acid sequences of KS domain ......... 29 16 Alignment of amino acid sequences of A domain from isolates SAB E-31, SAB E-41 and SAB E-57 w ith the reference strains in GenBank Database using ClustalW program ........................................ 31 17 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57 with the reference strains based on amino acid sequences of A do main.................................................................................................... 31 LIST OF APPENDIXES Page 1 The 16S rDNA sequences of three marine bacterial isolates..................... 55 2 DNA sequences of KS domain of PKS gene from two bacterial isolates........................................................................................................ 57 3 DNA sequences of A domain of NRPS gene from three bacterial isolates........................................................................................................ 58 4 Alignment of 16S rDNA sequences from isolate SAB E-31 using BlastN program ................................................................................ 60 5 Alignment of 16S rDNA sequences from isolate SAB E-41 using BlastN program ................................................................................ 62 6 Alignment of 16S rDNA sequences from isolate SAB E-57 using BlastN program ................................................................................ 64 7 Plasmid map of T-Vector pMD20 (TaKaRa Bio Inc.) .............................. 66 8 Alignment of DNA sequences encoding KS domain of PKS gene from isolate SAB E-41 using BlastX program .......................................... 67 9 Alignment of DNA sequences encoding KS domain of PKS gene from isolate SAB E-57 using BlastX program .......................................... 68 10 Alignment of DNA sequences encod ing A do main o f NRPS gene from isolate SAB E-31 using BlastX program .......................................... 69 11 Alignment of DNA sequences encoding A domain of NRPS gene from isolate SAB E-41 using BlastX program .......................................... 70 12 Alignment of DNA sequences encoding A domain of NRPS gene from isolate SAB E-57 using BlastX program .......................................... 71 INTRODUCTION Background The improper and uncontrolled uses of antibiotics against pathogenic bacteria induced and accelerated the occurrence of multi drugs resistant (MDR) strain. The number of infection cases by MDR strains in Indonesia remains high. Some of infection cases were tuberculosis, HIV, malaria, diarrhea and infection on upper respiratory system (Ditjen PP & PL Depkes RI 2011). In 2009, Indo nesia was ranked eighth of 27 countries with the highest of multi drugs resistant cases in the world (WHO 2010). The increase of MDR cases has encouraged many scientists to find the new bioactive compounds in order to solve the MDR problem. Nowadays, the exploration of bioactive compounds has been carried out in many kinds of resources such as medicinal plants, animals, aquatic organisms as well as microorganisms in unique ecosystem in order to find the new bioactive compounds which can treat the MDR strains. Indonesia was one of the hotspot countries possessing many natural resources and almost 70% of the area was covered by coastal area. Considering that, the exploration of new bioactive compounds in an aquatic area is very promising for the new inve ntion of chemotherape utic agents which can be developed and applied in pharmaceutical industry in the future. Marine sponges are one of the evolutionary multicellular organisms that have been reported very potential in producing many kinds of bioactive compounds. Some of the bioactive compounds showed antibacterial, antifungal, antiviral, anticancer, antifouling and cytotoxic properties (Taylor et al. 2007). The limitation of sponge biomass is the main factor for isolating the large scale of bioactive compounds. Therefore, alternative and ecologically sound sources of bioactive compounds are needed. Marine microorganisms have contributed to the majority of bioactive compounds. They can produce the same metabolite compo unds as their host (Proksch et al. 2002). The surfaces and internal spaces of marine sponges are unique microhabitat and more nutrient rich than seawater or most sediments, thus they would likely be a unique niche for the isolation of diverse microorganisms (Friedrich et al. 1999). Isolation and screening the potent bacterial isolates from marine sponge, identifying the antibiotic-encoding genes in active microorganisms, cloning in amenable host and characterize the bioactive compounds were the main strategy for producing large amounts of new metabolites (Webster & Hill 2001). Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 have been isolated from sponge Jaspis sp. at Waigeo Island, Raja Ampat District, West Papua Province. Each of these isolates indicated different activity against Staphylococcus aureus, Escherichia coli, enteropathogenic E. coli K1-1, Pseudomonas aeruginosa, Candida albicans and C. tropicalis by using multilayer technique (Abubakar 2009). Extraction, fractionation and purification of antimicrobial compounds for these isolates were important for this study in order to characterize their antimicrobial compounds. Analysis of 16S rDNA and detection of KS domain of PKS and A domain of NRPS genes were important for identifying these bacteria as well as ensuring their capability in synthesizing the bioactive compounds. Aims and Scope of the Study The aims of this study were to determine the antimicrobial activity of bioactive compounds, analyze the 16S rDNA and detect the occurrence of KS and A domain of PKS and NRPS genes of those three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 that symbiosis with sponge Jaspis sp. LITERATURES Marine Natural Products The ocean covers more than 70% of earth surface and is considered as a great reservoir of natural resources. However, the extent of marine biodiversity, especially of microorganisms, is barely known. Marine microbial communities are composed of ubiquitous members that can be found not only in the sur face waters of the sea, but also in the lower and abyssal depths from coastal to the offshore regions (Larsen et al. 2005). Several studies have repor ted the discovery of new bioactive compounds from marine organisms, focusing mainly on chemistry of secondary metabolites, which include now more than 15,000 structurally diverse bioactive compounds isolated during the last 30 years (Salomon et al. 2004). Given the diversity of marine or ganisms and habitats, marine natural products encompass a wide variety of chemical classes such as terpenoid, polyketides, acetogenins, peptides and alkaloids of varying structures representing biosynthetic schemes of stunning variety (Wright 1998). Marine sponges are one of the benthic organisms that play a potential role as natural compounds producer. They also became a host for a wide range of microbes. The role of these diverse microbes in sponge biology varies from the digestion of microbes as a food source to mutualistic symbiosis with the spo nge. On the other hand, sponge is believed to provide shelter from predators, a substrate for colonization, access to sunlight for photosynthetic microbes and a supply of nutrients (Taylor et al. 2007). The availability of sponge biomass is the main factor for isolating marine natural prod ucts. Therefore, marine microorganisms which associated with marine sponges became one of the alternative ways to solve that problem. They have contributed to the majority of marine natural prod ucts and produced the same metabolite compounds as their hos t. Friedrich et al. (2001) reported that many sponges contain enormous amounts of bacteria within their tissues, sometimes occupying 40 to 60% of the total biomass (equivalent to 108 to 1010 bacteria per gram). Considering to the rich diversity of microorganisms in their tissues and the growth of microorganisms were more rapidly, therefore isolation and cultivation of associated microorganism producer of bioactive compounds could help to solve the recognized problem of development of potential sponge-derived drugs. Convincing evidence for the involvement of microorganisms in natural product synthesis has been complied for the tropical sponges Dysidea herbacea and Theonella swinhoei, in which the producing microbe is a cynobacterium in the former and a bacterium in the latter (Proksch et al. 2002). Thus an alternative strategy targeting the microorganisms associated with sponges for the screening of bioactive natural products may prove to be an effective approach to circumvent the associated difficulties of dealing with the organism itself. Marine Sponge -Associated Bacteria Sponges are filter feeders animal which live in areas with strong c urrents or wave action. Most carnivorous animals avoid sponges because of the splinter-like spicules and toxic chemicals produced/sequestered by the sponge. Sponges are organized around a system of pores, ostia, canals and chambers that are used to canalize the large flow of water that is pumped through spo nges. The water enters the sponge through the inhalant canals and exits by the oscules. A sponge is constituted of three layers. The first layer comprises pinacocytes and is called the pinacoderm. Under the pinacoderm is the mesohyl region that contains canals and choanocyte chambers. This is where the sponge metabolisms, reproduction and nutrient transfer occur. The third layer is the choa nod erm and contains choanocytes. They are flagellated cells possessing a collar of cytoplasmic tentacles. It is through the movement of these tentacles that the flow of water is created bringing in nutrients (Wilkinson 1992). There are mainly three classes of sponges, namely the Calcarea (5 orders and 24 families), Demospongiae (15 orders and 92 families) and Hexactinellida (6 orders and 20 families). So far about 15.000 species of sponges have been described, but their true diversity may be higher (Fieseler et al. 2004). Most of the species are placed under the class Demospongiae. Since sponges are simple and sessile organisms; during e volution they have de velope d potent chemical defensive mechanism to protect themselves from competitors and predators as well as infectious microorganisms (Belarbi et al. 2003). Interaction between marine sponges and living aquatic microorganisms are so variously and had many important roles. Many microorganisms were found that growth commensally in the surface and also found inside of the other microorganisms such as in the food digestive system. Porus from sponges contain diverse bacteria. Reinheimer (1991) found that there were many kinds of bacteria such as Pseudomonas, Bacillus, Micrococcus, Aeromonas, Vibrio, Achromobacter, Flavobacterium and Corynebacterium in Microcionia prolifera sponge. There is a symbiotic connection between sponges and a number of bacteria and algae. Sponges give protection for the symbionts and the symbionts give nutrition for sponges. Algae that symbiotic with sponges give nutrient from their photosynthesis product (Taylor et al. 2007). Suryati et al. (2000) reported that the formation of bioactive compounds from sponges was depend on the precursor of enzyme, nutrient and product of symbiotic with another biota that contain bioactive compounds such as bacteria, mold and another kinds of dinoflagellata that can spur on producing bioactive. Suryati et al. (2000) found a number of sponge types living in Spermonde seawater, South Sulawesi, the diversity of mold and symbiotic bacterial with sponges are so variously and usually dominated by Aeromonas, Flavobacterium, Vibrio, Pseudomonas, Acinetobacter and Bacillus (Table 1). Table 1 Bacterial isolates identification from marine sponges (Suryati et al. 2000) No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Marine Sponges Acanthela clethera Aplisina sp. Callyspongia sp. Clathria bacilana Clathria reinwardhi Jaspis sp. Phakelia aruensis Phyllospongia sp. Reniochalina sp. Thionella cilindrica Stylotella aurantiorum Xestospongia sp. Bacterial species Flavobacterium sp., Aeromonas sp. Aeromonas sp. Pseudomonas sp. Aeromonas sp. Aeromonas sp. Flavobacterum sp. Bacillus sp., Aeromonas sp. Vibrio sp., Pseudomonas sp., Aeromonas sp. Acinetobacter sp. Aeromonas sp. Aeromonas sp., Vibrio sp. Enterobacteriaceae sp., Aeromonas sp. Experimental evidence suggests that there are qualitative and quantitative variations in secondary metabolites produced by some organisms. There is a tende ncy to explain these variations ecologically with environmental factors influencing the biochemical profile of the organisms. There are many examples of marine invertebrates with chemical defenses. The synthesis and storage of these substances favors the survival of such organisms in a complex environment and gives such species a selective advantage due to the genetic transmission of the capacity for chemical defense synthesis (Chr istop hersen 1991). Menezes et al. (2010 ) reported that microbial diversity associated with algae, ascidians and sponges from the north coast of Sao Paulo State, Brazil had been dominated by phylum Firmicutes, Bacillus spp., together with Ruegeria spp. Bacillus was the most abundant genus recovered, with 33 isolates, followed by Ruegeria with 31 isolates and Micrococcus with 23 isolates. All of them revealed broad distribution among the marine macroorganisms sampled. 16S rDNA sequencing-based analysis showed that marine-derived bacteria were related to 41 genera distributed among the phyla Proteobacteria (35.4%), Actinobacteria (30.4%), Firmicutes (28.7%) and Bacteroidetes (1.1%). Bioactive Compounds Marine sponges are pre-eminent producers of bioactive secondary metabolites and their repertoire includes peptides, terpenes and sterols. Many of these compounds showed a functional diversity of actions including antimicrobial, antiviral and cytotoxic activities (Table 2). Bioactive compounds of sponge origin have been used as basic for the synthesis of analogs, for example is glycolipids produced by bacteria that live associated with the marine spo nge Agelas sp. and the antibacterial agelasines isolated from the marine sponge Agelas nakamurai (Bakkestuen et al. 2005). Kimura et al. (1998) had isolated 1-Methyherbipoline from Halisulfate-1 and suvanin as a serine protease inhibitor from Coscinoderma mathewsi sponge. Bioactive compounds such as macrocyclic peptide had isolated from Theonella swinhoei comes from water area at Japan. These bioactive compounds known as Cyclotheonamida A and B that have inhibitory activity to serine protease like thrombin and contains vinylogous tyrosine (V-Tyr) and α-ketoarginine residu which is still unknown amino acid in nature. Table 2 Bioactive compounds produced by marine sponges (Soediro 1999; Simmons et al. 2005) Pharmaceutical Activity Cytotoxic Bioactive Compounds Types of Sponge 3,6 epoksieikosa acid Swinholida A Vaskulin Halisilindramida A Jasplakinolide Jaspicamides Hymeniacidon hauraki Theonella swinhoei Cribrocalina vasculum Halichondria caveolata Jaspis johnstoni Jaspis sp. Anticancer Agelasfin (AGL) Agelas muritianus Anti blood cancer Kurasin A Amfidinolid B1, B2, B3, N, Q Triangulinat acid Lingbya majuscule Amphidinium sp. Pellina triagulata Antiviral (HIV 1) Trikendiol Trikentrion loeve Antimicrobial Hormotamnim Diskodermin E-H Wondosterols Hormothamnion Discodermia kiiensis Jaspis wondoensis Antibacterial Lokisterolamin A and B Corticium sp. Antifungal kortikatat acid A,B,C Leukasandrolida Halisilindramida Petrosia corticata Leucasandra caveolata Halichondria cylindrical Imunomodulator Agelasflin 10 and 12 Agelas muritianus Anti-inflammatory Manualida Luffariella variabilis Unknown substances (still research) Halisiklamina A Bastadin A and B Klatirimin Halisiklamina B Haliclona sp. Lanthella basta Clathria basilana Xestrospongia sp. O’Keefe et al. (1998) had isolated Adociavirin from Adocia sp. sponge at Bay water area, New Zealand; extract that dissolved in distillation water potential as antisitopatic inside of CEM-SS cell which infection from HIV-1. Matsunaga et al. (1992) had been isolated 1-acid carboxymethylnicotinic from sponge Antosigmella raromicroscera which can be used as protease inhibitor. Li et al. (2006a) had been isolated 399 bacteria from the sponges Stelletta tenuis, Halichondria rugosa, Dysidea avara, and Craniella australiensis in the South China Sea, among which, 13 isolates from S. tenuis, 42 from H. rugosa, and 20 from D. avara showed pronounced broad-spectrum antimicrobial activities and enzymatic potentials. Many of the pharmacologically most promising natural products from sponges are complex polyketides. The fact that polyketide synthases (PKSs) are almost absent in metazoans suggests a microbial origin. PKSs are therefore a particularly good study object to investigate the role of symbionts in the chemistry of marine sponges (Castoe et al. 2007). Polyketide Synthas e and Nonribos omal Peptide Synthetas e The structural characteristics of marine natural products have revealed that they mainly belong to two important chemical families, namely, polyketides and cyclopeptides, and are synthesized by multifunctional enzymes called polyketide synthases (PKSs) and nonribosomal peptide synthetase (NRPSs). Polyketides are a group of secondary metabolites, exhibiting remarkable diversity in their structures and functions. Polyketide natural prod ucts are known for their wide range of pharmacologically important activities, including antimicrobial, antifungal, antiparasitic and antitumor properties (Hill 2005). Nonribosomal peptides are part of a family of complex natural products built from simple amino acid monomers. They can be found in bacteria and fungi where they are synthesized by nonribosomal peptide synthetase (NRPS) which are large multimodular and multifunc tional proteins. Nonribosomal peptides as well as the hybrid products are of much interest because of their pharmaceutical properties such as the immunosuppressant cyclosporine (Schwartzer et al. 2003). Schirmer et al. (2005) had already characterized PKS gene cluster in metagenomic libraries from Discordemia dissolute. The PKS gene cluster is 110 kb and contain of three open reading frames (ORF). The first PKS ORF codes for a remarkably large protein of 25.572 amino acids with a predicted molecular mass of 2.7 MDa. The most remarkable features of this large PKS gene cluster are the presence of a complete set of reductive domains (ketoreductase, dehydratase, and enoylreductase) in all except one module, which lacks the ER, and Cmethyltransferase domains in 8 of the 14 modules. The products of the PKS gene clusters have more similarity with fatty acids (Figure 1). Figure 1 Organization of the multimodular PKS gene cluster in metagenomic libraries from D. dissolute (Schirmer et al. 2005). Nguyen (2009) had already investigated the polyketides biosynthetic pathway in the spo nge Theonella swinhoei and the beetle Paederus fuscipes. The adenylation (A) domain of an NRPS module is responsible for specific selection and activation of a defined amino acid. Expression of A domains should help us gain insights into the biosynthetic pathways of pederin and onnamides. There were two A domains on the module PedF2 and PedH6 of the ped gene cluster and also two A domains on the module OnnI2 and OnnJ4 of the onn gene cluster. In agreement with the structure of onnamide B, the prediction results showed that glycine and arginine were the specific amino acids of NRPS modules on the onn gene cluster (Figure 2). Figure 2 Two A domains inside the biosynthetic gene cluster of Onnamide B (Nguyen 2009). MATERIALS AND METHODS Duration and Place of Study This research was carried out from July 2011 to March 2012. Extraction, fractionation and purification of antimicrobial compounds were carried out in the laboratory of Microbiology, Department of Biology and Biopharmaca Research Center, IPB, Indonesia. Molecular genetic analysis was carried out in Yohda Laboratory, Department of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology (TUAT), Japan. The flowchart of method s that used in this study is given in F igure 3. Materials Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 had been isolated from sponge Jaspis sp. at Waigeo Island, Raja Ampat District, West Papua Province by Abubakar (2009). These bacteria were used for the extraction, fractionation and purification of antimicrobial compounds. Specific primer 63 f (5CAGGCCTAACACATGCAAGTC-3) and 1387r (5-GGGCGGWGTGTACAAG GC-3) was used for analysis of 16S rDNA (Marchesi et al. 1998). Degenerate pr imer (f: 5-GCSATGGAYCCSCARCARCGSVT-3); (r: 5-GTSCCSGTSCCRTG SSCYTCSAC-3) for KS domain and degenerate primer (f: 5-AARDSIGGIGSIG SITAYBICC-3); (r: 5-CKRWAICCICKIAIYTTIAYYTG-3) for A domain (Schirmer et al. 2005). Extraction of Antimicrobial Compounds Each of those three isolates was sub-cultured in 500 ml Seawater Complete Broth media (bacto peptone 2.5 g, yeast extract 0.5 g, glycerol 1.5 ml, seawater 375 ml and distilled water 125 ml) and incubated in fluctuate incubator with 100 rpm at 300C until the culture reached the stationary phase. After that, 10% liquid bacterial inocula from previously incubation were cultured in 500 ml SWC broth and incubated in the same condition until reached the stationary phase for secondary metabolite production (Muller et al. 2004). Extraction of antimicrobial compounds was done by modifying the method from Sunaryanto et al. (2010). As much 500 ml of liquid bacterial culture were mixed with 500 ml of ethyl acetate solvent, incubated at room temperature for 24 hours and stirred for 2 hours with 250 rpm. These mixtures were separated and the ethyl acetate layers were evaporated with rotary evaporator until the drying residue was obtained as crude extract. The crude extract of bacterial cells were dissolved with ethyl acetate (pro analyze) to get 100 mg/ml concentration. Extraction of Antimicrobial Compounds DNA Extraction Antimicrobial Activity Test PCR Amplification of Ketos ynthase (KS) and Adenylation (A) Domain Detection of Antimicrobial Compounds Fractionation of Bacterial Crude Extract Cloning of DNA Fragments Encod ing KS and A Domain Sequencing and Bioinformatics Analysis of KS and A Domain Purification of Antimicrobial Compounds Morphological and Molecular Identification Figure 3 Flowchart of procedural steps used in this study. Antimicrobial Activity Test Antibacterial and antifungal activities from crude extracts were tested by using a gar diffusion method a gainst microbial test strains. As much 100 µl bacterial crude extracts dissolved in ethyl acetate (pro analyze) were applied carefully into 6 mm paper disks (Whatman) and at the same time, the disks were dried up using a hairdryer at 400 C. After that, the disks were sterilized under UV light for 2 hours and put into agar plate that have been seeded with 1% (v/v) of microbial test strains (conc entration 1x10 6 CFU/ml, OD 620 0.45). The plate was incubated at 40 C for 3 hours to optimize the diffusion of bacterial crude extract into media. This assay was carried out in triplicate. The diameters of inhibition zones were measured in millimeters after incubation for 24 hours at 370 C. Control disks were soaked with ethyl acetate solvent and prepared in the same manner (Sudirman 2010). Detection of Antimicrobial Compounds Antimicrobial compounds in each of the bacterial crude extract were detected using bioautography method. As much 10 µl of crude extracts were spotted on TLC plates (MERCK Silica Gel 60 F 254 ) and eluted with vertical chromatography using n-butanol : ethyl acetate solvent mixture with the ratio of 3:7 (v/v). The spots on TLC plate were detected under UV light at 254 nm and 366 nm wave- length. After that, the retardation factor (Rf) values were calculated. The spots on TLC plate were cut off and dried up in room temperature. The developed TLC plates were sterilized under UV light for 1 hour before covered by 15 ml of melting SWC (450 C) containing test strains, and incubated at 37 0 C for 24 hours. Diameter of inhibition zone around the chromatogram indicated that the spo t was an active fraction (Sudirman 2005) . Fractionation of Bacterial Crude Extract Bacterial crude extract of isolate SAB E-41 was fractionated using semi automated flash chromatography (Buchi Pump Controller C-610). As much 3 g of crude extract was dissolved with chloroform- methanol solvent mixture (90%-10%) and injected into silica gel-column chromatography (column dimension 0.40 x 150 mm, particle size of silica gel 40 x 63 µm). Chloroform- methanol solvent mixture (90%-10%) was flowed into silica gel-column chromatography with the flow rate of 3.5 ml/minutes. The polarity level in the column was increased slowly by changing the methanol concentration from 20%, 30%, 50%, 70% until 90%. Two hundred and five fractions were collected (5 ml/each fraction) from fraction collector and combined into thirty fractions based on the same chromatogram. These fractions were dried up and dissolved with chloroform- methanol-ethyl acetate solvent (1:1:1) to test their antimicrobial activity. Purification of Antimicrobial Compounds Fifteen active fractions were further purified using preparative thin layer chromatography (PTLC) technique. Each of the active fractions was spotted onto silica gel plate (MERCK Silica Gel 60 F 254 ; 0.1 mm thickness) and eluted with nbutanol-ethyl acetate solvent mixture (3:7). The active spots on the silica gel plate were detected under UV light at 254 nm and 366 nm wave-length. The active spots were extracted directly from the silica gel plate and dissolved with chloroformmethanol-ethyl acetate solvent mixture (1:1:1). The active fractions that were purified by preparative TLC were tested for their antimicrobial activity. Morphological and Molecular Identification Morphological characterization of those three isolates was performed using Gram staining p rocedure. Molecular analysis of 16S rDNA was done using specific primer, 63f (5-CAGGCCTAACACATGCAAGTC-3) and 1387r (5-GGG CGGWGTGTACAAGGC-3) (Marchesi et al. 1998). The PCR cycling condition for 16S rDNA was carried out under the following condition such as initial denaturation at 94 0 C for 5 min, followed by 30 cycles of denaturation at 940 C for 1 min, annealing at 550 C for 1 min, elongation at 720 C for 1 min and post PCR at 720C for 10 min. PCR products of 16S rDNA were purified using GENECLEAN ® II Kit. These PCR products were sub-cloned into T-Vector pMD20 and transformed into competent E. coli DH5-α using heat shock method (Sambrook & Russell 2001). Afterwards, several steps such as PCR colony, isolation and restriction of recombinant plasmid, PCR sequencing and purification of PCR products were done before the 16S rDNA sequence analysis. DNA Extraction Each of bacterial isolates were sub-cultured into SWC broth media and incubated at room temperature for 24 hours. After that, 1.5 ml bacteria isolate was drawn into microtube and centrifuged (18.000xg) for 10 min to obtain bacteria pellet that were used for DNA extraction. The supernatant was discarded and 250 µl Tris-EDTA (TE) buffer was added and centrifuged at 8000 rpm for 10 min. The supernatant was discarded and pellet was re-suspended three times in TE buffer. As much 250 µl TE and 5 µl lysozyme were added together and microtube slowly inverted to allow mixing and incubated at 370 C for 30 min. After incubation, the solution was added with 500 µl SDS 10% and 10 µl proteinase K and incubated again at 370 C for 60 min. Afterwards, as much 80 µl NaCl was added together with 100 µl CTAB 10% and incubated at 650 C for 20 min. After incubation, added again the solution with 650 µl PCI and shake n strongly then centrifuged at 14.000 rpm for 10 min. The upper solution was transferred into a new microtube then 650 µl CI was added and centrifuged again in same condition. DNA was precipitated using absolute ethanol (2x vol) and Na acetate 3 M 0.1 vol and incubated overnight in freezer. After that, 1 ml ethanol 70% was added for final washing and centrifuged at 12.000 rpm for 10 min. The supernatant was discarded and pellet was air dried overnight. After this step, 20 µl of TE was added and the extracted DNA was stored at -200C for further applications (Sambrook & Russel 2001). PCR Amplification of KS and A Domain KS domain of PKS and A do main of NRPS genes from those three isolates were amplified using PCR primers such as degenerate KS domain (f: 5-GCSATG GAYCCSCARCARCGSVT-3); (r: 5-GTSCCSGTSCCRTGSSCYTCSAC-3) and degenerate A domain (f: 5-AARDSIGGIGSIGSITAYBICC-3); (r: 5-CKRWAICC ICKIAIYTTIAYYTG-3) (Schirmer et al. 2005). The PCR cycling condition for KS domain was carried out in three steps such as initial denaturation at 94 0 C for 5 min, followed by 35 cycles of denaturation at 940 C for 1 min, annealing at 500 C for 1 min, elongation at 720 C for 1 min 10 sec and post PCR at 720 C for 10 min. The PCR cycling condition for A domain was the same as for KS domain except for annealing which was carried out at 550 C for 1 min. In all cases, the reaction mixtures contained 4 µl dNTP mix (2.5 mM), 5 µl 10X Ex Taq buffer, primer forward and reverse (10 µM; each of 5 µl), 2 µl DNA template (500 ng/µl), 1 µl TaKaRa Ex TaqTM (5 units/µl) and 28 µl milli Q. The total volume of reaction mixtures was 50 µl. PCR products were analyzed using agarose gel electrophoresis 1% (b/v). Cloning o f DNA Frag me nts Encoding KS and A Domain Purification of PCR products from KS domain of PKS gene (700 bp) and A domain of NRPS gene (1000 bp) were carried out using GENECLEAN ® II Kit. Each of the purification prod ucts was sub-cloned into T-Vector pMD20 (TaKaRa) and transformed into competent E. coli DH5α using heat shock method (Sambrook & Russell 2001). Isolation of recombinant plasmid was performed using Mag ExtractorT M Quick Plasmid Miniprep kit (Toyobo, Japan). Restriction of recombinant plasmid was conducted using the combination of restriction enzymes such as BamHI (BioLabs) and XbaI (TaKaRa). The reaction mixtures contained 0.2 µl BamHI (5 units/µl), 0.2 µl XbaI (5 units/µl), 1 µl 10X NE buffer 4, 0.5 µl DNA template (500 ng/µl) and 8.1 µl milli Q. The total volume of reaction mixtures was 10 µl. The reaction mixtures were incubated at 370 C for 24 hours and the restriction product was analyzed using agarose gel electrophoresis 1% (b/v). Sequencing a nd Bioinformatics Analysis of KS and A Domain DNA fragments that were inserted into plasmid T-Vector pMD20 named pMD20-KS domain and pMD20-A domain were used for the sequencing process. M13 primer RV and M13 primer M4 were used for PCR sequencing. The PCR cycling condition was carried in three steps such as initial denaturation at 960 C for 5 min, followed by 25 cycles of denaturation at 960 C for 1 min, annealing at 500 C for 30 sec, elongation at 60 0 C for 1 min and post PCR at 40 C for an unlimited time. The Big Dye® X TerminatorT M purification kit was used to purify the PCR product before DNA sequencing. The DNA was run in an automated DNA sequencer using ABI 3130 XL Genetic Analyzer. The DNA sequences were compared to the database available at NCBI using BlastN program for 16S rDNA and BlastX program for KS and A domain. Construction of phylogenetic tree was carried out using MEGA5 program with neighbor-joining method. RESULTS Antimicrobial Activity of Bacterial Crude Extracts Antimicrobial compounds of three bacterial isolates can be extracted using ethyl acetate solvent. During the extraction process, the ethyl acetate solvent will be formed two layers. First layer was aqueous phase that contained the antimicrobial compounds which have been extracted by ethyl acetate solvent. Second layer was organic phase that contained the bacterial cultures. The first layer was obtained and separated from bacterial cultures then evaporated with rotary evaporator until the drying residue was obtained as crude extract. Crude extracts of isolates SAB E-31, SAB E-41 and SAB E-57 showed different antimicrobial activity against non-pathogenic and pathogenic microbes. The bacterial crude extract of isolate SAB E-41 showed better antimicrobial activity than bacterial crude extracts of isolates SAB E-31 and SAB E-57. Crude extracts of those three isolates demonstrated the best activity against S. aureus (Table 3). Table 3 Diameter average of inhibition zone (mm) from three bacterial crude extracts (100 mg/ml) produced by sponge-associated bacteria Bacterial Isolates SAB E-31 SAB E-41 SAB E-57 Positive Control (Ampicilin 100 mg/ml) Negative Control (Ethyl acetate) BS* 8.3 10.5 9.5 Diameter Average of Inhibition Zone (mm) SA** EC* PA** EPEC K1-1** CA** CT** 10.5 5.3 4.8 5.6 2.5 2.5 14.5 7.5 6.7 8.7 5.7 4.5 13.2 6.4 5.8 6.5 3.8 2.8 14 30 9 27 - - - - - - - - - - BS = B. subtilis; SA = S. aureus; EC = E. coli; PA = P. aeruginosa; EPEC K1-1 = Enteropa thogenic E. coli K1-1; CA = C. albicans; CT = C. tropicalis; * = nonpathogen; ** = pa thogen. Crude extracts of those three isolates showed different antimicrobial activity against EPEC K1-1, C. albicans and C. tropicalis whereas for positive control with ampicilin 100 mg/ml, there was no inhibition a gainst these pathogenic microbe s (Figure 4). Ethyl acetate solvent that used as a negative control didn’t inhibit the growth of non-pathogenic or pathogenic microbes. Figure 4 Antimicrobial activity of three bacterial crude extracts isolates SAB E-31, SAB E-41 and SAB E-57 using agar diffusion method; C+ = positive control (ampicilin 100 mg/ml); C- = negative control (ethyl acetate). Thin Layer Chromatography and Bioautography TLC analysis for three bacterial crude extracts showed that there were many spots in silica gel plate with many k inds of retardation factor (Rf) values (Table 4). The solvent system, n-butanol-ethyl acetate with the ratio of 3:7 had successfully separated the component of bacterial crude extract. Six spots and many kinds of Rf values were obtained from this solvent system (Figure 5). Table 4 Variation of Rf values from three bacterial crude extracts eluted with different solvent systems Solvent Systems Crude Extracts n-but : CH 3 COOH : SAB E-31 ddH 2 O SAB E-41 SAB E-57 (3:1:1) n-but : EtOAc : ddH 2 O (2:3:1) n-but : EtOAc (3:7) Number of Spots λ 254 λ 366 nm nm 3 2 3 2 3 2 Rf Values λ 254 λ 366 nm nm 0.80; 0.61; 0.31 0.80; 0.61 0.91; 0.72; 0.35 0.91; 0.72 0.88; 0.72; 0.35 0.88; 0.72 SAB E-31 SAB E-41 SAB E-57 3 4 4 2 2 2 0.91; 0.53; 0.44 0.91; 0.83; 0.67; 0.55 0.92; 0.85; 0.61; 0.50 0.91; 0.53 0.91; 0.83 0.92; 0.85 SAB E-31 6 3 0.88; 0.55; 0.23 SAB E-41 6 3 SAB E-57 6 3 0.88; 0.72; 0.62; 0.55; 0.44; 0.23 0.92; 0.77; 0.66; 0.60; 0.46; 0.27 0.91; 0.75; 0.65; 0.56; 0.45; 0.31 0.92; 0.60; 0.27 0.91; 0.56; 0.31 Figure 5 Profile of each bacterial crude extract on silica gel plate merck 60 F 254 eluted with n-butanol and ethyl acetate mixture (3:7); Red box: active spots/fractions. The spots/fractions which were detected under UV light at 254 and 366 nm wave- length were further tested for their antimicrobial activity using bioautography method. This method was able to quickly detect which spots/fractions were the active fractions or pollutant compounds in bacterial crude extract. Part of the silica gel plate was cut based on the separated spots/fractions and the different of Rf values in order to make the visualization of inhibition zones clearer. Diameter of inhibition zone around the chromatogram indicated that the spot was an active fraction (Figure 6). Bioautography detection resulted that at least 4 active spots/fractions showed antimicrobial activity against P. aeruginosa and 2 active spots/fractions which inhibited the growth of S. aureus (Table 5). Figure 6 Antimicrobial activity of active spo ts/fractions using bioautography method. Table 5 Active spots/fractions of three bacterial crude extracts detected using bioautography method Bacterial Isolates SAB E-31 SAB E-41 SAB E-57 Rf Values Microbial Test Strains PA** + ++ ++ ++ CA** - Rf 1 Rf 2 Rf 3 Rf 4 Rf 5 Rf 6 = 0.88 = 0.72 = 0.62 = 0.55 = 0.44 = 0.23 SA** - Rf 1 Rf 2 Rf 3 Rf 4 Rf 5 Rf 6 = 0.92 = 0.77 = 0.66 = 0.60 = 0.46 = 0.27 +++ +++ - ++ +++ +++ ++ - Rf 1 Rf 2 Rf 3 Rf 4 Rf 5 Rf 6 = 0.91 = 0.75 = 0.65 = 0.56 = 0.45 = 0.31 +++ + - ++ +++ +++ ++ - + = Weak inhibition; ++ = Medium inhibition; +++ = Strong inhibition; SA = S. aureus; PA = P. aeruginosa; CA = C. albicans; ** = pathogen. Fractionation of Bacterial Crude Extract from Isolate SAB E-41 Two hundred and five fractions were successfully collected from the fractionation process and combined into thirty composite fractions based on the same chromatogram. These composite fractions were evaporated until the drying residue was obtained as crude extract. These crude extracts were dissolved with chloroform- methanol-ethyl acetate mixture (1:1:1) in concentration of 100 mg/ml. Antimicrobial activity of thirty compos ite fractions were tested to S. aureus, P. aeruginosa, EPEC K1-1, C. albicans and C. tropicalis. Fifteen composite fractions coded as BA-1, BA-2, BA-3, BA-4, BA-5, BA6, BA-7, BA-8, BA-11, BA-12, BA-13, BA-14, BA-15, BA-17 and BA-18 showed different antimicrobial activity against S. aureus, P. aeruginosa, EPEC K1-1 and C. albicans (Table 6). Fraction BA-2 that was eluted by chloroform- methanol (90%-10%) solvent system showed antifunga l activity against C. albicans whereas no active fractions showed antimicrobial activity against C. tropicalis. Table 6 Antimicrobial activity of thirty composite fractions collected from silica gel-column chromatography Solvent Systems and Fractions Chloroform-Methanol (90%-10%) Fraction BA-1 Fraction BA-2 Fraction BA-3 Fraction BA-4 Fraction BA-5 Fraction BA-6 Fraction BA-7 Chloroform-Methanol (80%-20%) Fraction BA-8 Fraction BA-9 Fraction BA-10 Chloroform-Methanol (70%-30%) Fraction BA-11 Fraction BA-12 Chloroform-Methanol (50%-50%) Fraction BA-13 Fraction BA-14 Fraction BA-15 Fraction BA-16 Fraction BA-17 Fraction BA-18 Chloroform-Methanol (30%-70%) Fraction BA-19 Fraction BA-20 Fraction BA-21 Fraction BA-22 Fraction BA-23 Chloroform-Methanol (20%-80%) Fraction BA-24 Fraction BA-25 Fraction BA-26 Fraction BA-27 Chloroform-Methanol (10%-90%) Fraction BA-28 Fraction BA-29 Fraction BA-30 Negative Control (Chloroform-Methanol) Positive Control (Ampicilin 100 mg/ml) Diameter of Inhibition zone (mm) SA** PA** EPEC K1-1** CA** CT** 7 8 7 3 5 4 4 2 6 4 3 4 4 2 7 10 3 10 3 2 3 3 - - 2 - - - - - 3 10 3 3 - - - 14 2 2 12 3 2 - 7 - - - - - - - - - - - - - 30 27 - - - SA = S. aureus; PA = P. aeruginosa; EPEC K1-1 = Enteropathogenic E. coli K1-1; CA = C. albicans; CT = C. tropicalis; ** = pathogen. Fraction BA-13 that was eluted by chloroform- methanol (50% -50%) solvent system demonstrated the highest inhibition against S. aureus followed by fraction BA-17. The diameter of inhibition zone that formed by these two active fractions were about 12 mm and 14 mm (Figure 7). Fraction BA-2 and BA-4 that was eluted by chloroform- methanol (90% -10%) showed the best activity against EPEC K1-1. The diameter of inhibition zone that formed by these two active fractions were about 10 mm (Figure 7). Figure 7 Antimicrobial activity of thirty compos ite fractions; C+ = positive control (ampicilin 100 mg/ml); C- = negative control (chloroform- methanol). Purification of Antimicrobial Compounds using Preparative TLC Fifteen composite fractions that had an active fraction were further purified using PTLC technique. Antimicrobial compounds in active fractions could be extracted directly from silica gel plate and dissolved with chloroform- methanolethyl acetate (1:1:1). Antimicrobial compounds of active fractions, obtained from PTLC technique were tested to S. aureus, EPEC K1-1 and C. albicans. Fraction BA-13 has demonstrated as the most antimicrobial compounds compared to the other fractions (Table 7). This fraction resulted four different of active compounds with Rf values of 0.87; 0.50; 0.41 and 0.12 (Figure 8). These four active compounds displayed significant activity against S. aureus and EPEC K1-1. Fractions BA-17 and BA-18 carried two kinds of active compounds with Rf values of 0.93; 0.12 for the BA-17 and 0.93; 0.25 for the BA-18 (Figure 8). Both of these active compounds showed activity against S. aureus. Fraction BA-2 carried one active compound (Rf 0.77) that showed activity against C. albicans (Figure 9). Table 7 Rf values of active compounds from fifteen composite fractions Active Compounds BA-1 BA-2 BA-3 BA-4 BA-5 BA-6 BA-7 BA-8 BA-11 BA-12 BA-13 BA-14 BA-15 BA-17 BA-18 Rf Values Rf 1 = 0.81 Rf 1 = 0.77 Rf 1 = 0.78 Rf 1 = 0.87 Rf 2 = 0.66 Rf 1 = 0.81 Rf 2 = 0.53 Rf 1 = 0.87 Rf 2 = 0.65 Rf 3 = 0.35 Rf 1 = 0.87 Rf 2 = 0.62 Rf 3 = 0.38 Rf 1 = 0.90 Rf 2 = 0.71 Rf 3 = 0.35 Rf 1 = 0.87 Rf 2 = 0.68 Rf 1 = 0.90 Rf 2 = 0.71 Rf 3 = 0.62 Rf 4 = 0.41 Rf 5 = 0.12 Rf 1 = 0.87 Rf 2 = 0.72 Rf 3 = 0.60 Rf 4 = 0.50 Rf 5 = 0.41 Rf 6 = 0.12 Rf 1 = 0.90 Rf 2 = 0.75 Rf 3 = 0.68 Rf 4 = 0.58 Rf 1 = 0.93 Rf 2 = 0.78 Rf 3 = 0.68 Rf 4 = 0.33 Rf 1 = 0.93 Rf 2 = 0.71 Rf 3 = 0.68 Rf 4 = 0.33 Rf 5 = 0.12 Rf 1 = 0.93 Rf 2 = 0.41 Rf 3 = 0.25 Rf 4 = 0.12 Diameter of Inhibition Zone (mm) SA** EPEC K1-1** CA** 4 4 8 14 2 2 2 6 4 10 2 10 2 6 4 8 3 10 3 10 4 6 8 4 12 2 2 2 14 2 2 2 - SA = S. aureus; EPEC K1-1 = Enteropathogenic E. coli K1-1; CA = C. albicans; ** = pathogen. λ 366 nm λ 366 nm Active Co mpounds (Rf 0.87) Active Co mpounds (Rf 0.93) λ 254 nm Active Co mpounds (Rf 0.50) Active Co mpounds (Rf 0.41) Active Co mpounds (Rf 0.12) BA-17 BA-13 Active Co mpounds (Rf 0.12) Active Co mpounds (Rf 0.93) Active Co mpounds (Rf 0.25) BA-18 Figure 8 Profile of active compounds from fraction BA-13, BA-17 and BA-18 on silica gel plate merck 60 F 254 (Arrow head). Figure 9 Antimicrobial activity of fifteen compos ite fractions purified using PTLC technique; C- = negative control (ethyl acetate). Morphological and Molecular Identification Bas ed on 16S rDNA Analysis Morphological characterization of isolates SAB E-31, SAB E-41 and SAB E-57 was carried out using Gram staining bacteria. Gram staining results showed that three marine bacterial isolates were rod-shaped, motile, formed spores and Gram-pos itive bacteria. These isolates have the same characteristics with the genus of Bacillus (Figure 10). 2 µm A. 2 µm B. 2 µm C. Figure 10 Gram staining of three bacterial isolates coded as: A). SAB E-31; B). SAB E-41 and C). SAB E-57. Molecular analysis of 16S rDNA was carried out for the further identification of those three isolates. PCR product of 16S rDNA sequences was about 1300 bp. Sequences analysis of 16S rDNA showed a high similarity level (97-98%) to various strains of Bacillus compared to those available in GenBank database (Table 8). Table 8 Similarity of 16S rDNA sequences from isolates SAB E-31, SAB E-41 and SAB E-57 compared with GenBank Database Bacterial Isolates SAB E-31 SAB E-41 SAB E-57 Similarity Bacillus pumilus strain KD3 Bacillus amyloliquefaciens strain zy2 Bacillus subtilis strain YRL02 Identity (%) 98 98 97 EValue 0.0 0.0 0.0 Accession EU500930.1 JN160740.1 EU373407.1 Phylogenetic analysis of 16S rDNA sequences showed that isolates SAB E31, SAB E-41 and SAB E-57 formed a different clade with the reference strains of Bacillus in GenBank database. Clade-1 was formed by the reference strains of Bacillus while clade-2 was formed by those three isolates with Bacillus sp. DF49 (Figure 11). Figure 11 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57 with the reference strains based on 16S rDNA sequences. Numbers at the node s indicate the levels of bootstrap support based on 1000 re-sampled data sets. The scale bar indicates 0.1 substitutions per nucleotide pos ition. Amplification of DNA Frag me nts Encoding KS and A Domain DNA fragments of three bacterial isolates encoding KS and A domain were successfully amplified using PCR. Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 had a DNA fragment encoding A do main whereas only two bacterial isolates coded as SAB E-41 and SAB E-57 had a DNA fragment encoding KS do main. Visualization of the PCR amplicon for DNA fragments was analyzed using agarose gel electrophoresis 1% (b/v). DNA fragment encoding KS domain was in a size of 700 bp while for A domain was in a size of 1000 bp SAB E-57 SAB E-41 SAB E-31 SAB E-57 SAB E-41 SAB E-31 bp 1 kb Ladder (Figure 12). 1 kb 1000 750 500 250 A Do main (~1000 bp) KS Do main (~700 bp) Figure 12 Agarose gel electrop horesis of DNA fragments encod ing KS Domain and A Domain. Cloning a nd Bioinformatics Analys is DNA fragments of those three isolates encoding KS and A domain were successfully cloned into T-Vector pMD20 (TaKaRa Bio Inc.) and named pMD20KS domain and pMD20-A domain. White colonies of E. coli DH5-α carrying the recombinant plasmid had been isolated and for the plasmid excision was do ne using a combination of restriction enzymes, Bam HI and XbaI. The cropped recombinant plasmid DNA resulted in two bands which were approximately of 2736 bp that showed the size of T- vector pMD20 and 700 bp was the size of DNA fragment encoding KS domain and 1000 bp for DNA fragment encoding A do main (Figure 13). 19329 7743 6223 4254 3472 T-Vector 2690 pMD20 1882 (2736 bp) 1489 19329 7743 6223 4254 3472 2690 1882 1489 KS Do main (~700 bp) 421 SAB E-57 T-Vector pMD20 (2736 bp) A Do main (~1000 bp) 925 925 SAB E-41 SAB E-31 bp Control λ Marker SAB E-57 SAB E-41 Control λ Marker bp 421 A B Figure 13 Restriction of recombinant plasmids digested with BamHI + XbaI; A). pMD20-KS domain and B). pMD20-A do main. Bioinformatics sequences analysis of DNA fragment encoding KS domain from two bacterial isolates coded as SAB E-41 and SAB E-57 using BlastX program showed that isolate SAB E-41 had a similarity level of 97 % with type I PKS from Bacillus amyloliquefaciens LL3 while isolate SAB E-57 had a similarity level of 98% with putative polyketide synthase pksL from B. amyloliquefaciens subsp. plantarum CAU-B946 (Table 9). Table 9 Bioinformatics sequences analysis of DNA fragment encoding KS domain using BlastX program Bacterial Isolates SAB E-41 SAB E-57 Similarity Type I PKS; B. amyloliquefaciens LL3 Putative polyketide synthase pksL; B. amyloliquefaciens subsp. plantarum CAU-B946 Identity (%) 97 EValue 2e-130 YP_005545643.1 98 2e-143 YP_005130937.1 Accession Sequences analysis of DNA fragment encoding A domain showed that isolate SAB E-31 had a similarity level of 81% with bacitracin synthetase 1 from B. pumilus ATCC 7061. Isolate SAB E-41 had a similarity level of 80% with surfactin synthetase B from B. amyloliquefaciens subsp. plantarum CAU-B946 while isolate SAB E-57 had a similarity level of 81% with surfactin synthetase A from B. amyloliquefaciens subsp. plantarum CAU-B946 (Table 10). Table 10 Bioinformatics sequences analysis of DNA fragment encoding A domain using BlastX program Bacterial Isolates SAB E-31 SAB E-41 SAB E-57 Similarity Bacitracin synthetase 1; B. pumilus ATCC 7061 Surfactin synthetase B; B. amyloliquefaciens subsp. plantarum CAU-B946 Surfactin synthetase A; B. amyloliquefaciens subsp. plantarum CAU-B946 Identity (%) 81 EValue 1e-154 ZP_03054623.1 80 1e-139 YP_005129035.1 81 1e-129 YP_005129034.1 Accession Amino acid sequences of KS domain from isolates SAB E-41 and SAB E57 were aligned and compared with the reference strains in GenBank database. Two bacterial isolates have a homology of conserved region with the reference strains (Figure 14). Phylogenetic analysis of amino acid sequences of KS domain showed that isolates SAB E-41 and SAB E-57 formed a different clade with the other reference strains (Figure 15). Figure 14 Alignment of amino acid sequences of KS domain from isolates SAB E41 and SAB E-57 with the reference strains in GenBank Database using ClustalW program. Shaded area showed the similarity of amino acid sequences. Black bo x showed a homology of conserved region. Clade-1 Clade-2 Figure 15 Phylogenetic tree of isolates SAB E-41 and S AB E-57 with the reference strains based on amino acid sequences of KS domain. Numbers at the nodes indicate the levels of bootstrap support based on 1000 re-sampled data sets. The scale bar indicates 0.1 substitutions per nucleotide pos ition. Amino acid sequences of A domain from isolates SAB E-31, SAB E-41 and SAB E-57 were also aligned and compared with the reference strains in GenBank database. These bacteria have a homology of conserved region with the other reference strains (Figure 16). Phylogenetic analysis of amino acid sequences of A domain showed that isolate SAB E-41 was formed a similar clade with B. amyloliquefaciens FZB42 while isolates SAB E-31 and SAB E-57 formed a same clade (Figure 17). Figure 16 Alignment of amino acid sequences of A domain from isolates SAB E31, SAB E-41 and SAB E-57 with the reference strains in GenBank Database using ClustalW Program. Black box showed a homology of conserved region for amino acid sequences. Figure 17 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57 with the reference strains based on amino acid sequences of A domain. Numbers at the nodes indicate the levels of bootstrap support based on 1000 re-sampled data sets. The scale bar indicates 0.2 substitutions per nucleotide pos ition. DISCUSSION Antimicrobial compounds of three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 can be directly extracted using ethyl acetate solvent. The selection of this solvent system for the extraction process is based on the polarity difference of the liquid culture of bacteria. This solvent is non-pola r so that the separation of liquid culture of bacteria from ethyl acetate layer was more easily done. Jeffery et al. (1989) stated that antimicrobial compounds contained in bacterial crude extract have different solubility prope rties in each of solvent system. The selection of an appropriate solvent in the extraction process is largely determined by the solubility properties of the antimicrobial compounds, type of substrate, the partition coefficient and the distribution ratio of solvent system. Crude extracts of those three isolates showed different antimicrobial activity against non-pathogenic and pathogenic microbes. The highest activity shown by the bacterial crude extracts of isolate SAB E-41 which indicated from the large diameter of the inhibition zone. Lay (1994) stated that physical and chemical properties of antimicrobial compounds will affect the resulting of clear zone. The greater of the molecular weight of bioactive compounds, the more enlarge of the inhibition zone. The other factors that also affecting the inhibition zone are the density of cells, the sensitivity of microbial test strains to antimicrobial compounds, the component of antimicrobial compounds, the diffusion rate of antimicrobial compounds into media and the expos ure time of microbial test strains to antimicrobial compounds. On the other hand, crude extracts of those three isolates showed the highest activity against S. aureus whereas the lowest activity shown by the test strains of C. albicans and C. tropicalis. The antimicrobial compounds from three bacterial crude extracts couldn’t optimally inhibit the growth of pa thogenic fungal because of the lower concentration. For the comparison in tested their antimicrobial activity, ampicillin was used as a positive control whereas ethyl acetate solvent was used as a negative control. Ampicillin which included in - lactam group of antibiotics had a broad spectrum activity so it was chos e as a positive control for this study. This ant ibiotic unable to inhibit the growth of enteropathogenic E. coli K1-1 because of the - lactamase enzyme that prod uced by this microbe while interestingly, our crude extracts from three bacterial isolates which associated with sponge Jaspis sp. are able to inhibit the growth of this microbe as seen from the diameter average of inhibition zone. Anand et al. (2006) reported that antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India which guided fractionation of the broth showed that the ethyl acetate extract of strain SC3 demonstrated activity against the bacterial and fungal test strains. The strain SC3 showed the highest activity against test strains of B. subtilis and E. coli with an inhibition zone of 26 mm and for C. albicans of 15 mm. TLC analysis and bioautography test were performed for three bacterial crude extracts. The TLC analysis aims to separate the constituent components of bacterial crude extract based on the difference of absorption, partition and solubility of the chemical components that will be moved with the polarity of eluent whereas bioautography test aims to quickly detect which spots were the active compounds or impurities compounds. Detection of the spots was performed by administering the chromatogram plates under UV light with 254 nm and 366 nm wave- length. All of the detection methods that used in this study are expected to quickly detect the presence of active compo unds without needed to detect the spots one by one on silica gel plate. In this study, detection of active compounds was only performed with these methods so that not all patches of the active compounds contained in bacterial crude extract can be detected. Sudirman (2005) reported that the rapid detection of active compounds contained in bacterial crude extract can be done using bioautography method besides the UV irradiation and the spraying of color-forming reaction. Bioautography techniq ue is a combination of chemical methods (chromatography) with the microbiological method, the chromatogram plates covered with media that contained microbial test strains and incubated according to the growth temperature of test strains. Betina (1964) stated that bioautography method can be directly detected the activity and the minimum number of active compounds that contained in the bacterial crude extract. In addition, this method can be directly detected the specify location of active compounds based on the position or the Rf values on the chromatogram plate. Four active spots/fractions with the Rf values of 0.62; 0.55; 0.44; 0.23 (isolate SAB E-31), Rf 0.66; 0.60; 0.46; 0.27 (isolate SAB E-41) and Rf 0.65; 0.56; 0.45; 0.31 (isolate SAB E-57) were successfully detected using these methods and respectively showed antimicrobial activity against P. aeruginosa, while two active spots/fractions with the Rf values of 0.77; 0.66 (isolate SAB E-41) and Rf 0.75; 0.56 (isolate SAB E-57) respectively displayed antimicrobial activity against S. aureus. No active spots/fractions from isolates SAB E-31 can inhibit the growth of S. aureus. Banoet (2011) reported that at least one active spot/fraction was successfully detected using TLC analysis and bioautography detection. Two spots/fractions with the Rf value of 0.31; 0.81 from ethyl acetate extract of isolate HAL-13 and one spot/fraction with the Rf value of 0.85 from n-butanol extract of isolate HAA-01 and Rf 0.28 from the same extract of isolate HAL-74 displayed antimicrobial activity against S. aureus and enterop athogenic E. coli K1-1. Bacterial crude extract of isolate SAB E-41 was further fractionated using column chromatography techniques. The selection of this crude extract for further testing due to the be st activity against non-pathogenic and pathogenic microbes compared to the other bacterial crude extracts. Purification of antimicrobial compounds via column chromatography techniques is based on the polarity of eluent. Crude extract was slowly injected into the silica gel-column chromatography and this extract will be separated based on the difference of the polarity of eluent system that used d uring the elucidation process. Hurtubise (2010) mentioned that chromatography column was a classic chromatography method which used to separate the constituent components of antimicrobial compounds for the large quantities by adsorption and partition mechanism. The principles of this process were based on the different polarity of active compounds that contained in the bacterial crude extract. The right selection of stationary phase (absorbance) and mobile phase (eluent) in fractionation process will be determined the successful of the separation of crude extract. The solvent was allowed to flow through the column due to the gravity or the pressure. The compounds will move through the column at the different rates, separated and collected as the fractions out of the column base. Two hundred and five fractions were collected from the fraction collector. TLC analys is was performed to know the fractions which have the same chromatogram. Fractions with the same chromatogram can be combined into one fraction. A total of 30 composite fractions were successfully obtained after TLC analysis. Antimicrobial activity of thirty composite fractions was tested to P. aeruginosa, S. aureus, enteropathogenic E coli K1-1, C. albicans and C. tropicalis. Fifteen composite fractions coded as BA-1, BA-2, BA-3, BA-4, BA-5, BA-6, BA7, BA-8, BA-11, BA-12, BA-13, BA-14, BA-15, BA-17 and BA-18 showed different antimicrobial activity against non-pathogenic and pa thogenic microbes. Fraction BA-2 demonstrated a broad spectrum of inhibition compared to the other fractions. Fraction BA-13 displayed significant activity against S. aureus followed by fraction BA-17. Anand et al. (2006) mentioned that further fractionation of the ethyl acetate extract from strain CS3 was undertaken by reverse phase HPLC. Fifteen fractions had been successfully detected based on the HPLC trace. Fraction 11 was found to be active against test strains of E. coli, B. subtilis and C. albicans. Fractions 5 and 7 were found to have trace activity against E. coli. The remaining fractions were found that not possess antimicrobial properties. Crude extract of isolate SAB E-41 showed antifungal activity against C. albicans and C. tropicalis. After the fractionation process, the fractions are only active against the test strain of C. albicans. The active compounds in these fractions are allegedly lost due to the purification process. Antifungal activity of crude extracts from isolate SAB E-41 was a combination factor of several active compounds which can be lost during the fractionation process. Sudirman (2005) stated that during the purification process, most of the active compounds separated from one to another or the active compounds are separated from the pollutant compound so that the antimicrobial activity from bacterial crude extract can be different between before or after the purification process. Fifteen composite fractions that showed antimicrobial activity were further purified using PTLC techniques. The basic selection of this technique as the purification method was due to the less quantity of active compo unds. The principle of this technique was the active compounds that had already known their position of Rf values were scraped off from silica gel plate and dissolved in chloroform- methanol-ethyl acetate solvent mixtures (1:1:1). Antimicrobial activity test for active compounds, obtained from PTLC technique were tested to a representative group of Gram-positive, Gram- negative bacteria and yeast. Three kinds of pathogenic microbes that selected to test their antimicrobial activity were S. aureus, enteropathogenic E coli K1-1 and C. albicans. These microbes have an important aspect in public health because of their pathogenicity to humans. S. aureus is a group of Gram pos itive bacteria, aerobic facultative and cause skin infections or infection on the upper respiratory system while enteropathogenic E. coli K1-1 is a group of Gram- negative bacteria that are resistant to -lactam class of antibiotics because it possess a -lactamase activity enzyme. C. albicans is the pa thogenic yeast that causes candidiasis disease. Liu (2009) stated that S. aureus is one of the bacteria that cause minor skin infections, pneumonia and meningitis. This strain can be found at skin mucosal surface, nasal passage and gastrointestinal system. Other bacteria that also cause a seriously infection disease was EPEC K1-1. This strain can cause infection at gastrointestinal system. Budiarti (1997 ) found that 55% of diarrhea disease in Indo nesia, mostly caused by EPEC K1-1. C. albicans was a pathogenic strain that caused a serious problem of infection disease. This strain can be found normally at mucosal surface and urogenital system as human microflora. Many of genitally diseases or related to human immune systems diseases such HIV-AIDS are mostly caused b y this strain (Kortig et al. 1999). Fraction BA-13 that was eluted by chloroform- methanol (50% -50%) solvent system demonstrated as the most antimicrobial compound followed by fraction BA-17 and BA-18. Four active compounds with the Rf values of 0.87; 0.50; 0.41 and 0.12 respectively obtained from fraction BA-13 and each of those displayed significant activity against S. aureus and enteropathogenic E. coli K1-1. Two active compounds with the Rf values of 0.93; 0.12 were successfully obtained from fraction BA-17 while two active compounds with the Rf values of 0.93; 0.25 were successfully obtained from fraction BA-18. Both of the active compounds from fraction BA-17 and BA-18 have antimicrobial activity against S. aureus. Banoet (2011) had purified active compounds for bacterial crude extract of isolate HAL-13 which associated with sponge Haliclona sp. Fraction BS13-5 displayed as the most active compounds. Four active compounds with the Rf values of 0.35; 0.41; 0.72 and 0.87 were collected using PTLC technique. Two of these active compounds with the Rf values of 0.35 and 0.41 showed the best activity against enteropathogenic E. coli K1-1. The diameter of inhibition zone that formed by these two active compounds was 12 mm. Morphology of isolates SAB E-31, SAB E-41 and SAB E-57 was characterized using Gram staining procedure. The results showed that these bacteria were rod-shaped, Gram positive, motile and formed spores. The characteristic of these isolates are same with the genus of Bacillus. Anand et al. (2006) reported that the morphological and physiological characterization of strain SC3 isolated from India waters showed that it to be a Gram-positive, motile, catalase and oxidase-positive rod. Molecular identification of this strain also indicated it to be a member of the Bacillus genera. Molecular genetic analysis of 16S rDNA sequences were done for the further identification of those three isolates. Three bacterial isolates which associated with sponge Jaspis sp. were included in the genus of Bacillus based on 16 rDNA analysis. These sequences were in a size of approximately 1300 bp and have conserved and variable regions (Appendix 1). This gene is quite large with the polymorphism between species that can be used as a tool to distinguish between species (Woese 2006; Clarridge 2004). Analysis of 16S rDNA is an important standard for bacterial identification. This identification method was based on the most suitable sequences of bacteria with all of the 16S rDNA sequences that are known in the GenBank database. Partial analysis of 16S rDNA sequences showed that isolate SAB E-31 had 98% of homology level with B. pumilus strain KD3 (Accession No. EU500930.1) (Appe ndix 4), isolate SAB E-41 had 98% of homology level with B. amyloliquefaciens strain zy2 (Accession No. JN160740.1) (Appe ndix 5) and isolate SAB E-57 had 97% of homology level with B. subtilis strain YRL02 (Accession No. EU373407.1) (Appendix 6). Phylogenetic analys is of 16S rDNA sequences showed that three bacterial isolates formed a different clade with the other reference strains. First clade was dominated formed by the reference strains of Bacillus while second clade was formed by those three isolates with Bacillus sp. DF49. This strain was in the same clade with isolate SAB E-41. Isolate SAB E-57 formed a closely relationship clade with isolates SAB E-31, SAB E-41 and Bacillus sp. DF49. This phylogenetic result was different from the BlastN result. Although the result was different but three bacterial isolates were still include in the ge nus of Bacillus. The formation of different clade between three bacterial isolates and other reference strains meant that these isolates were assumed in a new species of Bacillus. Santos et al. (2010) reported that molecular identification by partial 16S rRNA gene sequencing and phylogenetic analysis showed that the majority of bacterial isolates isolated from Brazilian spo nges could be subdivided into three phylogenetically different clusters. Five strains were affiliated with Firmicutes (genera Bacillus and Virgibacillus), three with α-Proteobacteria (Pseudovibrio sp.) and four with Proteobacteria (Pseudomonas and Stenotrophomonas). Marine Bacillus species are often isolated from sediments, invertebrates and marine sponges (Pabel et al. 2003). The species of this genus is known to generate spores under adverse conditions, such as those encountered in marine ecosystems (Hentschel et al. 2001). In the marine environment, members of the genus Bacillus are known for their production of metabolites with antimicrobial, antifungal or generally cytotoxic property. They were regularly isolated from invertebrates and thus display a high potential in the search for new antimicrobial substances (Muscholl-Silberhorn et al. 2008). Many antibiotics including cyclic peptides, cyclic lipopeptides and novel thiopeptides have been reported from this strain (Nagai et al. 2003). Most of the bioactive compounds that produced by marine bacteria didn’t getting loose from the invo lvement of two multifunc tional enzymes named polyketide synthases (PKS) and non-ribo somal peptide synthetases (NRPS). These two multifunctional enzymes mostly involved in the biosynthesis of bioactive compounds. The simplest functional PKS mod ule consists of a ketosynthase (KS), an acyltransferase (AT), an acyl carrier protein (ACP) and a thioesterase (TE) domain (Schirmer et al. 2005). Besides that, the simplest NRPS module consists of an adenylation (A), a thiolation (T), a peptidyl carrier protein (PCP) and a conde nsation (C) do main (Schwarzer et al. 2003). In this study, the presence of KS and A domain in the cluster of PKS and NRPS genes from three marine bacterial isolates became one of the most important domains to be investigated. These two domains were analyzed using PCR amplification. PCR products that indicated the presence of KS domain will show the DNA fragment with the length of 700 bp (Appendix 2) whereas for A domain will show the length of 1000 bp (Appe ndix 3). Kim & Fuerst (2006) mentioned that the common feature of complex PKS gene is ketosynthase (KS) domain that usually present in each module and exhibits the highest degree of conservation among all domains. Likewise, Schirmer et al. (2005) stated that adenylation (A) domain become the most conserved domain of NRPS gene compared to the others. Therefore, the KS and A do main are especially well suited for phylogenetic analyses of PKS and NRPS gene diversity. Isolates SAB E-41 and SAB E-57 possessed DNA fragments encod ing KS and A do main in the cluster of PKS and NRPS genes whereas only isolate SAB E31 possessed DNA fragment encoding A do main in the cluster of NRPS gene. These meant that, by detecting one of the domains, whether KS domain of PKS gene or A domain of NRPS gene could be ensured that they can synthesize the bioactive compounds. Zhao et al. (2008) stated that the modular PKS and NRPS have been involved in natural prod uct synt hesis in many microorganisms. On the other hand, the presence of bo th KS and A domain in the cluster of PKS and NRPS genes at isolates SAB E-41 and SAB E-57 meant that these isolates ha ve the much broader potential in generating many kinds of bioactive compounds. Besides that, we also assumed that they formed the complex hybrid of PKS-NRPS genes. Interestingly, the existence of these hybrid PKS-NRPS systems will enlarge the variation of each mod ule in forming an immense variety of bioactive compounds. Many kinds of natural prod ucts are for med through the combination of PKS-NRPS hybrid systems such as yersiniabactin, one of the iron transport systems of Yersinia pestis that acts as a virulence factor for pathogenic strains and generated from a hybrid assembly line containing 3 NRPS modules and 1 PKS module (Cane & Walsh 1999) and bleomycin (BLM), a family of anticancer antibiotics produced by Streptomyces verticillus and generated from BLM megasynthetase that consist of 10 NRPS modules and 1 PKS module (Shen et al. 2001). Donadio et al. (2007) stated that PKS, NRPS or both are molecular assembly lines that direct product formation on a protein template. Both systems accomplish their task by maintaining reaction intermediates covalently bound as thioesters on the same phosphopantetheine prosthetic group. In PKS assembly lines, the monomers are acetyl-CoA, malonyl-CoA or methylmalonyl-CoA whereas the monomers for NRPS assembly are proteinogenic and nonproteinogenic amino acids and other carboxylic acids such as aryl acids. DNA fragment of KS and A domain were sub-cloned into T-Vector pMD20 (Appendix 7) and transformed into competent E. coli DH5α. Recombinant plasmid that carrying the DNA fragment were named pMD20-KS domain and pMD20-A domain. This recombinant plasmid was isolated and digested with a combination of restriction enzymes, BamHI and XbaI. Several steps like PCR sequenc ing and purification of PCR product were done for DNA sequencing. M13 primer RV and M13 primer M4 were used for PCR sequencing and Big Dye® X TerminatorT M purification kit was used for the purification of PCR product. Sequences analysis of DNA fragment encod ing KS domain showed that isolates SAB E-41 and SAB E-57 have a feature of subject sequences that similar to polyketide synthase. Isolate SAB E-41 had 97% of homology level with type I PKS from B. amyloliquefaciens LL3 (Appe ndix 8). Weitao et al. (2011) found that the complete genome sequence of B. amyloliquefaciens LL3 that isolated from Korean fermented food presented the glutamic acid- independent production of poly- -glutamic acid. This compound is a capsular component or extracellular secretion of Bacillus and a few other organisms that widely used in medicine, cosmetics, food and wastewater treatment. -PGA is a natural polyamide consisting of D- and L-glutamic acid units connected by -amide linka ges (Ashiuchi & Misono 2002; Candela et al. 2009). Isolate SAB E-57 had 98% of homology level with putative polyketide synthase from B. amyloliquefaciens subsp. plantarum CAU-B946 (Appendix 9). Borriss et al. (2011) reported that strain CAU B946 that isolated from the rice rhizosphere, was identified by 16S rRNA gene and gyrA gene sequencing and by physiological and biochemical analysis as being B. amyloliquefaciens subsp. plantarum. Due to its capability to produce antibiotics, some products developed from strain CAU B946 had already been applied as biofungicides to control several plant diseases such as tobacco black shank, rice sheath blight, cotton fusarium wilt, cotton verticillium wilt, and wheat scab. Likewise, bioinformatics sequences analysis of DNA fragment encoding A domain showed that isolates SAB E-31, SAB E-41 and SAB E-57 have a feature of subject sequences that similar to nonribosomal peptide synthetase. Isolate SAB E31 had 81% of homology level with bacitracin synthetase 1 from B. pumilus ATCC 7061 (Appendix 10). Awais et al. (2008) isolated a Bacillus species from soil that collected from different areas and identified as B. pumilus according to Bergey’s Manual of Determinative Bacteriology. The antibiotic that prod uced by the identified B. pumilus strain was designated as bacitracin. This compound was active against Micrococcus luteus and S. aureus. Bottone and Peluso (2003) produced an antifungal compound from B. pumilus that is active against Mucoraceae and Aspergillus species. The active compound inhibited Mucor and Aspergillus spore germina tion, aborted elongating hyphae and presumable inducing a cell-wall lesion. Isolate SAB E-41 had 80% of homology level with surfactin synthetase B from B. amyloliquefaciens subsp. plantarum CAU-B946 (Appendix 11) while isolate SAB E-57 had 81% of homology level with surfactin synthetase A from the same strain of bacteria, CAU-B946 (Appendix 12). Prokof'yeva et al. (1996) mentioned that some of bacteria from Bacillus group produce biologically active lipopeptides that are modified by a fatty acid. One of them was surfactin that has a large spectrum of biological activity. Surfactin is a powerful lipopeptide that commonly used as an antibiotic. This antibiotic contains a - hydroxy fatty acid a nd synthesized by a linear nonribosomal peptide synthetase. Besides that, surfactin has surface active properties directed against microbial adhesion and disruptive the permeability of membrane cell of Gram pos itive and Gram negative bacteria. Besides the surfactin peptides, recently, Blom et al. (2012) reported that the genome of the rhizoba cterium B. amyloliquefaciens subsp. plantarum CAU B946 was 4.02 Mb in size and harbored 3,823 genes (coding sequences/CDS). Nine giant gene clusters were dedicated to nonribosomal synthesis of antimicrobial compounds. This strain also possessed a gene cluster that involved in synthesis of iturin A. Mizumoto et al. (2007) mentioned that iturin A is a cyclolipopeptide containing seven residues of α-amino acids (L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-DAsn-L-Ser-) and one residue of a β-amino acid is likely to be the active agents in biological control. The alignment of amino acid sequences encod ing KS domain showed that isolates SAB E-41 and SAB E-57 have a conserved region of amino acid sequences with the other reference strains but the similarity number of amino acid sequences from isolate SAB E-41 with B. amyloliquefaciens LL3 and isolate SAB E-57 with strain CAU B946 was very low. These meant that isolates SAB E-41 and SAB E57 have the new feature of amino acid sequences encoding polyketide synthase enzyme. Besides that, phylogenetic analysis also proved that these two bacterial isolates formed an own clade and differed to the other reference strains. Polyketide synthase enzyme from isolate SAB E-41 was closely related to isolate SAB E-57 and differ to the other reference strains based on phylogenetic tree result. Meanwhile, the alignment of amino acid sequences encod ing A domain also showed that isolates SAB E-31, SAB E-41 and SAB E-57 have a conserved region of amino acid sequences as shown in figure 16. The similarity number of amino acid sequences encoding A domain from three bacterial isolates was also low. Isolate SAB E-31 has a low similarity number of amino acid sequences with B. pumilus ATCC 7061 and so do isolates SAB E-41 and SAB E-57 with strain CAU B946. These meant that three bacterial isolates have a new feature of amino acid sequences encoding nonribosomal peptide synthetase enzyme. Phylogenetic analysis showed that isolates SAB E-31 and SAB E-57 formed a different clade with the other reference strains. Nonribosomal peptide synthetase enzyme from isolate SAB E-31 was closely related to isolate SAB E-57 while for isolate SAB E-41 was closely related to B. amyloliquefaciens FZB42 based on phylogenetic tree result. Fortman and Sherman (2005) stated that few marine NRPS genes have been revealed compared with PKS genes. Zhang et al. (2009) reported that the NRPS genes from the bacteria associated with South China Sea Sponges cluster together forming two groups , which means that these NRPS genes are different from the other marine NRPS genes. Twelve NRPS genes grouped together showed a high similarity to three NRPS relatives of spongeassociated bacteria. In summary, combining the antimicrobial activity test and detection the occurrence of KS and A domain of PKS and NRPS genes based on molecular approach could be applied to efficient screening of the potent marine bacterial isolates and also predicting their related compounds. These related compounds could be developed and applied in pharmaceutical industry in order to treat the resistant microbes. CONCLUSION AND SUGGESTION Conclusion Crude extracts of isolates SAB E-31, SAB E-41 and SAB E-57 showed different antimicrobial activity against non-pathogenic and pathogenic microbes. Bacterial crude extract of isolate SAB E-41 demonstrated the best antimicrobial activity compared to the other bacterial crude extracts. Three marine bacterial isolates were included in the ge nus of Bacillus based on molecular genetic analysis of 16S rDNA. Both isolates SAB E-41 and SAB E-57 possessed KS and A domain in the cluster of PKS and NRPS genes and only isolate SAB E-31 possessed A domain in the cluster of NRPS gene. Suggestion Further purification of active compounds for four active fractions (Rf 0.87, 0.50, 0.41 and 0.12) from fraction BA-13 was needed to be conducted in order to identify the group of these active compo unds and the molecule structure elucidation. 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SAB E-31 XbaI ↓ TCTAGAGGATCTACTAGTCATATGGATTGGGCGGTGTGTACAAGGCCCGGGAACGTATTC ACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGA CTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTAAACCTTGCGGTCTCGCAGCCC TTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGAC GTCATCCCCACCTCCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATG CTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACAC GAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCGAAGGGAAAGCCCCTATCTC TAGGGTTGTCAGAGGATGGTCAAGGACCCTGGTAAGGTTCTTCGCGTTGCTTCAGAAATT AAACCCCCACATGCTCCCACCCGCTTGTGCGGGCCCCCCGTCAATTCCTTTGAGTTTCAG TCTTGCGACCGTACTCCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGG CGGAAACCCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAA TCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTT CGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCT CCTCTTCTGCACTCAAGTTTCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGGCT TTCACATCAGACTTAAGAAAACCGCCTGCGAGCCCCTTTACGCCCAATAAATTCCGGACA ACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGGTAGTTAGCCGTGGCTTTCTGG TTAGGTACCGTCAAGGTGCAAGCAGTTACTCTTGCACTTGTTCTTCCCTAACAACAGAGC CTTTACGATTCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTACGTCCA TTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGT GTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCAC CAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGACAGCCGAAACCGTCTTTCATCCT TGAACCATGCGGTTCAAGGAACTATCCGGTATTAGCTCCGGTTTCCCGGAGTTATCCCAG TCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCCGGGAGCAAG CTCCCTTCTGTCCGCTACGACTTGCATGTGTTAGGCCCTGAATCGGATCC ↑ BamHI B. SAB E-41 XbaI ↓ TCTAGAGGACTACTAGTCATATGGATTGGGCGGTGTGTACAAGGCCCGGGAACGTATTCA CCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGAC CGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCT TTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACG TCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGC TGGCAACTAAGATCAAGGGTTGCGCATCGTTGCGGGACTTAACCCAACATCTCACGACAC GAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCT AGGATTGTCAGAGGATGTACAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAAAC CACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACC GTACTCCCCAGGGCGGAGTGCTTTAATGCGTTTAGCTGACAGCACTAAAGGGGCGGAAAC CCCCTAACAACACTATAGCAACTCTATCGTTTACGGGCGTGGACTACCAGGGTATCTAAT CCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTC GCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATATCCACTCT CCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGGTTGAGCCGGGGGCT TTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACG CTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAG GTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTT ACGATCCGAAAACAACTTCATCACTCACGCGGCGTTGCGTCCGTCAGACTTTCGTCCATT GCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGT GGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCA ACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTG AACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTCCCGGAGTTATCCCAGTCT TACAGGCAGTTACCCACGTTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCC ATCTGTCCGCTCGACTTGCATGTGTTAGGCCCTGAATCGGATCC ↑ BamHI C. SAB E-57 XbaI ↓ TCTAGAGGGATCTACTAGTCATATGGGATTGGGCGGGGTGGTACAGGCCCGGGGAACGTA TTCACCGCGGCATGCTGATCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCA GACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGC CCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTG ACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAA TGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGAC ACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATTC TCTAGGATTGTCAGAGGATGTCAAGACCCTGGTAAGGTTCTTCGCGTTGCTTACGAAATT AAACCCACATGCTCCCACCGCTTGTGCGGGCCCCCCGTCAATTCCTTTGAGTTTCAGTCT TGCGACCGTACTCCCCCAGGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGGCG GGAAACCCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAAT CCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTC GCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGGAATTCCACTCT CCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCCTCCCCCGGTTGAGCCGGGGGC TTTCACATCAGACTTAAGAAACCAGCCTGCGAGCCCTCTTACGCCCAATAATATCCGGAC AACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGG TTAGGTACCGTCAAGGTGCCGCCCTATTTGAATCGGCACTTGTTCTTCCCTAACAACAGA GCTATTACGATCCGAAAACCTTCATCACTCATCGCGGCGTTGCTCCGTCAGACTTTCGTC CATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCA GTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTC ACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACTTTTATTC TGAACCTTGCGGTCAGACAACCTCCGGTTTAGCCCCGGTTCCCGGAGTTTCCCAGTCTAC CAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGACCAAGCTCCC ATCTGTCCGCTCGACTTCCATGTGTTAGCCCTGAATCGGATCC ↑ BamHI Appendix 2 DNA sequences of KS domain of PKS gene from two bacterial isolates A. SAB E-41 XbaI ↓ TCTAGAGGATCTACTAGTCATATGGATTGTGCCGGTGCCGTGGGCCTCGACATAACTGAC GGTCCGCGGGCTGATACCTGCTTTTTTCAGACAGGCCTTAATGACTTCTGCCTGTGCAGC AGGACTCGGGACGGTAATTCCGCTTACTTTCCCGACGTGGTTAACGGCGCTTCCTTTAAT GACCGCGTAAATGCGGTCGCCGTCTTGTTCGGCTTTTTCCAGCGGCTTGAGCAAGACCGC ACCGACACCTTCTCCGGAAACGTAGCCGTCCCCGCCCTCGCCGAATGTGCGGCAGCGGCC GTCACTTGAGTGCATCCCTACGCTTCCGTAGCTGAGATATTTCGCCGGGTGCAGCGACAA GTTCACCCCTCCCGCAAGCGCGGCTTCACATTCGCCGCGGCGGATGCTTTCAATGGCCAG ATGAACGGCGGTCAATGATGAGGAACAAACGGTATCCACCGCGATGCTCGGCCCGTGGAA GTCACAATAATAGGACACTCTGTTGGCGATCTGCGCATAATTCAGTGAAACCGGAAAAGG ATCAGCTTCAGATAATTGTTCTGCGCCGATTAAGGAATAATCTTTATGCATCACCCCTGC AAATACGCCGATCGGATGCTGTTTCTCCCCTTTATTCCCCAGCGTTTCAGGCGTATACCC CGCATCTTCAATCGTTTCCCAGCATGTTTCTAAAAACAGCCGCTGCTGCGGATCCATCGC ATCGGATCC ↑ BamHI B. SAB E-57 XbaI ↓ TCTAGAGGATCTACTAGTCATATGGATTGTGCCCGTGCCATGCCCTTCCACCATTTGAAT GGTTTCCGGATTAATGTGAAAAGTATCATAGACATGCCGTTCCAATCGTTCTTGAGAAAG CGCGCTTGGAGCGGTTATTCCGTTTGTAGCGCCGTCCTGATTCATTGCAGAGCCTTTTAT GACGCCGTACACATGATCTCCGTCACTGACGGCGTCGCTAAGACGTTTCAGCACCACCGC TCCCACACCTTCACCCGGCACGAAGCCGTCCGCGCTTTGATCAAACGTATGGCAGCGCCC GGTCGGTGACAGCATATTCGCCTTATTTGACGACTGATAAAAAGCGGGAGTGGATTGAAT GAAAACCCCGCCCGCCACCGCCATTTCGGTTTCTTTCGTCCAGAGCCCCTGACATGCCAA ATGAATGGCTGTCAGCGAACTGGAACATGCTGTATCAACAGTGATCGCCGGCCCTTGTAG ATTAAGGTGATAGGCGATCCTTGCCGGAGTGACGGAATTGTGATTGCCCCAAAAAGCCTG CGCGGGCCCCTGCTGTTTAAAAATGGTCTGGTAATCTCCGCCGCAGGAGCCGGCATACAC GCCGCATTCCCGGCCTCTTACCGAGTCTCCCGCATATCCCGCATCTCCAAGCGCTTTCCA CGATTCTTCCAGAAACACCCGCTGCTGCGGGTCCCATCATCGGATCC ↑ BamHI Appe ndix 3 DNA sequences of A domain of NRPS gene from three bacterial isolates A. SAB E-31 XbaI ↓ TCTAGAGGATCTACTAGTCATATGGATTCCGCGGATTTTGACTTGATGATCGATTCGCCC AATGAATTCAATATCTCCATTTGACAGCCATCGTGCGAGATCGCCCGTTTTGTACATGAC TTCACCAGGTCGGAAAGGATTTTCAACAAACTCTTCGCTCGTTAACTCTGGCTGGCGATA ATACCCCTTGACCAGTCCGTCACCTGCGACACAAAGCTCTCCAGCTACTCCCGGCGGCTG CACATGTCCAAACTCATCAACAATATAAACAGCCGTTTGACTCACCGGCTTTCCGATCGG AATCGATAGTGCTTGTTCTTCAATGTGATTCACCGGATAATACGTTGTGAAAATCGTGCT TTCAGAAGGTCCATACATATGAATAAGTTTGTCTTCTCCAACCGTTTCAAGGGCTGCCAC AACATGCGGCACAGAAGCACGTTCCCCGCCAAACAGCACTTTTCTGACGTTTTTCAGGCT GCCTTTTTTCATATCAATCAGTAAGTGAAATAGAGCGGTCGTGATCATTAAAATACTGAC TTTTTCCTTCTCAATCGCGCCAGAAAGCTCATTCATATTTAAAATGTGATCCTTTGGCAA AACAATGAGTTTCGCTCCGTTCAATAAAGCGCCAAACACATCAAACATAAATGCATCAAA TACATAGTTTGAAAGGCTCATCACCGTGTCTTCATGATGAATGGTGAGATAATTCGACTG CTTCACTGTTCTCAAAATGTTCCGATGCGTCACCATGTTCCCCTTTAGGTTTTGCCTGTC GTACCAGCATGTGGTAGGTCAAAATTCGCCCAAGTCCCAAAGGCGAAACACATACAAACT GGGATTGCTCTCTGACTGCTGATCAACGGCTTGGATCTTCTGTTTCTATGATTTTCCCCC TTCAAACGCAGTAAGCACAGAGCGATGACGCAGCACCTGGATGGGGTCAGGGACAAACTG TGCACCACTATCTTGTCAAAAAGTGCCTTGAATGCGCTCATCCAGGGAAGTCCAGGATCG ATTGGGACACATACGCCACCCACCCGCCAATCGGATCC ↑ BamHI B. SAB E-41 XbaI ↓ TCTAGAGGATCTACCTAGTCCATATGGATTTGCGGGCGGTGCTTATGTGCCCGATTGATC CCGGTCTTTTGCCGGGAGGACCGTCTCCGCTTTATGGGCGGCAGACAGCTCGATTCGGCT CGTGCTGACAGTTCAGGACTATCAAAGAACAAGCGGGCACATTGCAAGTCCCGATTGTCA TGCTGGATGAAAAGCGCGGATGAAACGGTAAGCGGAACAGACTTGAATCTTCCGGCCGGC GGGCAACGACTTGGCGTATATCATGTATACATCCGGATCGACCGGCAAACCGAAAGGCGT CATGATTGAACCACAGAAATATCATCAGGCTCGTCAAACATTCGAATTACGTGCCGGTTC ATGAAGAAGACCGGATGGCGCAAACGGGAGCCGTCAGCTTTGATGCCGGAACCTTCGAAG TCTTCGGTGCATTGCTGAACGGAGCCGCGCTGCACCCGGTGAAAAAAGAGACACTGCTTG ACGCCGGACGATTCGCCCAATTTCTGAAAGAGCAGCGGATCACGACCATGTGGCTGACGT CTCCGCTGTTTAATCAGCTTGCCCAAAAGGATGCGGGCATGTTTAACACGCTCCGGCACC TCATCATCGGCGGTGATGCGCTTGTGCCGCATATCGTCAGCAAAGTGAGGAAGGCATCAC CGGAGCTGTCGCTTTGGAACGGCTACGGGCCGACGGAGAATACGACGTTTTCGACGAGTT TTCTCATTGATCAGGACTGCGACGGCTCGATCCCGATCGGCAAGCCGATCGGAAATTCCA CTGCGTACATTATGGACGAAAACCGCAACCTCCAGCCGATCGGCGCTCCCGGTGAGCTGT GCGTCGGCGGAAGCGGAGTGGCAAGAGGCTATGTGAATCTGCCTGAATTAACGGAGAAGC AGTTTGTCCGCGATCCGTTCAGACCGGAGAAAAGATATACCCGGACGGGGGACTTGGCGA AAGATGGCTTCCCGGCGGCCCGAACGAGTTTTTTGGCCGAAATGGCCACCCAAGAAAAAA TCGGATCC ↑ BamHI C. SAB E-57 XbaI ↓ TCTAGAGGATCTACTAGTCATATGGGATTGCCGCGGATTTTGACCTGGATCATCTTCACG GCCGAATATATTCGATCGTCCCGTCCGGCAGCCAGCGCGCCATATCACCCGGTGCGGTAC ATAGCGGCCGCTTCCGTTAAACGGATCTTGCAAAAAACTTCTCTGCGGGTCAAATCCGGA AAGATTTAAATAGCCGCGGCCTACACCGTCACCCGCGATATACAGCTCACCGGCCGTCCC GTCGGGCTGAAGCCTCTGGTGCTTATCCAATATATACAGACGGGCGTTGCCGAGCGGTTT TCCGATCGGAACGTACGCCGCCTGTTGATTCATTCCGTTATCGGCTGACACCTGATGCAC GGACGCATCTACGCACGTTTCTGTCGGCCCGTAGACATTCGTCAGACGCGGCGCCCTGCC CGATTGATGAAAGAGGTTCATCAGCTGTTCAGCAACAGCGGCGGACAGGCCCTCTCCCCC AATGAGCATGTGGCGCAATTCAATTCCGCTGACATCTCCCGCCGCAACCATCATCTGCAG ATGCGCAGGGGTTCCGTCAGTGGCTTCAATCCGGTTTTGACGATAATAGTCCAGCAGTGC CGAGCCATTCGTTACAGTCGTTTTCGGCACGATATAAAGCGTCTGTCCCAAAAGAAGCGA GGCAAAAATCTGTTTGACGGACGCATCAAAATGTAACGGAGCCAAAAGCGCCATTCTTAA TGTCTGCTCACCGCATTGATAAATCTCCTGCTGCAGTGATTGCACCAGATGATGAACATT TGGGCCGGTGCTCAATCATCACGCCCTTCGGCCGCCCCCGTTGTTACCTGACGTGTAGAT GATGTAAAGCCACGCCCGGTCTGATTTGTTGTTACTTGACAACCGTCCCGCCAGCCCGTT TTCAAAACTTGAAACAAGCCCTCCGTCAAAAATTCAAATACCGTTTCGGGCAATCCCGCC TGCCACGGCGCCCTGTTATTTCTCGGGCCCTCCTGTTCAGGAAACAAGACCTTACCGCCT TCCCGCCTTGTTCCCTTCCTCCAAAAATGGGTAACCTTGGAAATTCCCGGAATTCCCCGC CCCGGGAAAAAGGTCCAAAGGGAAAGTCTTAATTCGGGGAAACCAATTATATTGCCAACC CAACCCCCGCGCCAATCGGATCC ↑ BamHI Appendix 4 Alignment of 16S rDNA sequences from isolate SAB E-31 using BlastN program gb|EU500930.1| Bacillus pumilus strain KD3 16S ribosomal RNA gene, partial sequence Length=1502 Score = 2234 bits (1162), Expect = 0.0 Identities = 1294/1320 (98%), Gaps = 21/1320 (2%) Strand=Plus/Minus Query 1 Sbjct 1385 Query 61 Sbjct 1325 Query 121 Sbjct 1265 Query 181 Sbjct 1205 Query 241 Sbjct 1145 Query 301 Sbjct 1085 Query 361 Sbjct 1025 Query 421 Sbjct 969 Query 481 Sbjct 916 Query 541 Sbjct 857 Query 601 Sbjct 799 Query 661 Sbjct 739 Query 721 Sbjct 679 Query 781 Sbjct 619 Query 841 Sbjct 561 GGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTA 60 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTA 1326 GCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGT 120 GGGATTGGCTAAACCTTGCGGTCTCGCAGCCCTTTGTTCTGTCCATTGTAGCACGTGTGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GGGATTGGCTAAACCTTGCGGTCTCGCAGCCCTTTGTTCTGTCCATTGTAGCACGTGTGT 180 1266 1206 AGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTCCCTCCGGTTTGTCA ||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||| AGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCA 240 CCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTC 300 GTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGT 360 1146 1086 1026 CACTCTGTCCCCGAAGGGAAAGCCCCTATCTCTAGGGTTGTCAGAGGATGGTCAAGGACC |||||||||||||||||||||| |||||||||||||||||||||||||| |||||| | CACTCTGTCCCCGAAGGGAAAG-CCCTATCTCTAGGGTTGTCAGAGGAT-GTCAAG--AC 420 CTGGTAAGGTTCTTCGCGTTGCTTCAGAAATTAAACCCCCACATGCTCCCACCCGCTTGT ||||||||||||||||||||||||| ||||||| ||||||||||| |||||||| CTGGTAAGGTTCTTCGCGTTGCTTC--GAATTAAA---CCACATGCTCC--ACCGCTTGT 480 GCGGGCCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCAGGCGGAG ||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||| GCGGG-CCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCAGGCGGAG 540 TGCTTAATGCGTTAGCTGCAGCACTAAGGGGGCGGAAACCCCCCTAACACTTAGCACTCA ||||||||||||||||||||||||||| |||||||||| ||||||||||||||||||||| TGCTTAATGCGTTAGCTGCAGCACTAA-GGGGCGGAAA-CCCCCTAACACTTAGCACTCA 600 TCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCC 660 970 917 858 800 740 TCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTAC 720 GCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTT 780 680 620 CCAATGACCCTCCCCGGTTGAGCCGGGGGGCTTTCACATCAGACTTAAGAAAACCGCCTG |||||||||||||||||||||||| |||||||||||||||||||||||| |||||||||| CCAATGACCCTCCCCGGTTGAGCC-GGGGGCTTTCACATCAGACTTAAG-AAACCGCCTG 840 CGAGCCCCTTTACGCCCAATAAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGC |||| ||||||||||||||| ||||||||||||||||||||||||||||||||||||||| CGAG-CCCTTTACGCCCAAT-AATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGC 900 562 504 Query 901 Sbjct 503 Query 961 Sbjct 444 Query 1021 Sbjct 386 Query 1081 Sbjct 326 Query 1141 Sbjct 266 Query 1201 Sbjct 206 Query 1261 Sbjct 146 TGCTGGCACGGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCAAGCAGTTA ||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||| TGCTGGCAC-GTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCAAGCAGTTA 960 CTCTTGCACTTGTTCTTCCCTAACAACAGAGCCTTTACGATTCCGAAAACCTTCATCACT ||||||||||||||||||||||||||||||| |||||||| ||||||||||||||||||| CTCTTGCACTTGTTCTTCCCTAACAACAGAG-CTTTACGA-TCCGAAAACCTTCATCACT 1020 CACGCGGCGTTGCTCCGTCAGACTTACGTCCATTGCGGAAGATTCCCTACTGCTGCCTCC ||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||| CACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCC 1080 CGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTA 1140 CGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATC 1200 445 387 327 267 207 TGTAAGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TGTAAGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCG 1260 GTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTAC 1320 147 87 Appendix 5 Alignment of 16S rDNA sequences from isolate SAB E-41 using BlastN program gb|JN160740.1| Bacillus amyloliquefaciens strain zy2 16S ribosomal RNA gene, partial sequence Length=1449 Score = 2267 bits (1179), Expect = 0.0 Identities = 1320/1343 (98%), Gaps = 22/1343 (2%) Strand=Plus/Minus Query 1 Sbjct 1381 Query 61 Sbjct 1321 Query 121 Sbjct 1261 Query 181 Sbjct 1201 Query 241 Sbjct 1141 Query 301 Sbjct 1082 Query 361 Sbjct 1022 Query 421 Sbjct 963 Query 481 Sbjct 904 Query 541 Sbjct 846 Query 601 Sbjct 795 Query 661 Sbjct 736 Query 721 Sbjct 676 Query 781 Sbjct 617 Query 841 Sbjct 557 GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGA 60 TTCCAGCTTCACGCAGTCGAGTTGCAGACCGCGATCCGAACTGAGAACAGATTTGTGGGA ||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||| TTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGA 120 TTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCC 180 CAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGG 1322 1262 1202 240 1142 CAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCATCGTT |||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||| CAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGC-TCGTT 300 GCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCAC 360 1083 1023 TCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTACAAGACCTGGTA ||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||| TCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGT-CAAGACCTGGTA 420 AGGTTCTTCGCGTTGCTTCGAATTAAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGT ||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||| AGGTTCTTCGCGTTGCTTCGAATTAAA-CCACATGCTCCACCGCTTGTGCGGGCCCCCGT 480 CAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGGCGGAGTGCTTTAATGCGT ||||||||||||||||||||||||||||||||||||||||| |||||||||| ||||||| CAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGG-CGGAGTGCTT-AATGCGT 540 TTAGCTGACAGCACTAAAGGGGCGGAAACCCCCTAACAACACTATAGCAACTCTATCGTT | ||||| ||||||||| |||||||||||||||||||| || ||||| ||| |||||| T-AGCTG-CAGCACTAA-GGGGCGGAAACCCCCTAACA---CT-TAGCA-CTC-ATCGTT 600 TACGGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAG ||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||| TACGG-CGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAG 660 CGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCAT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCAT 720 TTCACCGCTACACGTGGAATATCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCA |||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||| TTCACCGCTACACGTGGAAT-TCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCA 780 ATGACCCTCCCCGGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ATGACCCTCCCCGGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAG 840 CCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGG 900 964 905 847 796 737 677 618 558 498 Query 901 Sbjct 497 Query 961 Sbjct 437 Query 1021 Sbjct 379 Query 1081 Sbjct 320 Query 1141 Sbjct 260 Query 1201 Sbjct 200 Query 1261 Sbjct 140 Query 1318 Sbjct 80 CACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGG 960 CACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACAACTTCATCACTCACGCG |||||||||||||||||||||||||||||||||||||||||| |||||||||||||||| CACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAAC--CTTCATCACTCACGCG 1020 GCGTTGCGTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAG ||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||| GCGTTGC-TCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAG 1080 GAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCAT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCAT 1140 CGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAA 1200 GTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATT 1260 AGCCCCGG-TTCCCGGAGTTATCCCAGTCTTACAGGCA-GTTACCCACGT-TTACTCACC |||||||| ||||||||||||||||||||||||||||| ||||||||||| ||||||||| AGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACC 1317 CGTCCGCCGCTAACATCAGGGAG ||||||||||||||||||||||| CGTCCGCCGCTAACATCAGGGAG 1340 58 438 380 321 261 201 141 81 Appendix 6 Alignment of 16S rDNA sequences from isolate SAB E-57 using BlastN program gb|EU373407.1| Bacillus gene, partial sequence Length=1518 subtilis strain YRL02 16S ribosomal Score = 2127 bits (1106), Expect = 0.0 Identities = 1298/1334 (97%), Gaps = 25/1334 (2%) Strand=Plus/Minus Query 14 Sbjct 1400 Query 73 Sbjct 1340 Query 133 Sbjct 1280 Query 193 Sbjct 1220 Query 253 Sbjct 1160 Query 313 Sbjct 1100 Query 373 Sbjct 1040 Query 433 Sbjct 982 Query 493 Sbjct 927 Query 553 Sbjct 868 Query 613 Sbjct 810 Query 673 Sbjct 750 Query 733 Sbjct 690 Query 793 Sbjct 631 GTACAGGCCCGGGGAACGTATTCACCGCGGCATGCTGATC-GCGATTACTAGCGATTCCA ||||| | || ||||||||||||||||||||||||||||| ||||||||||||||||||| GTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCA 72 1341 GCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGC 132 TTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGT ||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||| TTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATCGTAGCACGTGTGTAGCCCAGGT 192 1281 1221 CATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTC 252 ACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGA 312 CTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCC 1161 1101 372 1041 CCCGAAGGGGACGTCCTATTCTCTAGGATTGTCAGAGGATGTCAAGACCCTGGTAAGGTT |||||||||||||||||| |||||||||||||||||||||||||||| |||||||||||| CCCGAAGGGGACGTCCTA-TCTCTAGGATTGTCAGAGGATGTCAAGA-CCTGGTAAGGTT 432 CTTCGCGTTGCTTACGAAATTAAACCCACATGCTCCCACCGCTTGTGCGGGCCCCCCGTC ||||||||||||| || ||||||| ||||||||| |||||||||||||||| |||||||| CTTCGCGTTGCTT-CG-AATTAAA-CCACATGCT-CCACCGCTTGTGCGGG-CCCCCGTC 492 AATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCAGGGCGGAGTGCTTAATGCGTT ||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||| AATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCA-GGCGGAGTGCTTAATGCGTT 552 983 928 869 AGCTGCAGCACTAAGGGGGCGGGAAACCCCCCTAACACTTAGCACTCATCGTTTACGGCG |||||||||||||||||||||| || ||||||||||||||||||||||||||||||||| AGCTGCAGCACTAAGGGGGCGG--AAACCCCCTAACACTTAGCACTCATCGTTTACGGCG 612 TGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTT 672 ACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGC 732 811 751 691 TACACGTGGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCC ||||||| |||||||||||||||||||||||||||||||||||||||||||||||| ||| TACACGT-GGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGAACCC 792 TCCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCAGCCTGCGAGCCCTCT | |||||||||||||||||||||||||||||||||||||||||| ||||||||||||| | T-CCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACC-GCCTGCGAGCCCT-T 852 632 575 RNA Query 853 Sbjct 574 Query 913 Sbjct 515 Query 973 Sbjct 456 Query 1033 Sbjct 398 Query 1093 Sbjct 338 Query 1153 Sbjct 278 Query 1213 Sbjct 218 Query 1269 Sbjct 158 Query 1327 Sbjct 98 TACGCCCAATAATATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACG ||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||| TACGCCCAATAAT-TCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACG 912 TAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAATCGGCAC ||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||| TAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAA-CGGCAC 972 TTGTTCTTCCCTAACAACAGAGCTATTACGATCCGAAAACCTTCATCACTCATCGCGGCG |||||||||||||||||||||||| ||||||||||||||||||||||||||| ||||||| TTGTTCTTCCCTAACAACAGAGCT-TTACGATCCGAAAACCTTCATCACTCA-CGCGGCG 1032 TTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGT 1092 CTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTC 1152 GCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGG 1212 TAGCCGAAGCCA-CTTTTAT-TCTGAACCTTGCGG-TCAGACAACC-TCCGGTTTAGCCC |||||||||||| ||||||| |||||||| ||||| ||| |||||| |||||| |||||| TAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATAGCCC CGG-TTCCCGGAGTT-TCCCAGTCTACCAGGCAGGTTACCCACGTGTTACTCACCCGTCC ||| ||||||||||| ||||||||| ||||||||||||||||||||||||||||||||| CGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCC GCCGCTAACATCAG |||||||||||||| GCCGCTAACATCAG 1340 85 516 457 399 339 279 219 1268 159 1326 99 Appendix 7 Plasmid map of T-Vector pMD20 (TaKaRa Bio Inc.) Appendix 8 Alignment of DNA sequences encoding KS domain of PKS gene from isolate SAB E-41 using BlastX program ref|YP_005545643.1| baeL gene product [Bacillus amyloliquefaciens LL3] gb|AEB63415.1| bacillaene synthesis; polyketide synthase of type I [Bacillus amyloliquefaciens LL3] Length=3513 GENE ID: 12204614 baeL | bacillaene synthesis; polyketide synthase of type I [Bacillus amyloliquefaciens LL3] Score = 417 bits (1072), Expect = 2e-130 Identities = 221/229 (97%), Positives = 225/229 (98%) Gaps = 0/229 (0%), Frame = -3 Query 689 Sbjct 472 Query 509 Sbjct 532 Query 329 Sbjct 592 Query 149 Sbjct 652 MDPQQRLFLETCWETIEDAGYTPETLGNKGEKQHPIGVFAGVMHKDYSLIGAEQLSEADP MDPQ+RLFL+TCWETIEDAGYTPETLGNK KQ P+GVFAGVMHKDYSLIGAEQLSE DP MDPQERLFLQTCWETIEDAGYTPETLGNKKNKQRPVGVFAGVMHKDYSLIGAEQLSETDP 510 FPVSLNYAQIANRVSYYCDFHGPSIAVDTVCSSSLTAVHLAIESIRRGECEAALAGGVNL FPVSLNYAQIANRVSYYCDFHGPS+AVDTVCSSSLTAVHLAIESIRRGECEAALAGGVNL FPVSLNYAQIANRVSYYCDFHGPSLAVDTVCSSSLTAVHLAIESIRRGECEAALAGGVNL 330 SLHPAKYLSYGSVGMHSSDGRCRTFgeggdgyvsgegvGAVLLKPLEKAEQDGDRIYAVI SLHPAKYLSYGSVGMHSSDGRCRTFGEGGDGYVSGEGVGAVLLKPLEKAEQDGDRIYAVI SLHPAKYLSYGSVGMHSSDGRCRTFGEGGDGYVSGEGVGAVLLKPLEKAEQDGDRIYAVI 150 KGSAVNHVGKVSGITVPSPAAQAEVIKACLKKAGISPRTVSYVEAHGTG KGSAVNHVGKVSGITVPSPAAQAEVIKACLKKAGISPRTVSYVEAHGTG KGSAVNHVGKVSGITVPSPAAQAEVIKACLKKAGISPRTVSYVEAHGTG 3 700 531 591 651 Appendix 9 Alignment of DNA sequences encoding KS domain of PKS gene from isolate SAB E-57 using BlastX program ref|YP_005130937.1| putative polyketide synthase pksL PKS [Bacillus amyloliquefaciens subsp. plantarum CAU B946] emb|CCF05742.1| putative polyketide synthase pksL PKS [Bacillus amyloliquefaciens subsp. plantarum CAU B946] Length=2071 GENE ID: 11698078 dfnJ | putative polyketide synthase pksL PKS [Bacillus amyloliquefaciens CAU-B946] Score = 451 bits (1160), Expect = 2e-143 Identities = 218/222 (98%), Positives = 219/222 (99%) Gaps = 0/222 (0%), Frame = -2 Query 666 Sbjct 954 Query 486 Sbjct 1014 Query 306 Sbjct 1074 Query 126 Sbjct 1134 DPQQRVFLEESWKALGDAGYAGDSVRGRECGVYAGSCGGDYQTIFKQQGPAQAFWGNHNS DPQQR+FLEESWKAL DAGYAGDSVRGRECGVYAGSCGGDYQ IFKQQGPAQAFWGNHNS DPQQRLFLEESWKALEDAGYAGDSVRGRECGVYAGSCGGDYQAIFKQQGPAQAFWGNHNS 487 VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ 307 SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD GATNGITAPSALSQERLERHVYDTFHINPETIQMVEGHGTGT GATNGITAPSALSQERLERHVYDTFHINPETIQMVE HGTGT GATNGITAPSALSQERLERHVYDTFHINPETIQMVEAHGTGT 1 1175 1013 1073 127 1133 Appendix 10 Alignment of DNA sequences encoding A domain of NRPS gene from isolate SAB E-31 using BlastX program ref|ZP_03054623.1| bacitracin synthetase 1 (BA1) [Bacillus pumilus ATCC 7061] gb|EDW21930.1| bacitracin synthetase 1 (BA1) [Bacillus pumilus ATCC 7061] Length=3570 Score = 492 bits (1266), Expect = 1e-154 Identities = 263/323 (81%), Positives = 281/323 (87%) Gaps = 6/323 (2%), Frame = -1 Query 969 Sbjct 1588 Query 789 Sbjct 1645 Query 609 Sbjct 1702 Query 429 Sbjct 1762 Query 249 Sbjct 1822 Query 69 Sbjct 1882 DERIQGTF*QDSGAQFVPDPIQVLRHRSVLTAFEGGKS*KQKIQAVDQQSESNPSLYVFR DER++ F DSGAQF+ QVLRHRSVL +FEG + + + + QQS+SN + V DERVKH-FLTDSGAQFLLTH-QVLRHRSVLASFEGTII-ETEDRGIVQQSDSNIDIRVLP 790 LWDLGEF*PTTCWYDRQNLKGNMVTHRNILRTVKQSNYLTIHHEDTVMSLSNYVFDAFMF DL T+ + KGNMVTHRNILRTVKQSNYL IHHEDTVMSLSNYVFDAFMF E-DLANLTYTSGTTGKP--KGNMVTHRNILRTVKQSNYLAIHHEDTVMSLSNYVFDAFMF 610 DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKKGSLKNVR DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKK SLKNVR DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKKDSLKNVR 430 KVLFGGERASVPHVVAALETVGEDKLIHMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV KVLFGGERASVPHV+ ALETVGE KL+HMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV KVLFGGERASVPHVMTALETVGEGKLVHMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV SQTAVYIVDEFGHVQPPGVAGELCVAGDGLVKGYYRQPELTSEEFVENPFRPGEVMYKTG SQTAVYIVDEFG +QPPGVAGELCVAGDGLVKGYY QP+LTSE+FVENPFRPGEVMYKTG SQTAVYIVDEFGQLQPPGVAGELCVAGDGLVKGYYGQPKLTSEKFVENPFRPGEVMYKTG DLARWLSNGDIEFIGRIDHQVKI DLARWLSNG+IEFIGRIDHQVKI DLARWLSNGEIEFIGRIDHQVKI 1 1904 1644 1701 1761 250 1821 70 1881 Appendix 11 Alignment of DNA sequences encoding A do main of NRPS gene from isolate SAB E-41 using BlastX program ref|YP_005129035.1| surfactin synthetase B [Bacillus amyloliquefaciens subsp. plantarum CAU B946] emb|CCF03840.1| surfactin synthetase B [Bacillus amyloliquefaciens subsp. plantarum CAU B946] Length=3586 GENE ID: 11700595 srfAB | surfactin synthetase B [Bacillus amyloliquefaciens CAU-B946] Score = 449 bits (1154), Expect = 1e-139 Identities = 236/295 (80%), Positives = 246/295 (83%) Gaps = 6/295 (2%), Frame = +1 Query 112 Sbjct 573 Query 286 Sbjct 630 Query 466 Sbjct 690 Query 646 Sbjct 750 Query 826 Sbjct 810 KEQAGTLQVPIVMLDEKRG*--NGKRNRLESSGRRATTWRISCIHPDRPANRKAS*LNHR +EQAGTLQVPIVMLDE +G L + G + +P K + HR QEQAGTLQVPIVMLDESADETVSGTDLNLPAGGNDLAYIMYTSGSTGKP---KGVMIEHR 285 629 NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA 465 QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW 645 NGYGPTENTTFSTSFLIDQDCDGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG NGYGPTENTTFSTSFLIDQD DGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG NGYGPTENTTFSTSFLIDQDYDGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG 825 VARGYVNLPELTEKQFVRDPFRPEKRYTRTGDLAKDGFPAARTSFLAEMATQEKI VARGYVNLPELTEKQFVRDPFRP++ RTGDLAK P FL + Q K+ VARGYVNLPELTEKQFVRDPFRPDETIYRTGDLAK-WLPDGTIEFLGRIDNQVKV 990 863 689 749 809 Appendix 12 Alignment of DNA sequences encoding A do main of NRPS gene from isolate SAB E-57 using BlastX program ref|YP_005129034.1| surfactin synthetase A SrfA [Bacillus amyloliquefaciens subsp. plantarum CAU B946] emb|CCF03839.1| surfactin synthetase A SrfA [Bacillus amyloliquefaciens subsp. plantarum CAU B946] Length=3584 GENE ID: 11700594 srfAA | surfactin synthetase A SrfA [Bacillus amyloliquefaciens CAU-B946] Score = 420 bits (1080), Expect = 1e-129 Identities = 227/280 (81%), Positives = 236/280 (84%) Gaps = 15/280 (5%), Frame = -1 Query 845 Sbjct 593 Query 665 Sbjct 650 Query 485 Sbjct 710 Query 305 Sbjct 770 Query 125 Sbjct 829 NNKSDRAWLYIIYTSgnngggrRA**LSTGPNVHHLVQSLQQEIYQCGEQTLRMALLAPL + +SDR YIIYTSG G + + VHHLVQSLQQEIYQCGEQTLRMALLAP STQSDRL-AYIIYTSGTTGRPKGV--MIEHRQVHHLVQSLQQEIYQCGEQTLRMALLAPF 666 HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD 486 VSGIELRHMLIGGEGLSAAVAEQLMNLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG VSGIELRHMLIGGEGLSAAVAEQL+NLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG VSGIELRHMLIGGEGLSAAVAEQLLNLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG 306 MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNLSGFDPQRSF MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNL ++ F MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNLPDLTAEK-F LQDPFNGSGRYVPHRVIWRAG-----CRTGRSNIFGREDD LQDPFNGSGR ++R G G GREDD LQDPFNGSGR------MYRTGDMARWLPDGTIEYIGREDD 21 862 649 709 769 126 828 INTRODUCTION Background The improper and uncontrolled uses of antibiotics against pathogenic bacteria induced and accelerated the occurrence of multi drugs resistant (MDR) strain. The number of infection cases by MDR strains in Indonesia remains high. Some of infection cases were tuberculosis, HIV, malaria, diarrhea and infection on upper respiratory system (Ditjen PP & PL Depkes RI 2011). In 2009, Indo nesia was ranked eighth of 27 countries with the highest of multi drugs resistant cases in the world (WHO 2010). The increase of MDR cases has encouraged many scientists to find the new bioactive compounds in order to solve the MDR problem. Nowadays, the exploration of bioactive compounds has been carried out in many kinds of resources such as medicinal plants, animals, aquatic organisms as well as microorganisms in unique ecosystem in order to find the new bioactive compounds which can treat the MDR strains. Indonesia was one of the hotspot countries possessing many natural resources and almost 70% of the area was covered by coastal area. Considering that, the exploration of new bioactive compounds in an aquatic area is very promising for the new inve ntion of chemotherape utic agents which can be developed and applied in pharmaceutical industry in the future. Marine sponges are one of the evolutionary multicellular organisms that have been reported very potential in producing many kinds of bioactive compounds. Some of the bioactive compounds showed antibacterial, antifungal, antiviral, anticancer, antifouling and cytotoxic properties (Taylor et al. 2007). The limitation of sponge biomass is the main factor for isolating the large scale of bioactive compounds. Therefore, alternative and ecologically sound sources of bioactive compounds are needed. Marine microorganisms have contributed to the majority of bioactive compounds. They can produce the same metabolite compo unds as their host (Proksch et al. 2002). The surfaces and internal spaces of marine sponges are unique microhabitat and more nutrient rich than seawater or most sediments, thus they would likely be a unique niche for the isolation of diverse microorganisms (Friedrich et al. 1999). Isolation and screening the potent bacterial isolates from marine sponge, identifying the antibiotic-encoding genes in active microorganisms, cloning in amenable host and characterize the bioactive compounds were the main strategy for producing large amounts of new metabolites (Webster & Hill 2001). Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 have been isolated from sponge Jaspis sp. at Waigeo Island, Raja Ampat District, West Papua Province. Each of these isolates indicated different activity against Staphylococcus aureus, Escherichia coli, enteropathogenic E. coli K1-1, Pseudomonas aeruginosa, Candida albicans and C. tropicalis by using multilayer technique (Abubakar 2009). Extraction, fractionation and purification of antimicrobial compounds for these isolates were important for this study in order to characterize their antimicrobial compounds. Analysis of 16S rDNA and detection of KS domain of PKS and A domain of NRPS genes were important for identifying these bacteria as well as ensuring their capability in synthesizing the bioactive compounds. Aims and Scope of the Study The aims of this study were to determine the antimicrobial activity of bioactive compounds, analyze the 16S rDNA and detect the occurrence of KS and A domain of PKS and NRPS genes of those three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 that symbiosis with sponge Jaspis sp. LITERATURES Marine Natural Products The ocean covers more than 70% of earth surface and is considered as a great reservoir of natural resources. However, the extent of marine biodiversity, especially of microorganisms, is barely known. Marine microbial communities are composed of ubiquitous members that can be found not only in the sur face waters of the sea, but also in the lower and abyssal depths from coastal to the offshore regions (Larsen et al. 2005). Several studies have repor ted the discovery of new bioactive compounds from marine organisms, focusing mainly on chemistry of secondary metabolites, which include now more than 15,000 structurally diverse bioactive compounds isolated during the last 30 years (Salomon et al. 2004). Given the diversity of marine or ganisms and habitats, marine natural products encompass a wide variety of chemical classes such as terpenoid, polyketides, acetogenins, peptides and alkaloids of varying structures representing biosynthetic schemes of stunning variety (Wright 1998). Marine sponges are one of the benthic organisms that play a potential role as natural compounds producer. They also became a host for a wide range of microbes. The role of these diverse microbes in sponge biology varies from the digestion of microbes as a food source to mutualistic symbiosis with the spo nge. On the other hand, sponge is believed to provide shelter from predators, a substrate for colonization, access to sunlight for photosynthetic microbes and a supply of nutrients (Taylor et al. 2007). The availability of sponge biomass is the main factor for isolating marine natural prod ucts. Therefore, marine microorganisms which associated with marine sponges became one of the alternative ways to solve that problem. They have contributed to the majority of marine natural prod ucts and produced the same metabolite compounds as their hos t. Friedrich et al. (2001) reported that many sponges contain enormous amounts of bacteria within their tissues, sometimes occupying 40 to 60% of the total biomass (equivalent to 108 to 1010 bacteria per gram). Considering to the rich diversity of microorganisms in their tissues and the growth of microorganisms were more rapidly, therefore isolation and cultivation of associated microorganism producer of bioactive compounds could help to solve the recognized problem of development of potential sponge-derived drugs. Convincing evidence for the involvement of microorganisms in natural product synthesis has been complied for the tropical sponges Dysidea herbacea and Theonella swinhoei, in which the producing microbe is a cynobacterium in the former and a bacterium in the latter (Proksch et al. 2002). Thus an alternative strategy targeting the microorganisms associated with sponges for the screening of bioactive natural products may prove to be an effective approach to circumvent the associated difficulties of dealing with the organism itself. Marine Sponge -Associated Bacteria Sponges are filter feeders animal which live in areas with strong c urrents or wave action. Most carnivorous animals avoid sponges because of the splinter-like spicules and toxic chemicals produced/sequestered by the sponge. Sponges are organized around a system of pores, ostia, canals and chambers that are used to canalize the large flow of water that is pumped through spo nges. The water enters the sponge through the inhalant canals and exits by the oscules. A sponge is constituted of three layers. The first layer comprises pinacocytes and is called the pinacoderm. Under the pinacoderm is the mesohyl region that contains canals and choanocyte chambers. This is where the sponge metabolisms, reproduction and nutrient transfer occur. The third layer is the choa nod erm and contains choanocytes. They are flagellated cells possessing a collar of cytoplasmic tentacles. It is through the movement of these tentacles that the flow of water is created bringing in nutrients (Wilkinson 1992). There are mainly three classes of sponges, namely the Calcarea (5 orders and 24 families), Demospongiae (15 orders and 92 families) and Hexactinellida (6 orders and 20 families). So far about 15.000 species of sponges have been described, but their true diversity may be higher (Fieseler et al. 2004). Most of the species are placed under the class Demospongiae. Since sponges are simple and sessile organisms; during e volution they have de velope d potent chemical defensive mechanism to protect themselves from competitors and predators as well as infectious microorganisms (Belarbi et al. 2003). Interaction between marine sponges and living aquatic microorganisms are so variously and had many important roles. Many microorganisms were found that growth commensally in the surface and also found inside of the other microorganisms such as in the food digestive system. Porus from sponges contain diverse bacteria. Reinheimer (1991) found that there were many kinds of bacteria such as Pseudomonas, Bacillus, Micrococcus, Aeromonas, Vibrio, Achromobacter, Flavobacterium and Corynebacterium in Microcionia prolifera sponge. There is a symbiotic connection between sponges and a number of bacteria and algae. Sponges give protection for the symbionts and the symbionts give nutrition for sponges. Algae that symbiotic with sponges give nutrient from their photosynthesis product (Taylor et al. 2007). Suryati et al. (2000) reported that the formation of bioactive compounds from sponges was depend on the precursor of enzyme, nutrient and product of symbiotic with another biota that contain bioactive compounds such as bacteria, mold and another kinds of dinoflagellata that can spur on producing bioactive. Suryati et al. (2000) found a number of sponge types living in Spermonde seawater, South Sulawesi, the diversity of mold and symbiotic bacterial with sponges are so variously and usually dominated by Aeromonas, Flavobacterium, Vibrio, Pseudomonas, Acinetobacter and Bacillus (Table 1). Table 1 Bacterial isolates identification from marine sponges (Suryati et al. 2000) No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Marine Sponges Acanthela clethera Aplisina sp. Callyspongia sp. Clathria bacilana Clathria reinwardhi Jaspis sp. Phakelia aruensis Phyllospongia sp. Reniochalina sp. Thionella cilindrica Stylotella aurantiorum Xestospongia sp. Bacterial species Flavobacterium sp., Aeromonas sp. Aeromonas sp. Pseudomonas sp. Aeromonas sp. Aeromonas sp. Flavobacterum sp. Bacillus sp., Aeromonas sp. Vibrio sp., Pseudomonas sp., Aeromonas sp. Acinetobacter sp. Aeromonas sp. Aeromonas sp., Vibrio sp. Enterobacteriaceae sp., Aeromonas sp. Experimental evidence suggests that there are qualitative and quantitative variations in secondary metabolites produced by some organisms. There is a tende ncy to explain these variations ecologically with environmental factors influencing the biochemical profile of the organisms. There are many examples of marine invertebrates with chemical defenses. The synthesis and storage of these substances favors the survival of such organisms in a complex environment and gives such species a selective advantage due to the genetic transmission of the capacity for chemical defense synthesis (Chr istop hersen 1991). Menezes et al. (2010 ) reported that microbial diversity associated with algae, ascidians and sponges from the north coast of Sao Paulo State, Brazil had been dominated by phylum Firmicutes, Bacillus spp., together with Ruegeria spp. Bacillus was the most abundant genus recovered, with 33 isolates, followed by Ruegeria with 31 isolates and Micrococcus with 23 isolates. All of them revealed broad distribution among the marine macroorganisms sampled. 16S rDNA sequencing-based analysis showed that marine-derived bacteria were related to 41 genera distributed among the phyla Proteobacteria (35.4%), Actinobacteria (30.4%), Firmicutes (28.7%) and Bacteroidetes (1.1%). Bioactive Compounds Marine sponges are pre-eminent producers of bioactive secondary metabolites and their repertoire includes peptides, terpenes and sterols. Many of these compounds showed a functional diversity of actions including antimicrobial, antiviral and cytotoxic activities (Table 2). Bioactive compounds of sponge origin have been used as basic for the synthesis of analogs, for example is glycolipids produced by bacteria that live associated with the marine spo nge Agelas sp. and the antibacterial agelasines isolated from the marine sponge Agelas nakamurai (Bakkestuen et al. 2005). Kimura et al. (1998) had isolated 1-Methyherbipoline from Halisulfate-1 and suvanin as a serine protease inhibitor from Coscinoderma mathewsi sponge. Bioactive compounds such as macrocyclic peptide had isolated from Theonella swinhoei comes from water area at Japan. These bioactive compounds known as Cyclotheonamida A and B that have inhibitory activity to serine protease like thrombin and contains vinylogous tyrosine (V-Tyr) and α-ketoarginine residu which is still unknown amino acid in nature. Table 2 Bioactive compounds produced by marine sponges (Soediro 1999; Simmons et al. 2005) Pharmaceutical Activity Cytotoxic Bioactive Compounds Types of Sponge 3,6 epoksieikosa acid Swinholida A Vaskulin Halisilindramida A Jasplakinolide Jaspicamides Hymeniacidon hauraki Theonella swinhoei Cribrocalina vasculum Halichondria caveolata Jaspis johnstoni Jaspis sp. Anticancer Agelasfin (AGL) Agelas muritianus Anti blood cancer Kurasin A Amfidinolid B1, B2, B3, N, Q Triangulinat acid Lingbya majuscule Amphidinium sp. Pellina triagulata Antiviral (HIV 1) Trikendiol Trikentrion loeve Antimicrobial Hormotamnim Diskodermin E-H Wondosterols Hormothamnion Discodermia kiiensis Jaspis wondoensis Antibacterial Lokisterolamin A and B Corticium sp. Antifungal kortikatat acid A,B,C Leukasandrolida Halisilindramida Petrosia corticata Leucasandra caveolata Halichondria cylindrical Imunomodulator Agelasflin 10 and 12 Agelas muritianus Anti-inflammatory Manualida Luffariella variabilis Unknown substances (still research) Halisiklamina A Bastadin A and B Klatirimin Halisiklamina B Haliclona sp. Lanthella basta Clathria basilana Xestrospongia sp. O’Keefe et al. (1998) had isolated Adociavirin from Adocia sp. sponge at Bay water area, New Zealand; extract that dissolved in distillation water potential as antisitopatic inside of CEM-SS cell which infection from HIV-1. Matsunaga et al. (1992) had been isolated 1-acid carboxymethylnicotinic from sponge Antosigmella raromicroscera which can be used as protease inhibitor. Li et al. (2006a) had been isolated 399 bacteria from the sponges Stelletta tenuis, Halichondria rugosa, Dysidea avara, and Craniella australiensis in the South China Sea, among which, 13 isolates from S. tenuis, 42 from H. rugosa, and 20 from D. avara showed pronounced broad-spectrum antimicrobial activities and enzymatic potentials. Many of the pharmacologically most promising natural products from sponges are complex polyketides. The fact that polyketide synthases (PKSs) are almost absent in metazoans suggests a microbial origin. PKSs are therefore a particularly good study object to investigate the role of symbionts in the chemistry of marine sponges (Castoe et al. 2007). Polyketide Synthas e and Nonribos omal Peptide Synthetas e The structural characteristics of marine natural products have revealed that they mainly belong to two important chemical families, namely, polyketides and cyclopeptides, and are synthesized by multifunctional enzymes called polyketide synthases (PKSs) and nonribosomal peptide synthetase (NRPSs). Polyketides are a group of secondary metabolites, exhibiting remarkable diversity in their structures and functions. Polyketide natural prod ucts are known for their wide range of pharmacologically important activities, including antimicrobial, antifungal, antiparasitic and antitumor properties (Hill 2005). Nonribosomal peptides are part of a family of complex natural products built from simple amino acid monomers. They can be found in bacteria and fungi where they are synthesized by nonribosomal peptide synthetase (NRPS) which are large multimodular and multifunc tional proteins. Nonribosomal peptides as well as the hybrid products are of much interest because of their pharmaceutical properties such as the immunosuppressant cyclosporine (Schwartzer et al. 2003). Schirmer et al. (2005) had already characterized PKS gene cluster in metagenomic libraries from Discordemia dissolute. The PKS gene cluster is 110 kb and contain of three open reading frames (ORF). The first PKS ORF codes for a remarkably large protein of 25.572 amino acids with a predicted molecular mass of 2.7 MDa. The most remarkable features of this large PKS gene cluster are the presence of a complete set of reductive domains (ketoreductase, dehydratase, and enoylreductase) in all except one module, which lacks the ER, and Cmethyltransferase domains in 8 of the 14 modules. The products of the PKS gene clusters have more similarity with fatty acids (Figure 1). Figure 1 Organization of the multimodular PKS gene cluster in metagenomic libraries from D. dissolute (Schirmer et al. 2005). Nguyen (2009) had already investigated the polyketides biosynthetic pathway in the spo nge Theonella swinhoei and the beetle Paederus fuscipes. The adenylation (A) domain of an NRPS module is responsible for specific selection and activation of a defined amino acid. Expression of A domains should help us gain insights into the biosynthetic pathways of pederin and onnamides. There were two A domains on the module PedF2 and PedH6 of the ped gene cluster and also two A domains on the module OnnI2 and OnnJ4 of the onn gene cluster. In agreement with the structure of onnamide B, the prediction results showed that glycine and arginine were the specific amino acids of NRPS modules on the onn gene cluster (Figure 2). Figure 2 Two A domains inside the biosynthetic gene cluster of Onnamide B (Nguyen 2009). MATERIALS AND METHODS Duration and Place of Study This research was carried out from July 2011 to March 2012. Extraction, fractionation and purification of antimicrobial compounds were carried out in the laboratory of Microbiology, Department of Biology and Biopharmaca Research Center, IPB, Indonesia. Molecular genetic analysis was carried out in Yohda Laboratory, Department of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology (TUAT), Japan. The flowchart of method s that used in this study is given in F igure 3. Materials Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 had been isolated from sponge Jaspis sp. at Waigeo Island, Raja Ampat District, West Papua Province by Abubakar (2009). These bacteria were used for the extraction, fractionation and purification of antimicrobial compounds. Specific primer 63 f (5CAGGCCTAACACATGCAAGTC-3) and 1387r (5-GGGCGGWGTGTACAAG GC-3) was used for analysis of 16S rDNA (Marchesi et al. 1998). Degenerate pr imer (f: 5-GCSATGGAYCCSCARCARCGSVT-3); (r: 5-GTSCCSGTSCCRTG SSCYTCSAC-3) for KS domain and degenerate primer (f: 5-AARDSIGGIGSIG SITAYBICC-3); (r: 5-CKRWAICCICKIAIYTTIAYYTG-3) for A domain (Schirmer et al. 2005). Extraction of Antimicrobial Compounds Each of those three isolates was sub-cultured in 500 ml Seawater Complete Broth media (bacto peptone 2.5 g, yeast extract 0.5 g, glycerol 1.5 ml, seawater 375 ml and distilled water 125 ml) and incubated in fluctuate incubator with 100 rpm at 300C until the culture reached the stationary phase. After that, 10% liquid bacterial inocula from previously incubation were cultured in 500 ml SWC broth and incubated in the same condition until reached the stationary phase for secondary metabolite production (Muller et al. 2004). Extraction of antimicrobial compounds was done by modifying the method from Sunaryanto et al. (2010). As much 500 ml of liquid bacterial culture were mixed with 500 ml of ethyl acetate solvent, incubated at room temperature for 24 hours and stirred for 2 hours with 250 rpm. These mixtures were separated and the ethyl acetate layers were evaporated with rotary evaporator until the drying residue was obtained as crude extract. The crude extract of bacterial cells were dissolved with ethyl acetate (pro analyze) to get 100 mg/ml concentration. Extraction of Antimicrobial Compounds DNA Extraction Antimicrobial Activity Test PCR Amplification of Ketos ynthase (KS) and Adenylation (A) Domain Detection of Antimicrobial Compounds Fractionation of Bacterial Crude Extract Cloning of DNA Fragments Encod ing KS and A Domain Sequencing and Bioinformatics Analysis of KS and A Domain Purification of Antimicrobial Compounds Morphological and Molecular Identification Figure 3 Flowchart of procedural steps used in this study. Antimicrobial Activity Test Antibacterial and antifungal activities from crude extracts were tested by using a gar diffusion method a gainst microbial test strains. As much 100 µl bacterial crude extracts dissolved in ethyl acetate (pro analyze) were applied carefully into 6 mm paper disks (Whatman) and at the same time, the disks were dried up using a hairdryer at 400 C. After that, the disks were sterilized under UV light for 2 hours and put into agar plate that have been seeded with 1% (v/v) of microbial test strains (conc entration 1x10 6 CFU/ml, OD 620 0.45). The plate was incubated at 40 C for 3 hours to optimize the diffusion of bacterial crude extract into media. This assay was carried out in triplicate. The diameters of inhibition zones were measured in millimeters after incubation for 24 hours at 370 C. Control disks were soaked with ethyl acetate solvent and prepared in the same manner (Sudirman 2010). Detection of Antimicrobial Compounds Antimicrobial compounds in each of the bacterial crude extract were detected using bioautography method. As much 10 µl of crude extracts were spotted on TLC plates (MERCK Silica Gel 60 F 254 ) and eluted with vertical chromatography using n-butanol : ethyl acetate solvent mixture with the ratio of 3:7 (v/v). The spots on TLC plate were detected under UV light at 254 nm and 366 nm wave- length. After that, the retardation factor (Rf) values were calculated. The spots on TLC plate were cut off and dried up in room temperature. The developed TLC plates were sterilized under UV light for 1 hour before covered by 15 ml of melting SWC (450 C) containing test strains, and incubated at 37 0 C for 24 hours. Diameter of inhibition zone around the chromatogram indicated that the spo t was an active fraction (Sudirman 2005) . Fractionation of Bacterial Crude Extract Bacterial crude extract of isolate SAB E-41 was fractionated using semi automated flash chromatography (Buchi Pump Controller C-610). As much 3 g of crude extract was dissolved with chloroform- methanol solvent mixture (90%-10%) and injected into silica gel-column chromatography (column dimension 0.40 x 150 mm, particle size of silica gel 40 x 63 µm). Chloroform- methanol solvent mixture (90%-10%) was flowed into silica gel-column chromatography with the flow rate of 3.5 ml/minutes. The polarity level in the column was increased slowly by changing the methanol concentration from 20%, 30%, 50%, 70% until 90%. Two hundred and five fractions were collected (5 ml/each fraction) from fraction collector and combined into thirty fractions based on the same chromatogram. These fractions were dried up and dissolved with chloroform- methanol-ethyl acetate solvent (1:1:1) to test their antimicrobial activity. Purification of Antimicrobial Compounds Fifteen active fractions were further purified using preparative thin layer chromatography (PTLC) technique. Each of the active fractions was spotted onto silica gel plate (MERCK Silica Gel 60 F 254 ; 0.1 mm thickness) and eluted with nbutanol-ethyl acetate solvent mixture (3:7). The active spots on the silica gel plate were detected under UV light at 254 nm and 366 nm wave-length. The active spots were extracted directly from the silica gel plate and dissolved with chloroformmethanol-ethyl acetate solvent mixture (1:1:1). The active fractions that were purified by preparative TLC were tested for their antimicrobial activity. Morphological and Molecular Identification Morphological characterization of those three isolates was performed using Gram staining p rocedure. Molecular analysis of 16S rDNA was done using specific primer, 63f (5-CAGGCCTAACACATGCAAGTC-3) and 1387r (5-GGG CGGWGTGTACAAGGC-3) (Marchesi et al. 1998). The PCR cycling condition for 16S rDNA was carried out under the following condition such as initial denaturation at 94 0 C for 5 min, followed by 30 cycles of denaturation at 940 C for 1 min, annealing at 550 C for 1 min, elongation at 720 C for 1 min and post PCR at 720C for 10 min. PCR products of 16S rDNA were purified using GENECLEAN ® II Kit. These PCR products were sub-cloned into T-Vector pMD20 and transformed into competent E. coli DH5-α using heat shock method (Sambrook & Russell 2001). Afterwards, several steps such as PCR colony, isolation and restriction of recombinant plasmid, PCR sequencing and purification of PCR products were done before the 16S rDNA sequence analysis. DNA Extraction Each of bacterial isolates were sub-cultured into SWC broth media and incubated at room temperature for 24 hours. After that, 1.5 ml bacteria isolate was drawn into microtube and centrifuged (18.000xg) for 10 min to obtain bacteria pellet that were used for DNA extraction. The supernatant was discarded and 250 µl Tris-EDTA (TE) buffer was added and centrifuged at 8000 rpm for 10 min. The supernatant was discarded and pellet was re-suspended three times in TE buffer. As much 250 µl TE and 5 µl lysozyme were added together and microtube slowly inverted to allow mixing and incubated at 370 C for 30 min. After incubation, the solution was added with 500 µl SDS 10% and 10 µl proteinase K and incubated again at 370 C for 60 min. Afterwards, as much 80 µl NaCl was added together with 100 µl CTAB 10% and incubated at 650 C for 20 min. After incubation, added again the solution with 650 µl PCI and shake n strongly then centrifuged at 14.000 rpm for 10 min. The upper solution was transferred into a new microtube then 650 µl CI was added and centrifuged again in same condition. DNA was precipitated using absolute ethanol (2x vol) and Na acetate 3 M 0.1 vol and incubated overnight in freezer. After that, 1 ml ethanol 70% was added for final washing and centrifuged at 12.000 rpm for 10 min. The supernatant was discarded and pellet was air dried overnight. After this step, 20 µl of TE was added and the extracted DNA was stored at -200C for further applications (Sambrook & Russel 2001). PCR Amplification of KS and A Domain KS domain of PKS and A do main of NRPS genes from those three isolates were amplified using PCR primers such as degenerate KS domain (f: 5-GCSATG GAYCCSCARCARCGSVT-3); (r: 5-GTSCCSGTSCCRTGSSCYTCSAC-3) and degenerate A domain (f: 5-AARDSIGGIGSIGSITAYBICC-3); (r: 5-CKRWAICC ICKIAIYTTIAYYTG-3) (Schirmer et al. 2005). The PCR cycling condition for KS domain was carried out in three steps such as initial denaturation at 94 0 C for 5 min, followed by 35 cycles of denaturation at 940 C for 1 min, annealing at 500 C for 1 min, elongation at 720 C for 1 min 10 sec and post PCR at 720 C for 10 min. The PCR cycling condition for A domain was the same as for KS domain except for annealing which was carried out at 550 C for 1 min. In all cases, the reaction mixtures contained 4 µl dNTP mix (2.5 mM), 5 µl 10X Ex Taq buffer, primer forward and reverse (10 µM; each of 5 µl), 2 µl DNA template (500 ng/µl), 1 µl TaKaRa Ex TaqTM (5 units/µl) and 28 µl milli Q. The total volume of reaction mixtures was 50 µl. PCR products were analyzed using agarose gel electrophoresis 1% (b/v). Cloning o f DNA Frag me nts Encoding KS and A Domain Purification of PCR products from KS domain of PKS gene (700 bp) and A domain of NRPS gene (1000 bp) were carried out using GENECLEAN ® II Kit. Each of the purification prod ucts was sub-cloned into T-Vector pMD20 (TaKaRa) and transformed into competent E. coli DH5α using heat shock method (Sambrook & Russell 2001). Isolation of recombinant plasmid was performed using Mag ExtractorT M Quick Plasmid Miniprep kit (Toyobo, Japan). Restriction of recombinant plasmid was conducted using the combination of restriction enzymes such as BamHI (BioLabs) and XbaI (TaKaRa). The reaction mixtures contained 0.2 µl BamHI (5 units/µl), 0.2 µl XbaI (5 units/µl), 1 µl 10X NE buffer 4, 0.5 µl DNA template (500 ng/µl) and 8.1 µl milli Q. The total volume of reaction mixtures was 10 µl. The reaction mixtures were incubated at 370 C for 24 hours and the restriction product was analyzed using agarose gel electrophoresis 1% (b/v). Sequencing a nd Bioinformatics Analysis of KS and A Domain DNA fragments that were inserted into plasmid T-Vector pMD20 named pMD20-KS domain and pMD20-A domain were used for the sequencing process. M13 primer RV and M13 primer M4 were used for PCR sequencing. The PCR cycling condition was carried in three steps such as initial denaturation at 960 C for 5 min, followed by 25 cycles of denaturation at 960 C for 1 min, annealing at 500 C for 30 sec, elongation at 60 0 C for 1 min and post PCR at 40 C for an unlimited time. The Big Dye® X TerminatorT M purification kit was used to purify the PCR product before DNA sequencing. The DNA was run in an automated DNA sequencer using ABI 3130 XL Genetic Analyzer. The DNA sequences were compared to the database available at NCBI using BlastN program for 16S rDNA and BlastX program for KS and A domain. Construction of phylogenetic tree was carried out using MEGA5 program with neighbor-joining method. RESULTS Antimicrobial Activity of Bacterial Crude Extracts Antimicrobial compounds of three bacterial isolates can be extracted using ethyl acetate solvent. During the extraction process, the ethyl acetate solvent will be formed two layers. First layer was aqueous phase that contained the antimicrobial compounds which have been extracted by ethyl acetate solvent. Second layer was organic phase that contained the bacterial cultures. The first layer was obtained and separated from bacterial cultures then evaporated with rotary evaporator until the drying residue was obtained as crude extract. Crude extracts of isolates SAB E-31, SAB E-41 and SAB E-57 showed different antimicrobial activity against non-pathogenic and pathogenic microbes. The bacterial crude extract of isolate SAB E-41 showed better antimicrobial activity than bacterial crude extracts of isolates SAB E-31 and SAB E-57. Crude extracts of those three isolates demonstrated the best activity against S. aureus (Table 3). Table 3 Diameter average of inhibition zone (mm) from three bacterial crude extracts (100 mg/ml) produced by sponge-associated bacteria Bacterial Isolates SAB E-31 SAB E-41 SAB E-57 Positive Control (Ampicilin 100 mg/ml) Negative Control (Ethyl acetate) BS* 8.3 10.5 9.5 Diameter Average of Inhibition Zone (mm) SA** EC* PA** EPEC K1-1** CA** CT** 10.5 5.3 4.8 5.6 2.5 2.5 14.5 7.5 6.7 8.7 5.7 4.5 13.2 6.4 5.8 6.5 3.8 2.8 14 30 9 27 - - - - - - - - - - BS = B. subtilis; SA = S. aureus; EC = E. coli; PA = P. aeruginosa; EPEC K1-1 = Enteropa thogenic E. coli K1-1; CA = C. albicans; CT = C. tropicalis; * = nonpathogen; ** = pa thogen. Crude extracts of those three isolates showed different antimicrobial activity against EPEC K1-1, C. albicans and C. tropicalis whereas for positive control with ampicilin 100 mg/ml, there was no inhibition a gainst these pathogenic microbe s (Figure 4). Ethyl acetate solvent that used as a negative control didn’t inhibit the growth of non-pathogenic or pathogenic microbes. Figure 4 Antimicrobial activity of three bacterial crude extracts isolates SAB E-31, SAB E-41 and SAB E-57 using agar diffusion method; C+ = positive control (ampicilin 100 mg/ml); C- = negative control (ethyl acetate). Thin Layer Chromatography and Bioautography TLC analysis for three bacterial crude extracts showed that there were many spots in silica gel plate with many k inds of retardation factor (Rf) values (Table 4). The solvent system, n-butanol-ethyl acetate with the ratio of 3:7 had successfully separated the component of bacterial crude extract. Six spots and many kinds of Rf values were obtained from this solvent system (Figure 5). Table 4 Variation of Rf values from three bacterial crude extracts eluted with different solvent systems Solvent Systems Crude Extracts n-but : CH 3 COOH : SAB E-31 ddH 2 O SAB E-41 SAB E-57 (3:1:1) n-but : EtOAc : ddH 2 O (2:3:1) n-but : EtOAc (3:7) Number of Spots λ 254 λ 366 nm nm 3 2 3 2 3 2 Rf Values λ 254 λ 366 nm nm 0.80; 0.61; 0.31 0.80; 0.61 0.91; 0.72; 0.35 0.91; 0.72 0.88; 0.72; 0.35 0.88; 0.72 SAB E-31 SAB E-41 SAB E-57 3 4 4 2 2 2 0.91; 0.53; 0.44 0.91; 0.83; 0.67; 0.55 0.92; 0.85; 0.61; 0.50 0.91; 0.53 0.91; 0.83 0.92; 0.85 SAB E-31 6 3 0.88; 0.55; 0.23 SAB E-41 6 3 SAB E-57 6 3 0.88; 0.72; 0.62; 0.55; 0.44; 0.23 0.92; 0.77; 0.66; 0.60; 0.46; 0.27 0.91; 0.75; 0.65; 0.56; 0.45; 0.31 0.92; 0.60; 0.27 0.91; 0.56; 0.31 Figure 5 Profile of each bacterial crude extract on silica gel plate merck 60 F 254 eluted with n-butanol and ethyl acetate mixture (3:7); Red box: active spots/fractions. The spots/fractions which were detected under UV light at 254 and 366 nm wave- length were further tested for their antimicrobial activity using bioautography method. This method was able to quickly detect which spots/fractions were the active fractions or pollutant compounds in bacterial crude extract. Part of the silica gel plate was cut based on the separated spots/fractions and the different of Rf values in order to make the visualization of inhibition zones clearer. Diameter of inhibition zone around the chromatogram indicated that the spot was an active fraction (Figure 6). Bioautography detection resulted that at least 4 active spots/fractions showed antimicrobial activity against P. aeruginosa and 2 active spots/fractions which inhibited the growth of S. aureus (Table 5). Figure 6 Antimicrobial activity of active spo ts/fractions using bioautography method. Table 5 Active spots/fractions of three bacterial crude extracts detected using bioautography method Bacterial Isolates SAB E-31 SAB E-41 SAB E-57 Rf Values Microbial Test Strains PA** + ++ ++ ++ CA** - Rf 1 Rf 2 Rf 3 Rf 4 Rf 5 Rf 6 = 0.88 = 0.72 = 0.62 = 0.55 = 0.44 = 0.23 SA** - Rf 1 Rf 2 Rf 3 Rf 4 Rf 5 Rf 6 = 0.92 = 0.77 = 0.66 = 0.60 = 0.46 = 0.27 +++ +++ - ++ +++ +++ ++ - Rf 1 Rf 2 Rf 3 Rf 4 Rf 5 Rf 6 = 0.91 = 0.75 = 0.65 = 0.56 = 0.45 = 0.31 +++ + - ++ +++ +++ ++ - + = Weak inhibition; ++ = Medium inhibition; +++ = Strong inhibition; SA = S. aureus; PA = P. aeruginosa; CA = C. albicans; ** = pathogen. Fractionation of Bacterial Crude Extract from Isolate SAB E-41 Two hundred and five fractions were successfully collected from the fractionation process and combined into thirty composite fractions based on the same chromatogram. These composite fractions were evaporated until the drying residue was obtained as crude extract. These crude extracts were dissolved with chloroform- methanol-ethyl acetate mixture (1:1:1) in concentration of 100 mg/ml. Antimicrobial activity of thirty compos ite fractions were tested to S. aureus, P. aeruginosa, EPEC K1-1, C. albicans and C. tropicalis. Fifteen composite fractions coded as BA-1, BA-2, BA-3, BA-4, BA-5, BA6, BA-7, BA-8, BA-11, BA-12, BA-13, BA-14, BA-15, BA-17 and BA-18 showed different antimicrobial activity against S. aureus, P. aeruginosa, EPEC K1-1 and C. albicans (Table 6). Fraction BA-2 that was eluted by chloroform- methanol (90%-10%) solvent system showed antifunga l activity against C. albicans whereas no active fractions showed antimicrobial activity against C. tropicalis. Table 6 Antimicrobial activity of thirty composite fractions collected from silica gel-column chromatography Solvent Systems and Fractions Chloroform-Methanol (90%-10%) Fraction BA-1 Fraction BA-2 Fraction BA-3 Fraction BA-4 Fraction BA-5 Fraction BA-6 Fraction BA-7 Chloroform-Methanol (80%-20%) Fraction BA-8 Fraction BA-9 Fraction BA-10 Chloroform-Methanol (70%-30%) Fraction BA-11 Fraction BA-12 Chloroform-Methanol (50%-50%) Fraction BA-13 Fraction BA-14 Fraction BA-15 Fraction BA-16 Fraction BA-17 Fraction BA-18 Chloroform-Methanol (30%-70%) Fraction BA-19 Fraction BA-20 Fraction BA-21 Fraction BA-22 Fraction BA-23 Chloroform-Methanol (20%-80%) Fraction BA-24 Fraction BA-25 Fraction BA-26 Fraction BA-27 Chloroform-Methanol (10%-90%) Fraction BA-28 Fraction BA-29 Fraction BA-30 Negative Control (Chloroform-Methanol) Positive Control (Ampicilin 100 mg/ml) Diameter of Inhibition zone (mm) SA** PA** EPEC K1-1** CA** CT** 7 8 7 3 5 4 4 2 6 4 3 4 4 2 7 10 3 10 3 2 3 3 - - 2 - - - - - 3 10 3 3 - - - 14 2 2 12 3 2 - 7 - - - - - - - - - - - - - 30 27 - - - SA = S. aureus; PA = P. aeruginosa; EPEC K1-1 = Enteropathogenic E. coli K1-1; CA = C. albicans; CT = C. tropicalis; ** = pathogen. Fraction BA-13 that was eluted by chloroform- methanol (50% -50%) solvent system demonstrated the highest inhibition against S. aureus followed by fraction BA-17. The diameter of inhibition zone that formed by these two active fractions were about 12 mm and 14 mm (Figure 7). Fraction BA-2 and BA-4 that was eluted by chloroform- methanol (90% -10%) showed the best activity against EPEC K1-1. The diameter of inhibition zone that formed by these two active fractions were about 10 mm (Figure 7). Figure 7 Antimicrobial activity of thirty compos ite fractions; C+ = positive control (ampicilin 100 mg/ml); C- = negative control (chloroform- methanol). Purification of Antimicrobial Compounds using Preparative TLC Fifteen composite fractions that had an active fraction were further purified using PTLC technique. Antimicrobial compounds in active fractions could be extracted directly from silica gel plate and dissolved with chloroform- methanolethyl acetate (1:1:1). Antimicrobial compounds of active fractions, obtained from PTLC technique were tested to S. aureus, EPEC K1-1 and C. albicans. Fraction BA-13 has demonstrated as the most antimicrobial compounds compared to the other fractions (Table 7). This fraction resulted four different of active compounds with Rf values of 0.87; 0.50; 0.41 and 0.12 (Figure 8). These four active compounds displayed significant activity against S. aureus and EPEC K1-1. Fractions BA-17 and BA-18 carried two kinds of active compounds with Rf values of 0.93; 0.12 for the BA-17 and 0.93; 0.25 for the BA-18 (Figure 8). Both of these active compounds showed activity against S. aureus. Fraction BA-2 carried one active compound (Rf 0.77) that showed activity against C. albicans (Figure 9). Table 7 Rf values of active compounds from fifteen composite fractions Active Compounds BA-1 BA-2 BA-3 BA-4 BA-5 BA-6 BA-7 BA-8 BA-11 BA-12 BA-13 BA-14 BA-15 BA-17 BA-18 Rf Values Rf 1 = 0.81 Rf 1 = 0.77 Rf 1 = 0.78 Rf 1 = 0.87 Rf 2 = 0.66 Rf 1 = 0.81 Rf 2 = 0.53 Rf 1 = 0.87 Rf 2 = 0.65 Rf 3 = 0.35 Rf 1 = 0.87 Rf 2 = 0.62 Rf 3 = 0.38 Rf 1 = 0.90 Rf 2 = 0.71 Rf 3 = 0.35 Rf 1 = 0.87 Rf 2 = 0.68 Rf 1 = 0.90 Rf 2 = 0.71 Rf 3 = 0.62 Rf 4 = 0.41 Rf 5 = 0.12 Rf 1 = 0.87 Rf 2 = 0.72 Rf 3 = 0.60 Rf 4 = 0.50 Rf 5 = 0.41 Rf 6 = 0.12 Rf 1 = 0.90 Rf 2 = 0.75 Rf 3 = 0.68 Rf 4 = 0.58 Rf 1 = 0.93 Rf 2 = 0.78 Rf 3 = 0.68 Rf 4 = 0.33 Rf 1 = 0.93 Rf 2 = 0.71 Rf 3 = 0.68 Rf 4 = 0.33 Rf 5 = 0.12 Rf 1 = 0.93 Rf 2 = 0.41 Rf 3 = 0.25 Rf 4 = 0.12 Diameter of Inhibition Zone (mm) SA** EPEC K1-1** CA** 4 4 8 14 2 2 2 6 4 10 2 10 2 6 4 8 3 10 3 10 4 6 8 4 12 2 2 2 14 2 2 2 - SA = S. aureus; EPEC K1-1 = Enteropathogenic E. coli K1-1; CA = C. albicans; ** = pathogen. λ 366 nm λ 366 nm Active Co mpounds (Rf 0.87) Active Co mpounds (Rf 0.93) λ 254 nm Active Co mpounds (Rf 0.50) Active Co mpounds (Rf 0.41) Active Co mpounds (Rf 0.12) BA-17 BA-13 Active Co mpounds (Rf 0.12) Active Co mpounds (Rf 0.93) Active Co mpounds (Rf 0.25) BA-18 Figure 8 Profile of active compounds from fraction BA-13, BA-17 and BA-18 on silica gel plate merck 60 F 254 (Arrow head). Figure 9 Antimicrobial activity of fifteen compos ite fractions purified using PTLC technique; C- = negative control (ethyl acetate). Morphological and Molecular Identification Bas ed on 16S rDNA Analysis Morphological characterization of isolates SAB E-31, SAB E-41 and SAB E-57 was carried out using Gram staining bacteria. Gram staining results showed that three marine bacterial isolates were rod-shaped, motile, formed spores and Gram-pos itive bacteria. These isolates have the same characteristics with the genus of Bacillus (Figure 10). 2 µm A. 2 µm B. 2 µm C. Figure 10 Gram staining of three bacterial isolates coded as: A). SAB E-31; B). SAB E-41 and C). SAB E-57. Molecular analysis of 16S rDNA was carried out for the further identification of those three isolates. PCR product of 16S rDNA sequences was about 1300 bp. Sequences analysis of 16S rDNA showed a high similarity level (97-98%) to various strains of Bacillus compared to those available in GenBank database (Table 8). Table 8 Similarity of 16S rDNA sequences from isolates SAB E-31, SAB E-41 and SAB E-57 compared with GenBank Database Bacterial Isolates SAB E-31 SAB E-41 SAB E-57 Similarity Bacillus pumilus strain KD3 Bacillus amyloliquefaciens strain zy2 Bacillus subtilis strain YRL02 Identity (%) 98 98 97 EValue 0.0 0.0 0.0 Accession EU500930.1 JN160740.1 EU373407.1 Phylogenetic analysis of 16S rDNA sequences showed that isolates SAB E31, SAB E-41 and SAB E-57 formed a different clade with the reference strains of Bacillus in GenBank database. Clade-1 was formed by the reference strains of Bacillus while clade-2 was formed by those three isolates with Bacillus sp. DF49 (Figure 11). Figure 11 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57 with the reference strains based on 16S rDNA sequences. Numbers at the node s indicate the levels of bootstrap support based on 1000 re-sampled data sets. The scale bar indicates 0.1 substitutions per nucleotide pos ition. Amplification of DNA Frag me nts Encoding KS and A Domain DNA fragments of three bacterial isolates encoding KS and A domain were successfully amplified using PCR. Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 had a DNA fragment encoding A do main whereas only two bacterial isolates coded as SAB E-41 and SAB E-57 had a DNA fragment encoding KS do main. Visualization of the PCR amplicon for DNA fragments was analyzed using agarose gel electrophoresis 1% (b/v). DNA fragment encoding KS domain was in a size of 700 bp while for A domain was in a size of 1000 bp SAB E-57 SAB E-41 SAB E-31 SAB E-57 SAB E-41 SAB E-31 bp 1 kb Ladder (Figure 12). 1 kb 1000 750 500 250 A Do main (~1000 bp) KS Do main (~700 bp) Figure 12 Agarose gel electrop horesis of DNA fragments encod ing KS Domain and A Domain. Cloning a nd Bioinformatics Analys is DNA fragments of those three isolates encoding KS and A domain were successfully cloned into T-Vector pMD20 (TaKaRa Bio Inc.) and named pMD20KS domain and pMD20-A domain. White colonies of E. coli DH5-α carrying the recombinant plasmid had been isolated and for the plasmid excision was do ne using a combination of restriction enzymes, Bam HI and XbaI. The cropped recombinant plasmid DNA resulted in two bands which were approximately of 2736 bp that showed the size of T- vector pMD20 and 700 bp was the size of DNA fragment encoding KS domain and 1000 bp for DNA fragment encoding A do main (Figure 13). 19329 7743 6223 4254 3472 T-Vector 2690 pMD20 1882 (2736 bp) 1489 19329 7743 6223 4254 3472 2690 1882 1489 KS Do main (~700 bp) 421 SAB E-57 T-Vector pMD20 (2736 bp) A Do main (~1000 bp) 925 925 SAB E-41 SAB E-31 bp Control λ Marker SAB E-57 SAB E-41 Control λ Marker bp 421 A B Figure 13 Restriction of recombinant plasmids digested with BamHI + XbaI; A). pMD20-KS domain and B). pMD20-A do main. Bioinformatics sequences analysis of DNA fragment encoding KS domain from two bacterial isolates coded as SAB E-41 and SAB E-57 using BlastX program showed that isolate SAB E-41 had a similarity level of 97 % with type I PKS from Bacillus amyloliquefaciens LL3 while isolate SAB E-57 had a similarity level of 98% with putative polyketide synthase pksL from B. amyloliquefaciens subsp. plantarum CAU-B946 (Table 9). Table 9 Bioinformatics sequences analysis of DNA fragment encoding KS domain using BlastX program Bacterial Isolates SAB E-41 SAB E-57 Similarity Type I PKS; B. amyloliquefaciens LL3 Putative polyketide synthase pksL; B. amyloliquefaciens subsp. plantarum CAU-B946 Identity (%) 97 EValue 2e-130 YP_005545643.1 98 2e-143 YP_005130937.1 Accession Sequences analysis of DNA fragment encoding A domain showed that isolate SAB E-31 had a similarity level of 81% with bacitracin synthetase 1 from B. pumilus ATCC 7061. Isolate SAB E-41 had a similarity level of 80% with surfactin synthetase B from B. amyloliquefaciens subsp. plantarum CAU-B946 while isolate SAB E-57 had a similarity level of 81% with surfactin synthetase A from B. amyloliquefaciens subsp. plantarum CAU-B946 (Table 10). Table 10 Bioinformatics sequences analysis of DNA fragment encoding A domain using BlastX program Bacterial Isolates SAB E-31 SAB E-41 SAB E-57 Similarity Bacitracin synthetase 1; B. pumilus ATCC 7061 Surfactin synthetase B; B. amyloliquefaciens subsp. plantarum CAU-B946 Surfactin synthetase A; B. amyloliquefaciens subsp. plantarum CAU-B946 Identity (%) 81 EValue 1e-154 ZP_03054623.1 80 1e-139 YP_005129035.1 81 1e-129 YP_005129034.1 Accession Amino acid sequences of KS domain from isolates SAB E-41 and SAB E57 were aligned and compared with the reference strains in GenBank database. Two bacterial isolates have a homology of conserved region with the reference strains (Figure 14). Phylogenetic analysis of amino acid sequences of KS domain showed that isolates SAB E-41 and SAB E-57 formed a different clade with the other reference strains (Figure 15). Figure 14 Alignment of amino acid sequences of KS domain from isolates SAB E41 and SAB E-57 with the reference strains in GenBank Database using ClustalW program. Shaded area showed the similarity of amino acid sequences. Black bo x showed a homology of conserved region. Clade-1 Clade-2 Figure 15 Phylogenetic tree of isolates SAB E-41 and S AB E-57 with the reference strains based on amino acid sequences of KS domain. Numbers at the nodes indicate the levels of bootstrap support based on 1000 re-sampled data sets. The scale bar indicates 0.1 substitutions per nucleotide pos ition. Amino acid sequences of A domain from isolates SAB E-31, SAB E-41 and SAB E-57 were also aligned and compared with the reference strains in GenBank database. These bacteria have a homology of conserved region with the other reference strains (Figure 16). Phylogenetic analysis of amino acid sequences of A domain showed that isolate SAB E-41 was formed a similar clade with B. amyloliquefaciens FZB42 while isolates SAB E-31 and SAB E-57 formed a same clade (Figure 17). Figure 16 Alignment of amino acid sequences of A domain from isolates SAB E31, SAB E-41 and SAB E-57 with the reference strains in GenBank Database using ClustalW Program. Black box showed a homology of conserved region for amino acid sequences. Figure 17 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57 with the reference strains based on amino acid sequences of A domain. Numbers at the nodes indicate the levels of bootstrap support based on 1000 re-sampled data sets. The scale bar indicates 0.2 substitutions per nucleotide pos ition. DISCUSSION Antimicrobial compounds of three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 can be directly extracted using ethyl acetate solvent. The selection of this solvent system for the extraction process is based on the polarity difference of the liquid culture of bacteria. This solvent is non-pola r so that the separation of liquid culture of bacteria from ethyl acetate layer was more easily done. Jeffery et al. (1989) stated that antimicrobial compounds contained in bacterial crude extract have different solubility prope rties in each of solvent system. The selection of an appropriate solvent in the extraction process is largely determined by the solubility properties of the antimicrobial compounds, type of substrate, the partition coefficient and the distribution ratio of solvent system. Crude extracts of those three isolates showed different antimicrobial activity against non-pathogenic and pathogenic microbes. The highest activity shown by the bacterial crude extracts of isolate SAB E-41 which indicated from the large diameter of the inhibition zone. Lay (1994) stated that physical and chemical properties of antimicrobial compounds will affect the resulting of clear zone. The greater of the molecular weight of bioactive compounds, the more enlarge of the inhibition zone. The other factors that also affecting the inhibition zone are the density of cells, the sensitivity of microbial test strains to antimicrobial compounds, the component of antimicrobial compounds, the diffusion rate of antimicrobial compounds into media and the expos ure time of microbial test strains to antimicrobial compounds. On the other hand, crude extracts of those three isolates showed the highest activity against S. aureus whereas the lowest activity shown by the test strains of C. albicans and C. tropicalis. The antimicrobial compounds from three bacterial crude extracts couldn’t optimally inhibit the growth of pa thogenic fungal because of the lower concentration. For the comparison in tested their antimicrobial activity, ampicillin was used as a positive control whereas ethyl acetate solvent was used as a negative control. Ampicillin which included in - lactam group of antibiotics had a broad spectrum activity so it was chos e as a positive control for this study. This ant ibiotic unable to inhibit the growth of enteropathogenic E. coli K1-1 because of the - lactamase enzyme that prod uced by this microbe while interestingly, our crude extracts from three bacterial isolates which associated with sponge Jaspis sp. are able to inhibit the growth of this microbe as seen from the diameter average of inhibition zone. Anand et al. (2006) reported that antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India which guided fractionation of the broth showed that the ethyl acetate extract of strain SC3 demonstrated activity against the bacterial and fungal test strains. The strain SC3 showed the highest activity against test strains of B. subtilis and E. coli with an inhibition zone of 26 mm and for C. albicans of 15 mm. TLC analysis and bioautography test were performed for three bacterial crude extracts. The TLC analysis aims to separate the constituent components of bacterial crude extract based on the difference of absorption, partition and solubility of the chemical components that will be moved with the polarity of eluent whereas bioautography test aims to quickly detect which spots were the active compounds or impurities compounds. Detection of the spots was performed by administering the chromatogram plates under UV light with 254 nm and 366 nm wave- length. All of the detection methods that used in this study are expected to quickly detect the presence of active compo unds without needed to detect the spots one by one on silica gel plate. In this study, detection of active compounds was only performed with these methods so that not all patches of the active compounds contained in bacterial crude extract can be detected. Sudirman (2005) reported that the rapid detection of active compounds contained in bacterial crude extract can be done using bioautography method besides the UV irradiation and the spraying of color-forming reaction. Bioautography techniq ue is a combination of chemical methods (chromatography) with the microbiological method, the chromatogram plates covered with media that contained microbial test strains and incubated according to the growth temperature of test strains. Betina (1964) stated that bioautography method can be directly detected the activity and the minimum number of active compounds that contained in the bacterial crude extract. In addition, this method can be directly detected the specify location of active compounds based on the position or the Rf values on the chromatogram plate. Four active spots/fractions with the Rf values of 0.62; 0.55; 0.44; 0.23 (isolate SAB E-31), Rf 0.66; 0.60; 0.46; 0.27 (isolate SAB E-41) and Rf 0.65; 0.56; 0.45; 0.31 (isolate SAB E-57) were successfully detected using these methods and respectively showed antimicrobial activity against P. aeruginosa, while two active spots/fractions with the Rf values of 0.77; 0.66 (isolate SAB E-41) and Rf 0.75; 0.56 (isolate SAB E-57) respectively displayed antimicrobial activity against S. aureus. No active spots/fractions from isolates SAB E-31 can inhibit the growth of S. aureus. Banoet (2011) reported that at least one active spot/fraction was successfully detected using TLC analysis and bioautography detection. Two spots/fractions with the Rf value of 0.31; 0.81 from ethyl acetate extract of isolate HAL-13 and one spot/fraction with the Rf value of 0.85 from n-butanol extract of isolate HAA-01 and Rf 0.28 from the same extract of isolate HAL-74 displayed antimicrobial activity against S. aureus and enterop athogenic E. coli K1-1. Bacterial crude extract of isolate SAB E-41 was further fractionated using column chromatography techniques. The selection of this crude extract for further testing due to the be st activity against non-pathogenic and pathogenic microbes compared to the other bacterial crude extracts. Purification of antimicrobial compounds via column chromatography techniques is based on the polarity of eluent. Crude extract was slowly injected into the silica gel-column chromatography and this extract will be separated based on the difference of the polarity of eluent system that used d uring the elucidation process. Hurtubise (2010) mentioned that chromatography column was a classic chromatography method which used to separate the constituent components of antimicrobial compounds for the large quantities by adsorption and partition mechanism. The principles of this process were based on the different polarity of active compounds that contained in the bacterial crude extract. The right selection of stationary phase (absorbance) and mobile phase (eluent) in fractionation process will be determined the successful of the separation of crude extract. The solvent was allowed to flow through the column due to the gravity or the pressure. The compounds will move through the column at the different rates, separated and collected as the fractions out of the column base. Two hundred and five fractions were collected from the fraction collector. TLC analys is was performed to know the fractions which have the same chromatogram. Fractions with the same chromatogram can be combined into one fraction. A total of 30 composite fractions were successfully obtained after TLC analysis. Antimicrobial activity of thirty composite fractions was tested to P. aeruginosa, S. aureus, enteropathogenic E coli K1-1, C. albicans and C. tropicalis. Fifteen composite fractions coded as BA-1, BA-2, BA-3, BA-4, BA-5, BA-6, BA7, BA-8, BA-11, BA-12, BA-13, BA-14, BA-15, BA-17 and BA-18 showed different antimicrobial activity against non-pathogenic and pa thogenic microbes. Fraction BA-2 demonstrated a broad spectrum of inhibition compared to the other fractions. Fraction BA-13 displayed significant activity against S. aureus followed by fraction BA-17. Anand et al. (2006) mentioned that further fractionation of the ethyl acetate extract from strain CS3 was undertaken by reverse phase HPLC. Fifteen fractions had been successfully detected based on the HPLC trace. Fraction 11 was found to be active against test strains of E. coli, B. subtilis and C. albicans. Fractions 5 and 7 were found to have trace activity against E. coli. The remaining fractions were found that not possess antimicrobial properties. Crude extract of isolate SAB E-41 showed antifungal activity against C. albicans and C. tropicalis. After the fractionation process, the fractions are only active against the test strain of C. albicans. The active compounds in these fractions are allegedly lost due to the purification process. Antifungal activity of crude extracts from isolate SAB E-41 was a combination factor of several active compounds which can be lost during the fractionation process. Sudirman (2005) stated that during the purification process, most of the active compounds separated from one to another or the active compounds are separated from the pollutant compound so that the antimicrobial activity from bacterial crude extract can be different between before or after the purification process. Fifteen composite fractions that showed antimicrobial activity were further purified using PTLC techniques. The basic selection of this technique as the purification method was due to the less quantity of active compo unds. The principle of this technique was the active compounds that had already known their position of Rf values were scraped off from silica gel plate and dissolved in chloroform- methanol-ethyl acetate solvent mixtures (1:1:1). Antimicrobial activity test for active compounds, obtained from PTLC technique were tested to a representative group of Gram-positive, Gram- negative bacteria and yeast. Three kinds of pathogenic microbes that selected to test their antimicrobial activity were S. aureus, enteropathogenic E coli K1-1 and C. albicans. These microbes have an important aspect in public health because of their pathogenicity to humans. S. aureus is a group of Gram pos itive bacteria, aerobic facultative and cause skin infections or infection on the upper respiratory system while enteropathogenic E. coli K1-1 is a group of Gram- negative bacteria that are resistant to -lactam class of antibiotics because it possess a -lactamase activity enzyme. C. albicans is the pa thogenic yeast that causes candidiasis disease. Liu (2009) stated that S. aureus is one of the bacteria that cause minor skin infections, pneumonia and meningitis. This strain can be found at skin mucosal surface, nasal passage and gastrointestinal system. Other bacteria that also cause a seriously infection disease was EPEC K1-1. This strain can cause infection at gastrointestinal system. Budiarti (1997 ) found that 55% of diarrhea disease in Indo nesia, mostly caused by EPEC K1-1. C. albicans was a pathogenic strain that caused a serious problem of infection disease. This strain can be found normally at mucosal surface and urogenital system as human microflora. Many of genitally diseases or related to human immune systems diseases such HIV-AIDS are mostly caused b y this strain (Kortig et al. 1999). Fraction BA-13 that was eluted by chloroform- methanol (50% -50%) solvent system demonstrated as the most antimicrobial compound followed by fraction BA-17 and BA-18. Four active compounds with the Rf values of 0.87; 0.50; 0.41 and 0.12 respectively obtained from fraction BA-13 and each of those displayed significant activity against S. aureus and enteropathogenic E. coli K1-1. Two active compounds with the Rf values of 0.93; 0.12 were successfully obtained from fraction BA-17 while two active compounds with the Rf values of 0.93; 0.25 were successfully obtained from fraction BA-18. Both of the active compounds from fraction BA-17 and BA-18 have antimicrobial activity against S. aureus. Banoet (2011) had purified active compounds for bacterial crude extract of isolate HAL-13 which associated with sponge Haliclona sp. Fraction BS13-5 displayed as the most active compounds. Four active compounds with the Rf values of 0.35; 0.41; 0.72 and 0.87 were collected using PTLC technique. Two of these active compounds with the Rf values of 0.35 and 0.41 showed the best activity against enteropathogenic E. coli K1-1. The diameter of inhibition zone that formed by these two active compounds was 12 mm. Morphology of isolates SAB E-31, SAB E-41 and SAB E-57 was characterized using Gram staining procedure. The results showed that these bacteria were rod-shaped, Gram positive, motile and formed spores. The characteristic of these isolates are same with the genus of Bacillus. Anand et al. (2006) reported that the morphological and physiological characterization of strain SC3 isolated from India waters showed that it to be a Gram-positive, motile, catalase and oxidase-positive rod. Molecular identification of this strain also indicated it to be a member of the Bacillus genera. Molecular genetic analysis of 16S rDNA sequences were done for the further identification of those three isolates. Three bacterial isolates which associated with sponge Jaspis sp. were included in the genus of Bacillus based on 16 rDNA analysis. These sequences were in a size of approximately 1300 bp and have conserved and variable regions (Appendix 1). This gene is quite large with the polymorphism between species that can be used as a tool to distinguish between species (Woese 2006; Clarridge 2004). Analysis of 16S rDNA is an important standard for bacterial identification. This identification method was based on the most suitable sequences of bacteria with all of the 16S rDNA sequences that are known in the GenBank database. Partial analysis of 16S rDNA sequences showed that isolate SAB E-31 had 98% of homology level with B. pumilus strain KD3 (Accession No. EU500930.1) (Appe ndix 4), isolate SAB E-41 had 98% of homology level with B. amyloliquefaciens strain zy2 (Accession No. JN160740.1) (Appe ndix 5) and isolate SAB E-57 had 97% of homology level with B. subtilis strain YRL02 (Accession No. EU373407.1) (Appendix 6). Phylogenetic analys is of 16S rDNA sequences showed that three bacterial isolates formed a different clade with the other reference strains. First clade was dominated formed by the reference strains of Bacillus while second clade was formed by those three isolates with Bacillus sp. DF49. This strain was in the same clade with isolate SAB E-41. Isolate SAB E-57 formed a closely relationship clade with isolates SAB E-31, SAB E-41 and Bacillus sp. DF49. This phylogenetic result was different from the BlastN result. Although the result was different but three bacterial isolates were still include in the ge nus of Bacillus. The formation of different clade between three bacterial isolates and other reference strains meant that these isolates were assumed in a new species of Bacillus. Santos et al. (2010) reported that molecular identification by partial 16S rRNA gene sequencing and phylogenetic analysis showed that the majority of bacterial isolates isolated from Brazilian spo nges could be subdivided into three phylogenetically different clusters. Five strains were affiliated with Firmicutes (genera Bacillus and Virgibacillus), three with α-Proteobacteria (Pseudovibrio sp.) and four with Proteobacteria (Pseudomonas and Stenotrophomonas). Marine Bacillus species are often isolated from sediments, invertebrates and marine sponges (Pabel et al. 2003). The species of this genus is known to generate spores under adverse conditions, such as those encountered in marine ecosystems (Hentschel et al. 2001). In the marine environment, members of the genus Bacillus are known for their production of metabolites with antimicrobial, antifungal or generally cytotoxic property. They were regularly isolated from invertebrates and thus display a high potential in the search for new antimicrobial substances (Muscholl-Silberhorn et al. 2008). Many antibiotics including cyclic peptides, cyclic lipopeptides and novel thiopeptides have been reported from this strain (Nagai et al. 2003). Most of the bioactive compounds that produced by marine bacteria didn’t getting loose from the invo lvement of two multifunc tional enzymes named polyketide synthases (PKS) and non-ribo somal peptide synthetases (NRPS). These two multifunctional enzymes mostly involved in the biosynthesis of bioactive compounds. The simplest functional PKS mod ule consists of a ketosynthase (KS), an acyltransferase (AT), an acyl carrier protein (ACP) and a thioesterase (TE) domain (Schirmer et al. 2005). Besides that, the simplest NRPS module consists of an adenylation (A), a thiolation (T), a peptidyl carrier protein (PCP) and a conde nsation (C) do main (Schwarzer et al. 2003). In this study, the presence of KS and A domain in the cluster of PKS and NRPS genes from three marine bacterial isolates became one of the most important domains to be investigated. These two domains were analyzed using PCR amplification. PCR products that indicated the presence of KS domain will show the DNA fragment with the length of 700 bp (Appendix 2) whereas for A domain will show the length of 1000 bp (Appe ndix 3). Kim & Fuerst (2006) mentioned that the common feature of complex PKS gene is ketosynthase (KS) domain that usually present in each module and exhibits the highest degree of conservation among all domains. Likewise, Schirmer et al. (2005) stated that adenylation (A) domain become the most conserved domain of NRPS gene compared to the others. Therefore, the KS and A do main are especially well suited for phylogenetic analyses of PKS and NRPS gene diversity. Isolates SAB E-41 and SAB E-57 possessed DNA fragments encod ing KS and A do main in the cluster of PKS and NRPS genes whereas only isolate SAB E31 possessed DNA fragment encoding A do main in the cluster of NRPS gene. These meant that, by detecting one of the domains, whether KS domain of PKS gene or A domain of NRPS gene could be ensured that they can synthesize the bioactive compounds. Zhao et al. (2008) stated that the modular PKS and NRPS have been involved in natural prod uct synt hesis in many microorganisms. On the other hand, the presence of bo th KS and A domain in the cluster of PKS and NRPS genes at isolates SAB E-41 and SAB E-57 meant that these isolates ha ve the much broader potential in generating many kinds of bioactive compounds. Besides that, we also assumed that they formed the complex hybrid of PKS-NRPS genes. Interestingly, the existence of these hybrid PKS-NRPS systems will enlarge the variation of each mod ule in forming an immense variety of bioactive compounds. Many kinds of natural prod ucts are for med through the combination of PKS-NRPS hybrid systems such as yersiniabactin, one of the iron transport systems of Yersinia pestis that acts as a virulence factor for pathogenic strains and generated from a hybrid assembly line containing 3 NRPS modules and 1 PKS module (Cane & Walsh 1999) and bleomycin (BLM), a family of anticancer antibiotics produced by Streptomyces verticillus and generated from BLM megasynthetase that consist of 10 NRPS modules and 1 PKS module (Shen et al. 2001). Donadio et al. (2007) stated that PKS, NRPS or both are molecular assembly lines that direct product formation on a protein template. Both systems accomplish their task by maintaining reaction intermediates covalently bound as thioesters on the same phosphopantetheine prosthetic group. In PKS assembly lines, the monomers are acetyl-CoA, malonyl-CoA or methylmalonyl-CoA whereas the monomers for NRPS assembly are proteinogenic and nonproteinogenic amino acids and other carboxylic acids such as aryl acids. DNA fragment of KS and A domain were sub-cloned into T-Vector pMD20 (Appendix 7) and transformed into competent E. coli DH5α. Recombinant plasmid that carrying the DNA fragment were named pMD20-KS domain and pMD20-A domain. This recombinant plasmid was isolated and digested with a combination of restriction enzymes, BamHI and XbaI. Several steps like PCR sequenc ing and purification of PCR product were done for DNA sequencing. M13 primer RV and M13 primer M4 were used for PCR sequencing and Big Dye® X TerminatorT M purification kit was used for the purification of PCR product. Sequences analysis of DNA fragment encod ing KS domain showed that isolates SAB E-41 and SAB E-57 have a feature of subject sequences that similar to polyketide synthase. Isolate SAB E-41 had 97% of homology level with type I PKS from B. amyloliquefaciens LL3 (Appe ndix 8). Weitao et al. (2011) found that the complete genome sequence of B. amyloliquefaciens LL3 that isolated from Korean fermented food presented the glutamic acid- independent production of poly- -glutamic acid. This compound is a capsular component or extracellular secretion of Bacillus and a few other organisms that widely used in medicine, cosmetics, food and wastewater treatment. -PGA is a natural polyamide consisting of D- and L-glutamic acid units connected by -amide linka ges (Ashiuchi & Misono 2002; Candela et al. 2009). Isolate SAB E-57 had 98% of homology level with putative polyketide synthase from B. amyloliquefaciens subsp. plantarum CAU-B946 (Appendix 9). Borriss et al. (2011) reported that strain CAU B946 that isolated from the rice rhizosphere, was identified by 16S rRNA gene and gyrA gene sequencing and by physiological and biochemical analysis as being B. amyloliquefaciens subsp. plantarum. Due to its capability to produce antibiotics, some products developed from strain CAU B946 had already been applied as biofungicides to control several plant diseases such as tobacco black shank, rice sheath blight, cotton fusarium wilt, cotton verticillium wilt, and wheat scab. Likewise, bioinformatics sequences analysis of DNA fragment encoding A domain showed that isolates SAB E-31, SAB E-41 and SAB E-57 have a feature of subject sequences that similar to nonribosomal peptide synthetase. Isolate SAB E31 had 81% of homology level with bacitracin synthetase 1 from B. pumilus ATCC 7061 (Appendix 10). Awais et al. (2008) isolated a Bacillus species from soil that collected from different areas and identified as B. pumilus according to Bergey’s Manual of Determinative Bacteriology. The antibiotic that prod uced by the identified B. pumilus strain was designated as bacitracin. This compound was active against Micrococcus luteus and S. aureus. Bottone and Peluso (2003) produced an antifungal compound from B. pumilus that is active against Mucoraceae and Aspergillus species. The active compound inhibited Mucor and Aspergillus spore germina tion, aborted elongating hyphae and presumable inducing a cell-wall lesion. Isolate SAB E-41 had 80% of homology level with surfactin synthetase B from B. amyloliquefaciens subsp. plantarum CAU-B946 (Appendix 11) while isolate SAB E-57 had 81% of homology level with surfactin synthetase A from the same strain of bacteria, CAU-B946 (Appendix 12). Prokof'yeva et al. (1996) mentioned that some of bacteria from Bacillus group produce biologically active lipopeptides that are modified by a fatty acid. One of them was surfactin that has a large spectrum of biological activity. Surfactin is a powerful lipopeptide that commonly used as an antibiotic. This antibiotic contains a - hydroxy fatty acid a nd synthesized by a linear nonribosomal peptide synthetase. Besides that, surfactin has surface active properties directed against microbial adhesion and disruptive the permeability of membrane cell of Gram pos itive and Gram negative bacteria. Besides the surfactin peptides, recently, Blom et al. (2012) reported that the genome of the rhizoba cterium B. amyloliquefaciens subsp. plantarum CAU B946 was 4.02 Mb in size and harbored 3,823 genes (coding sequences/CDS). Nine giant gene clusters were dedicated to nonribosomal synthesis of antimicrobial compounds. This strain also possessed a gene cluster that involved in synthesis of iturin A. Mizumoto et al. (2007) mentioned that iturin A is a cyclolipopeptide containing seven residues of α-amino acids (L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-DAsn-L-Ser-) and one residue of a β-amino acid is likely to be the active agents in biological control. The alignment of amino acid sequences encod ing KS domain showed that isolates SAB E-41 and SAB E-57 have a conserved region of amino acid sequences with the other reference strains but the similarity number of amino acid sequences from isolate SAB E-41 with B. amyloliquefaciens LL3 and isolate SAB E-57 with strain CAU B946 was very low. These meant that isolates SAB E-41 and SAB E57 have the new feature of amino acid sequences encoding polyketide synthase enzyme. Besides that, phylogenetic analysis also proved that these two bacterial isolates formed an own clade and differed to the other reference strains. Polyketide synthase enzyme from isolate SAB E-41 was closely related to isolate SAB E-57 and differ to the other reference strains based on phylogenetic tree result. Meanwhile, the alignment of amino acid sequences encod ing A domain also showed that isolates SAB E-31, SAB E-41 and SAB E-57 have a conserved region of amino acid sequences as shown in figure 16. The similarity number of amino acid sequences encoding A domain from three bacterial isolates was also low. Isolate SAB E-31 has a low similarity number of amino acid sequences with B. pumilus ATCC 7061 and so do isolates SAB E-41 and SAB E-57 with strain CAU B946. These meant that three bacterial isolates have a new feature of amino acid sequences encoding nonribosomal peptide synthetase enzyme. Phylogenetic analysis showed that isolates SAB E-31 and SAB E-57 formed a different clade with the other reference strains. Nonribosomal peptide synthetase enzyme from isolate SAB E-31 was closely related to isolate SAB E-57 while for isolate SAB E-41 was closely related to B. amyloliquefaciens FZB42 based on phylogenetic tree result. Fortman and Sherman (2005) stated that few marine NRPS genes have been revealed compared with PKS genes. Zhang et al. (2009) reported that the NRPS genes from the bacteria associated with South China Sea Sponges cluster together forming two groups , which means that these NRPS genes are different from the other marine NRPS genes. Twelve NRPS genes grouped together showed a high similarity to three NRPS relatives of spongeassociated bacteria. In summary, combining the antimicrobial activity test and detection the occurrence of KS and A domain of PKS and NRPS genes based on molecular approach could be applied to efficient screening of the potent marine bacterial isolates and also predicting their related compounds. These related compounds could be developed and applied in pharmaceutical industry in order to treat the resistant microbes. CONCLUSION AND SUGGESTION Conclusion Crude extracts of isolates SAB E-31, SAB E-41 and SAB E-57 showed different antimicrobial activity against non-pathogenic and pathogenic microbes. Bacterial crude extract of isolate SAB E-41 demonstrated the best antimicrobial activity compared to the other bacterial crude extracts. Three marine bacterial isolates were included in the ge nus of Bacillus based on molecular genetic analysis of 16S rDNA. Both isolates SAB E-41 and SAB E-57 possessed KS and A domain in the cluster of PKS and NRPS genes and only isolate SAB E-31 possessed A domain in the cluster of NRPS gene. Suggestion Further purification of active compounds for four active fractions (Rf 0.87, 0.50, 0.41 and 0.12) from fraction BA-13 was needed to be conducted in order to identify the group of these active compo unds and the molecule structure elucidation. 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SAB E-31 XbaI ↓ TCTAGAGGATCTACTAGTCATATGGATTGGGCGGTGTGTACAAGGCCCGGGAACGTATTC ACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGA CTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTAAACCTTGCGGTCTCGCAGCCC TTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGAC GTCATCCCCACCTCCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATG CTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACAC GAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCGAAGGGAAAGCCCCTATCTC TAGGGTTGTCAGAGGATGGTCAAGGACCCTGGTAAGGTTCTTCGCGTTGCTTCAGAAATT AAACCCCCACATGCTCCCACCCGCTTGTGCGGGCCCCCCGTCAATTCCTTTGAGTTTCAG TCTTGCGACCGTACTCCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGG CGGAAACCCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAA TCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTT CGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCT CCTCTTCTGCACTCAAGTTTCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGGCT TTCACATCAGACTTAAGAAAACCGCCTGCGAGCCCCTTTACGCCCAATAAATTCCGGACA ACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGGTAGTTAGCCGTGGCTTTCTGG TTAGGTACCGTCAAGGTGCAAGCAGTTACTCTTGCACTTGTTCTTCCCTAACAACAGAGC CTTTACGATTCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTACGTCCA TTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGT GTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCAC CAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGACAGCCGAAACCGTCTTTCATCCT TGAACCATGCGGTTCAAGGAACTATCCGGTATTAGCTCCGGTTTCCCGGAGTTATCCCAG TCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCCGGGAGCAAG CTCCCTTCTGTCCGCTACGACTTGCATGTGTTAGGCCCTGAATCGGATCC ↑ BamHI B. SAB E-41 XbaI ↓ TCTAGAGGACTACTAGTCATATGGATTGGGCGGTGTGTACAAGGCCCGGGAACGTATTCA CCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGAC CGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCT TTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACG TCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGC TGGCAACTAAGATCAAGGGTTGCGCATCGTTGCGGGACTTAACCCAACATCTCACGACAC GAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCT AGGATTGTCAGAGGATGTACAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAAAC CACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACC GTACTCCCCAGGGCGGAGTGCTTTAATGCGTTTAGCTGACAGCACTAAAGGGGCGGAAAC CCCCTAACAACACTATAGCAACTCTATCGTTTACGGGCGTGGACTACCAGGGTATCTAAT CCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTC GCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATATCCACTCT CCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGGTTGAGCCGGGGGCT TTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACG CTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAG GTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTT ACGATCCGAAAACAACTTCATCACTCACGCGGCGTTGCGTCCGTCAGACTTTCGTCCATT GCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGT GGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCA ACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTG AACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTCCCGGAGTTATCCCAGTCT TACAGGCAGTTACCCACGTTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCC ATCTGTCCGCTCGACTTGCATGTGTTAGGCCCTGAATCGGATCC ↑ BamHI C. SAB E-57 XbaI ↓ TCTAGAGGGATCTACTAGTCATATGGGATTGGGCGGGGTGGTACAGGCCCGGGGAACGTA TTCACCGCGGCATGCTGATCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCA GACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGC CCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTG ACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAA TGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGAC ACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATTC TCTAGGATTGTCAGAGGATGTCAAGACCCTGGTAAGGTTCTTCGCGTTGCTTACGAAATT AAACCCACATGCTCCCACCGCTTGTGCGGGCCCCCCGTCAATTCCTTTGAGTTTCAGTCT TGCGACCGTACTCCCCCAGGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGGCG GGAAACCCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAAT CCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTC GCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGGAATTCCACTCT CCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCCTCCCCCGGTTGAGCCGGGGGC TTTCACATCAGACTTAAGAAACCAGCCTGCGAGCCCTCTTACGCCCAATAATATCCGGAC AACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGG TTAGGTACCGTCAAGGTGCCGCCCTATTTGAATCGGCACTTGTTCTTCCCTAACAACAGA GCTATTACGATCCGAAAACCTTCATCACTCATCGCGGCGTTGCTCCGTCAGACTTTCGTC CATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCA GTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTC ACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACTTTTATTC TGAACCTTGCGGTCAGACAACCTCCGGTTTAGCCCCGGTTCCCGGAGTTTCCCAGTCTAC CAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGACCAAGCTCCC ATCTGTCCGCTCGACTTCCATGTGTTAGCCCTGAATCGGATCC ↑ BamHI Appendix 2 DNA sequences of KS domain of PKS gene from two bacterial isolates A. SAB E-41 XbaI ↓ TCTAGAGGATCTACTAGTCATATGGATTGTGCCGGTGCCGTGGGCCTCGACATAACTGAC GGTCCGCGGGCTGATACCTGCTTTTTTCAGACAGGCCTTAATGACTTCTGCCTGTGCAGC AGGACTCGGGACGGTAATTCCGCTTACTTTCCCGACGTGGTTAACGGCGCTTCCTTTAAT GACCGCGTAAATGCGGTCGCCGTCTTGTTCGGCTTTTTCCAGCGGCTTGAGCAAGACCGC ACCGACACCTTCTCCGGAAACGTAGCCGTCCCCGCCCTCGCCGAATGTGCGGCAGCGGCC GTCACTTGAGTGCATCCCTACGCTTCCGTAGCTGAGATATTTCGCCGGGTGCAGCGACAA GTTCACCCCTCCCGCAAGCGCGGCTTCACATTCGCCGCGGCGGATGCTTTCAATGGCCAG ATGAACGGCGGTCAATGATGAGGAACAAACGGTATCCACCGCGATGCTCGGCCCGTGGAA GTCACAATAATAGGACACTCTGTTGGCGATCTGCGCATAATTCAGTGAAACCGGAAAAGG ATCAGCTTCAGATAATTGTTCTGCGCCGATTAAGGAATAATCTTTATGCATCACCCCTGC AAATACGCCGATCGGATGCTGTTTCTCCCCTTTATTCCCCAGCGTTTCAGGCGTATACCC CGCATCTTCAATCGTTTCCCAGCATGTTTCTAAAAACAGCCGCTGCTGCGGATCCATCGC ATCGGATCC ↑ BamHI B. SAB E-57 XbaI ↓ TCTAGAGGATCTACTAGTCATATGGATTGTGCCCGTGCCATGCCCTTCCACCATTTGAAT GGTTTCCGGATTAATGTGAAAAGTATCATAGACATGCCGTTCCAATCGTTCTTGAGAAAG CGCGCTTGGAGCGGTTATTCCGTTTGTAGCGCCGTCCTGATTCATTGCAGAGCCTTTTAT GACGCCGTACACATGATCTCCGTCACTGACGGCGTCGCTAAGACGTTTCAGCACCACCGC TCCCACACCTTCACCCGGCACGAAGCCGTCCGCGCTTTGATCAAACGTATGGCAGCGCCC GGTCGGTGACAGCATATTCGCCTTATTTGACGACTGATAAAAAGCGGGAGTGGATTGAAT GAAAACCCCGCCCGCCACCGCCATTTCGGTTTCTTTCGTCCAGAGCCCCTGACATGCCAA ATGAATGGCTGTCAGCGAACTGGAACATGCTGTATCAACAGTGATCGCCGGCCCTTGTAG ATTAAGGTGATAGGCGATCCTTGCCGGAGTGACGGAATTGTGATTGCCCCAAAAAGCCTG CGCGGGCCCCTGCTGTTTAAAAATGGTCTGGTAATCTCCGCCGCAGGAGCCGGCATACAC GCCGCATTCCCGGCCTCTTACCGAGTCTCCCGCATATCCCGCATCTCCAAGCGCTTTCCA CGATTCTTCCAGAAACACCCGCTGCTGCGGGTCCCATCATCGGATCC ↑ BamHI Appe ndix 3 DNA sequences of A domain of NRPS gene from three bacterial isolates A. SAB E-31 XbaI ↓ TCTAGAGGATCTACTAGTCATATGGATTCCGCGGATTTTGACTTGATGATCGATTCGCCC AATGAATTCAATATCTCCATTTGACAGCCATCGTGCGAGATCGCCCGTTTTGTACATGAC TTCACCAGGTCGGAAAGGATTTTCAACAAACTCTTCGCTCGTTAACTCTGGCTGGCGATA ATACCCCTTGACCAGTCCGTCACCTGCGACACAAAGCTCTCCAGCTACTCCCGGCGGCTG CACATGTCCAAACTCATCAACAATATAAACAGCCGTTTGACTCACCGGCTTTCCGATCGG AATCGATAGTGCTTGTTCTTCAATGTGATTCACCGGATAATACGTTGTGAAAATCGTGCT TTCAGAAGGTCCATACATATGAATAAGTTTGTCTTCTCCAACCGTTTCAAGGGCTGCCAC AACATGCGGCACAGAAGCACGTTCCCCGCCAAACAGCACTTTTCTGACGTTTTTCAGGCT GCCTTTTTTCATATCAATCAGTAAGTGAAATAGAGCGGTCGTGATCATTAAAATACTGAC TTTTTCCTTCTCAATCGCGCCAGAAAGCTCATTCATATTTAAAATGTGATCCTTTGGCAA AACAATGAGTTTCGCTCCGTTCAATAAAGCGCCAAACACATCAAACATAAATGCATCAAA TACATAGTTTGAAAGGCTCATCACCGTGTCTTCATGATGAATGGTGAGATAATTCGACTG CTTCACTGTTCTCAAAATGTTCCGATGCGTCACCATGTTCCCCTTTAGGTTTTGCCTGTC GTACCAGCATGTGGTAGGTCAAAATTCGCCCAAGTCCCAAAGGCGAAACACATACAAACT GGGATTGCTCTCTGACTGCTGATCAACGGCTTGGATCTTCTGTTTCTATGATTTTCCCCC TTCAAACGCAGTAAGCACAGAGCGATGACGCAGCACCTGGATGGGGTCAGGGACAAACTG TGCACCACTATCTTGTCAAAAAGTGCCTTGAATGCGCTCATCCAGGGAAGTCCAGGATCG ATTGGGACACATACGCCACCCACCCGCCAATCGGATCC ↑ BamHI B. SAB E-41 XbaI ↓ TCTAGAGGATCTACCTAGTCCATATGGATTTGCGGGCGGTGCTTATGTGCCCGATTGATC CCGGTCTTTTGCCGGGAGGACCGTCTCCGCTTTATGGGCGGCAGACAGCTCGATTCGGCT CGTGCTGACAGTTCAGGACTATCAAAGAACAAGCGGGCACATTGCAAGTCCCGATTGTCA TGCTGGATGAAAAGCGCGGATGAAACGGTAAGCGGAACAGACTTGAATCTTCCGGCCGGC GGGCAACGACTTGGCGTATATCATGTATACATCCGGATCGACCGGCAAACCGAAAGGCGT CATGATTGAACCACAGAAATATCATCAGGCTCGTCAAACATTCGAATTACGTGCCGGTTC ATGAAGAAGACCGGATGGCGCAAACGGGAGCCGTCAGCTTTGATGCCGGAACCTTCGAAG TCTTCGGTGCATTGCTGAACGGAGCCGCGCTGCACCCGGTGAAAAAAGAGACACTGCTTG ACGCCGGACGATTCGCCCAATTTCTGAAAGAGCAGCGGATCACGACCATGTGGCTGACGT CTCCGCTGTTTAATCAGCTTGCCCAAAAGGATGCGGGCATGTTTAACACGCTCCGGCACC TCATCATCGGCGGTGATGCGCTTGTGCCGCATATCGTCAGCAAAGTGAGGAAGGCATCAC CGGAGCTGTCGCTTTGGAACGGCTACGGGCCGACGGAGAATACGACGTTTTCGACGAGTT TTCTCATTGATCAGGACTGCGACGGCTCGATCCCGATCGGCAAGCCGATCGGAAATTCCA CTGCGTACATTATGGACGAAAACCGCAACCTCCAGCCGATCGGCGCTCCCGGTGAGCTGT GCGTCGGCGGAAGCGGAGTGGCAAGAGGCTATGTGAATCTGCCTGAATTAACGGAGAAGC AGTTTGTCCGCGATCCGTTCAGACCGGAGAAAAGATATACCCGGACGGGGGACTTGGCGA AAGATGGCTTCCCGGCGGCCCGAACGAGTTTTTTGGCCGAAATGGCCACCCAAGAAAAAA TCGGATCC ↑ BamHI C. SAB E-57 XbaI ↓ TCTAGAGGATCTACTAGTCATATGGGATTGCCGCGGATTTTGACCTGGATCATCTTCACG GCCGAATATATTCGATCGTCCCGTCCGGCAGCCAGCGCGCCATATCACCCGGTGCGGTAC ATAGCGGCCGCTTCCGTTAAACGGATCTTGCAAAAAACTTCTCTGCGGGTCAAATCCGGA AAGATTTAAATAGCCGCGGCCTACACCGTCACCCGCGATATACAGCTCACCGGCCGTCCC GTCGGGCTGAAGCCTCTGGTGCTTATCCAATATATACAGACGGGCGTTGCCGAGCGGTTT TCCGATCGGAACGTACGCCGCCTGTTGATTCATTCCGTTATCGGCTGACACCTGATGCAC GGACGCATCTACGCACGTTTCTGTCGGCCCGTAGACATTCGTCAGACGCGGCGCCCTGCC CGATTGATGAAAGAGGTTCATCAGCTGTTCAGCAACAGCGGCGGACAGGCCCTCTCCCCC AATGAGCATGTGGCGCAATTCAATTCCGCTGACATCTCCCGCCGCAACCATCATCTGCAG ATGCGCAGGGGTTCCGTCAGTGGCTTCAATCCGGTTTTGACGATAATAGTCCAGCAGTGC CGAGCCATTCGTTACAGTCGTTTTCGGCACGATATAAAGCGTCTGTCCCAAAAGAAGCGA GGCAAAAATCTGTTTGACGGACGCATCAAAATGTAACGGAGCCAAAAGCGCCATTCTTAA TGTCTGCTCACCGCATTGATAAATCTCCTGCTGCAGTGATTGCACCAGATGATGAACATT TGGGCCGGTGCTCAATCATCACGCCCTTCGGCCGCCCCCGTTGTTACCTGACGTGTAGAT GATGTAAAGCCACGCCCGGTCTGATTTGTTGTTACTTGACAACCGTCCCGCCAGCCCGTT TTCAAAACTTGAAACAAGCCCTCCGTCAAAAATTCAAATACCGTTTCGGGCAATCCCGCC TGCCACGGCGCCCTGTTATTTCTCGGGCCCTCCTGTTCAGGAAACAAGACCTTACCGCCT TCCCGCCTTGTTCCCTTCCTCCAAAAATGGGTAACCTTGGAAATTCCCGGAATTCCCCGC CCCGGGAAAAAGGTCCAAAGGGAAAGTCTTAATTCGGGGAAACCAATTATATTGCCAACC CAACCCCCGCGCCAATCGGATCC ↑ BamHI Appendix 4 Alignment of 16S rDNA sequences from isolate SAB E-31 using BlastN program gb|EU500930.1| Bacillus pumilus strain KD3 16S ribosomal RNA gene, partial sequence Length=1502 Score = 2234 bits (1162), Expect = 0.0 Identities = 1294/1320 (98%), Gaps = 21/1320 (2%) Strand=Plus/Minus Query 1 Sbjct 1385 Query 61 Sbjct 1325 Query 121 Sbjct 1265 Query 181 Sbjct 1205 Query 241 Sbjct 1145 Query 301 Sbjct 1085 Query 361 Sbjct 1025 Query 421 Sbjct 969 Query 481 Sbjct 916 Query 541 Sbjct 857 Query 601 Sbjct 799 Query 661 Sbjct 739 Query 721 Sbjct 679 Query 781 Sbjct 619 Query 841 Sbjct 561 GGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTA 60 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTA 1326 GCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGT 120 GGGATTGGCTAAACCTTGCGGTCTCGCAGCCCTTTGTTCTGTCCATTGTAGCACGTGTGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GGGATTGGCTAAACCTTGCGGTCTCGCAGCCCTTTGTTCTGTCCATTGTAGCACGTGTGT 180 1266 1206 AGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTCCCTCCGGTTTGTCA ||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||| AGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCA 240 CCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTC 300 GTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGT 360 1146 1086 1026 CACTCTGTCCCCGAAGGGAAAGCCCCTATCTCTAGGGTTGTCAGAGGATGGTCAAGGACC |||||||||||||||||||||| |||||||||||||||||||||||||| |||||| | CACTCTGTCCCCGAAGGGAAAG-CCCTATCTCTAGGGTTGTCAGAGGAT-GTCAAG--AC 420 CTGGTAAGGTTCTTCGCGTTGCTTCAGAAATTAAACCCCCACATGCTCCCACCCGCTTGT ||||||||||||||||||||||||| ||||||| ||||||||||| |||||||| CTGGTAAGGTTCTTCGCGTTGCTTC--GAATTAAA---CCACATGCTCC--ACCGCTTGT 480 GCGGGCCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCAGGCGGAG ||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||| GCGGG-CCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCAGGCGGAG 540 TGCTTAATGCGTTAGCTGCAGCACTAAGGGGGCGGAAACCCCCCTAACACTTAGCACTCA ||||||||||||||||||||||||||| |||||||||| ||||||||||||||||||||| TGCTTAATGCGTTAGCTGCAGCACTAA-GGGGCGGAAA-CCCCCTAACACTTAGCACTCA 600 TCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCC 660 970 917 858 800 740 TCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTAC 720 GCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTT 780 680 620 CCAATGACCCTCCCCGGTTGAGCCGGGGGGCTTTCACATCAGACTTAAGAAAACCGCCTG |||||||||||||||||||||||| |||||||||||||||||||||||| |||||||||| CCAATGACCCTCCCCGGTTGAGCC-GGGGGCTTTCACATCAGACTTAAG-AAACCGCCTG 840 CGAGCCCCTTTACGCCCAATAAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGC |||| ||||||||||||||| ||||||||||||||||||||||||||||||||||||||| CGAG-CCCTTTACGCCCAAT-AATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGC 900 562 504 Query 901 Sbjct 503 Query 961 Sbjct 444 Query 1021 Sbjct 386 Query 1081 Sbjct 326 Query 1141 Sbjct 266 Query 1201 Sbjct 206 Query 1261 Sbjct 146 TGCTGGCACGGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCAAGCAGTTA ||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||| TGCTGGCAC-GTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCAAGCAGTTA 960 CTCTTGCACTTGTTCTTCCCTAACAACAGAGCCTTTACGATTCCGAAAACCTTCATCACT ||||||||||||||||||||||||||||||| |||||||| ||||||||||||||||||| CTCTTGCACTTGTTCTTCCCTAACAACAGAG-CTTTACGA-TCCGAAAACCTTCATCACT 1020 CACGCGGCGTTGCTCCGTCAGACTTACGTCCATTGCGGAAGATTCCCTACTGCTGCCTCC ||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||| CACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCC 1080 CGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTA 1140 CGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATC 1200 445 387 327 267 207 TGTAAGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TGTAAGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCG 1260 GTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTAC 1320 147 87 Appendix 5 Alignment of 16S rDNA sequences from isolate SAB E-41 using BlastN program gb|JN160740.1| Bacillus amyloliquefaciens strain zy2 16S ribosomal RNA gene, partial sequence Length=1449 Score = 2267 bits (1179), Expect = 0.0 Identities = 1320/1343 (98%), Gaps = 22/1343 (2%) Strand=Plus/Minus Query 1 Sbjct 1381 Query 61 Sbjct 1321 Query 121 Sbjct 1261 Query 181 Sbjct 1201 Query 241 Sbjct 1141 Query 301 Sbjct 1082 Query 361 Sbjct 1022 Query 421 Sbjct 963 Query 481 Sbjct 904 Query 541 Sbjct 846 Query 601 Sbjct 795 Query 661 Sbjct 736 Query 721 Sbjct 676 Query 781 Sbjct 617 Query 841 Sbjct 557 GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGA 60 TTCCAGCTTCACGCAGTCGAGTTGCAGACCGCGATCCGAACTGAGAACAGATTTGTGGGA ||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||| TTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGA 120 TTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCC 180 CAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGG 1322 1262 1202 240 1142 CAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCATCGTT |||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||| CAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGC-TCGTT 300 GCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCAC 360 1083 1023 TCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTACAAGACCTGGTA ||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||| TCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGT-CAAGACCTGGTA 420 AGGTTCTTCGCGTTGCTTCGAATTAAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGT ||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||| AGGTTCTTCGCGTTGCTTCGAATTAAA-CCACATGCTCCACCGCTTGTGCGGGCCCCCGT 480 CAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGGCGGAGTGCTTTAATGCGT ||||||||||||||||||||||||||||||||||||||||| |||||||||| ||||||| CAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGG-CGGAGTGCTT-AATGCGT 540 TTAGCTGACAGCACTAAAGGGGCGGAAACCCCCTAACAACACTATAGCAACTCTATCGTT | ||||| ||||||||| |||||||||||||||||||| || ||||| ||| |||||| T-AGCTG-CAGCACTAA-GGGGCGGAAACCCCCTAACA---CT-TAGCA-CTC-ATCGTT 600 TACGGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAG ||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||| TACGG-CGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAG 660 CGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCAT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCAT 720 TTCACCGCTACACGTGGAATATCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCA |||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||| TTCACCGCTACACGTGGAAT-TCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCA 780 ATGACCCTCCCCGGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ATGACCCTCCCCGGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAG 840 CCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGG 900 964 905 847 796 737 677 618 558 498 Query 901 Sbjct 497 Query 961 Sbjct 437 Query 1021 Sbjct 379 Query 1081 Sbjct 320 Query 1141 Sbjct 260 Query 1201 Sbjct 200 Query 1261 Sbjct 140 Query 1318 Sbjct 80 CACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGG 960 CACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACAACTTCATCACTCACGCG |||||||||||||||||||||||||||||||||||||||||| |||||||||||||||| CACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAAC--CTTCATCACTCACGCG 1020 GCGTTGCGTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAG ||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||| GCGTTGC-TCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAG 1080 GAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCAT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCAT 1140 CGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAA 1200 GTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATT 1260 AGCCCCGG-TTCCCGGAGTTATCCCAGTCTTACAGGCA-GTTACCCACGT-TTACTCACC |||||||| ||||||||||||||||||||||||||||| ||||||||||| ||||||||| AGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACC 1317 CGTCCGCCGCTAACATCAGGGAG ||||||||||||||||||||||| CGTCCGCCGCTAACATCAGGGAG 1340 58 438 380 321 261 201 141 81 Appendix 6 Alignment of 16S rDNA sequences from isolate SAB E-57 using BlastN program gb|EU373407.1| Bacillus gene, partial sequence Length=1518 subtilis strain YRL02 16S ribosomal Score = 2127 bits (1106), Expect = 0.0 Identities = 1298/1334 (97%), Gaps = 25/1334 (2%) Strand=Plus/Minus Query 14 Sbjct 1400 Query 73 Sbjct 1340 Query 133 Sbjct 1280 Query 193 Sbjct 1220 Query 253 Sbjct 1160 Query 313 Sbjct 1100 Query 373 Sbjct 1040 Query 433 Sbjct 982 Query 493 Sbjct 927 Query 553 Sbjct 868 Query 613 Sbjct 810 Query 673 Sbjct 750 Query 733 Sbjct 690 Query 793 Sbjct 631 GTACAGGCCCGGGGAACGTATTCACCGCGGCATGCTGATC-GCGATTACTAGCGATTCCA ||||| | || ||||||||||||||||||||||||||||| ||||||||||||||||||| GTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCA 72 1341 GCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGC 132 TTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGT ||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||| TTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATCGTAGCACGTGTGTAGCCCAGGT 192 1281 1221 CATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTC 252 ACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGA 312 CTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCC 1161 1101 372 1041 CCCGAAGGGGACGTCCTATTCTCTAGGATTGTCAGAGGATGTCAAGACCCTGGTAAGGTT |||||||||||||||||| |||||||||||||||||||||||||||| |||||||||||| CCCGAAGGGGACGTCCTA-TCTCTAGGATTGTCAGAGGATGTCAAGA-CCTGGTAAGGTT 432 CTTCGCGTTGCTTACGAAATTAAACCCACATGCTCCCACCGCTTGTGCGGGCCCCCCGTC ||||||||||||| || ||||||| ||||||||| |||||||||||||||| |||||||| CTTCGCGTTGCTT-CG-AATTAAA-CCACATGCT-CCACCGCTTGTGCGGG-CCCCCGTC 492 AATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCAGGGCGGAGTGCTTAATGCGTT ||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||| AATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCA-GGCGGAGTGCTTAATGCGTT 552 983 928 869 AGCTGCAGCACTAAGGGGGCGGGAAACCCCCCTAACACTTAGCACTCATCGTTTACGGCG |||||||||||||||||||||| || ||||||||||||||||||||||||||||||||| AGCTGCAGCACTAAGGGGGCGG--AAACCCCCTAACACTTAGCACTCATCGTTTACGGCG 612 TGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTT 672 ACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGC 732 811 751 691 TACACGTGGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCC ||||||| |||||||||||||||||||||||||||||||||||||||||||||||| ||| TACACGT-GGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGAACCC 792 TCCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCAGCCTGCGAGCCCTCT | |||||||||||||||||||||||||||||||||||||||||| ||||||||||||| | T-CCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACC-GCCTGCGAGCCCT-T 852 632 575 RNA Query 853 Sbjct 574 Query 913 Sbjct 515 Query 973 Sbjct 456 Query 1033 Sbjct 398 Query 1093 Sbjct 338 Query 1153 Sbjct 278 Query 1213 Sbjct 218 Query 1269 Sbjct 158 Query 1327 Sbjct 98 TACGCCCAATAATATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACG ||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||| TACGCCCAATAAT-TCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACG 912 TAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAATCGGCAC ||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||| TAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAA-CGGCAC 972 TTGTTCTTCCCTAACAACAGAGCTATTACGATCCGAAAACCTTCATCACTCATCGCGGCG |||||||||||||||||||||||| ||||||||||||||||||||||||||| ||||||| TTGTTCTTCCCTAACAACAGAGCT-TTACGATCCGAAAACCTTCATCACTCA-CGCGGCG 1032 TTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGT 1092 CTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTC 1152 GCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGG 1212 TAGCCGAAGCCA-CTTTTAT-TCTGAACCTTGCGG-TCAGACAACC-TCCGGTTTAGCCC |||||||||||| ||||||| |||||||| ||||| ||| |||||| |||||| |||||| TAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATAGCCC CGG-TTCCCGGAGTT-TCCCAGTCTACCAGGCAGGTTACCCACGTGTTACTCACCCGTCC ||| ||||||||||| ||||||||| ||||||||||||||||||||||||||||||||| CGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCC GCCGCTAACATCAG |||||||||||||| GCCGCTAACATCAG 1340 85 516 457 399 339 279 219 1268 159 1326 99 Appendix 7 Plasmid map of T-Vector pMD20 (TaKaRa Bio Inc.) Appendix 8 Alignment of DNA sequences encoding KS domain of PKS gene from isolate SAB E-41 using BlastX program ref|YP_005545643.1| baeL gene product [Bacillus amyloliquefaciens LL3] gb|AEB63415.1| bacillaene synthesis; polyketide synthase of type I [Bacillus amyloliquefaciens LL3] Length=3513 GENE ID: 12204614 baeL | bacillaene synthesis; polyketide synthase of type I [Bacillus amyloliquefaciens LL3] Score = 417 bits (1072), Expect = 2e-130 Identities = 221/229 (97%), Positives = 225/229 (98%) Gaps = 0/229 (0%), Frame = -3 Query 689 Sbjct 472 Query 509 Sbjct 532 Query 329 Sbjct 592 Query 149 Sbjct 652 MDPQQRLFLETCWETIEDAGYTPETLGNKGEKQHPIGVFAGVMHKDYSLIGAEQLSEADP MDPQ+RLFL+TCWETIEDAGYTPETLGNK KQ P+GVFAGVMHKDYSLIGAEQLSE DP MDPQERLFLQTCWETIEDAGYTPETLGNKKNKQRPVGVFAGVMHKDYSLIGAEQLSETDP 510 FPVSLNYAQIANRVSYYCDFHGPSIAVDTVCSSSLTAVHLAIESIRRGECEAALAGGVNL FPVSLNYAQIANRVSYYCDFHGPS+AVDTVCSSSLTAVHLAIESIRRGECEAALAGGVNL FPVSLNYAQIANRVSYYCDFHGPSLAVDTVCSSSLTAVHLAIESIRRGECEAALAGGVNL 330 SLHPAKYLSYGSVGMHSSDGRCRTFgeggdgyvsgegvGAVLLKPLEKAEQDGDRIYAVI SLHPAKYLSYGSVGMHSSDGRCRTFGEGGDGYVSGEGVGAVLLKPLEKAEQDGDRIYAVI SLHPAKYLSYGSVGMHSSDGRCRTFGEGGDGYVSGEGVGAVLLKPLEKAEQDGDRIYAVI 150 KGSAVNHVGKVSGITVPSPAAQAEVIKACLKKAGISPRTVSYVEAHGTG KGSAVNHVGKVSGITVPSPAAQAEVIKACLKKAGISPRTVSYVEAHGTG KGSAVNHVGKVSGITVPSPAAQAEVIKACLKKAGISPRTVSYVEAHGTG 3 700 531 591 651 Appendix 9 Alignment of DNA sequences encoding KS domain of PKS gene from isolate SAB E-57 using BlastX program ref|YP_005130937.1| putative polyketide synthase pksL PKS [Bacillus amyloliquefaciens subsp. plantarum CAU B946] emb|CCF05742.1| putative polyketide synthase pksL PKS [Bacillus amyloliquefaciens subsp. plantarum CAU B946] Length=2071 GENE ID: 11698078 dfnJ | putative polyketide synthase pksL PKS [Bacillus amyloliquefaciens CAU-B946] Score = 451 bits (1160), Expect = 2e-143 Identities = 218/222 (98%), Positives = 219/222 (99%) Gaps = 0/222 (0%), Frame = -2 Query 666 Sbjct 954 Query 486 Sbjct 1014 Query 306 Sbjct 1074 Query 126 Sbjct 1134 DPQQRVFLEESWKALGDAGYAGDSVRGRECGVYAGSCGGDYQTIFKQQGPAQAFWGNHNS DPQQR+FLEESWKAL DAGYAGDSVRGRECGVYAGSCGGDYQ IFKQQGPAQAFWGNHNS DPQQRLFLEESWKALEDAGYAGDSVRGRECGVYAGSCGGDYQAIFKQQGPAQAFWGNHNS 487 VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ 307 SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD GATNGITAPSALSQERLERHVYDTFHINPETIQMVEGHGTGT GATNGITAPSALSQERLERHVYDTFHINPETIQMVE HGTGT GATNGITAPSALSQERLERHVYDTFHINPETIQMVEAHGTGT 1 1175 1013 1073 127 1133 Appendix 10 Alignment of DNA sequences encoding A domain of NRPS gene from isolate SAB E-31 using BlastX program ref|ZP_03054623.1| bacitracin synthetase 1 (BA1) [Bacillus pumilus ATCC 7061] gb|EDW21930.1| bacitracin synthetase 1 (BA1) [Bacillus pumilus ATCC 7061] Length=3570 Score = 492 bits (1266), Expect = 1e-154 Identities = 263/323 (81%), Positives = 281/323 (87%) Gaps = 6/323 (2%), Frame = -1 Query 969 Sbjct 1588 Query 789 Sbjct 1645 Query 609 Sbjct 1702 Query 429 Sbjct 1762 Query 249 Sbjct 1822 Query 69 Sbjct 1882 DERIQGTF*QDSGAQFVPDPIQVLRHRSVLTAFEGGKS*KQKIQAVDQQSESNPSLYVFR DER++ F DSGAQF+ QVLRHRSVL +FEG + + + + QQS+SN + V DERVKH-FLTDSGAQFLLTH-QVLRHRSVLASFEGTII-ETEDRGIVQQSDSNIDIRVLP 790 LWDLGEF*PTTCWYDRQNLKGNMVTHRNILRTVKQSNYLTIHHEDTVMSLSNYVFDAFMF DL T+ + KGNMVTHRNILRTVKQSNYL IHHEDTVMSLSNYVFDAFMF E-DLANLTYTSGTTGKP--KGNMVTHRNILRTVKQSNYLAIHHEDTVMSLSNYVFDAFMF 610 DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKKGSLKNVR DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKK SLKNVR DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKKDSLKNVR 430 KVLFGGERASVPHVVAALETVGEDKLIHMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV KVLFGGERASVPHV+ ALETVGE KL+HMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV KVLFGGERASVPHVMTALETVGEGKLVHMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV SQTAVYIVDEFGHVQPPGVAGELCVAGDGLVKGYYRQPELTSEEFVENPFRPGEVMYKTG SQTAVYIVDEFG +QPPGVAGELCVAGDGLVKGYY QP+LTSE+FVENPFRPGEVMYKTG SQTAVYIVDEFGQLQPPGVAGELCVAGDGLVKGYYGQPKLTSEKFVENPFRPGEVMYKTG DLARWLSNGDIEFIGRIDHQVKI DLARWLSNG+IEFIGRIDHQVKI DLARWLSNGEIEFIGRIDHQVKI 1 1904 1644 1701 1761 250 1821 70 1881 Appendix 11 Alignment of DNA sequences encoding A do main of NRPS gene from isolate SAB E-41 using BlastX program ref|YP_005129035.1| surfactin synthetase B [Bacillus amyloliquefaciens subsp. plantarum CAU B946] emb|CCF03840.1| surfactin synthetase B [Bacillus amyloliquefaciens subsp. plantarum CAU B946] Length=3586 GENE ID: 11700595 srfAB | surfactin synthetase B [Bacillus amyloliquefaciens CAU-B946] Score = 449 bits (1154), Expect = 1e-139 Identities = 236/295 (80%), Positives = 246/295 (83%) Gaps = 6/295 (2%), Frame = +1 Query 112 Sbjct 573 Query 286 Sbjct 630 Query 466 Sbjct 690 Query 646 Sbjct 750 Query 826 Sbjct 810 KEQAGTLQVPIVMLDEKRG*--NGKRNRLESSGRRATTWRISCIHPDRPANRKAS*LNHR +EQAGTLQVPIVMLDE +G L + G + +P K + HR QEQAGTLQVPIVMLDESADETVSGTDLNLPAGGNDLAYIMYTSGSTGKP---KGVMIEHR 285 629 NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA 465 QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW 645 NGYGPTENTTFSTSFLIDQDCDGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG NGYGPTENTTFSTSFLIDQD DGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG NGYGPTENTTFSTSFLIDQDYDGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG 825 VARGYVNLPELTEKQFVRDPFRPEKRYTRTGDLAKDGFPAARTSFLAEMATQEKI VARGYVNLPELTEKQFVRDPFRP++ RTGDLAK P FL + Q K+ VARGYVNLPELTEKQFVRDPFRPDETIYRTGDLAK-WLPDGTIEFLGRIDNQVKV 990 863 689 749 809 Appendix 12 Alignment of DNA sequences encoding A do main of NRPS gene from isolate SAB E-57 using BlastX program ref|YP_005129034.1| surfactin synthetase A SrfA [Bacillus amyloliquefaciens subsp. plantarum CAU B946] emb|CCF03839.1| surfactin synthetase A SrfA [Bacillus amyloliquefaciens subsp. plantarum CAU B946] Length=3584 GENE ID: 11700594 srfAA | surfactin synthetase A SrfA [Bacillus amyloliquefaciens CAU-B946] Score = 420 bits (1080), Expect = 1e-129 Identities = 227/280 (81%), Positives = 236/280 (84%) Gaps = 15/280 (5%), Frame = -1 Query 845 Sbjct 593 Query 665 Sbjct 650 Query 485 Sbjct 710 Query 305 Sbjct 770 Query 125 Sbjct 829 NNKSDRAWLYIIYTSgnngggrRA**LSTGPNVHHLVQSLQQEIYQCGEQTLRMALLAPL + +SDR YIIYTSG G + + VHHLVQSLQQEIYQCGEQTLRMALLAP STQSDRL-AYIIYTSGTTGRPKGV--MIEHRQVHHLVQSLQQEIYQCGEQTLRMALLAPF 666 HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD 486 VSGIELRHMLIGGEGLSAAVAEQLMNLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG VSGIELRHMLIGGEGLSAAVAEQL+NLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG VSGIELRHMLIGGEGLSAAVAEQLLNLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG 306 MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNLSGFDPQRSF MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNL ++ F MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNLPDLTAEK-F LQDPFNGSGRYVPHRVIWRAG-----CRTGRSNIFGREDD LQDPFNGSGR ++R G G GREDD LQDPFNGSGR------MYRTGDMARWLPDGTIEYIGREDD 21 862 649 709 769 126 828 SUMMARY EFFENDI. Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analys is. Under direction of ARIS TRI WAHYUDI and MUNTI YUHANA. The increase of global resistance of the pathogenic microbes against various antibiotics becomes a serious concern in public health. Many efforts are conducted in order to solve this problem, such as finding the new bioactive compounds from marine bacteria which associated with marine sponges. Three marine bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 that associated with sponge Jaspis sp. showed their capability in producing antimicrobial compounds. In this study, extraction of antimicrobial compounds from these bacteria was conducted using ethyl acetate solvent. Each of bacterial crude extract displayed significant activity against B. subtilis, E. coli as non-pathogenic microbes and S. aureus, P. aeruginosa, enterop athogenic E. coli K1-1, C. albicans and C. tropicalis as pathogenic microbes. Bacterial crude extract of isolate SAB E-41 demonstrated better activity than bacterial crude extracts of isolates SAB E-31 and SAB E-57. Analysis of constituent component for each bacterial crude extract was conducted using thin layer chromatography (TLC). Each of bacterial crude extract was spotted onto silica gel plate and eluted with n-butanol-ethyl acetate solvent mixture (3:7). Six spots/fractions were successfully detected by viewing under UV light at 254 nm and 366 nm wave- length. Active spots/fractions from each bacterial crude extract were detected using bioautography method. Four spots from each bacterial crude extract showed antimicrobial activity against P. aeruginosa and two spots showed antimicrobial activity against S. aureus. Fractionation of bacterial crude extract from isolate SAB E-41 was carried out using silica gel-column chromatography. Two hundred and five fractions were successfully collected from fraction collector and combined into thirty compos ite fractions based on the same chromatogram by using TLC ana lysis. The antimicrobial activity of thirty compos ite fractions was tested to S. aureus, P. aeruginosa, enteropathogenic E. coli K1-1, C. albicans and C. tropicalis. Fifteen of the m named by BA-1, BA-2, BA-3, BA-4, BA-5, BA-6, BA-7, BA-8, BA-11, BA12, BA-13, BA-14, BA-15, BA-17 and BA-18 have different antimicrobial activity against S. aureus, P. aeruginosa, EPEC K1-1 and C. albicans. Fraction BA-2 that was eluted by chloroform- methanol (90% -10%) solvent system showed antifungal activity against C. albicans while fraction BA-13 that was eluted with chloroformmethanol (50% -50%) solvent system showed the highest inhibition against S. aureus followed by fraction BA-17. The diameter of inhibition zone that formed by these two active fractions were about 12 mm and 14 mm. Fraction BA-2 and BA-4 that was eluted with chloroform- methanol (90%-10%) showed the best activity against enteropathogenic E. coli K1-1. The diameter of inhibition zone that formed by these two active fractions were about 10 mm. Purification of fifteen compos ite fractions was conducted using preparative thin layer chromatography (PTLC) technique. Four active fractions with the Rf values of 0.87; 0.50; 0.41 and 0.12 were successfully collected from fraction BA- 13 and displayed significant activity against S. aureus and EPEC K1-1. Fractions BA-17 and BA-18 carried two kinds of active compounds with the Rf values of 0.93; 0.12 for the BA-17 and 0.93; 0.25 for the BA-18. Both of these active compounds were successfully collected and showed antimicrobial activity against S. aureus. Morphological and molecular identification of those three isolates were carried out in order to identify these bacteria. Sequences analysis of 16S rDNA showed that three isolates were included in the genus of Bacillus. Isolate SAB E-31 had 98% of homology level with B. pumilus strain KD3 while isolate SAB E-41 had 98 % of homology level with B. amyloliquefaciens strain zy2 and isolate SAB E-57 had 97% of homology level with B. subtilis strain YRL02. PCR amplification of ketosynthase (KS) domain of PKS and adenylation (A) domain of NRPS were successfully amplified and sub-cloned into T-Vector pMD20. All isolates coded as SAB E-31, SAB E-41 and SAB E-57 possessed A domain and only two isolates coded as SAB E-41 and SAB E-57 possessed KS domain. DNA fragment encoding KS domain was in a size of 700 bp while for A domain was in a size of 1000 bp. Sequences analysis of DNA fragment encoding KS domain using BlastX program indicated that isolate SAB E-41 showed a similarity level of 97% with type I PKS from Bacillus amyloliquefaciens LL3 and isolate SAB E-57 showed a similarity level of 98% with putative polyketide synthase pksL from B. amyloliquefaciens subsp. plantarum CAU-B946 whereas for A domain indicated that isolate SAB E-31 showed a similarity level of 81% with bacitracin synthetase 1 from B. pumilus ATCC 7061. Isolate SAB E-41 s howed a similarity level of 80% with sur factin synthetase B from B. amyloliquefaciens subsp. plantarum CAUB946 and isolate SAB E-57 showed a similarity level of 81% with surfactin synthetase A from the same strain of Bacillus, CAU-B946. Keywords : Antimicrobial compound, fractionation, cloning, 16S rDNA, KS and A domain, Jaspis sp.
Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analys is. B. Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analys is.
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Antimicrobial Activity of Bioactive Compounds Isolated from Marine Bacteria Associated with Sponge Jaspis sp. and Their Genetics Analys is.

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