Functional Genomics of Soil Bacteria using a Metagenomics Approach by Kavita S. Kakirde A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 4, 2012 Keywords: soil, metagenomics, shuttle BAC vector, metagenomic library construction, high molecular weight DNA, antibacterial compounds, Copyright 2012 by Kavita S. Kakirde Approved by Mark R. Liles, Chair, Associate Professor of Biological Sciences Paul A. Cobine, Assistant Professor of Biological Sciences Eduardus Duin, Associate Professor of Biochemistry Omar A. Oyarzabal, Associate Professor of Biological Sciences Abstract Soil microbial communities are an abundant resource for natural product discovery. Traditional methods such as cultivation of soil microorganisms from soil under laboratory conditions have lead to discovery of new compounds but the vast majority of microorganisms are as yet unculturable and hence many prokaryotic phyla have yet to be explored for bioactive secondary metabolites. One of the significant breakthroughs to overcome this limitation is the application of metagenomics to investigate the genetic and functional diversity of as-yet- uncultured microorganisms from natural environments. Metagenomic analyses can provide extensive information on the structure, composition, and predicted gene functions of diverse environmental microbial assemblages. Our studies used a metagenomic approach to identify large-insert clones that express an antimicrobial activity. Bacterial artificial chromosome (BAC) vectors have been used to clone and express DNA fragments from single genomes and from entire microbial communities. Cloning and expression of large insert DNA in different host organisms can be of significance in the functional analysis and is facilitated by shuttle BAC vectors which permit the transfer and replication of BAC genomic libraries in the host organism of choice. In the first study, we designed and constructed a novel Gram negative shuttle BAC vector that enables enables stable replication of cloned DNA in diverse Gram-negative species. This vector possesses an inducible copy system to increase the number of plasmids per cell. Thus, the vector that is maintained as a single copy can be induced by addition of arabinose thereby getting ii a ~100-fold amplification of the DNA and potentially better expression of the cloned DNA due to a gene dosage affect. The pGNS-BAC vector can be used for high efficiency cloning of large fragments of genomic DNA transferred from Escherichia coli to other Gram-negative bacteria. The second study describes screening a soil metagenomic library to identify recombinant clones producing an antimicrobial activity. Here we used a culture-independent and function based method to characterize the soil “metagenome” to access novel antibiotics of potential medical importance. Three different libraries were screened using various tester strains. After multiple rounds of screening and validation tests we identified several clones with antimicrobial activity. Clones of interest were further characterized using preliminary biochemical studies and genetic analysis. The third study focused on detailed characterization of one of the clones (clone P6L4) identified from the screening of the large-insert library. The anti-MRSA activity derived from this clone was consistent and reproducible in all the bioassays that were performed. Basic biochemical and genetic analysis revealed that the anti-MRSA activity is likely due to the esterase produced by this clone which counteracts the action of the chloramphenicol acetyl transferase which in turn leads to growth inhibition of the MRSA by chloramphenicol. iii Acknowledgments I would like to give a heartfelt thanks to Dr. Mark Liles, who has been an excellent mentor during my research work over the past five years. This dissertation work would not be possible without his continued guidance and encouragement. I highly value his support and advice that have been vital to my development as a graduate student. Special thanks to my advisory committee members, Drs. Evert Duin, Paul Cobine, and Omar Oyzarbal for sharing their wisdom, expertise, and advice throughout the years. I would also like to thank Dr. Peter Panizzi for reviewing my dissertation. I would like to acknowledge Nancy Capps, Andrew Wiggins, Paul Bergen, Shamima Nasrin, Molly Staley, Dr. Larissa Parsley, Dr. Molli Newman, Dr. Abel Carrias, Jahangir Hossain, Chao Ran, Malachi Williams, Katherine Vest and Ann Marie Goode for their contribution to the research within this dissertation and for helping me to wade through tough times. I am thankful to all our colleagues at Lucigen Corp. for their collaborative work in these research projects. I am forever grateful to my parents Shodhan and Gauravi Kakirde, my grandmother Savita Kakirde, and my sister Namrata for their unconditional love and patience; and for the sacrifices they made to enable me to pursue my Doctor of Philosophy degree. Most importantly, I would like to thank my husband Sree Menon, who has been my strength, support and motivation through it all and has always driven me to give my best. I will always be indebted to God for giving me an opportunity to achieve my desired goals. iv Table of Contents Abstract............................................................................................................................................ii Acknowledgments..........................................................................................................................iv List of Tables.................................................................................................................................vii List of Figures...............................................................................................................................viii I. Literature Review........................................................................................................................1 A. Metagenomics for Characterization of Soil Microbial Communities.....................................1 1. Introduction…………….….……..……………….…………………..…………….........2 2. Exploring the soil environment ……..………………………………..….…………........3 3. Metagenomic Applications.…………..………….….…....………………………...….....8 4. Analyzying the soil metagenome…...…...............….…………………………...……...16 5. Metagenomic Library screening……………...…….....……………….………….…….28 6. Conclusions...…...…....…………...………….....…….…………...…...….....................33 B. Antibiotics: Modes of Action…………………………………………………………….34 II. Gram-negative Shuttle BAC Vector for Heterologous Expression of Metagenomic Libraries …………………………………………………............................. 38 A. Abstract…….…...………….………...……………………………………………....…...38 B. Introduction……..…...……..….…………………………………………………….……39 C. Materials and Methods......…...….......................................................................................41 D. Results…………...…………….....………………………………………….……………45 v E. Discussion……..…………..…...………………………………………..………………...48 III. Screening Soil Metagenomic Libraries to Identify Recombinant Clones Producing an Antimicrobial Activity.............................................................................................................55 A. Abstract…….…....….…………..………………………………………….…………….55 B. Introduction........................................................................................................................56 C. Materials and Methods..................................................................................………...…..57 D. Results…........................................................................................................................,,..61 E. Discussion…………....………………………..…..……………………………….....…..63 IV. Characterization of metagenomic clonesidentified from the screening of metagenomic BAC libraries .................................................................................................72 A. Abstract…….…………………………………………………………………………...72 B. Introduction………………….……………………………………………...………......73 C. Materials and Methods……………………..………………………………….………..74 D. Results……….…………………………………..……………………………………...82 E. Discussion…………………………………………………………………………...…..89 F. Future Work……………………………………………………………………….…….92 Comprehensive bibliography …………………………………………………………………..199 vi List of Tables Table 2.1. Bacterial strains and plasmids.……….………………………...………………........50 Table 2.2. MIC for Cm and Gm conferred by pGNS-BAC.........................................................51 Table 3.1. Details of different metagenomic libraries used for screening .................................65 Table 3.2. Shortlisted clones after validation experiments..........................................................66 Table 4.1. Preliminary charcterization of active metagenomic clones........................................94 Table 4.2. Summary of anti-MRSA BAC clone annotations ………........................................190 vii List of Figures Figure 1. Schematic of microbial metagenomic library construction and screening………………...…………………………………………………….…...37 Figure 2.1. Isolation of BAC vector DNA from E. coli and S. marcescens.…………….……..52 Figure 2.2. Annotated plasmid map for pGNS-BAC-1 and pGNS-BAC ………………….......53 Figure 2.3. Growth pattern of E. coli and S. marcescens on a Cm gradient agar with and without arabinose.….........………………………………………………………....54 Figure 3.1. Examples of metagenomic clones exhibiting inhibition of tester