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By SAMANTHA MARIE WATERS a DISSERTATION PRESENTED TO HOW DOES BACILLUS SUBTILIS RESPOND AND ADAPT TO LOW-PRESSURE GROWTH? By SAMANTHA MARIE WATERS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014 1 © 2014 Samantha Marie Waters 2 ACKNOWLEDGMENTS I thank my mother for providing a shining example of what inner strength and will power may accomplish in life. Sarah and Samuel, thank you for being supportive younger siblings. I would like to express my gratitude to Dr. Wayne L. Nicholson for granting me the opportunity to work in his lab. I would also like to thank my graduate committee, Drs. de Crecy, Kolaczkowski, Ferl and Romeo for their guidance during my research these past four years, as well as my graduate student cohort, Rafael Oliveira. For their candid advice, I thank Drs. Mobberley, Foster, Mueller and Koenig. I thank the University of Florida’s High Performance Computing Center for all the support with software and Galaxy tool errors. A special thank you to Justin, for being super awesome. All of my friends who have been the solid ground under my feet: Christine, Samantha, Alia, Zinzi, Kimberly, Jon, Julia, and Jennifer; thank you for always believing in me. And to David, Monte, Josh and Beth, thank you for all the spectacular movie nights that were much appreciated and greatly needed to stay sane during these past few years. 3 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................3 LIST OF TABLES ...........................................................................................................................7 LIST OF ABBREVIATIONS ..........................................................................................................9 ABSTRACT ...................................................................................................................................10 1 LITERATURE REVIEW .......................................................................................................12 Pressure ...................................................................................................................................12 High Hydrostatic Pressure: Protein Volume ...........................................................................12 High Hydrostatic Pressure: Protein Denaturation ...................................................................14 High Hydrostatic Pressure: Membrane Fluidity .....................................................................16 High Hydrostatic Pressure: Mesophilic Organisms ................................................................18 Low Pressure ..........................................................................................................................19 Bacillus subtilis Strain 168 .....................................................................................................20 Experimental Evolution ..........................................................................................................21 2 EXPOSURE OF BACILLUS SUBTILIS TO LOW PRESSURE (5 KPA) INDUCES SEVERAL GLOBAL REGULONS INCLUDING THE SIGB-MEDIATED GENERAL STRESS RESPONSE .............................................................................................................23 Introduction .............................................................................................................................23 Bacterial Strains, Media, and Growth Conditions ...........................................................25 Isolation and Labeling of Total RNA ..............................................................................26 Transcription Microarray Experiments ...........................................................................26 Microarray Data Analysis and Normalization .................................................................27 β-Galactosidase Assay .....................................................................................................27 Microarray Data Accession Number ...............................................................................28 Results and Discussion ...........................................................................................................28 Transcriptome Analysis of Strain WN624 at 5 kPa and ~ 101 kPa ................................28 Induction of ctc-lacZ Expression by LP ..........................................................................30 Inactivation of sigB Does Not Alter Fitness at ~ 101 kPa or at 5 kPa .............................31 Towards an Understanding of the LP Response. .............................................................33 3 MICROARRAY ANALYSIS OF DIFFERENTIAL GENE EXPRESSION IN A BACILLUS SUBTILIS STRAIN ADAPTED TO ENHANCED GROWTH AT 5 KPA .......60 Introduction .............................................................................................................................60 Material and Methods .............................................................................................................62 Bacillus subtilis, Media, and Growth Conditions ............................................................62 RNA Extraction ...............................................................................................................62 Microarray Experiments ..................................................................................................63 4 Microarray Data Analysis and Normalization .................................................................63 BLASTp Analysis of Unknown and Putative Function mRNA Signals .........................64 Sporulation Frequency .....................................................................................................64 β-galactosidase Assays ....................................................................................................64 Competition Experiments ................................................................................................65 Microarray Data Accession Number ...............................................................................65 Results .....................................................................................................................................66 Microarray Analysis of Global Gene Expression of Bacillus subtilis Strains WN624 and WN1106 at 5 kPa ..................................................................................................66 Anaerobic Response at 5 kPa ..........................................................................................67 Membrane Fluidity at Low-Pressure ...............................................................................69 Transporter Genes are Differentially Expressed at LP ....................................................70 DNA Binding Proteins at LP ...........................................................................................73 Genes of Unknown Function were Differentially Expressed at LP ................................74 The General Stress Response at LP .................................................................................75 Inactivation of sigB Does Not Alter Fitness at ~101 kPa or at 5 kPa ..............................77 Sporulation at 5 kPa .........................................................................................................79 Discussion ...............................................................................................................................79 4 WHOLE GENOME RE-SEQUENCING REVEALS MUTATIONAL CHANGES IN BACILLUS SUBTILIS AFTER A 1,000 GENERATION 5 KPA EVOLUTION EXPERIMENT .....................................................................................................................126 Introduction ...........................................................................................................................126 Materials and Methods .........................................................................................................127 Bacterial Strains, Media and Growth Conditions ..........................................................127 DNA Extraction and Quality Control for Re-Sequencing .............................................128 Whole Genome Re-Sequencing and Mutation Identification .......................................128 In silico Analysis of Mutations ......................................................................................130 Rifampicin Resistance Assay ........................................................................................130 Competition Experiments ..............................................................................................131 RNA Isolation ................................................................................................................131 Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) .............132 Results and Discussion .........................................................................................................132 Whole Genome Re-Sequencing and Alignment ............................................................132 Mutational Analysis During the 5 kPa Evolution Experiment ......................................133 bacD Analysis ...............................................................................................................135 fliI Analysis ...................................................................................................................136
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