USING SHEWANELLA BALTICA ECOTYPES AS A MODEL FOR TRANSCRIPTIONAL VARIATION AT THE POPULATION LEVEL by WILLIAM SEALY HAMBRIGHT Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE IN BIOLOGY THE UNIVERSITY OF TEXAS AT ARLINGTON DECEMBER 2010 Copyright © by Sealy Hambright 2010 All Rights Reserved ACKNOWLEDGEMENTS First I would like to thank my family, and my amazing girlfriend Mandi. Without their support I would not be where I am at today. Next, I would like to thank Dr. Jorge Rodrigues for his guidance and undaunted patience and support throughout this project. Thank you to my committee, Dr. Thomas Chrzanowski and Dr. Woo-Suk Chang. Dr. Chrzanowski is not only an advisor, but has been a professor, mentor, and friend during my tenure at UTA. I am in debt to all of my fellow colleagues for their immense help and ability to put up with my caffeine driven antics. To name a few: Drs Sridev Mohapatra, Babur Mirza, Atcha Boonmee, as well as Aditya Ranjan M.S., Jantiya Isanapong M.S., Bogar Garcia, Fabiana da Silva Paula, Blaine Thompson, Christi Hull M.S., Briony Foster, and the entire Melotto lab (including Dr. Melotto). I especially want to thank Austin Willis M.S., for our work was often a collaborative effort in learning which I believe is the foundation of research. November 19, 2010 iii ABSTRACT USING SHEWANELLA BALTICA ECOTYPES AS A MODEL FOR TRANSCRIPTIONAL VARIATION AT THE POPULATION LEVEL Sealy Hambright M.S. The University of Texas at Arlington, 2010 Supervising Professor: Jorge Rodrigues Eukaryotic studies have shown considerable transcriptional variation among individuals of the same population. Owing to the cost of sequencing entire eukaryotic genomes, tested organisms were assumed to be genomically similar or even identical. We overcame this necessary assumption by using four sequenced strains of the bacterium Shewanella baltica, (OS155, OS185, OS195, OS223), as models to assess transcriptional variation and ecotype formation within a prokaryotic population. The strains were isolated from various depths throughout a water column of the Baltic Sea occupying different ecological niches characterized by various abiotic parameters. Although their genome sequences are strikingly similar, when grown in the laboratory under identical conditions, all strains exhibited statistically significantly different growth rates suggesting global expressional variation. To confirm the findings, custom microarray slides containing probes representing all four of the sequenced genomes were hybridized with two strains at a time, in a two color manner, using a loop design. A one way ANOVA designated 415 core genes to be differentially expressed between the four strains at a stringent P value of 0.001. Furthermore, when analyzing common gene sequences shared iv among 32 other strains within the water column, Ecotype Simulation software consistently grouped all four model strains into discrete ecotypes. Transcriptional pattern variations such as the ones highlighted here may be used as indicators of short-term evolution emerging from the formation of bacterial ecotypes. v TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................................iii ABSTRACT ..................................................................................................................................... iv LIST OF ILLUSTRATIONS............................................................................................................. viii LIST OF TABLES ............................................................................................................................ ix Chapter Page 1. LITERATURE REVIEW……………………………………..………..….. ........................... 1 1.1 The Shewanella genus ..................................................................................... 1 1.2 Mechanisms for genetic diversity: Sexually active bacteria ............................. 8 1.3 The bacterial species concept: Molecular standards to define species ........... 9 1.4 Bacterial evolution: The transcriptome ........................................................... 13 2. USING SHEWANELLA BALTICA ECOTYPES AS A MODEL FOR TRANSCRIPTIONAL VARIATION AT THE POPULATION LEVEL…………………...15 2.1 Introduction..................................................................................................... 15 2.2 Materials and Methods ................................................................................... 17 2.2.1 Cultivation and isolation ................................................................. 17 2.2.2 Ecotype Simulation (ES) ................................................................ 17 2.2.3 RNA extraction, enrichment and labeling ....................................... 18 2.2.4 Oligonucleotide Microarray design ................................................. 18 2.2.5 Microarray hybridization ................................................................. 19 2.2.6 Global expression profile analyses ................................................ 20 2.2.7 Experimental standards ................................................................. 21 2.3 Results ........................................................................................................... 21 2.3.1 Standardized growth assays .......................................................... 21 vi 2.3.2 Ecotype simulation .......................................................................... 23 2.3.3 Global expression analysis ............................................................ 26 2.4 Discussion ...................................................................................................... 37 2.4.1 Scope of the study ......................................................................... 37 2.4.2 Ecotype demarcations .................................................................... 37 2.4.3 Genotypic and phenotypic differences among strains ................... 38 2.4.4 Foundations for analysis scheme .................................................. 39 2.4.5 Expressional relatedness among strains ....................................... 41 APPENDIX A. COMPLETE PROTOCOL: CELL TO ANALYSIS ......................................................... 45 B. SUPPLEMENTAL MATERIALS .................................................................................... 54 REFERENCES ............................................................................................................................... 69 BIOGRAPHICAL INFORMATION .................................................................................................. 74 vii LIST OF ILLUSTRATIONS Figure Page 1.1 16S rRNA gene based phylogenetic tree of the Shewanella genus .......................................... 2 1.2 16S rRNA gene based phylogenetic tree of the Shewanella baltica isolates obtained from Gotland deep water column. ................................................................. 4 2.1 Microarray hybridization loop design for all possible pairwise comparisons ............................ 20 2.2 Shewanella baltica growth in defined media containing glucose as single carbon source ............................................................................................................... 22 2.3 Shewanella baltica growth in defined media containing maltose as single carbon source ............................................................................................................... 23 2.4 Ecotype Simulation (ES) using gyrB sequences for 36 Shewanella baltica strains within the water column ............................................................................................... 25 2.5 Comparative differentially expressed gene profiles per strain ................................................. 27 2.6 Global gene distribution profile indicating range of expressional variation of all gene-individuals .............................................................................................................. 29 2.7 Experimental variation versus significance between strains .................................................... 30 2.8 Number of genes found per COG designation ......................................................................... 31 2.9 Pathway predictions for the OS155 total transcriptome using the cellular omics-viewer from Biocyc.org ............................................................................... 33 2.10 Pathway predictions for the OS195 total transcriptome using the cellular omics-viewer from Biocyc.org ............................................................................... 34 2.11 Pathway predictions for the OS185 total transcriptome using the cellular omics-viewer from Biocyc.org ............................................................................... 35 2.12 Pathway predictions for the OS223 total transcriptome using the cellular omics-viewer from Biocyc.org ............................................................................... 36 viii LIST OF TABLES Table Page 1.1 Abiotic parameters within the Gotland Deep water column for each strain isolation depth ................................................................................................... 5 1.2 Genome features and gene content of sequenced Shewanella baltica strains ......................... 7 B.1 Genes identified by Genespring GX 11 to be significantly differentially