Examination of Metabolic and Regulatory Networks Of
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EXAMINATION OF METABOLIC AND REGULATORY NETWORKS OF DESULFOVIBRIO SPECIES A Dissertation Presented to The Faculty of the Graduate School University of Missouri-Columbia In Partial Fulfillment Of the Requirement for the Degree Doctor of Philosophy by CHRISTOPHER LEE HEMME Judy D. Wall, Dissertation Supervisor DECEMBER 2004 Acknowledgements I would like to thank my dissertation advisor Dr. Judy Wall for giving me the opportunity to work in her laboratory and for being patient with me during the difficult times. I would also like to thank my colleagues and friends in the Wall laboratory: Bill Yen, Brett Emo, Grant Zane, Joe Ringbauer, Laurie Casalot, Kate Hart, Suzanne Miller, Leena Pattarkine, Kelly Bender, Elliot Drury and especially Barbara Giles. Thank you all for your help and friendship. I would like to acknowledge the members of my dissertation committee, Drs. Dave Emerich, David Eide, Arun Chatterjee, Toni Kazic and Peter Tipton, for the advice and aid they offered over the course of this project. Finally, I would like to thank my parents for always believing in me, and my brother for giving me three wonderful nephews who helped keep my spirits up. ii EXAMINATION OF METABOLIC AND REGULATORY NETWORKS OF DESULFOVIBRIO SPECIES Christopher L. Hemme Dr. Judy D. Wall, Dissertation Supervisor Abstract The sulfate-reducing bacteria are a morphologically diverse group of organisms characterized by the ability to couple the enzymatic reduction of sulfate to energy production and growth. This metabolic activity has profound economic and environmental consequences such as the corrosion of metal structures and the souring of petroleum reserves. The sulfate-reducing bacteria are also among a select group of organisms that may be used as tools for the bioremediation of toxic heavy metal contaminants from the environment. To understand the mechanisms through which these bacteria impact our environment both positively and negatively, genomic studies have been undertaken to predict the metabolic and regulatory networks of two species of the genus Desulfovibrio. Studies have focused on the elucidation of carbon metabolic pathways, the role of CRP-FNR proteins in the regulation of Desulfovibrio metabolic pathways, and the prediction of global regulatory networks using bioinformatics techniques. Surprisingly, several hexose metabolic genes were found despite the fact that biochemical evidence suggests that these bacteria do not use hexose sugars as growth substrates. This physiological paradox was explored. Secondary pathways for the metabolism of galactose and the synthesis of α,α- trehalose were observed in Desulfovibrio desulfuricans G20 but not Desulfovibrio vulgaris Hildenborough. Physiological experiments showed that despite the presence of a complete set of galactose metabolism genes, Dv. desulfuricans was unable to utilize galactose as the sole carbon source for growth. Growth experiments using [14C]-labeled galactose in the presence of lactate suggested that galactose was incorporated into the cell. This result coupled with the published observation that the extracellular polymeric substances (EPS) of Dv. desulfuricans strains contained detectable levels of galactose suggests that metabolism of galactose occurs for the purposes of production of EPS and possibly lipopolysaccharide (LPS). iii To explore the possible hierarchical regulation of substrate utilization, global regulators responding to redox signals were sought. Sequencing revealed multiple orthologs of the CRP-FNR genes in Desulfovibrio. A mutant strain of Dv. vulgaris was constructed that was interrupted in a gene encoding a putative CRP-FNR protein. A phenotypic analysis of the mutant strain showed no significant differences in the growth rates and growth yields of the cells compared to wild type when grown on lactate-sulfate, pyruvate-sulfate (respiration), pyruvate alone (fermentation) or formate-sulfate. However, the mutant was shown to be impaired in growth on ethanol-sulfate compared to wild type, suggesting the involvement of this protein in either the cellular ability to use ethanol or maintain cellular integrity. A computational analysis of the promoter regions of putative transcription units of Desulfovibrio revealed possible regulatory protein binding motifs homologous to the E. coli CRP and GalR binding sites as well as a motif common to genes encoding phosphate homeostasis proteins. Further examination revealed a set of statistically significant motifs not immediately identified as E. coli homologs. This set of motifs may represent unique regulatory motifs of Desulfovibrio. iv Table of Contents Acknowledgements ..............................................................................................................ii ABSTRACT ........................................................................................................................iii Table of Contents .................................................................................................................v List of Tables ........................................................................................................................viii List of Figures.......................................................................................................................ix 1. General Background.......................................................................................................1 1.1 Overview ........................................................................................................................1 1.2 Introduction to the Sulfate-Reducing Bacteria ..........................................................1 1.3 Classical Metabolism of Desulfovibrio ........................................................................7 1.3.1 Central Carbon Metabolism .....................................................................................7 1.3.2 Energetics of the SRB ................................................................................................9 1.3.2.1 Respiration by Reduction of Sulfate .....................................................................9 1.3.2.2 Growth with Alternative Terminal Electron Acceptors......................................13 1.3.2.3 Dissimilatory Metal Reduction ..............................................................................13 1.3.2.4 Oxygen Metabolism ................................................................................................14 1.4 Computational Methods for Sequence Analysis ........................................................14 1.4.1 Determination of Open Reading Frames.................................................................15 1.4.2 Elucidation of Protein Function by Sequence Homology.......................................15 1.4.3 Detection of Orthologs...............................................................................................18 1.4.4 Functional Assignment by Contextual Analysis......................................................19 1.4.5 Pitfalls of Functional Assignment by Sequence Homology Methods ....................20 1.4.6 Constructing Biochemical Networks from Genomic Sequence Data....................22 v 1.4.6.1 Metabolic Networks................................................................................................22 1.4.6.2 Regulatory Networks..............................................................................................23 1.5 Summary of Project......................................................................................................25 2. Comparative Carbon Metabolism of Desulfovibrio vulgaris Hildenborough and Desulfovibrio desulfuricans G20..............................................................................26 2.1 Introduction/Rationale .................................................................................................26 2.2 Materials and Methods.................................................................................................28 2.3 Results ........................................................................................................................38 2.3.1 Genomic and Physiological Insights into Carbon Metabolism of Desulfovibrio..38 2.3.1.1 Determination of BLAST Parameters and Statistical Cutoffs ...........................38 2.3.1.2 Central Carbon Metabolism ..................................................................................39 2.3.1.2.1 Glycolysis and Gluconeogenesis..........................................................................39 2.3.1.2.2 Organic Acid Metabolism ...................................................................................40 2.3.1.2.3 TCA Cycle.............................................................................................................41 2.3.1.2.4 Secondary Carbohydrate Metabolism Pathways..............................................43 2.3.1.3 Galactose Metabolism by Dv. desulfuricans G20 .................................................58 2.3.1.3.1 Genomic Analysis of Galactose Metabolism Genes ..........................................58 2.3.1.3.2 Physiological Studies of Galactose Metabolism by Dv. desulfuricans G20.....70 2.4 Discussion.......................................................................................................................80 2.4.1 Computational Prediction of Central Carbon Metabolism ...................................80 2.4.2 Galactose Metabolism of Desulfovibrio desulfuricans G20 ....................................82 3. Regulatory