Phylogenetic Studies on Pathogenicity in Proteobacter Family Suneel Chhatre*, Ramu Kakumanu, Neela Gamini, Newton Hilliard¶ Department of Chemistry, Eastern New Mexico University, Portales, NM 88130 The Model Organism Halothiobacillus neapolitanus, originally isolated from a shallow marine vent in the Aegean Sea is an obligate chemolithoautotroph. We choose this species due to: Its ability to thrive across a wide range of pH It’s a member of the gamma class of the proteobacteria (which also hasa large number of pathogenic species) The proposed extracytoplasmic oxidation of several substrates allows direct observation of pH effects on metabolic and energy conservation pathways in addition to stress response pathways In addition, since this species grows well at 30C, moderate salinity and pH >3, no specialized equipment or techniques will be required and complications due high temperature and extremely low pH i.e. <2.0 can be avoided. Sulfur Oxidation Pathway in Halothiobacillus sp. Oxygen Consumption Studies Oxygen consumption was found to be substrate concentration dependent over the 0 to 100nmol range for the substrates investigated. Maximum oxygen consumption occurred when utilizing either sulfide or tetrathionate as substrate. Both substrates displayed approximately 300 nmol oxygen consumption yielding an ~ 3:1 oxygen to substrate ratio. Thiosulfate showed only ~2/3 the oxygen consumption as that of sulfide with ~200nmol of oxygen consumed yielding an ~2:1 ratio. Sulfur showed less than one-half` of the oxygen consumption activity of sulfide yielding a final ratio of ~1.2:1. There was no apparent difference in oxygen consumption using different forms of elemental sulfur such as colloidal sulfur, sublimed sulfur or flowers of sulfur. Background The phylum Proteobacter contains a number of pathogenic species ranging from E. coli and Yersinia pestis in the gamma class to Bordetella in Beta class and Helicobacter pylori and Campylobacter jejuni in the delta/epsilon class. In addition to these heterotrophic pathogenic species the phylum also contains a number of species from more extreme environments such as deep sea thermal vents and acid sulfur springs. Recent interest has arisen in the relationship between deep sea thermal vent microbes and pathogenic species within same phylum. The ability of these microbes to survive in such hostile environments has evolutionary implications in the stress response adaptations. Although they are non-pathogenic, some deep sea vent Proteobacteria share many virulence genes with pathogenic proteobacteria, including genes for virulence factor MviN, hemolysin, invasion antigen CiaB. This comparative genomic study between deep sea vent -proteobacteria and pathogens of the same class has shown insights into the origins and evolution of pathogenesis. Sequence Alignment using C-554 Data Cytochrome 554 of H. neapolitanus sequence was compared with cytovhromes sequences of pathogenic species of Proteobacter. All the sequences showed the consensus signature motif of c-type cytochromes (CXXCHG). Lane 1: Molecular Marker Lane 2: End-filled DNA before ligation Cloning Relevant Genes Degenerate primers have been designed to “fish out” gene sequences of the key enzymes of sulfur oxidation pathway Two genomic libraries have been constructed (4Kb and 30-40 Kb) to be used as template DNA. First strategy is to PCR amplify the genes using forward primer based on the N-terminal sequence of C554 and the reverse primer would be from the the vector Several primers have been designed based on these approaches for the amplification of C554, tetrathionate hydrolase and thiosulfate oxidase and experiments are underway for cloning of these key enzymes Acknowledgements This work was supported by NIH NCRR grant number R-16480. The contents of this poster are solely the responsibility of the authors and do not necessarily represent the official view of NIH. pH Dependent Gene Expression From 2-D polyacrylamide gel electrophoresis (PAGE) analysis, it is clear that even the moderate decrease from pH 7 to pH 6 results in changes of expression of several proteins. Most noteable in the gels shown is a substantial increase in intensity of a protein in the upper left quadrant of the pH 6 gel. Numerous other less substantial changes appear across the gel. The fact that Halothiobacillus neapolitanus shows remarkable acid tolerance, is another link between the pathogenic species of Proteobacterand this organism. Phylogenetic Tree based on C-554 The sequence alignment with c554 from various organisms. The highlighted residues are conserved and make a consensus sequence in all c type cytochromes. Halothiobacillus neapolitanus contains ~ 20 c type cytochromes and this alignment can be used to design a common primer set to pull out them from the library Sievert et al. 2007