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Boynton T.O.,University of Georgia | Gerdes S.,Fellowship for Interpretation of Genomes | Craven S.H.,University of Georgia | Neidle E.L.,University of Georgia | And 2 more authors.
Applied and Environmental Microbiology | Year: 2011

Tetrapyrroles are ubiquitous molecules in nearly all living organisms. Heme, an iron-containing tetrapyrrole, is widely distributed in nature, including most characterized aerobic and facultative bacteria. A large majority of bacteria that contain heme possess the ability to synthesize it. Despite this capability and the fact that the biosynthetic pathway has been well studied, enzymes catalyzing at least three steps have remained "missing" in many bacteria. In the current work, we have employed comparative genomics via the SEED genomic platform, coupled with experimental verification utilizing Acinetobacter baylyi ADP1, to identify one of the missing enzymes, a new protoporphyrinogen oxidase, the penultimate enzyme in heme biosynthesis. COG1981 was identified by genomic analysis as a candidate protein family for the missing enzyme in bacteria that lacked HemG or HemY, two known protoporphyrinogen oxidases. The predicted amino acid sequence of COG1981 is unlike those of the known enzymes HemG and HemY, but in some genomes, the gene encoding it is found neighboring other heme biosynthetic genes. When the COG1981 gene was deleted from the genome of A. baylyi, a bacterium that lacks both hemG and hemY, the organism became auxotrophic for heme. Cultures accumulated porphyrin intermediates, and crude cell extracts lacked protoporphyrinogen oxidase activity. The heme auxotrophy was rescued by the presence of a plasmid-borne protoporphyrinogen oxidase gene from a number of different organisms, such as hemG from Escherichia coli, hemY from Myxococcus xanthus, or the human gene for protoporphyrinogen oxidase. © 2011, American Society for Microbiology. Source

Romanowski K.,University of Chicago | Zaborin A.,University of Chicago | Fernandez H.,University of Chicago | Fernandez H.,Medical Center | And 6 more authors.
BMC Microbiology | Year: 2011

Background: During extreme physiological stress, the intestinal tract can be transformed into a harsh environment characterized by regio- spatial alterations in oxygen, pH, and phosphate concentration. When the human intestine is exposed to extreme medical interventions, the normal flora becomes replaced by pathogenic species whose virulence can be triggered by various physico-chemical cues leading to lethal sepsis. We previously demonstrated that phosphate depletion develops in the mouse intestine following surgical injury and triggers intestinal P. aeruginosa to express a lethal phenotype that can be prevented by oral phosphate ([Pi]) supplementation. Results: In this study we examined the role of pH in the protective effect of [Pi] supplementation as it has been shown to be increased in the distal gut following surgical injury. Surgically injured mice drinking 25 mM [Pi] at pH 7.5 and intestinally inoculated with P. aeruginosa had increased mortality compared to mice drinking 25 mM [Pi] at pH 6.0 (p < 0.05). This finding was confirmed in C. elegans. Transcriptional analysis of P. aeruginosa demonstrated enhanced expression of various genes involved in media alkalization at pH 6.0 and a global increase in the expression of all iron-related genes at pH 7.5. Maintaining the pH at 6.0 via phosphate supplementation led to significant attenuation of iron-related genes as demonstrated by microarray and confirmed by QRT-PCR analyses. Conclusion: Taken together, these data demonstrate that increase in pH in distal intestine of physiologically stressed host colonized by P. aeruginosa can lead to the expression of siderophore-related virulence in bacteria that can be prevented without providing iron by maintaining local phosphate abundance at pH 6.0. This finding is particularly important as provision of exogenous iron has been shown to have untoward effects when administered to critically ill and septic patients. Given that phosphate, pH, and iron are near universal cues that dictate the virulence status of a broad range of microorganisms relevant to serious gut origin infection and sepsis in critically ill patients, the maintenance of phosphate and pH at appropriate physiologic levels to prevent virulence activation in a site specific manner can be considered as a novel anti-infective therapy in at risk patients. © 2011 Romanowski et al; licensee BioMed Central Ltd. Source

Zhang Y.,Sanford Burnham Institute for Medical Research | Zhang Y.,Woods Hole Oceanographic Institution | Zagnitko O.,Fellowship for Interpretation of Genomes | Rodionova I.,Sanford Burnham Institute for Medical Research | And 2 more authors.
PLoS Computational Biology | Year: 2011

Function diversification in large protein families is a major mechanism driving expansion of cellular networks, providing organisms with new metabolic capabilities and thus adding to their evolutionary success. However, our understanding of the evolutionary mechanisms of functional diversity in such families is very limited, which, among many other reasons, is due to the lack of functionally well-characterized sets of proteins. Here, using the FGGY carbohydrate kinase family as an example, we built a confidently annotated reference set (CARS) of proteins by propagating experimentally verified functional assignments to a limited number of homologous proteins that are supported by their genomic and functional contexts. Then, we analyzed, on both the phylogenetic and the molecular levels, the evolution of different functional specificities in this family. The results show that the different functions (substrate specificities) encoded by FGGY kinases have emerged only once in the evolutionary history following an apparently simple divergent evolutionary model. At the same time, on the molecular level, one isofunctional group (L-ribulokinase, AraB) evolved at least two independent solutions that employed distinct specificity-determining residues for the recognition of a same substrate (L-ribulose). Our analysis provides a detailed model of the evolution of the FGGY kinase family. It also shows that only combined molecular and phylogenetic approaches can help reconstruct a full picture of functional diversifications in such diverse families. © 2011 Zhang et al. Source

Davis J.J.,University of Illinois at Urbana - Champaign | Xia F.,Argonne National Laboratory | Overbeek R.A.,Fellowship for Interpretation of Genomes | Olsen G.J.,University of Illinois at Urbana - Champaign
International Journal of Systematic and Evolutionary Microbiology | Year: 2013

The tree of life is paramount for achieving an integrated understanding of microbial evolution and the relationships between physiology, genealogy and genomics. It provides the framework for interpreting environmental sequence data, whether applied to microbial ecology or to human health. However, there remain many instances where there is ambiguity in our understanding of the phylogeny of major lineages, and/or confounding nomenclature. Here we apply recent genomic sequence data to examine the evolutionary history of members of the classes Mollicutes (phylum Tenericutes) and Erysipelotrichia (phylum Firmicutes). Consistent with previous analyses, we find evidence of a specific relationship between them in molecular phylogenies and signatures of the 16S rRNA, 23S rRNA, ribosomal proteins and aminoacyl-tRNA synthetase proteins. Furthermore, by mapping functions over the phylogenetic tree we find that the erysipelotrichia lineages are involved in various stages of genomic reduction, having lost (often repeatedly) a variety of metabolic functions and the ability to form endospores. Although molecular phylogeny has driven numerous taxonomic revisions, we find it puzzling that the most recent taxonomic revision of the phyla Firmicutes and Tenericutes has further separated them into distinct phyla, rather than reflecting their common roots. © 2013 IUMS. Source

Dailey T.A.,University of Georgia | Boynton T.O.,University of Georgia | Albetel A.-N.,University of Georgia | Gerdes S.,Fellowship for Interpretation of Genomes | And 2 more authors.
Journal of Biological Chemistry | Year: 2010

Here we identify a previously undescribed protein, HemQ, that is required for heme synthesis in Gram-positive bacteria. Wehave characterized HemQ from Bacillus subtilis and a number of Actinobacteria. HemQ is a multimeric heme-binding protein. Spectroscopic studies indicate that this heme is high spin ferric iron and is ligated by a conserved histidine with the sixth coordination site available for binding a small molecule. The presence of HemQ along with the terminal two pathway enzymes, protoporphyrinogen oxidase (HemY) and ferrochelatase, is required to synthesize heme in vivo and in vitro. Although the exact role played by HemQ remains to be characterized, to be fully functional in vitro it requires the presence of a bound heme. HemQ possesses minimal peroxidase activity, but as a catalase it has a turnover of over 10 4 min-1. We propose that this activity may be required to eliminate hydrogen peroxide that is generated by each turnover of HemY. Given the essential nature of heme synthesis and the restricted distribution of HemQ, this protein is a potential antimicrobial target for pathogens such as Mycobacterium tuberculosis. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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