1986 — 2010 |
Stewart, Valley J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genetic Control of Nitrate Respiration in E. Coli @ University of California At Davis
Enterobacteria such as Escherichia coli and Salmonella enterica are facultative aerobes, and when cultured in the absence of oxygen can use a variety of alternative respiratory oxidants. Nitrate and nitrite are the preferred alternatives, and anaerobic respiratory enzyme gene expression is tightly controlled in response to their availability. This control is mediated by dual interacting two-component regulatory systems. Nitrate and nitrite control the autophosphorylation of two cytoplasmic membrane-bound sensor-kinases, the NarX and NarQ proteins, which in turn control the phosphorylation of two DNA-binding response regulators, the NarL and NarP proteins. Thus, transcription initiation at more than one dozen operons is activated or repressed according to electron acceptor availability. The Nar regulatory network is unique to enterobacteria;other species of proteobacteria (including several human pathogens) contain only the NarQ-NarP system (e.g., Vibrio cholerae) or the NarX-NarL system (e.g., Pseudomonas aeruginosa). The proposed studies therefore broadly inform our understanding of anaerobic physiology for many species within the gamma and beta subdivisions of the proteobacteria. Work proposed here comprises five specific aims: (1) We must characterize cross-regulation in vitro in biochemical detail in order to evaluate and extend models based primarily on in vivo observations. (2) In collaboration with E. P. Baldwin, we will study cooperative DMA binding by the response regulator NarL. (3) In collaboration with P. J. Kiley, we will study transcription activation by the response regulator NarP. (4) We continue to explore response to defined regulatory signals by the sensors NarX and NarQ. (5) In collaboration with M. M. Igo, we will identify previously- unknown target genes whose expression is controlled by the NarX-NarL or NarQ-NarP systems. Our overall goal is to integrate both in vivo and in vitro approaches to better understand the physiology of anaerobic metabolism. The relevance to public health of this fundamental research is in the realm of anaerobic physiology and metabolism. Most pathogenic species within the proteobacteria are facultative aerobes or facultative anaerobes, and many infectious diseases involve colonization of anaerobic environments such as the mammalian intestine. Thus, results from the model species E. coli enhance understanding for a broad range of pathogens that significantly impact public health.
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1989 — 2004 |
Stewart, Valley J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genetic Control of Nitrate Respiration in E Coli @ Cornell University Ithaca
Enterobacteria efficiently regulate their respiratory metabolism in response to availability of alternate electron acceptors such as oxygen, nitrate and fumarate. This regulation is coordinated to ensure use of the most efficient respiratory pathway. In the absence of oxygen, nitrate, the preferred anaerobic electron acceptor, induces synthesis of enzymes for nitrate respiration (formate dehydrogenase-N and nitrate reductase) while repressing the synthesis of other anaerobic respiratory enzymes such as fumarate reductase. The long-term goals of this project are to understand the physiological and genetic mechanisms by which nitrate coordinately regulates the synthesis of different anaerobic pathways in different ways. Previous work has identified two genes, narL and narX, which are required for nitrate induction of nitrate reductase synthesis and nitrate repression of fumarate reductase synthesis. The narL gene product, NARL, is hypothesized to be a DNA-binding protein that regulates transcription in response to nitrate. The narX gene product, NARX, is hypothesized to convert NARL to its repressor form. NARL and NARX show sequence similarity to the group of "two-component regulatory systems" involved in signal transduction. This project will define the roles of narL and narX in regulation by isolating and characterizing altered function mutations in these genes, by expressing narL and narX independently of each other under various growth conditions, and by analyzing binding of NARL to its target DNA sites. The structure and expression of the narL complex operon will be analyzed by operon and gene fusions and by transcript mapping, in order to understand the regulation of narX and narL expression. A search will be made for a hypothetical gene, "narQ", that may be required for conversion of NARL to its activator form. Finally, the structural genes for formate dehydrogenase-N will be identified, and their regulation by nitrate and NARL will be analyzed by genetic and molecular biological methods. Together, these studies will provide a more detailed view of how nitrate regulates and coordinates anaerobic metabolism.
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2009 — 2010 |
Stewart, Valley J. |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Genetic Control of Denitrification in Pseudomonas Aeruginosa @ University of California At Davis
DESCRIPTION (provided by applicant): Pseudomonas aeruginosa infection is the major cause of morbidity and mortality in cystic fibrosis. The organism persists in airways in large part through its ability to generate energy under the oxygen-limited conditions that define the dehydrated mucus in diseased lungs. This small-grant project will advance knowledge of transcription regulatory mechanisms that control energy metabolism. The Anr and Dnr proteins, members of the Crp-Fnr superfamily of transcription activators, are thought to differentially regulate expression of genes whose products are required for microaerobic or anaerobic respiration. The mechanism for this differential regulation is unclear. One hypothesis is that distinct specificity determinants for Anr and Dnr binding serve to direct each regulator to the appropriate operons. An alternative hypothesis is that Anr and Dnr have similar binding specificity determinants, but that these regulators function under different physiological conditions. I propose to distinguish between these two hypotheses by comparing expression from two representative transcription control regions. In previous studies by others, the arcD operon encoding arginine fermentation enzymes has been suggested to be regulated exclusively by Anr, whereas the nosR operon encoding nitrous oxide reductase has been suggested to be regulated exclusively by Dnr. Experiments will monitor arcD- and nosR-directed transcription under different growth conditions, in anr and dnr null mutants, and in strains in which anr and dnr are expressed constitutively. Results from these tests will provide the basis for further analysis of Anr and Dnr control of microaerobic and anaerobic respiration. If the first hypothesis is correct, the determinants for differential binding specificity will be identified. If the second hypothesis is correct, specific roles for Anr- and Dnr- regulated gene expression will be examined further in order to document specific responses to physiological parameters such as oxygen limitation and nitric oxide. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa infection is the major cause of morbidity and mortality in cystic fibrosis. The organism persists in airways in large part through its ability to generate energy under the oxygen-limited conditions that define the dehydrated mucus in diseased lungs. This small-grant project will advance knowledge of transcription regulatory mechanisms that control energy metabolism.
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2011 — 2012 |
Stewart, Valley J. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Rmpa, a Unique Virulence Regulator in Emerging Klebsiella Pneumoniae @ University of California At Davis
DESCRIPTION (provided by applicant): The family Enterobacteriaceae includes an array of human, animal and plant pathogens. Human intestinal and extraintestinal infections are caused by specific strains of the closely-related species Escherichia, Shigella, Salmonella and Klebsiella. Although relatively little-studied, Klebsiella pneumoniae nevertheless is a notorious nosocomial pathogen responsible for widespread dissemination of antibiotic resistance. Recently, K. pneumoniae has been identified as the cause of an emerging community-acquired pyogenic liver infection termed KLA (K. pneumoniae liver abscess). This infection is due to specific clones expressing the Hmv (hypermucoviscosity) phenotype, which results from unknown modifications of capsule composition or synthesis. Capsule is an essential pathogenicity factor for K. pneumoniae infections, and Hmv is important for virulence of KLA strains. The Hmv phenotype depends on the auxiliary regulator RmpA, a unique protein that likely forms heterodimers with the global RcsB response regulator. Characterized strains contain multiple rmpA alleles on mobile genetic elements. The relevance to NIAID, and the long-term objectives for this project, are to identify and understand genetic determinants for K. pneumoniae pathogenicity and virulence. The goal of this proposal is to initiate genetic and biochemical analysis in a virulent KLA strain of K. pneumoniae. Studies are designed to identify the functions of RmpA and interacting regulators in the control of virulence factors including capsule and the Hmv phenotype. Research will be in collaboration with a leader in the study of KLA, Prof. Jin-Town Wang at the National Taiwan University College of Medicine. Specific aims will apply a variety of in vivo and in vitro approaches to test five hypotheses: (1a) Duplicate rmpA alleles are at least partially redundant;(1b) RmpA is integrated into the RcsB regulatory network;(2) The RmpA regulon includes virulence genes beyond those involved directly in capsule synthesis and assembly;(3a) RcsB-RmpA heterodimers control capsule biosynthetic operon expression;and (3b) RmpA activity is modulated by phosphorylation. Impacts of the proposed research are: (i) identification and analysis of virulence factors in an emerging pathogen notorious for developing antibiotic resistance;(ii) improved understanding of the unique Hmv phenotype and its control by the RmpA regulon;and (iii) modified concepts regarding global RcsB regulatory network interactions with auxiliary regulators. PUBLIC HEALTH RELEVANCE: Klebsiella pneumoniae, a notorious nosocomial pathogen, has emerged recently as the cause of pyogenic liver abscess in otherwise healthy adults. Hypermucoviscous capsule, a protective layer surrounding the cell surface, is important for this pathology. The public health relevance of this research is to understand a unique regulator that controls hypermucoviscous capsule formation and composition.
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