Management of key metabolic intersections is a critical issue in bacterial economy. One such intersection, the interconversion of a-ketoglutarate and glutamate, is particularly important and interesting because it represents the point of coupling between central carbon metabolism and central nitrogen metabolism. Synthesis of the enzymes of the tricarboxylic acid branch of the Krebs cycle (citrate synthase, aconitase and isocitrate dehydrogenase) that produce a-ketoglutarate and the enzymes of nitrogen assimilation (glutamine synthetase, glutamate synthase and glutamate dehydrogenase) are encoded by genes that are subject to multiple forms of regulation. This project seeks to understand in detail the molecular mechanisms that regulate these genes in Bacillus subtilis, a well studied model Gram-positive bacterium, and Listeria monocytogenes, a model intracellular pathogen whose metabolic regulation is relatively unexplored. The proposal focuses on three transcriptional regulators (CcpC, GltC and GlnR) and two enzymes (glutamate dehydrogenase and aconitase) that have post-transcriptional regulatory functions.
The aims of this proposal are: (a) to determine the molecular mechanisms that control the synthesis and activity of the tricarboxylic acid branch enzymes;(b) to reveal the molecular mechanisms that control the synthesis of glutamate and glutamine;and, (c) to determine the role of aconitase as a post-transcriptional regulator of sporulation in B. subtilis and of iron metabolism and virulence genes in L. monocytogenes. These issues will be addressed using a combination of genetic and biochemical techniques. The phenotypes of mutants defective in each of the regulatory factors will be determined and the proteins will be purified and tested using in vitro transcription and DNA and RNA binding experiments. Since L. monocytogenes mutants that lack two repressors of the Krebs cycle genes have a significant defect in virulence in a mouse model, the molecular basis for this defect will be explored.

Public Health Relevance

Maintaining the proper balance in intracellular metabolism is a critical factor in bacterial growth, survival and pathogenesis. This proposal aims to unravel the intricate mechanisms by which two related bacterial species, Bacillus subtilis (a non-pathogen) and Listeria monocytogenes (the causative agent of listeriosis), control the flow of metabolites between central carbon and central nitrogen pathways. The knowledge gained will provide a deeper understanding of pathogenic mechanisms and may aid in designing novel inhibitors of bacterial growth and pathogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036718-25
Application #
8119688
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Hagan, Ann A
Project Start
1986-09-01
Project End
2013-07-31
Budget Start
2011-08-01
Budget End
2013-07-31
Support Year
25
Fiscal Year
2011
Total Cost
$283,005
Indirect Cost
Name
Tufts University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02111
Mittal, Meghna; Pechter, Kieran B; Picossi, Silvia et al. (2013) Dual role of CcpC protein in regulation of aconitase gene expression in Listeria monocytogenes and Bacillus subtilis. Microbiology 159:68-76
Pechter, Kieran B; Meyer, Frederik M; Serio, Alisa W et al. (2013) Two roles for aconitase in the regulation of tricarboxylic acid branch gene expression in Bacillus subtilis. J Bacteriol 195:1525-37
Moses, Susanne; Sinner, Tatjana; Zaprasis, Adrienne et al. (2012) Proline utilization by Bacillus subtilis: uptake and catabolism. J Bacteriol 194:745-58
Belitsky, Boris R (2011) Indirect repression by Bacillus subtilis CodY via displacement of the activator of the proline utilization operon. J Mol Biol 413:321-36
Mittal, Meghna; Picossi, Silvia; Sonenshein, Abraham L (2009) CcpC-dependent regulation of citrate synthase gene expression in Listeria monocytogenes. J Bacteriol 191:862-72
Picossi, Silvia; Belitsky, Boris R; Sonenshein, Abraham L (2007) Molecular mechanism of the regulation of Bacillus subtilis gltAB expression by GltC. J Mol Biol 365:1298-313
Sonenshein, Abraham L (2007) Control of key metabolic intersections in Bacillus subtilis. Nat Rev Microbiol 5:917-27
Serio, Alisa W; Pechter, Kieran B; Sonenshein, Abraham L (2006) Bacillus subtilis aconitase is required for efficient late-sporulation gene expression. J Bacteriol 188:6396-405
Serio, Alisa W; Sonenshein, Abraham L (2006) Expression of yeast mitochondrial aconitase in Bacillus subtilis. J Bacteriol 188:6406-10
Kim, Hyun-Jin; Mittal, Meghna; Sonenshein, Abraham L (2006) CcpC-dependent regulation of citB and lmo0847 in Listeria monocytogenes. J Bacteriol 188:179-90

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