The initiation of sporulation in Bacillus subtilis is regulated by the phosphorelay signal transduction system. The goal of the phosphorelay is the production of SpoOA~P. The level of SpoOA~P reflects the recognition and integration of a series of metabolic, environmental and cell cycle signals which regulates the initiation of sporulation. A family of aspartate-phosphatases (Rap) provides a mechanism for regulating the phosphate flow in the phosphorelay by specifically dephosphorylating the SpoOF~P intermediate response regulator. Rap phosphatases are induced by physiological states which are alternative to sporulation. RapA activity is controlled by the product of the phrAgene. The PhrA C-terminal pentapeptide directly inhibts RapA phosphatase activity. Production of the pentapeptide inhibitor is regulated by an export-import control circuit which yields the pentapeptide inhibitor form internalized by the OppA system. The focus of this proposal is the characterization of the RapA-PhrA system modulating the phosphorelay. The role of the export-import circuit that controls the formation of active peptide inhibitor will be investigated by characterization of the enzymes involved in peptide processing. We will investigate whether a regulatory hierarchy is the basis for timing modulation of phosphatase activity by the PhrA peptide. The enzymes involved in peptide processing will be identified and characterized. Peptide localization studies will be carried out in order to define the peptide role in cell communication. The interaction of PhrA peptide with RapA will be investigated biochemically via competitive binding assays and surface plasmon resonance, and genetically by generating RapA-RapB hybrid proteins. The mechanisms of interaction of RapA with SpoOF will be studied by the surface plasmon resonance method and with a genetic approach. Genetic and biochemical approaches will be used for the identification of the target of RapC.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055594-03
Application #
6138560
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Ikeda, Richard A
Project Start
1998-01-01
Project End
2001-12-31
Budget Start
2000-01-01
Budget End
2000-12-31
Support Year
3
Fiscal Year
2000
Total Cost
$330,800
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Diaz, Alejandra R; Core, Leighton J; Jiang, Min et al. (2012) Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide. J Bacteriol 194:1378-88
Hobbs, Carey A; Bobay, Benjamin G; Thompson, Richele J et al. (2010) NMR solution structure and DNA-binding model of the DNA-binding domain of competence protein A. J Mol Biol 398:248-63
Lehmann, Mandy; Noack, Deborah; Wood, Malcolm et al. (2009) Lung epithelial injury by B. anthracis lethal toxin is caused by MKK-dependent loss of cytoskeletal integrity. PLoS One 4:e4755
Wilson, Adam C; Soyer, Magali; Hoch, James A et al. (2008) The bicarbonate transporter is essential for Bacillus anthracis lethality. PLoS Pathog 4:e1000210
Fukushima, Tatsuya; Szurmant, Hendrik; Kim, Eun-Ja et al. (2008) A sensor histidine kinase co-ordinates cell wall architecture with cell division in Bacillus subtilis. Mol Microbiol 69:621-32
Bongiorni, Cristina; Fukushima, Tatsuya; Wilson, Adam C et al. (2008) Dual promoters control expression of the Bacillus anthracis virulence factor AtxA. J Bacteriol 190:6483-92
Perego, Marta; Hoch, James A (2008) Commingling regulatory systems following acquisition of virulence plasmids by Bacillus anthracis. Trends Microbiol 16:215-21
Diaz, Alejandra R; Stephenson, Sophie; Green, J Michael et al. (2008) Functional role for a conserved aspartate in the Spo0E signature motif involved in the dephosphorylation of the Bacillus subtilis sporulation regulator Spo0A. J Biol Chem 283:2962-72
Wilson, Adam C; Perego, Marta; Hoch, James A (2007) New transposon delivery plasmids for insertional mutagenesis in Bacillus anthracis. J Microbiol Methods 71:332-5
Kao, Mou-Chieh; Di Bernardo, Salvatore; Perego, Marta et al. (2004) Functional roles of four conserved charged residues in the membrane domain subunit NuoA of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli. J Biol Chem 279:32360-6