The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a complex enzyme system which in Salmonella typhimurium and Escherichia coli catalyzes the concomitant transmembrane transport and phosphorylation of its sugar substrates. It also regulates the utilization of a variety of carbon sources not taken up via the PTS-catalyzed group translocation mechanism. Studies in our laboratory have provided information regarding the mechanisms by which the PTS functions in catalysis and regulation of carbon metabolism. We have cloned and sequenced many of the gene encoding PTS proteins and proteins which regulate their expression, and have conducted extensive molecular genetic and biochemical analyses to define their mechanisms of action. These studies have allowed us to define the allosteric mechanism by which PTS-mediated protein phosphorylation regulates the activities of non-PTS carbohydrate permeases, catabolic enzymes and adenylate cyclase. Recently, our studies have shown that the fructose (fru) regulon is exceptionally complex and that the fru repressor (FruR) together with other fructose-specific proteins regulates transcription of hundreds of genes encoding enzymes involved in Salmonella pathogenesis and central pathways of carbon metabolism (glycolysis, the Krebs cycle, the glyoxylate shunt, gluconeogenesis, and electron transfer). The purpose of the proposed research is to extend our understanding of the fru regulon, the fructose-specific PTS proteins and their involvement in sugar transport and transcriptional regulation. Employing Salmonella typhimurium, a combined biochemical and molecular genetic approach will be taken as follows: 1. We will sequence the fruR gene and overproduce and purify FruR for physicochemical, DNA binding and in vitro transcription studies. Other proteins believed to be involved in regulation will similarly be prepared. Target genes to be studied include those of the fru regulon which are negatively regulated by FruR as well as those encoding enzymes of central metabolic pathways which are positively regulated by FruR. The possible involvement of the enzymes of the fructose PTS and protein phosphorylation will be tested. 2. We will characterize the various components of the fru regulon including cryptic genes encoding duplicate copies of PTS energy coupling proteins and permeases. We will also characterize the Enzyme IIIfru-MR-FPr and the Enzyme IIfru proteins in order to define the structure-function relationships of their various domains. X-ray diffraction studies (to be conducted collaboratively) will complement the molecular genetic and biochemical studies proposed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI014176-17
Application #
2060053
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1977-09-01
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
17
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Quan, John A; Schneider, Barbara L; Paulsen, Ian T et al. (2002) Regulation of carbon utilization by sulfur availability in Escherichia coli and Salmonella typhimurium. Microbiology 148:123-31
Saier Jr, M H (2001) Evolution of transport proteins. Genet Eng (N Y) 23:1-10
Jack, D L; Yang, N M; Saier Jr, M H (2001) The drug/metabolite transporter superfamily. Eur J Biochem 268:3620-39
Chung, Y J; Saier Jr, M H (2001) SMR-type multidrug resistance pumps. Curr Opin Drug Discov Devel 4:237-45
Chung, Y J; Krueger, C; Metzgar, D et al. (2001) Size comparisons among integral membrane transport protein homologues in bacteria, Archaea, and Eucarya. J Bacteriol 183:1012-21
Tchieu, J H; Norris, V; Edwards, J S et al. (2001) The complete phosphotranferase system in Escherichia coli. J Mol Microbiol Biotechnol 3:329-46
Jack, D L; Paulsen, I T; Saier, M H (2000) The amino acid/polyamine/organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations. Microbiology 146 ( Pt 8):1797-814
Saier Jr, M H (2000) Families of transmembrane transporters selective for amino acids and their derivatives. Microbiology 146 ( Pt 8):1775-95
Jack, D L; Storms, M L; Tchieu, J H et al. (2000) A broad-specificity multidrug efflux pump requiring a pair of homologous SMR-type proteins. J Bacteriol 182:2311-3
Saier Jr, M H (2000) Vectorial metabolism and the evolution of transport systems. J Bacteriol 182:5029-35

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