The major goal of this proposal is to analyze the components of E. coli which are required for secretion of proteins across the cytoplasmic membrane. We will continue genetic approaches to defining the genes of the bacteria which code for components of the export machinery. We will develop new strategies for selecting mutants pleiotropically defective in secretion. We will continue in vivo genetic and biochemical characterization of two secretion genes which we have recently identified, secD and secE. This characterization involves, cloning, sequencing, the isolation of null mutations, the preparation of antibodies to the gene products and the determination of the cellular location of these products. We are analyzing the role of the sec gene products in an in vitro protein secretion system. This work is being done in collaboration with Dr. P.-C. Tai and his colleagues, who have established the essential role of the secY and secA genes for the functioning of this system. We will study the mechanism of regulation of the sec genes. We have shown that the secA and secD genes are regulated by the secretion needs of the cell. Mutations which block secretion cause a derepression in the synthesis of these proteins. We will use genetic approaches to define the site of regulation of these genes and the regulatory factors involved. We will do studies to determine whether the properties of the export process will allow us to develop a genetic approach to the problem of protein folding. Results with B-galactosidase suggest the possibility that the folding of proteins which, are normally cytoplasmic may interfere with their transfer across the membrane. If this is so, a genetic selection for mutants that interfere with folding would be possible. At least one well-studied protein, the B subunit of tryptophan synthetase, will be used as a model system for these studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM041883-01
Application #
3300334
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1989-06-01
Project End
1994-05-31
Budget Start
1989-06-01
Budget End
1990-05-31
Support Year
1
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Cristina, Landeta; McPartland, Laura; Tran, Ngoc Q et al. (2018) Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes. Mol Microbiol :
Meehan, Brian M; Landeta, Cristina; Boyd, Dana et al. (2017) The essential cell division protein FtsN contains a critical disulfide bond in a non-essential domain. Mol Microbiol 103:413-422
Landeta, Cristina; Meehan, Brian M; McPartland, Laura et al. (2017) Inhibition of virulence-promoting disulfide bond formation enzyme DsbB is blocked by mutating residues in two distinct regions. J Biol Chem 292:6529-6541
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Hatahet, Feras; Blazyk, Jessica L; Martineau, Eugenie et al. (2015) Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase. Proc Natl Acad Sci U S A 112:15184-9
Chatelle, Claire; Kraemer, Stéphanie; Ren, Guoping et al. (2015) Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase. Antioxid Redox Signal 23:945-57
Hatahet, Feras; Boyd, Dana; Beckwith, Jon (2014) Disulfide bond formation in prokaryotes: history, diversity and design. Biochim Biophys Acta 1844:1402-14
Dwyer, Robert S; Malinverni, Juliana C; Boyd, Dana et al. (2014) Folding LacZ in the periplasm of Escherichia coli. J Bacteriol 196:3343-50
Beckwith, Jon (2014) Mission possible: getting to yes with François Jacob. Res Microbiol 165:348-50
Li, Zaoping; Boyd, Dana; Reindl, Martin et al. (2014) Identification of YidC residues that define interactions with the Sec Apparatus. J Bacteriol 196:367-77

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