The objective of this project is to study at the molecular level the mechanism by which the genes responsible for the phosphate global metabolism exert their function. In E. coli, 7-10 regulatory genes interact in a cascade fashion to control the expression of 15-20 structural genes (the Pho Regulon). One of these is the gene phoA of alkaline phosphatase, whose expression will be monitored. The project entails the reconstitution in vitro of the interacting regulatory gene products. It requires: 1) genetic manipulations to obtain efficient multicopy plasmids of each gene in order to overproduce and isolate the proteins; 2) preparation of specific DNA templates from plasmids carrying phoA and the regulatory genes (phoB, phoR, phoM); 3) isolation of specific nucleotide cofactors identified as positive and negative effectors. In vivo it will be possible to analyze the physiological effect of various levels of regulatory products by preparing gene fusions (like tacp-phoB) whose promoter will be regulated by the concentration of the inducer (in this case, IPTG). The genetic approach used will also allow the isolation of novel mutants of phoB and phoR.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024009-12
Application #
3272001
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1977-09-01
Project End
1992-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
12
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Rao, N N; Torriani, A (1990) Molecular aspects of phosphate transport in Escherichia coli. Mol Microbiol 4:1083-90
Rao, N N; Wang, E; Yashphe, J et al. (1986) Nucleotide pool in pho regulon mutants and alkaline phosphatase synthesis in Escherichia coli. J Bacteriol 166:205-11