We have shown previously that the ecf gene of E. coli is intimately associated with the active transport of amino acids and certain simple saccharides. Because of its importance in cellular processes, biochemical and genetic characterization of this energy coupling factor (ECF) will be pursued. The structural relationship of the ECF to beta-cystathionase will be investigated and elucidated at the nucleotide level, by DNA sequencing. Clones carrying both ecf and metC (cystathionase) gens have been isolated and will be used for these studies. Experiments are proposed to tackle the problem of the energetics of the osmotic shock-sensitive transport systems. Attempts will be made to identify the direct energy donor for these transport systems by finding the third reaction that makes acetyl phosphate and by investigating the fate of acetyl phosphate. The glutamine transport system of E. coli has been cloned using the plasmid pBR322. Using this, the genetic structure as well as the molecular organization of this transport system will be determined and elucidated. Reconstitution of glutamine transport will spheroplasts will be carried out with glutamine transport mutants as an in vitro complementation test to sort out the functional role of the transport components involved. The physiological effects of the glutamine transport defect on the catabolism of glutamate will be purused genetically in the initial phase, followed by biochemical study to find out what is the biochemical basis of the effect. The main objective of this research proposal is to broaden our knowledge concerning the molecular and genetic organization of the energy transduction machinery and the molecular mechanisms of the coupling of metabolic energy to active transport.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029843-05
Application #
3277549
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-12-01
Project End
1986-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
5
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Boston Biomedical Research Institute
Department
Type
DUNS #
058893371
City
Watertown
State
MA
Country
United States
Zip Code
02472