Grantee Research Project Results
Carbon and Nitrogen Repression of Branched Chain Keto Acid Dehydrogenase in Pseudomonas putidaEPA Grant Number: U915028
Title: Carbon and Nitrogen Repression of Branched Chain Keto Acid Dehydrogenase in Pseudomonas putida
Investigators: Hester, Kathryn L.
Institution: University of Oklahoma
EPA Project Officer: Edwards, Jason
Project Period: January 1, 1996 through March 10, 2000
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1996)
Research Category: Fellowship - Microbiology , Academic Fellowships , Biology/Life Sciences
The objective of this research project is to study the carbon and nitrogen regulation of amino acid metabolism in Pseudomonas putida using branched chain keto acid dehydrogenase (BCKAD) as a model system. My research will focus on the regulation of the expression of the bkd operon in P. putida. My hypothesis is that the product of the crc gene is responsible for glucose repression of BCKAD, while ammonium ion repression of BCKAD involves regulation of the metabolic effector glutamine by glutamine synthetase and the products of the nitrogen regulation control genes ntrB(NTRB) and ntrC(NTRC), and also might require an additional transacting factor that acts as a bridge between the 70-dependent bkd operon and the 54-dependent nitrogen regulatory system (NTR).Approach:
I began the study on ammonum ion of BCKAD by screening a P. putida chromosomal DNA library with the Klebsiella aerogenes nac gene to determine if a NAC analogue in P. putida exists. Unfortunately, no hybridization was seen; however, the negative result also could be due to differences in codon preference and G+C content between the two species, and does not rule out the possibility of a NAC homolog in P. putida. In addition to searching for a NAC homolog in P. putida, I will examine the roles of the three products of the glnAntrBntrC operon to prove that they function in the same way in the nitrogen regulation of BCKAD as they do in the model system proposed above.
Currently, I am isolating and identifying the glnAntrBntrC operon in P. putida. I have obtained these genes from Azotobacter vinelandii, and based on the high-sequence homology between the genes of these two species, I will use the A. vinelandii genes as DNA probes for screening a P. putida chromosomal DNA Sphl library. Once the genes have been isolated, cloned, and sequenced, I will make a knockout mutant of glnA (by inserting a tetracycline cassette into its coding region), and examine the possibility of derepression of BCKAD in this mutant. Knockout mutants of the ntrBntrC genes also will be created and examined for their ability to activate transcription of the bkd operon; their involvement in ammonium ion repression of BCKAD will be evidenced by the inability of the mutant to activate transcription of the operon.
Finally, I will use chemical mutagenesis to create P. putida mutants relieved of ammonium ion repression of BCKAD (these colonies will be identified by their large increase in growth over wild type colonies on plates containing valine, isoleucine, and ammonium ion). These mutants will be transformed with a P. putida chromosomal DNA library to isolate and identify protein(s) that complement the mutation. I hope that characterization of this putative protein will lead to identification of a transacting factor, which links the 70-dependent bkd operon and the 54-dependent NTR system.Supplemental Keywords:
fellowship, carbon and nitrogen repression, branched chain keto acid dehydrogenase, BCKAD, Pseudomonas putida, Azotobacter vinelandii, protein, gene.