Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Jun;172(6):3250–3256. doi: 10.1128/jb.172.6.3250-3256.1990

In vivo cloning and characterization of the gabCTDP gene cluster of Escherichia coli K-12.

E Metzer 1, Y S Halpern 1
PMCID: PMC209132  PMID: 2188954

Abstract

The gabCTDP gene cluster, which specifies and regulates synthesis of the gamma-aminobutyrate (GABA) transport carrier, of glutamate-succinic semialdehyde transaminase, and of succinic semialdehyde dehydrogenase, responsible for the uptake and metabolism of gamma-aminobutyric acid in Escherichia coli K-12, was cloned in vivo, using the mini-Mu replicon bacteriophage Mu dI5086 as the vector. A subclone containing a 7.8-kilobase (kb) EcoRI-HindIII fragment complemented all of our Gab- mutants. By restriction mapping, this DNA fragment was located at kb 2800.5 to 2808.5 on the physical map of the E. coli K-12 chromosome. A subclone containing a 1.8-kb EcoRI-SalI fragment complemented the gab-repressed strain CS101A (wild-type gabC) but did not complement any gab structural gene mutants. The gab genes are divergently transcribed from promoters located in the vicinity of the unique BamHI site. Transcription in both directions is under dual control of catabolite repression and nitrogen regulation. Using a procaryotic DNA-directed translation system, we observed three insert-coded polypeptide bands of 53 to 55, 45 to 48, and 40 to 43 kilodaltons (kDa). In vivo studies with subcloned fragments of the gab DNA identified the 53- to 55- and 45- to 48-kDa bands as products of the BamHI-SalI fragment and the 40- to 43-kDa band as the product of the EcoRI-SalI fragment. An additional 26- to 28-kDa band was identified as the product of the BamHI-HindIII fragment. Furthermore, the BamHI-SalI fragment was shown to specify synthesis of the two GABA enzymes, whereas synthesis of the GABA carrier was specified by the BamHI-HindIII fragment. No catalytic function in addition to its regulatory role could be attributed to the EcoRI-SalI gene product.

Full text

PDF
3250

Images in this article

3254Image
on p.3254
3254Image
on p.3254

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beck C. F., Warren R. A. Divergent promoters, a common form of gene organization. Microbiol Rev. 1988 Sep;52(3):318–326. doi: 10.1128/mr.52.3.318-326.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
  4. Dover S., Halpern Y. S. Control of the pathway of -aminobutyrate breakdown in Escherichia coli K-12. J Bacteriol. 1972 Apr;110(1):165–170. doi: 10.1128/jb.110.1.165-170.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dover S., Halpern Y. S. Novel type of catabolite repression in the pathway of gamma-aminobutyrate breakdown in Escherichia coli K-12. FEBS Lett. 1973 Dec 1;37(2):207–211. doi: 10.1016/0014-5793(73)80460-0. [DOI] [PubMed] [Google Scholar]
  6. Dover S., Halpern Y. S. Utilization of -aminobutyric acid as the sole carbon and nitrogen source by Escherichia coli K-12 mutants. J Bacteriol. 1972 Feb;109(2):835–843. doi: 10.1128/jb.109.2.835-843.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Groisman E. A., Casadaban M. J. Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing. J Bacteriol. 1986 Oct;168(1):357–364. doi: 10.1128/jb.168.1.357-364.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kahane S., Levitz R., Halpern Y. S. Specificity and regulation of gamma-aminobutyrate transport in Escherichia coli. J Bacteriol. 1978 Aug;135(2):295–299. doi: 10.1128/jb.135.2.295-299.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Klotsky R. A., Schwartz I. Measurement of cat expression from growth-rate-regulated promoters employing beta-lactamase activity as an indicator of plasmid copy number. Gene. 1987;55(1):141–146. doi: 10.1016/0378-1119(87)90257-5. [DOI] [PubMed] [Google Scholar]
  10. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  11. Metzer E., Levitz R., Halpern Y. S. Isolation and properties of Escherichia coli K-12 mutants impaired in the utilization of gamma-aminobutyrate. J Bacteriol. 1979 Mar;137(3):1111–1118. doi: 10.1128/jb.137.3.1111-1118.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Popham D. L., Szeto D., Keener J., Kustu S. Function of a bacterial activator protein that binds to transcriptional enhancers. Science. 1989 Feb 3;243(4891):629–635. doi: 10.1126/science.2563595. [DOI] [PubMed] [Google Scholar]
  13. Raleigh E. A., Wilson G. Escherichia coli K-12 restricts DNA containing 5-methylcytosine. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9070–9074. doi: 10.1073/pnas.83.23.9070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Schneider K., Beck C. F. New expression vectors for identifying and testing signal structures for initiation and termination of transcription. Methods Enzymol. 1987;153:452–461. doi: 10.1016/0076-6879(87)53071-3. [DOI] [PubMed] [Google Scholar]
  15. Schneider K., Beck C. F. Promoter-probe vectors for the analysis of divergently arranged promoters. Gene. 1986;42(1):37–48. doi: 10.1016/0378-1119(86)90148-4. [DOI] [PubMed] [Google Scholar]
  16. Shaibe E., Metzer E., Halpern Y. S. Control of utilization of L-arginine, L-ornithine, agmatine, and putrescine as nitrogen sources in Escherichia coli K-12. J Bacteriol. 1985 Sep;163(3):938–942. doi: 10.1128/jb.163.3.938-942.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shaibe E., Metzer E., Halpern Y. S. Metabolic pathway for the utilization of L-arginine, L-ornithine, agmatine, and putrescine as nitrogen sources in Escherichia coli K-12. J Bacteriol. 1985 Sep;163(3):933–937. doi: 10.1128/jb.163.3.933-937.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  19. Zaboura M., Halpern Y. S. Regulation of gamma-aminobutyric acid degradation in Escherichia coli by nitrogen metabolism enzymes. J Bacteriol. 1978 Feb;133(2):447–451. doi: 10.1128/jb.133.2.447-451.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES