Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Feb;169(2):600–604. doi: 10.1128/jb.169.2.600-604.1987

Effects of deletion and insertion mutations in the ilvM gene of Escherichia coli.

M F Lu, H E Umbarger
PMCID: PMC211820  PMID: 3027038

Abstract

A plasmid was constructed that carried the ilvG and ilvM genes and the associated promoter and leader regions derived from the K-12 strain of Escherichia coli. The ilvG gene contained a + 1 frameshift mutation that enabled the plasmid to specify acetohydroxyacid synthase II. The plasmid was modified by deletions in the terminus of and within the ilvM gene and by insertions into the ilvM gene. The effects of these modifications on the phenotypes of the plasmids were examined in a host strain that lacked all three isozymes of acetohydroxyacid synthase. Most of the ilvM mutant plasmids so obtained permitted growth of the host strain in the absence of isoleucine but not in the absence of valine. Growth in the presence of valine, however, was very slow. No significant acetohydroxyacid synthase activity could be detected even when the cells were grown in a valine-supplemented minimal medium. It thus appears that, at most, only a very low level of acetohydroxyacid synthase activity occurred with ilvG in the absence of ilvM and that low activity was more effective for acetohydroxy butyrate formation than for acetolactate formation. The ilvM gene product could be formed under the control of the lac promoter in the presence of a plasmid that carried an in-frame gene fusion between lacZ and the downstream portion of ilvG. Extracts from the host strain that contained such an IlvG(-)-IlvM+ plasmid could be combined with extracts from cells that contained one of the IlvG+-IlvM- plasmids to yield acetohydroxyacid synthase activity. Thus, the ilvM and ilvG genes could be expressed independently of each other.

Full text

PDF
600

Selected References

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

  1. BERTANI G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol. 1951 Sep;62(3):293–300. doi: 10.1128/jb.62.3.293-300.1951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett D. C., Umbarger H. E. Isolation and analysis of two Escherichia coli K-12 ilv attenuator deletion mutants with high-level constitutive expression of an ilv-lac fusion operon. J Bacteriol. 1984 Mar;157(3):839–845. doi: 10.1128/jb.157.3.839-845.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Felice M., Guardiola J., Esposito B., Iaccarino M. Structural genes for a newly recognized acetolactate synthase in Escherichia coli K-12. J Bacteriol. 1974 Dec;120(3):1068–1077. doi: 10.1128/jb.120.3.1068-1077.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eoyang L., Silverman P. M. Purification and subunit composition of acetohydroxyacid synthase I from Escherichia coli K-12. J Bacteriol. 1984 Jan;157(1):184–189. doi: 10.1128/jb.157.1.184-189.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eoyang L., Silverman P. M. Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition. J Bacteriol. 1986 Jun;166(3):901–904. doi: 10.1128/jb.166.3.901-904.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Friden P., Donegan J., Mullen J., Tsui P., Freundlich M., Eoyang L., Weber R., Silverman P. M. The ilvB locus of Escherichia coli K-12 is an operon encoding both subunits of acetohydroxyacid synthase I. Nucleic Acids Res. 1985 Jun 11;13(11):3979–3993. doi: 10.1093/nar/13.11.3979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Greener A., Hill C. W. Identification of a novel genetic element in Escherichia coli K-12. J Bacteriol. 1980 Oct;144(1):312–321. doi: 10.1128/jb.144.1.312-321.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Harms E., Hsu J. H., Subrahmanyam C. S., Umbarger H. E. Comparison of the regulatory regions of ilvGEDA operons from several enteric organisms. J Bacteriol. 1985 Oct;164(1):207–216. doi: 10.1128/jb.164.1.207-216.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hsu J. H., Harms E., Umbarger H. E. Leucine regulation of the ilvGEDA operon of Serratia marcescens by attenuation is modulated by a single leucine codon. J Bacteriol. 1985 Oct;164(1):217–222. doi: 10.1128/jb.164.1.217-222.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lawther R. P., Calhoun D. H., Adams C. W., Hauser C. A., Gray J., Hatfield G. W. Molecular basis of valine resistance in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1981 Feb;78(2):922–925. doi: 10.1073/pnas.78.2.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lawther R. P., Calhoun D. H., Gray J., Adams C. W., Hauser C. A., Hatfield G. W. DNA sequence fine-structure analysis of ilvG (IlvG+) mutations of Escherichia coli K-12. J Bacteriol. 1982 Jan;149(1):294–298. doi: 10.1128/jb.149.1.294-298.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  15. Schloss J. V., Van Dyk D. E., Vasta J. F., Kutny R. M. Purification and properties of Salmonella typhimurium acetolactate synthase isozyme II from Escherichia coli HB101/pDU9. Biochemistry. 1985 Aug 27;24(18):4952–4959. doi: 10.1021/bi00339a034. [DOI] [PubMed] [Google Scholar]
  16. Squires C. H., De Felice M., Devereux J., Calvo J. M. Molecular structure of ilvIH and its evolutionary relationship to ilvG in Escherichia coli K12. Nucleic Acids Res. 1983 Aug 11;11(15):5299–5313. doi: 10.1093/nar/11.15.5299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Squires C. H., De Felice M., Lago C. T., Calvo J. M. IlvHI locus of Salmonella typhimurium. J Bacteriol. 1983 Jun;154(3):1054–1063. doi: 10.1128/jb.154.3.1054-1063.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Squires C. H., De Felice M., Wessler S. R., Calvo J. M. Physical characterization of the ilvHI operon of Escherichia coli K-12. J Bacteriol. 1981 Sep;147(3):797–804. doi: 10.1128/jb.147.3.797-804.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Watson M. D., Wild J., Umbarger H. E. Positive control of ilvC expression in Escherichia coli K-12; identification and mapping of regulatory gene ilvY. J Bacteriol. 1979 Sep;139(3):1014–1020. doi: 10.1128/jb.139.3.1014-1020.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wek R. C., Hauser C. A., Hatfield G. W. The nucleotide sequence of the ilvBN operon of Escherichia coli: sequence homologies of the acetohydroxy acid synthase isozymes. Nucleic Acids Res. 1985 Jun 11;13(11):3995–4010. doi: 10.1093/nar/13.11.3995. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES