Skip to main content
SpringerLink
Log in

Physical and genetic structure of the glpK-cpxA interval of the Escherichia coli K-12 chromosome

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

Mutations at the cpxA locus of Escherichia coli K-12 affect cellular processes that are not otherwise related. We have now determined the physical and genetic structure of the E. coli chromosome in the region of cpxA (87.5 min). Our results indicate that cpxA is a single gene. Previous studies showed cpxA to be linked to tpiA. We therefore isolated two tpiA + recombinant plasmids, pRA200 and pRA300, from EcoRI and BamHI digests of F′133, respectively. By genetic complementation or enzyme overproduction, the 9.5 kb EcoRI fragment in pRA200 was shown to include glpK, tpiA and cdh. The 13.6 kb BamHI fragment of pRA300 lacks glpK, but includes tpiA, pfkA and cpxA. Neither fragment complemented a deletion of the rha operon. These data indicate the chromosomal gene order: 87 min-rha-cpxA-pfkA-cdh-tpiA-glpK-88 min. The EcoRI and BamHI fragments overlap in an interval corresponding to about 8.2 kb of DNA. The total region of the E. coli K12 chromosome covered by the two fragments is about 15 kb. A terminal 2 kb EcoRI-BamHI fragment from pRA300 complemented the chromosomal cpxA2[Ts] allele with respect to isoleucine and valine synthesis, RNA bacteriophage sensitivity and surface exclusion in Hfr strains, and envelope protein composition. Complementation occurred when the fragment was subcloned in pBR325 but not when it was subcloned in pBR322, suggesting that the 2 kb fragment lacks expression sequences that are supplied by cat (chloramphenicol acetyltransferase gene) expression sequences of pBR325. The cpxA locus on the E. coli chromosome was established with respect to two chromosomal Tn10 insertions by a combination of genetic and physical analyses. The locus established by those analyses was consistent with the location of the 2 kb EcoRI-BamHI fragment in the physical map of the region. Physical analyses of (rha-pfkA) and (rha-tpiA) deletion strains showed that they lack cpxA and surrounding genes. Since these strains were viable, cpxA is not essential under all growth conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Albin R, Silverman P (1984) Identification of the Escherichia coli K-12 cpxA locus as a single gene: construction and analysis of biologically-active cpxA gene fusions. Mol Gen Genet 197:272–279

    Google Scholar 

  • Armstrong K, Fan D (1976) Essential genes in the metB-malB region of Escherichia coli K-12. J Bacteriol 126:48–55

    Google Scholar 

  • Babul J (1978) Phosphor fructokinases from Escherichia coli. J Biol Chem 253:4350–4355

    Google Scholar 

  • Bachmann B (1983) Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev 47:180–230

    Google Scholar 

  • Cohen S, Chang A, Hsu L (1972) Non-chromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114

    Google Scholar 

  • Conrad C, Stearns G, Prater W, Rheiner J, Johnson J (1984) Characterization of a glpK transducing phage. Mol Gen Genet 193:376–378

    Google Scholar 

  • Cuozzo M, Silverman P, Minkley E, Jr (1984) Overproduction in Escherichia coli K-12 and purification of the TraJ protein encoded by the conjugative plasmid F. J Biol Chem 258:6659–6666

    Google Scholar 

  • Eoyang L, Silverman P (1984) Purification and subunit composition of acetohydroxyacid synthase I from Escherichia coli K-12. J Bacteriol 157:184–189

    Google Scholar 

  • Fowler T, Taylor L, Thompson R (1983) The control region of the F-plasmid transfer operon: DNA sequence of the traJ and traY genes and characterization of the traY-Z promoter. Gene 26:79–89

    Google Scholar 

  • Gaffney D, Skurray R, Willetts N (1983) Regulation of the F connjugation genes studies by hybridization and tra-lacZ fusion. J Mol Biol 168:103–122

    Google Scholar 

  • Hansen J, Olsen R (1978) Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids PMG1 and PMG5. J Bacteriol 135:227–238

    Google Scholar 

  • Humphreys G, Willshaw G, Anderson E (1975) A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta 383:457–463

    Google Scholar 

  • Jorgensen R, Berg D, Allet B, Reznikoff W (1979) Restriction enzyme cleavage map of Tn10, a transposon which encodes tetracycline resistance. J Bacteriol 137:681–685

    Google Scholar 

  • Maniatis T, Fritsch E, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, New York, pp 366–369

    Google Scholar 

  • McDonell M, Simon M, Studier FW (1977) Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol 110:119–146

    Google Scholar 

  • McEwen J, Silverman P (1980a) Chromosomal mutations of Escherichia coli K-12 that alter the expression of conjugative plasmid function. Proc Natl Acad Sci USA 77:513–517

    Google Scholar 

  • McEwen J, Silverman P (1980b) Genetic analysis of Escherichia coli K-12 chromosomal mutants defective in the expression of F-plasmid functions: identification of genes cpxA and cpxB. J Bacteriol 144:60–67

    Google Scholar 

  • McEwen J, Silverman P (1980c) Mutations in genes cpxA and cpxB of Escherichia coli K-12 cause a defect in isoleucine and valine synthesis. J Bacteriol 144:68–73

    Google Scholar 

  • McEwen J, Silverman P (1982) Mutations in genes cpxA and cpxB alter the protein composition of the Escherichia coli K-12 inner and outer membranes. J Bacteriol 144:1553–1559

    Google Scholar 

  • McEwen J, Sambucetti L, Silverman P (1983) Synthesis and translocation of outer membrane proteins in cpxA cpxB mutants of Escherichia coli K-12. J Bacteriol 154:375–382

    Google Scholar 

  • Morris J, Newman E (1980) Map location of the ssd mutation in Escherichia coli K-12. J Bacteriol 143:1504–1505

    Google Scholar 

  • Newman E, Malik N, Walker C (1982) L-serine degradation in Escherichia coli K-12: directly isolated ssd mutants and their intragenic revertants. J Bacteriol 150:710–715

    Google Scholar 

  • Pahel G, Bloom F, Tyler B (1979) Deletion mapping of the polA-metB region of the Escherichia coli chromosome. J Bacteriol 138:653–656

    Google Scholar 

  • Pouyssegur J, Lagarde A (1973) Systeine de transport du 2-ceto-3-desoxygluconate chez E. coli K-12: localisation d'un gene du structure et de son operateur. Mol Gen Genet 121:163–180

    Google Scholar 

  • Pouyssegur J, Stoeber F (1974) Genetic control of the 2-keto-3-deoxy-d-gluconate metabolism in Escherichia coli K-12: kdgR regulon. J Bacteriol 117:641–651

    Google Scholar 

  • Prentki P, Karch F, Iida S, Meyer J (1981) The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication. Gene 14:289–299

    Google Scholar 

  • Rigby P, Dieckmann M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick-translation. J Mol Biol 113:237–251

    Google Scholar 

  • Sambucetti L, Eoyang L, Silverman P (1982) Cellular control of conjugation in Escherichia coli K-12: effect of chromosomal mutations on F-plasmid gene expression. J Mol Biol 161:13–31

    Google Scholar 

  • Shimosaka M, Fukuda Y, Murata K, Kimura A (1982) Application of hybrid plasmids carrying glycolysis genes to ATP production by Escherichia coli. J Bacteriol 152:98–103

    Google Scholar 

  • Silverman P (1982) Gene cpxA is a new addition to the linkage map of Escherichia coli K-12. J Bacteriol 150:425–428

    Google Scholar 

  • Sutton A, Newman T, McEwen J, Silverman P, Freundlich M (1982) Mutations in genes cpxA and cpxB of Escherichia coli K-12 cause a posttranslational defect in acetohydroxyacid synthase I function in vivo. J Bacteriol 151:976–982

    Google Scholar 

  • Thomson J, Gerstenberger P, Goldberg D, Gociar E, Silva A, Fraenkel D (1979) ColE1 hybrid plasmids for Escherichia coli genes of glycolysis and the hexose monophosphate shunt. J Bacteriol 137:502–506

    Google Scholar 

  • Touati D (1983) Cloning and mapping of the manganese superoxide dismutase gene (sodA) of Escherichia coli K-12. J Bacteriol 155:1078–1087

    Google Scholar 

  • Umbarger HE (1978) Amino acid biosynthesis and its regulation. Annu Rev Biochem 47:533–606

    Google Scholar 

  • Vogel H, Bonner D (1956) Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 218:97–106

    Google Scholar 

  • Willetts N (1977) The transcriptional control of fertility in F-like plasmids. J Mol Biol 112:141–148

    Google Scholar 

  • Womble D, Taylor D, Rownd R (1977) Method for obtaining more accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation. J Bacteriol 130:148–153

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Biology, Division of Biological Sciences, Albert Einstein College of Medicine, 10461, Bronx, NY, USA

    Randi Albin & Philip M. Silverman

Authors
  1. Randi Albin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Philip M. Silverman
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by W. Arber

Rights and permissions

Reprints and permissions

About this article

Cite this article

Albin, R., Silverman, P.M. Physical and genetic structure of the glpK-cpxA interval of the Escherichia coli K-12 chromosome. Mol Gen Genet 197, 261–271 (1984). https://doi.org/10.1007/BF00330972

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00330972

Keywords

Search

Navigation