Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Nov;174(22):7407–7418. doi: 10.1128/jb.174.22.7407-7418.1992

The lethal phenotype caused by null mutations in the Escherichia coli htrB gene is suppressed by mutations in the accBC operon, encoding two subunits of acetyl coenzyme A carboxylase.

M Karow 1, O Fayet 1, C Georgopoulos 1
PMCID: PMC207437  PMID: 1358874

Abstract

Insertion mutations in the Escherichia coli htrB gene result in the unique phenotype of not affecting growth at temperatures below 32.5 degrees C but leading to a loss of viability at temperatures above this in rich media. When htrB bacteria growing in rich media were shifted to the nonpermissive temperature of 42 degrees C, they continued to grow at a rate similar to that at 30 degrees C but they produced phospholipids at the rate required for growth at 42 degrees C. This led to the accumulation of more than twice as much phospholipid per milligram of protein compared with that in wild-type bacteria. Consistent with HtrB playing a role in phospholipid biosynthesis, one complementation group of spontaneously arising mutations that suppressed htrB-induced lethality were mapped to the accBC operon. This operon codes for the biotin carboxyl carrier protein and biotin carboxylase subunits of the acetyl coenzyme A carboxylase enzyme complex, which catalyzes the first step in fatty acid biosynthesis. Four suppressor mutations mapped to this operon. Two alleles were identified as mutations in the accC gene, the third allele was identified as a mutation in the accB gene, and the fourth allele was shown to be an insertion of an IS1 transposable element in the promoter region of the operon, resulting in reduced transcription. The suppressor mutations caused a decrease in the rate of phospholipid biosynthesis, restoring the balance between the biosynthesis of phospholipids and growth rate, thus enabling htrB bacteria to grow at high temperatures.

Full text

PDF

Images in this article

Selected References

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

  1. Alix J. H. A rapid procedure for cloning genes from lambda libraries by complementation of E. coli defective mutants: application to the fabE region of the E. coli chromosome. DNA. 1989 Dec;8(10):779–789. doi: 10.1089/dna.1989.8.779. [DOI] [PubMed] [Google Scholar]
  2. Ames G. F. Lipids of Salmonella typhimurium and Escherichia coli: structure and metabolism. J Bacteriol. 1968 Mar;95(3):833–843. doi: 10.1128/jb.95.3.833-843.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ang D., Georgopoulos C. The heat-shock-regulated grpE gene of Escherichia coli is required for bacterial growth at all temperatures but is dispensable in certain mutant backgrounds. J Bacteriol. 1989 May;171(5):2748–2755. doi: 10.1128/jb.171.5.2748-2755.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  5. Bardwell J. C., Craig E. A. Ancient heat shock gene is dispensable. J Bacteriol. 1988 Jul;170(7):2977–2983. doi: 10.1128/jb.170.7.2977-2983.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Begg K. J., Donachie W. D. Cell shape and division in Escherichia coli: experiments with shape and division mutants. J Bacteriol. 1985 Aug;163(2):615–622. doi: 10.1128/jb.163.2.615-622.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bouloc P., Jaffé A., D'Ari R. The Escherichia coli lov gene product connects peptidoglycan synthesis, ribosomes and growth rate. EMBO J. 1989 Jan;8(1):317–323. doi: 10.1002/j.1460-2075.1989.tb03379.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Churchward G., Belin D., Nagamine Y. A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. Gene. 1984 Nov;31(1-3):165–171. doi: 10.1016/0378-1119(84)90207-5. [DOI] [PubMed] [Google Scholar]
  9. Fall R. R., Vagelos P. R. Acetyl coenzyme A carboxylase. Molecular forms and subunit composition of biotin carboxyl carrier protein. J Biol Chem. 1972 Dec 25;247(24):8005–8015. [PubMed] [Google Scholar]
  10. Galloway S. M., Raetz C. R. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis. J Biol Chem. 1990 Apr 15;265(11):6394–6402. [PubMed] [Google Scholar]
  11. Garwin J. L., Klages A. L., Cronan J. E., Jr Structural, enzymatic, and genetic studies of beta-ketoacyl-acyl carrier protein synthases I and II of Escherichia coli. J Biol Chem. 1980 Dec 25;255(24):11949–11956. [PubMed] [Google Scholar]
  12. Georgopoulos C. P. Bacterial mutants in which the gene N function of bacteriophage lambda is blocked have an altered RNA polymerase. Proc Natl Acad Sci U S A. 1971 Dec;68(12):2977–2981. doi: 10.1073/pnas.68.12.2977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Harder M. E., Beacham I. R., Cronan J. E., Jr, Beacham K., Honegger J. L., Silbert D. F. Temperature-sensitive mutants of Escherichia coli requiring saturated and unsaturated fatty acids for growth: isolation and properties. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3105–3109. doi: 10.1073/pnas.69.11.3105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harder M. E., Ladenson R. C., Schimmel S. D., Silbert D. F. Mutants of Escherichia coli with temperature-sensitive malonyl coenzyme A-acyl carrier protein transacylase. J Biol Chem. 1974 Dec 10;249(23):7468–7475. [PubMed] [Google Scholar]
  16. Herrin D. L., Schmidt G. W. Rapid, reversible staining of northern blots prior to hybridization. Biotechniques. 1988 Mar;6(3):196-7, 199-200. [PubMed] [Google Scholar]
  17. Hong G. F. A systemic DNA sequencing strategy. J Mol Biol. 1982 Jul 5;158(3):539–549. doi: 10.1016/0022-2836(82)90213-3. [DOI] [PubMed] [Google Scholar]
  18. Jackowski S., Alix J. H. Cloning, sequence, and expression of the pantothenate permease (panF) gene of Escherichia coli. J Bacteriol. 1990 Jul;172(7):3842–3848. doi: 10.1128/jb.172.7.3842-3848.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Karow M., Fayet O., Cegielska A., Ziegelhoffer T., Georgopoulos C. Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media. J Bacteriol. 1991 Jan;173(2):741–750. doi: 10.1128/jb.173.2.741-750.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Karow M., Georgopoulos C. Isolation and characterization of the Escherichia coli msbB gene, a multicopy suppressor of null mutations in the high-temperature requirement gene htrB. J Bacteriol. 1992 Feb;174(3):702–710. doi: 10.1128/jb.174.3.702-710.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Karow M., Georgopoulos C. Sequencing, mutational analysis, and transcriptional regulation of the Escherichia coli htrB gene. Mol Microbiol. 1991 Sep;5(9):2285–2292. doi: 10.1111/j.1365-2958.1991.tb02159.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Kondo H., Shiratsuchi K., Yoshimoto T., Masuda T., Kitazono A., Tsuru D., Anai M., Sekiguchi M., Tanabe T. Acetyl-CoA carboxylase from Escherichia coli: gene organization and nucleotide sequence of the biotin carboxylase subunit. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9730–9733. doi: 10.1073/pnas.88.21.9730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kusukawa N., Uemori T., Asada K., Kato I. Rapid and reliable protocol for direct sequencing of material amplified by the polymerase chain reaction. Biotechniques. 1990 Jul;9(1):66-8, 70, 72. [PubMed] [Google Scholar]
  25. Marr A. G., Ingraham J. L. EFFECT OF TEMPERATURE ON THE COMPOSITION OF FATTY ACIDS IN ESCHERICHIA COLI. J Bacteriol. 1962 Dec;84(6):1260–1267. doi: 10.1128/jb.84.6.1260-1267.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mizuno T. Random cloning of bent DNA segments from Escherichia coli chromosome and primary characterization of their structures. Nucleic Acids Res. 1987 Sep 11;15(17):6827–6841. doi: 10.1093/nar/15.17.6827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mulligan M. E., Hawley D. K., Entriken R., McClure W. R. Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):789–800. doi: 10.1093/nar/12.1part2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Muramatsu S., Mizuno T. Nucleotide sequence of the fabE gene and flanking regions containing a bent DNA sequence of Escherichia coli. Nucleic Acids Res. 1989 May 25;17(10):3982–3982. doi: 10.1093/nar/17.10.3982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nikaido H., Vaara M. Molecular basis of bacterial outer membrane permeability. Microbiol Rev. 1985 Mar;49(1):1–32. doi: 10.1128/mr.49.1.1-32.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. STERN I., SHAPIRO B. A rapid and simple method for the determination of esterified fatty acids and for total fatty acids in blood. J Clin Pathol. 1953 May;6(2):158–160. doi: 10.1136/jcp.6.2.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sell S. M., Eisen C., Ang D., Zylicz M., Georgopoulos C. Isolation and characterization of dnaJ null mutants of Escherichia coli. J Bacteriol. 1990 Sep;172(9):4827–4835. doi: 10.1128/jb.172.9.4827-4835.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Slater S. C., Voige W. H., Dennis D. E. Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway. J Bacteriol. 1988 Oct;170(10):4431–4436. doi: 10.1128/jb.170.10.4431-4436.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ulrich A. K., de Mendoza D., Garwin J. L., Cronan J. E., Jr Genetic and biochemical analyses of Escherichia coli mutants altered in the temperature-dependent regulation of membrane lipid composition. J Bacteriol. 1983 Apr;154(1):221–230. doi: 10.1128/jb.154.1.221-230.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vaara M. Increased outer membrane resistance to ethylenediaminetetraacetate and cations in novel lipid A mutants. J Bacteriol. 1981 Nov;148(2):426–434. doi: 10.1128/jb.148.2.426-434.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vaara M., Vaara T., Jensen M., Helander I., Nurminen M., Rietschel E. T., Mäkelä P. H. Characterization of the lipopolysaccharide from the polymyxin-resistant pmrA mutants of Salmonella typhimurium. FEBS Lett. 1981 Jun 29;129(1):145–149. doi: 10.1016/0014-5793(81)80777-6. [DOI] [PubMed] [Google Scholar]
  37. WEISSBACH A., HURWITZ J. The formation of 2-keto-3-deoxyheptonic acid in extracts of Escherichia coli B. I. Identification. J Biol Chem. 1959 Apr;234(4):705–709. [PubMed] [Google Scholar]
  38. Wachi M., Doi M., Tamaki S., Park W., Nakajima-Iijima S., Matsuhashi M. Mutant isolation and molecular cloning of mre genes, which determine cell shape, sensitivity to mecillinam, and amount of penicillin-binding proteins in Escherichia coli. J Bacteriol. 1987 Nov;169(11):4935–4940. doi: 10.1128/jb.169.11.4935-4940.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wall D., Delaney J. M., Fayet O., Lipinska B., Yamamoto T., Georgopoulos C. arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochrome d operon. J Bacteriol. 1992 Oct;174(20):6554–6562. doi: 10.1128/jb.174.20.6554-6562.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Way J. C., Davis M. A., Morisato D., Roberts D. E., Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. doi: 10.1016/0378-1119(84)90012-x. [DOI] [PubMed] [Google Scholar]
  41. Westling-Häggström B., Normark S. Genetic and physiological analysis of an envB spherelike mutant of Escherichia coli K-12 and characterization of its transductants. J Bacteriol. 1975 Jul;123(1):75–82. doi: 10.1128/jb.123.1.75-82.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. de Mendoza D., Garwin J. L., Cronan J. E., Jr Overproduction of cis-vaccenic acid and altered temperature control of fatty acid synthesis in a mutant of Escherichia coli. J Bacteriol. 1982 Sep;151(3):1608–1611. doi: 10.1128/jb.151.3.1608-1611.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES