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. 1992 Feb;174(3):702–710. doi: 10.1128/jb.174.3.702-710.1992

Isolation and characterization of the Escherichia coli msbB gene, a multicopy suppressor of null mutations in the high-temperature requirement gene htrB.

M Karow 1, C Georgopoulos 1
PMCID: PMC206146  PMID: 1732206

Abstract

Previous work established that the htrB gene of Escherichia coli is required for growth in rich media at temperatures above 32.5 degrees C but not at lower temperatures. In an effort to determine the functional role of the htrB gene product, we have isolated a multicopy suppressor of htrB, called msbB. The msbB gene has been mapped to 40.5 min on the E. coli genetic map, in a 12- to 15-kb gap of the genomic library made by Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Mapping data show that the order of genes in the region is eda-edd-zwf-pykA-msbB. The msbB gene codes for a protein of 37,410 Da whose amino acid sequence is similar to that of HtrB and, like HtrB, the protein is very basic in nature. The similarity of the HtrB and MsbB proteins could indicate that they play functionally similar roles. Mutational analysis of msbB shows that the gene is not essential for E. coli growth; however, the htrB msbB double mutant exhibits a unique morphological phenotype at 30 degrees C not seen with either of the single mutants. Analysis of both msbB and htrB mutants shows that these bacteria are resistant to four times more deoxycholate than wild-type bacteria but not to other hydrophobic substances. The addition of quaternary ammonium compounds rescues the temperature-sensitive phenotype of htrB bacteria, and this rescue is abolished by the simultaneous addition of Mg2+ or Ca2+. These results suggest that MsbB and HtrB play an important role in outer membrane structure and/or function.

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Selected References

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

  1. 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]
  2. Birkenbihl R. P., Vielmetter W. Cosmid-derived map of E. coli strain BHB2600 in comparison to the map of strain W3110. Nucleic Acids Res. 1989 Jul 11;17(13):5057–5069. doi: 10.1093/nar/17.13.5057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Conway T., Yi K. C., Egan S. E., Wolf R. E., Jr, Rowley D. L. Locations of the zwf, edd, and eda genes on the Escherichia coli physical map. J Bacteriol. 1991 Sep;173(17):5247–5248. doi: 10.1128/jb.173.17.5247-5248.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dame J. B., Shapiro B. M. Use of polymyxin B, levallorphan, and tetracaine to isolate novel envelope mutants of Escherichia coli. J Bacteriol. 1976 Aug;127(2):961–972. doi: 10.1128/jb.127.2.961-972.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fellay R., Frey J., Krisch H. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene. 1987;52(2-3):147–154. doi: 10.1016/0378-1119(87)90041-2. [DOI] [PubMed] [Google Scholar]
  9. Feng D. F., Johnson M. S., Doolittle R. F. Aligning amino acid sequences: comparison of commonly used methods. J Mol Evol. 1984;21(2):112–125. doi: 10.1007/BF02100085. [DOI] [PubMed] [Google Scholar]
  10. Fraenkel D. G., Banerjee S. Deletion mapping of zwf, the gene for a constitutive enzyme, glucose 6-phosphate dehydrogenase in Escherichia coli. Genetics. 1972 Aug;71(4):481–489. doi: 10.1093/genetics/71.4.481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heinrich P., Rosenstein R., Böhmer M., Sonner P., Götz F. The molecular organization of the lysostaphin gene and its sequences repeated in tandem. Mol Gen Genet. 1987 Oct;209(3):563–569. doi: 10.1007/BF00331163. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Ishino F., Matsuhashi M. Peptidoglycan synthetic enzyme activities of highly purified penicillin-binding protein 3 in Escherichia coli: a septum-forming reaction sequence. Biochem Biophys Res Commun. 1981 Aug 14;101(3):905–911. doi: 10.1016/0006-291x(81)91835-0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Knott V., Blake D. J., Brownlee G. G. Completion of the detailed restriction map of the E. coli genome by the isolation of overlapping cosmid clones. Nucleic Acids Res. 1989 Aug 11;17(15):5901–5912. doi: 10.1093/nar/17.15.5901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Kushner S. R., Nagaishi H., Templin A., Clark A. J. Genetic recombination in Escherichia coli: the role of exonuclease I. Proc Natl Acad Sci U S A. 1971 Apr;68(4):824–827. doi: 10.1073/pnas.68.4.824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  21. Lacroix J. M., Loubens I., Tempête M., Menichi B., Bohin J. P. The mdoA locus of Escherichia coli consists of an operon under osmotic control. Mol Microbiol. 1991 Jul;5(7):1745–1753. doi: 10.1111/j.1365-2958.1991.tb01924.x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Oden K. L., DeVeaux L. C., Vibat C. R., Cronan J. E., Jr, Gennis R. B. Genomic replacement in Escherichia coli K-12 using covalently closed circular plasmid DNA. Gene. 1990 Nov 30;96(1):29–36. doi: 10.1016/0378-1119(90)90337-q. [DOI] [PubMed] [Google Scholar]
  24. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  26. Recsei P. A., Gruss A. D., Novick R. P. Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1127–1131. doi: 10.1073/pnas.84.5.1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rowley D. L., Wolf R. E., Jr Molecular characterization of the Escherichia coli K-12 zwf gene encoding glucose 6-phosphate dehydrogenase. J Bacteriol. 1991 Feb;173(3):968–977. doi: 10.1128/jb.173.3.968-977.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shyamala V., Schneider E., Ames G. F. Tandem chromosomal duplications: role of REP sequences in the recombination event at the join-point. EMBO J. 1990 Mar;9(3):939–946. doi: 10.1002/j.1460-2075.1990.tb08192.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  30. Spratt B. G. Distinct penicillin binding proteins involved in the division, elongation, and shape of Escherichia coli K12. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2999–3003. doi: 10.1073/pnas.72.8.2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stern M. J., Ames G. F., Smith N. H., Robinson E. C., Higgins C. F. Repetitive extragenic palindromic sequences: a major component of the bacterial genome. Cell. 1984 Jul;37(3):1015–1026. doi: 10.1016/0092-8674(84)90436-7. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. Yang Y., Ames G. F. DNA gyrase binds to the family of prokaryotic repetitive extragenic palindromic sequences. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8850–8854. doi: 10.1073/pnas.85.23.8850. [DOI] [PMC free article] [PubMed] [Google Scholar]

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