Abstract
A novel genetic scheme allowed us to isolate extragenic suppressor mutations that restored mutant OmpF assembly. One group of these mutations, termed asmB for assembly suppressor mutation B, permitted mutant OmpF assembly in a non-allele-specific manner. Genetic mapping analyses placed the asmB mutations at the 2-min region of the Escherichia coli K-12 chromosome. Further analyses revealed that the asmB mutations map within the envA (lpxC) gene, which encodes an enzyme needed for the synthesis of the lipid A moiety of lipopolysaccharide (LPS). Nucleotide sequence analysis showed that the asmB mutations caused a change from F-50 to S (F50S substitution) (asmB2 and asmB3) or a G210S substitution (asmB1) in EnvA. Cells bearing the asmB alleles displayed increased sensitivity to various hydrophobic compounds and detergents, suggesting an alteration within the outer membrane. Direct examination (of the LPS showed that its amounts were reduced by the asmB mutations, with asmB1 exerting a greater effect than asmB2 or asmB3. Thus, it appears that the asmB mutations achieve mutant OmpF assembly suppression by reducing LPS levels, which in turn may alter membrane fluidity.
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- Apicella M. A., Griffiss J. M., Schneider H. Isolation and characterization of lipopolysaccharides, lipooligosaccharides, and lipid A. Methods Enzymol. 1994;235:242–252. doi: 10.1016/0076-6879(94)35145-7. [DOI] [PubMed] [Google Scholar]
- Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baker K., Mackman N., Holland I. B. Genetics and biochemistry of the assembly of proteins into the outer membrane of E. coli. Prog Biophys Mol Biol. 1987;49(2-3):89–115. doi: 10.1016/0079-6107(87)90010-1. [DOI] [PubMed] [Google Scholar]
- Bardwell J. C., McGovern K., Beckwith J. Identification of a protein required for disulfide bond formation in vivo. Cell. 1991 Nov 1;67(3):581–589. doi: 10.1016/0092-8674(91)90532-4. [DOI] [PubMed] [Google Scholar]
- Beall B., Lutkenhaus J. Sequence analysis, transcriptional organization, and insertional mutagenesis of the envA gene of Escherichia coli. J Bacteriol. 1987 Dec;169(12):5408–5415. doi: 10.1128/jb.169.12.5408-5415.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Borck K., Beggs J. D., Brammar W. J., Hopkins A. S., Murray N. E. The construction in vitro of transducing derivatives of phage lambda. Mol Gen Genet. 1976 Jul 23;146(2):199–207. doi: 10.1007/BF00268089. [DOI] [PubMed] [Google Scholar]
- Coleman J. Characterization of Escherichia coli cells deficient in 1-acyl-sn-glycerol-3- phosphate acyltransferase activity. J Biol Chem. 1990 Oct 5;265(28):17215–17221. [PubMed] [Google Scholar]
- Fourel D., Mizushima S., Pagès J. M. Dynamics of the exposure of epitopes on OmpF, an outer membrane protein of Escherichia coli. Eur J Biochem. 1992 May 15;206(1):109–114. doi: 10.1111/j.1432-1033.1992.tb16907.x. [DOI] [PubMed] [Google Scholar]
- 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]
- Grundström T., Normark S., Magnusson K. E. Overproduction of outer membrane protein suppresses envA-induced hyperpermeability. J Bacteriol. 1980 Dec;144(3):884–890. doi: 10.1128/jb.144.3.884-890.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanahan D., Jessee J., Bloom F. R. Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 1991;204:63–113. doi: 10.1016/0076-6879(91)04006-a. [DOI] [PubMed] [Google Scholar]
- 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]
- Laird M. W., Kloser A. W., Misra R. Assembly of LamB and OmpF in deep rough lipopolysaccharide mutants of Escherichia coli K-12. J Bacteriol. 1994 Apr;176(8):2259–2264. doi: 10.1128/jb.176.8.2259-2264.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lazar S. W., Kolter R. SurA assists the folding of Escherichia coli outer membrane proteins. J Bacteriol. 1996 Mar;178(6):1770–1773. doi: 10.1128/jb.178.6.1770-1773.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
- Misra R., Benson S. A. Isolation and characterization of OmpC porin mutants with altered pore properties. J Bacteriol. 1988 Feb;170(2):528–533. doi: 10.1128/jb.170.2.528-533.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Misra R., Miao Y. Molecular analysis of asmA, a locus identified as the suppressor of OmpF assembly mutants of Escherichia coli K-12. Mol Microbiol. 1995 May;16(4):779–788. doi: 10.1111/j.1365-2958.1995.tb02439.x. [DOI] [PubMed] [Google Scholar]
- Misra R. OmpF assembly mutants of Escherichia coli K-12: isolation, characterization, and suppressor analysis. J Bacteriol. 1993 Aug;175(16):5049–5056. doi: 10.1128/jb.175.16.5049-5056.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Misra R., Peterson A., Ferenci T., Silhavy T. J. A genetic approach for analyzing the pathway of LamB assembly into the outer membrane of Escherichia coli. J Biol Chem. 1991 Jul 25;266(21):13592–13597. [PubMed] [Google Scholar]
- Mohan S., Kelly T. M., Eveland S. S., Raetz C. R., Anderson M. S. An Escherichia coli gene (FabZ) encoding (3R)-hydroxymyristoyl acyl carrier protein dehydrase. Relation to fabA and suppression of mutations in lipid A biosynthesis. J Biol Chem. 1994 Dec 30;269(52):32896–32903. [PubMed] [Google Scholar]
- Mühlradt P. F., Golecki J. R. Asymmetrical distribution and artifactual reorientation of lipopolysaccharide in the outer membrane bilayer of Salmonella typhimurium. Eur J Biochem. 1975 Feb 21;51(2):343–352. doi: 10.1111/j.1432-1033.1975.tb03934.x. [DOI] [PubMed] [Google Scholar]
- Normark S. Genetics of a chain-forming mutant of Escherichia coli. Transduction and dominance of the envA gene mediating increased penetration to some antibacterial agents. Genet Res. 1970 Aug;16(1):63–78. doi: 10.1017/s0016672300002287. [DOI] [PubMed] [Google Scholar]
- Normark S. Mutation in Escherichia coli K-12 mediating spherelike envelopes and changes tolerance to ultraviolet irradiation and some antibiotics. J Bacteriol. 1969 Jun;98(3):1274–1277. doi: 10.1128/jb.98.3.1274-1277.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Normark S., Wolf-Watz H. Cell division and permeability of unbalanced envelope mutants of Escherichia coli K12. Ann Microbiol (Paris) 1974 Sep;125 B(2):211–226. [PubMed] [Google Scholar]
- Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
- Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ried G., Hindennach I., Henning U. Role of lipopolysaccharide in assembly of Escherichia coli outer membrane proteins OmpA, OmpC, and OmpF. J Bacteriol. 1990 Oct;172(10):6048–6053. doi: 10.1128/jb.172.10.6048-6053.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rudd K. E., Sofia H. J., Koonin E. V., Plunkett G., 3rd, Lazar S., Rouviere P. E. A new family of peptidyl-prolyl isomerases. Trends Biochem Sci. 1995 Jan;20(1):12–14. doi: 10.1016/s0968-0004(00)88940-9. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sen K., Nikaido H. Lipopolysaccharide structure required for in vitro trimerization of Escherichia coli OmpF porin. J Bacteriol. 1991 Jan;173(2):926–928. doi: 10.1128/jb.173.2.926-928.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sullivan N. F., Donachie W. D. Transcriptional organization within an Escherichia coli cell division gene cluster: direction of transcription of the cell separation gene envA. J Bacteriol. 1984 Nov;160(2):724–732. doi: 10.1128/jb.160.2.724-732.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Young K., Silver L. L., Bramhill D., Cameron P., Eveland S. S., Raetz C. R., Hyland S. A., Anderson M. S. The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase. J Biol Chem. 1995 Dec 22;270(51):30384–30391. doi: 10.1074/jbc.270.51.30384. [DOI] [PubMed] [Google Scholar]
- Yura T., Mori H., Nagai H., Nagata T., Ishihama A., Fujita N., Isono K., Mizobuchi K., Nakata A. Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region. Nucleic Acids Res. 1992 Jul 11;20(13):3305–3308. doi: 10.1093/nar/20.13.3305. [DOI] [PMC free article] [PubMed] [Google Scholar]