Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1974 Feb;117(2):527–543. doi: 10.1128/jb.117.2.527-543.1974

Alterations in the Outer Membrane of the Cell Envelope of Heptose-Deficient Mutants of Escherichia coli

Jane Koplow 1, Howard Goldfine 1
PMCID: PMC285543  PMID: 4590475

Abstract

The composition of the cell envelope of a heptose-deficient lipopolysaccharide mutant of Escherichia coli, GR467, was studied after fractionation into its outer and cytoplasmic membrane components by means of sucrose density gradient centrifugation. The outer membrane of GR467 had a lower density than that of its parent strain, CR34. Analysis of the fractionated membranes of GR467 indicated that the phospholipid-to-protein ratio had increased 2.4-fold in the outer membrane. The ratio in the mutant cytoplasmic membrane was also increased, although to a lesser extent. By employing a third parameter, the lipid A content of the outer membrane, it was found that the observed phospholipid-to-protein change in the outer membrane was due predominantly to a decrease in the relative amount of protein. This decrease in protein was particularly significant, since it was concomitant with a 68% decrease in the lipid A recovered in the outer membrane of GR467 relative to the lipid A recovered in the outer membrane of CR34. Similar findings were observed in a second heptose-deficient mutant of E. coli, RC-59. The apparent protein deficiency in GR467 was further studied by subjecting solubilized envelope proteins to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that major envelope proteins which were localized in the outer membrane were greatly diminished in GR467. Two revertants of GR467 with the wild-type amounts of heptose had wild-type relative levels of protein in their outer membranes. A partial heptose revertant had a relative level of protein in its outer membrane between those of the mutant and wild type.

Full text

PDF
527

Images in this article

536Image
on p.536
537Image
on p.537
538Image
on p.538

Selected References

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

  1. Ames G. F., Spudich E. N., Nikaido H. Protein composition of the outer membrane of Salmonella typhimurium: effect of lipopolysaccharide mutations. J Bacteriol. 1974 Feb;117(2):406–416. doi: 10.1128/jb.117.2.406-416.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  3. 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]
  4. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boman H. G., Jonsson S., Monner D., Normark S., Bloom G. D. Cell-surface alterations in Escherichia coli K-12 with chromosmal mutations changing ampicillin resistance. Ann N Y Acad Sci. 1971 Jun 11;182:342–357. doi: 10.1111/j.1749-6632.1971.tb30670.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Dröge W., Ruschmann E., Lüderitz O., Westphal O. Biochemical studies on lipopolysaccharides of Salmonella R mutants. 4. Phosphate groups linked to heptose units and their absence in some R lipopolysaccharides. Eur J Biochem. 1968 Mar;4(1):134–138. doi: 10.1111/j.1432-1033.1968.tb00183.x. [DOI] [PubMed] [Google Scholar]
  8. EIDLIC L., NEIDHARDT F. C. PROTEIN AND NUCLEIC ACID SYNTHESIS IN TWO MUTANTS OF ESCHERICHIA COLI WITH TEMPERATURE-SENSITIVE AMINOACYL RIBONUCLEIC ACID SYNTHETASES. J Bacteriol. 1965 Mar;89:706–711. doi: 10.1128/jb.89.3.706-711.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eidels L., Osborn M. J. Lipopolysaccharide and aldoheptose biosynthesis in transketolase mutants of Salmonella typhimurium. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1673–1677. doi: 10.1073/pnas.68.8.1673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Inouye M., Yee M. L. Homogeneity of envelope proteins of Escherichia coli separated by gel electrophoresis in sodium dodecyl sulfate. J Bacteriol. 1973 Jan;113(1):304–312. doi: 10.1128/jb.113.1.304-312.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Josephson B. L., Fraenkel D. G. Transketolase mutants of Escherichia coli. J Bacteriol. 1969 Dec;100(3):1289–1295. doi: 10.1128/jb.100.3.1289-1295.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kalckar H. M., Laursen P., Rapin A. M. Inactivation of phage c21 by various preparations from lipopolysaccharide of e. Coli k-12. Proc Natl Acad Sci U S A. 1966 Dec;56(6):1852–1858. doi: 10.1073/pnas.56.6.1852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lehmann V., Hämmerling G., Nurminen M., Minner I., Ruschmann E., Lüderitz O., Kuo T. T., Stocker B. A. A new class of heptose-defective mutant of Salmonella typhimurium. Eur J Biochem. 1973 Jan 15;32(2):268–275. doi: 10.1111/j.1432-1033.1973.tb02607.x. [DOI] [PubMed] [Google Scholar]
  16. MASTER R. W. POSSIBLE SYNTHESIS OF POLYRIBONUCLEOTIDES OF KNOWN BASE-TRIPLET SEQUENCES. Nature. 1965 Apr 3;206:93–93. doi: 10.1038/206093b0. [DOI] [PubMed] [Google Scholar]
  17. MURRAY R. G., STEED P., ELSON H. E. THE LOCATION OF THE MUCOPEPTIDE IN SECTIONS OF THE CELL WALL OF ESCHERICHIA COLI AND OTHER GRAM-NEGATIVE BACTERIA. Can J Microbiol. 1965 Jun;11:547–560. doi: 10.1139/m65-072. [DOI] [PubMed] [Google Scholar]
  18. Miura T., Mizushima S. Separation by density gradient centrifugation of two types of membranes from spheroplast membrane of Escherichia coli K12. Biochim Biophys Acta. 1968 Jan 3;150(1):159–161. doi: 10.1016/0005-2736(68)90020-5. [DOI] [PubMed] [Google Scholar]
  19. Monner D. A., Jonsson S., Boman H. G. Ampicillin-resistant mutants of Escherichia coli K-12 with lipopolysaccharide alterations affecting mating ability and susceptibility to sex-specific bacteriophages. J Bacteriol. 1971 Aug;107(2):420–432. doi: 10.1128/jb.107.2.420-432.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. NESBITT J. A., 3rd, LENNARZ W. J. COMPARISON OF LIPIDS AND LIPOPOLYSACCHARIDE FROM THE BACILLARY AND L FORMS OF PROTEUS P18. J Bacteriol. 1965 Apr;89:1020–1025. doi: 10.1128/jb.89.4.1020-1025.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. OSBORN M. J. STUDIES ON THE GRAM-NEGATIVE CELL WALL. I. EVIDENCE FOR THE ROLE OF 2-KETO- 3-DEOXYOCTONATE IN THE LIPOPOLYSACCHARIDE OF SALMONELLA TYPHIMURIUM. Proc Natl Acad Sci U S A. 1963 Sep;50:499–506. doi: 10.1073/pnas.50.3.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Osborn M. J., Gander J. E., Parisi E. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Site of synthesis of lipopolysaccharide. J Biol Chem. 1972 Jun 25;247(12):3973–3986. [PubMed] [Google Scholar]
  24. Rapin A. M., Kalckar H. M., Alberico L. The metabolic basis for masking of receptor-sites on E. coli K-12 for C21, a lipopolysaccharide core-specific phage. Arch Biochem Biophys. 1968 Oct;128(1):95–105. doi: 10.1016/0003-9861(68)90011-8. [DOI] [PubMed] [Google Scholar]
  25. Rietschel E. T., Gottert H., Lüderitz O., Westphal O. Nature and linkages of the fatty acids present in the lipid-A component of Salmonella lipopolysaccharides. Eur J Biochem. 1972 Jul 13;28(2):166–173. doi: 10.1111/j.1432-1033.1972.tb01899.x. [DOI] [PubMed] [Google Scholar]
  26. Rooney S. A., Goldfine H. Isolation and characterization of 2-keto-3-deoxyoctonate-lipid A from a heptose-deficient mutant of Escherichia coli. J Bacteriol. 1972 Aug;111(2):531–541. doi: 10.1128/jb.111.2.531-541.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rooney S. A., Goldfine H., Sweeley C. C. The identification of trans-2-tetradecenoic acid in hydrolysates of lipid A from Escherichia coli. Biochim Biophys Acta. 1972 Jul 7;270(3):289–295. doi: 10.1016/0005-2760(72)90192-0. [DOI] [PubMed] [Google Scholar]
  28. Schmidt G., Jann B., Jann K. Immunochemistry of R lipopolysaccharides of Escherichia coli. Different core regions in the lipopolysaccharides of O group 8. Eur J Biochem. 1969 Oct;10(3):501–510. doi: 10.1111/j.1432-1033.1969.tb00717.x. [DOI] [PubMed] [Google Scholar]
  29. Schmidt G., Jann B., Jann K. Immunochemistry of R lipopolysaccharides of Escherichia coli. Studies on R mutants with an incomplete core, derived from E. coli O8:K27. Eur J Biochem. 1970 Oct;16(2):382–392. doi: 10.1111/j.1432-1033.1970.tb01092.x. [DOI] [PubMed] [Google Scholar]
  30. Schnaitman C. A. Protein composition of the cell wall and cytoplasmic membrane of Escherichia coli. J Bacteriol. 1970 Nov;104(2):890–901. doi: 10.1128/jb.104.2.890-901.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shands J. W., Jr, Graham J. A., Nath K. The morphologic structure of isolated bacterial lipopolysaccharide. J Mol Biol. 1967 Apr 14;25(1):15–21. doi: 10.1016/0022-2836(67)90275-6. [DOI] [PubMed] [Google Scholar]
  32. Siccardi A. G., Lazdunski A., Shapiro B. M. Interrelationship between membrane protein composition and deoxyribonucleic acid synthesis in Escherichia coli. Biochemistry. 1972 Apr 25;11(9):1573–1582. doi: 10.1021/bi00759a005. [DOI] [PubMed] [Google Scholar]
  33. Tamaki S., Matsuhashi M. Increase in sensitivity to antibiotics and lysozyme on deletion of lipopolysaccharides in Escherichia coli strains. J Bacteriol. 1973 Apr;114(1):453–454. doi: 10.1128/jb.114.1.453-454.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tamaki S., Sato T., Matsuhashi M. Role of lipopolysaccharides in antibiotic resistance and bacteriophage adsorption of Escherichia coli K-12. J Bacteriol. 1971 Mar;105(3):968–975. doi: 10.1128/jb.105.3.968-975.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. White D. A., Lennarz W. J., Schnaitman C. A. Distribution of lipids in the wall and cytoplasmic membrane subfractions of the cell envelope of Escherichia coli. J Bacteriol. 1972 Feb;109(2):686–690. doi: 10.1128/jb.109.2.686-690.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wu H. C. Isolation and characterization of an Escherichia coli mutant with alteration in the outer membrane porteins of the cell envelope. Biochim Biophys Acta. 1972 Dec 1;290(1):274–289. doi: 10.1016/0005-2736(72)90070-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES