Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Sep;179(17):5521–5533. doi: 10.1128/jb.179.17.5521-5533.1997

Mutation of the htrB gene in a virulent Salmonella typhimurium strain by intergeneric transduction: strain construction and phenotypic characterization.

M G Sunshine 1, B W Gibson 1, J J Engstrom 1, W A Nichols 1, B D Jones 1, M A Apicella 1
PMCID: PMC179425  PMID: 9287009

Abstract

The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A beta-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV(A), which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a well-defined murine typhoid model of animal pathogenesis. To this end, an r- m+ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter- and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.

Full Text

The Full Text of this article is available as a PDF (969.6 KB).

Selected References

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

  1. Biek D. P., Cohen S. N. Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli. J Bacteriol. 1986 Aug;167(2):594–603. doi: 10.1128/jb.167.2.594-603.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bochner B. R., Huang H. C., Schieven G. L., Ames B. N. Positive selection for loss of tetracycline resistance. J Bacteriol. 1980 Aug;143(2):926–933. doi: 10.1128/jb.143.2.926-933.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brozek K. A., Bulawa C. E., Raetz C. R. Biosynthesis of lipid A precursors in Escherichia coli. A membrane-bound enzyme that transfers a palmitoyl residue from a glycerophospholipid to lipid X. J Biol Chem. 1987 Apr 15;262(11):5170–5179. [PubMed] [Google Scholar]
  4. Bullas L. R., Ryu J. I. Salmonella typhimurium LT2 strains which are r- m+ for all three chromosomally located systems of DNA restriction and modification. J Bacteriol. 1983 Oct;156(1):471–474. doi: 10.1128/jb.156.1.471-474.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chan S., Reinhold V. N. Detailed structural characterization of lipid A: electrospray ionization coupled with tandem mass spectrometry. Anal Biochem. 1994 Apr;218(1):63–73. doi: 10.1006/abio.1994.1141. [DOI] [PubMed] [Google Scholar]
  6. Clementz T., Bednarski J. J., Raetz C. R. Function of the htrB high temperature requirement gene of Escherichia coli in the acylation of lipid A: HtrB catalyzed incorporation of laurate. J Biol Chem. 1996 May 17;271(20):12095–12102. doi: 10.1074/jbc.271.20.12095. [DOI] [PubMed] [Google Scholar]
  7. Hitchcock P. J., Brown T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol. 1983 Apr;154(1):269–277. doi: 10.1128/jb.154.1.269-277.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Horne S. M., Kottom T. J., Nolan L. K., Young K. D. Decreased intracellular survival of an fkpA mutant of Salmonella typhimurium Copenhagen. Infect Immun. 1997 Feb;65(2):806–810. doi: 10.1128/iai.65.2.806-810.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Johnson R. S., Her G. R., Grabarek J., Hawiger J., Reinhold V. N. Structural characterization of monophosphoryl lipid A homologs obtained from Salmonella minnesota Re595 lipopolysaccharide. J Biol Chem. 1990 May 15;265(14):8108–8116. [PubMed] [Google Scholar]
  10. 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]
  11. Karow M., Fayet O., Georgopoulos C. 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. J Bacteriol. 1992 Nov;174(22):7407–7418. doi: 10.1128/jb.174.22.7407-7418.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Karow M., Georgopoulos C. The essential Escherichia coli msbA gene, a multicopy suppressor of null mutations in the htrB gene, is related to the universally conserved family of ATP-dependent translocators. Mol Microbiol. 1993 Jan;7(1):69–79. doi: 10.1111/j.1365-2958.1993.tb01098.x. [DOI] [PubMed] [Google Scholar]
  15. Kitchens R. L., Ulevitch R. J., Munford R. S. Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated pathway. J Exp Med. 1992 Aug 1;176(2):485–494. doi: 10.1084/jem.176.2.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee N. G., Sunshine M. G., Engstrom J. J., Gibson B. W., Apicella M. A. Mutation of the htrB locus of Haemophilus influenzae nontypable strain 2019 is associated with modifications of lipid A and phosphorylation of the lipo-oligosaccharide. J Biol Chem. 1995 Nov 10;270(45):27151–27159. [PubMed] [Google Scholar]
  17. Melaugh W., Phillips N. J., Campagnari A. A., Karalus R., Gibson B. W. Partial characterization of the major lipooligosaccharide from a strain of Haemophilus ducreyi, the causative agent of chancroid, a genital ulcer disease. J Biol Chem. 1992 Jul 5;267(19):13434–13439. [PubMed] [Google Scholar]
  18. Mojica T. Transduction by phage P1CM clr-100 in Salmonella typhimurium. Mol Gen Genet. 1975;138(2):113–126. doi: 10.1007/BF02428116. [DOI] [PubMed] [Google Scholar]
  19. Neal B. L., Brown P. K., Reeves P. R. Use of Salmonella phage P22 for transduction in Escherichia coli. J Bacteriol. 1993 Nov;175(21):7115–7118. doi: 10.1128/jb.175.21.7115-7118.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Qureshi N., Takayama K., Heller D., Fenselau C. Position of ester groups in the lipid A backbone of lipopolysaccharides obtained from Salmonella typhimurium. J Biol Chem. 1983 Nov 10;258(21):12947–12951. [PubMed] [Google Scholar]
  21. Rayssiguier C., Dohet C., Radman M. Interspecific recombination between Escherichia coli and Salmonella typhimurium occurs by the RecABCD pathway. Biochimie. 1991 Apr;73(4):371–374. doi: 10.1016/0300-9084(91)90103-8. [DOI] [PubMed] [Google Scholar]
  22. Rayssiguier C., Thaler D. S., Radman M. The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature. 1989 Nov 23;342(6248):396–401. doi: 10.1038/342396a0. [DOI] [PubMed] [Google Scholar]
  23. Schnaitman C. A., Klena J. D. Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol Rev. 1993 Sep;57(3):655–682. doi: 10.1128/mr.57.3.655-682.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Somerville J. E., Jr, Cassiano L., Bainbridge B., Cunningham M. D., Darveau R. P. A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide. J Clin Invest. 1996 Jan 15;97(2):359–365. doi: 10.1172/JCI118423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Strain S. M., Armitage I. M., Anderson L., Takayama K., Qureshi N., Raetz C. R. Location of polar substituents and fatty acyl chains on lipid A precursors from a 3-deoxy-D-manno-octulosonic acid-deficient mutant of Salmonella typhimurium. Studies by 1H, 13C, and 31P nuclear magnetic resonance. J Biol Chem. 1985 Dec 25;260(30):16089–16098. [PubMed] [Google Scholar]
  26. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  27. Van Alphen L., Lugtenberg B., Rietschel E. T., Mombers C. Architecture of the outer membrane of Escherichia coli K12. Phase transitions of the bacteriophage K3 receptor complex. Eur J Biochem. 1979 Nov;101(2):571–579. doi: 10.1111/j.1432-1033.1979.tb19752.x. [DOI] [PubMed] [Google Scholar]
  28. Wollenweber H. W., Schlecht S., Lüderitz O., Rietschel E. T. Fatty acid in lipopolysaccharides of Salmonella species grown at low temperature. Identification and position. Eur J Biochem. 1983 Jan 17;130(1):167–171. doi: 10.1111/j.1432-1033.1983.tb07132.x. [DOI] [PubMed] [Google Scholar]
  29. Wray C., Sojka W. J. Experimental Salmonella typhimurium infection in calves. Res Vet Sci. 1978 Sep;25(2):139–143. [PubMed] [Google Scholar]

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

RESOURCES