Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Aug;177(15):4488–4500. doi: 10.1128/jb.177.15.4488-4500.1995

Expression of genes kdsA and kdsB involved in 3-deoxy-D-manno-octulosonic acid metabolism and biosynthesis of enterobacterial lipopolysaccharide is growth phase regulated primarily at the transcriptional level in Escherichia coli K-12.

H Strohmaier 1, P Remler 1, W Renner 1, G Högenauer 1
PMCID: PMC177201  PMID: 7543480

Abstract

We have cloned and sequenced a cluster of six open reading frames containing gene kdsA from Escherichia coli K-12. The gene encodes 3-deoxy-D-manno-octulosonate 8-phosphate synthetase (KDO-8-phosphate synthetase), which catalyzes formation of 3-deoxy-D-manno-octulosonic acid (KDO), an essential component of enterobacterial lipopolysaccharide. We have also identified two other genes, hemA and prfA, at the beginning of the cluster. Deletion analysis shows that kdsA, the terminal gene of this putative operon, is transcribed from its own promoter located within the cluster rather than from two promoters preceding this group of six open reading frames. Northern (RNA) blot analysis as well as lacZ operon fusion experiments reveal that the expression of gene kdsA occurs maximally in the early log phase and falls to a low level in the late log and stationary phases. Hence, this gene is subjected to growth phase-dependent regulation at the transcriptional level. Similarly, we show that expression of gene kdsB, which codes for the CTP:CMP-3-deoxy-D-manno-octulosonate cytidyltransferase (CMP-KDO-synthetase), is also growth regulated. This enzyme catalyzes the activation of KDO via formation of CMP-KDO, which is necessary for the incorporation of KDO into lipid A. We have identified the promoter of gene kdsB, whose expression is growth regulated in the same way as that of kdsA. Despite the fact that transcription of genes kdsA and kdsB is shut off as cells enter stationary phase, KDO-8-phosphate synthetase as well as CMP-KDO-synthetase activities are still present at various levels during stationary-phase growth of an E. coli K-12 culture.

Full Text

The Full Text of this article is available as a PDF (641.5 KB).

Selected References

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

  1. Aldea M., Garrido T., Hernández-Chico C., Vicente M., Kushner S. R. Induction of a growth-phase-dependent promoter triggers transcription of bolA, an Escherichia coli morphogene. EMBO J. 1989 Dec 1;8(12):3923–3931. doi: 10.1002/j.1460-2075.1989.tb08573.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ball C. A., Osuna R., Ferguson K. C., Johnson R. C. Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli. J Bacteriol. 1992 Dec;174(24):8043–8056. doi: 10.1128/jb.174.24.8043-8056.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Clementz T., Raetz C. R. A gene coding for 3-deoxy-D-manno-octulosonic-acid transferase in Escherichia coli. Identification, mapping, cloning, and sequencing. J Biol Chem. 1991 May 25;266(15):9687–9696. [PubMed] [Google Scholar]
  6. Coleman J., Raetz C. R. First committed step of lipid A biosynthesis in Escherichia coli: sequence of the lpxA gene. J Bacteriol. 1988 Mar;170(3):1268–1274. doi: 10.1128/jb.170.3.1268-1274.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cooper T. G., Magasanik B. Transcription of the lac operon of Escherichia coli. J Biol Chem. 1974 Oct 25;249(20):6556–6561. [PubMed] [Google Scholar]
  8. Drolet M., Péloquin L., Echelard Y., Cousineau L., Sasarman A. Isolation and nucleotide sequence of the hemA gene of Escherichia coli K12. Mol Gen Genet. 1989 Apr;216(2-3):347–352. doi: 10.1007/BF00334375. [DOI] [PubMed] [Google Scholar]
  9. Elliott T. A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon. J Bacteriol. 1992 Jan;174(1):245–253. doi: 10.1128/jb.174.1.245-253.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elliott T. Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium. J Bacteriol. 1989 Jul;171(7):3948–3960. doi: 10.1128/jb.171.7.3948-3960.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Elliott T., Wang X. Salmonella typhimurium prfA mutants defective in release factor 1. J Bacteriol. 1991 Jul;173(13):4144–4154. doi: 10.1128/jb.173.13.4144-4154.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Emory S. A., Belasco J. G. The ompA 5' untranslated RNA segment functions in Escherichia coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency. J Bacteriol. 1990 Aug;172(8):4472–4481. doi: 10.1128/jb.172.8.4472-4481.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  14. Goldman R. C., Bolling T. J., Kohlbrenner W. E., Kim Y., Fox J. L. Primary structure of CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from Escherichia coli. J Biol Chem. 1986 Dec 5;261(34):15831–15835. [PubMed] [Google Scholar]
  15. Goldman R. C., Kohlbrenner W. E. Molecular cloning of the structural gene coding for CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase from Escherichia coli K-12. J Bacteriol. 1985 Jul;163(1):256–261. doi: 10.1128/jb.163.1.256-261.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hengge-Aronis R. Survival of hunger and stress: the role of rpoS in early stationary phase gene regulation in E. coli. Cell. 1993 Jan 29;72(2):165–168. doi: 10.1016/0092-8674(93)90655-a. [DOI] [PubMed] [Google Scholar]
  17. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [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. Kolter R., Siegele D. A., Tormo A. The stationary phase of the bacterial life cycle. Annu Rev Microbiol. 1993;47:855–874. doi: 10.1146/annurev.mi.47.100193.004231. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Lehmann V., Rupprecht E., Osborn M. J. Isolation of mutants conditionally blocked in the biosynthesis of the 3-deoxy-D-manno-octulosonic-acid--lipid-A part of lipopolysaccharides derived from Salmonella typhimurium. Eur J Biochem. 1977 Jun 1;76(1):41–49. doi: 10.1111/j.1432-1033.1977.tb11568.x. [DOI] [PubMed] [Google Scholar]
  22. Li J. M., Russell C. S., Cosloy S. D. Cloning and structure of the hem A gene of Escherichia coli K-12. Gene. 1989 Oct 30;82(2):209–217. doi: 10.1016/0378-1119(89)90046-2. [DOI] [PubMed] [Google Scholar]
  23. Li S. J., Cronan J. E., Jr Growth rate regulation of Escherichia coli acetyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis. J Bacteriol. 1993 Jan;175(2):332–340. doi: 10.1128/jb.175.2.332-340.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Parker C. T., Pradel E., Schnaitman C. A. Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12. J Bacteriol. 1992 Feb;174(3):930–934. doi: 10.1128/jb.174.3.930-934.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Raetz C. R., Purcell S., Meyer M. V., Qureshi N., Takayama K. Isolation and characterization of eight lipid A precursors from a 3-deoxy-D-manno-octylosonic acid-deficient mutant of Salmonella typhimurium. J Biol Chem. 1985 Dec 25;260(30):16080–16088. [PubMed] [Google Scholar]
  26. Ray P. H., Benedict C. D. CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase). Methods Enzymol. 1982;83:535–540. doi: 10.1016/0076-6879(82)83049-8. [DOI] [PubMed] [Google Scholar]
  27. Ray P. H., Benedict C. D., Grasmuk H. Purification and characterization of cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase. J Bacteriol. 1981 Mar;145(3):1273–1280. doi: 10.1128/jb.145.3.1273-1280.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ray P. H., Benedict C. D. Purification and characterization of specific 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase from Escherichia coli B. J Bacteriol. 1980 Apr;142(1):60–68. doi: 10.1128/jb.142.1.60-68.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ray P. H. Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli. J Bacteriol. 1980 Feb;141(2):635–644. doi: 10.1128/jb.141.2.635-644.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rick P. D., Fung L. W., Ho C., Osborn M. J. Lipid A mutants of Salmonella typhimurium. Purification and characterization of a lipid A precursor produced by a mutant in 3-deoxy-D-mannooctulosonate-8-phosphate synthetase. J Biol Chem. 1977 Jul 25;252(14):4904–4912. [PubMed] [Google Scholar]
  31. Rick P. D., Young D. A. Isolation and characterization of a temperature-sensitive lethal mutant of Salmonella typhimurium that is conditionally defective in 3-deoxy-D-manno-octulosonate-8-phosphate synthesis. J Bacteriol. 1982 May;150(2):447–455. doi: 10.1128/jb.150.2.447-455.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. Stirling D. A., Hulton C. S., Waddell L., Park S. F., Stewart G. S., Booth I. R., Higgins C. F. Molecular characterization of the proU loci of Salmonella typhimurium and Escherichia coli encoding osmoregulated glycine betaine transport systems. Mol Microbiol. 1989 Aug;3(8):1025–1038. doi: 10.1111/j.1365-2958.1989.tb00253.x. [DOI] [PubMed] [Google Scholar]
  36. Sørensen M. A., Kurland C. G., Pedersen S. Codon usage determines translation rate in Escherichia coli. J Mol Biol. 1989 May 20;207(2):365–377. doi: 10.1016/0022-2836(89)90260-x. [DOI] [PubMed] [Google Scholar]
  37. Verkamp E., Chelm B. K. Isolation, nucleotide sequence, and preliminary characterization of the Escherichia coli K-12 hemA gene. J Bacteriol. 1989 Sep;171(9):4728–4735. doi: 10.1128/jb.171.9.4728-4735.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  39. Wanner B. L., Kodaira R., Neidhardt F. C. Physiological regulation of a decontrolled lac operon. J Bacteriol. 1977 Apr;130(1):212–222. doi: 10.1128/jb.130.1.212-222.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Weiss R. B., Murphy J. P., Gallant J. A. Genetic screen for cloned release factor genes. J Bacteriol. 1984 Apr;158(1):362–364. doi: 10.1128/jb.158.1.362-364.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Woisetschläger M., Hödl-Neuhofer A., Högenauer G. Localization of the kdsA gene with the aid of the physical map of the Escherichia coli chromosome. J Bacteriol. 1988 Nov;170(11):5382–5384. doi: 10.1128/jb.170.11.5382-5384.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Woisetschläger M., Högenauer G. Cloning and characterization of the gene encoding 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli. J Bacteriol. 1986 Oct;168(1):437–439. doi: 10.1128/jb.168.1.437-439.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Woisetschläger M., Högenauer G. The kdsA gene coding for 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase is part of an operon in Escherichia coli. Mol Gen Genet. 1987 May;207(2-3):369–373. doi: 10.1007/BF00331603. [DOI] [PubMed] [Google Scholar]

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

RESOURCES