1887

Microbiology Society logo

Volume 144, Issue 3

Expression of the second lysine decarboxylase gene of Escherichia coli Free

Abstract

Certain amino acids are substrates for two decarboxylase enzymes in , one inducible by anaerobic growth at low pH and the other constitutive. In the case of lysine, an inducible decarboxylase (CadA) has been extensively characterized, but evidence for the existence of a second lysine decarboxylase is fragmentary and uncertain. This paper confirms that a second lysine decarboxylase is encoded by a locus () previously suggested to be a lysine decarboxylase gene on the basis of sequence comparisons. Overexpression of the cloned gene provided sufficient quantities of enzyme in cell-free extracts for preliminary examination of the properties of the gene product, Ldc. The enzyme is active over a broad range of pH with an optimum at 7.6, much higher than that of CadA, about 5.5. The temperature optimum for both enzymes is similar, at about 52 °C, but Ldc is more readily inactivated by heat than CadA. Expression of from its own promoter was very weak for cells growing in a variety of media, although a low level of lysine decarboxylase was present in cells that carried the region on an oligo-copy plasmid when these were grown in minimal-glucose medium. Northern analysis of RNA extracted from such cells revealed a transcript whose length corresponded to that of the gene, suggesting that is normally transcribed from a promoter immediately upstream. However, most of the mRNA was shorter, indicating degradation or premature termination. The upstream sequence promoted transcription of a gene to which it was fused. Introduction of the upstream sequence as an insert in a multicopy vector increased transcription of the resident fusion. The low level of expression in single copy, the emergence of expression when the gene is present at moderate copy number, and the derepression by the upstream sequence imply that this second lysine decarboxylase gene may not be constitutive but subject to specific repression by a factor which remains to be identified.

  • Published Online:
Keyword(s): gene expression and lysine decarboxylase
© Society for General Mcrobiology 1998
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-144-3-751
1998-03-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/144/3/mic-144-3-751.html?itemId=/content/journal/micro/10.1099/00221287-144-3-751&mimeType=html&fmt=ahah

References

  1. Adams M.H. 1959 Bacteriophages. New York: Interscience;
     [Google Scholar]
  2. Aiba H., Adhya S., de Crombrugge B. 1981; Evidence for two functional gal promoters in intact Escherichia coli cells.. J Biol Chem 256:11905–11910
     [Google Scholar]
  3. Applebaum D.M., Dunlop J.C., Morris D.R. 1977; Comparison of the biosynthetic and degradative ornithine decarboxylases of Escherichia coli. . Biochemistry 16:1580–1584
     [Google Scholar]
  4. Auger E.A., Redding K.E., Plumb T., Childs L.C., Meng S.-Y., Bennett G.N. 1989; Construction of lac fusions to the inducible arginine- and lysine decarboxylase genes of Escherichia coli K12.. Mol Microbiol 3:609–620
     [Google Scholar]
  5. Bird R., Louarn J., Martuscelli J., Caro L. 1972; Origin and sequence of chromosome replication in Escherichia coli. . J Mol Biol 70:549–566
     [Google Scholar]
  6. Boeker E.A., Snell E.E. 1972; Amino acid decarboxylases. . In The Enzymes, 3rd edn. 6 pp. 217–253 Boyer P.D. Edited by New York: Academic Press;
     [Google Scholar]
  7. Brown T. 1994 In Current Protocols in Molecular Biology Supplement 23 pp. 4.9.2–4.9.3 Ausubel F.M. Edited by others New York: Greene Publishing Associates/Wiley;
     [Google Scholar]
  8. Churchward G., Belin D., Nagamine Y. 1984; A pSClOl- derived plasmid which shows no sequence homology with other commonly used cloning vectors.. Gene 31:165–171
     [Google Scholar]
  9. Falkow S. 1958; Activity of lysine decarboxylase as an aid in the identification of Salmonellae and Shigellae. . Am J Clin Path 29:598–600
     [Google Scholar]
  10. Fellay R., Frey J., Krisch H. 1987; Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria.. Gene 52:147–154
     [Google Scholar]
  11. Goldemberg S.H. 1980; Lysine decarboxylase mutants of Escherichia coli: evidence for two enzyme forms.. J Bacteriol 141:1428–1431
     [Google Scholar]
  12. Guzman L.-M., Belin D., Carson M.J., Beckwith J. 1995; Tight regulation, modulation and high level expression by vectors containing the PBAD promoter.. J Bacteriol 177:4121–4130
     [Google Scholar]
  13. Hafner E.W., Tabor C.W., Tabor H. 1979; Mutants of Escherichia coli that do not contain 1,4-diaminobutane (pu- trescine) or spermidine.. J Biol Chem 254:12419–12426
     [Google Scholar]
  14. Igarishi K., Kashiwagi K., Hamasaki H., Miura A., Kakegawa T., Hirose S., Matsuzaki S. 1986; Formation of a compensatory polyamine by Escherichia coli polyamine-requiring mutants during growth in the absence of polyamines.. J. Bacteriol 166:128–134
     [Google Scholar]
  15. Kikuchi Y., Kojima H., Tanaka T., Takatsuka Y., Kamio Y. 1997; Characterization of a second lysine decarboxylase gene from Escherichia coli. . J Bacteriol 179:4486–4492
     [Google Scholar]
  16. Kushner S.R., Nagaishi H., Templin A., Clark A.J. 1971; Genetic recombination in Escherichia coli: the role of exonuclease I.. Proc Natl Acad Sei USA 68824–827
     [Google Scholar]
  17. Lane D., Cavaillé J., Chandler M. 1994; Induction of the SOS response by IS1 transposase.. J Mol Biol 242:339–350
     [Google Scholar]
  18. Lennox E.S. 1955; Transduction of linked genetic characters of the host by bacteriophage PI.. Virology 1:190–206
     [Google Scholar]
  19. Maniatis T., Fritsch E.F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  20. Meng S.-Y., Bennett G.N. 1992; Nucleotide sequence of the Escherichia coli cad operon: a system for neutralization of low extracellular pH.. J Bacteriol 174:2659–2669
     [Google Scholar]
  21. Miller J.H. 1972 Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  22. Pardee A.B., Jacob F., Monod J. 1959; The genetic control and cytoplasmic expression of ̒inducibility〓 in the synthesis of fi- galactosidase by Escherichia coli. . J Mol Biol 1:165–178
     [Google Scholar]
  23. Phan A.P.H., Ngo T.T., Lenhoff H.M. 1982; Spectro- photometric assay for lysine decarboxylase.. Anal Biochem 120:193–197
     [Google Scholar]
  24. Popkin P.S., Maas W.K. 1980; Escherichia coli regulatory mutation affecting lysine transport and lysine decarboxylase.. J Bacteriol 141:485–492
     [Google Scholar]
  25. Raleigh E.A., Kleckner N. 1986; Quantitation of insertion sequence IS10 transposase gene expression by a method generally applicable to any rarely expressed gene.. Proc Natl Acad Sci USA 831787–1791
     [Google Scholar]
  26. Sabo D.L., Boeker E.A., Byers B., Waron H., Fischer E.H. 1974; Purification and physical properties of inducible Escherichia coli lysine decarboxylase.. Biochemistry 13:662–670
     [Google Scholar]
  27. Silhavy T.J., Berman M.L., Enquist L.W. 1984 Experiments with Gene Fusions. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  28. Simons R.W., Houman F., Kleckner N. 1987; Improved single and multicopy lac-based cloning vectors for protein and operon fusions.. Gene 53:85–96
     [Google Scholar]
  29. Sugino A., Hirose S., Okazaki R. 1972; RNA-linked nascent DNA fragments in Escherichia coli. . Proc Natl Acad Sci USA 691863–1867
     [Google Scholar]
  30. Tabor C.W., Tabor H. 1985; Polyamines in microorganisms.. Microbiol Rev 49:81–99
     [Google Scholar]
  31. Tabor H., Hafner E.W., Tabor C.W. 1980; Construction of an Escherichia coli strain unable to synthesize putrescine, spermidine or cadaverine: characterization of two genes controlling lysine decarboxylase.. J Bacteriol 144:952–956
     [Google Scholar]
  32. Takayama M., Ohyama T., Igarishi K., Kobayashi H. 1994; Escherichia coli cad operon functions as a supplier of carbon dioxide.. Mol Microbiol 11:913–918
     [Google Scholar]
  33. Vieira J., Messing J. 1982; The pUC plasmids, an M13mp7- derived system for insertion mutagenesis and sequencing with synthetic universal primers.. Gene 19:259–268
     [Google Scholar]
  34. Watson N., Dunyak D.S., Rosey E.L., Slonczewski J.L., Olson E.R. 1992; Identification of elements involved in transcriptional regulation of the Escherichia coli cad operon by external pH.. J Bacteriol 174:530–540
     [Google Scholar]
  35. Wertheimer S.J., Leifer Z. 1983; Putrescine and spermidine sensitivity of lysine decarboxylase in Escherichia coli: evidence for a constitutive enzyme and its mode of regulation.. Biochem Biophys Res Commun 114:882–888
     [Google Scholar]
  36. Yamamoto Y, Miwa Y., Ohrnori H. 1995; Genbank submission, accession number D49445..
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-144-3-751
Loading
Expression of the second lysine decarboxylase gene of Escherichia coli
Microbiology 144, 751 (1998); https://doi.org/10.1099/00221287-144-3-751
/content/journal/micro/10.1099/00221287-144-3-751
/content/journal/micro/10.1099/00221287-144-3-751
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error