1887

Abstract

Summary: The inducible acetamidase of NCTC 8159 is expressed at high levels in the presence of a suitable inducer, such as acetamide. The gene and 1.5 kb of upstream sequence had previously been sequenced. A further 1.4 kb of upstream sequence has now been determined, containing an additional ORF on the opposite strand to the acetamidase gene. This ORF has significant homologies to genes encoding regulatory proteins involved in amidase expression in other organisms. Restriction fragments from the 4 kb region were subcloned into a promoter-probe shuttle vector to locate the approximate region of the acetamidase promoter and investigate the mechanism of regulation. An inducible promoter was found to lie in the 1.4 kb region situated 1.5 kb upstream from the acetamidase coding region. Expression of the acetamidase was studied at the protein and mRNA levels. Using immunoblotting, induction of the enzyme was demonstrated in minimal medium containing succinate plus acetamide, but not in a richer medium (Lemco broth) plus acetamide, confirming that regulation of acetamidase expression is mediated by both positive and negative control elements. After induction by acetamide, an increase above basal level could be detected after 1 h for both protein levels (using ELISA) and mRNA levels (using Northern blot analysis), indicating that control of expression is at the mRNA level. The size of the mRNA transcript detected was approximately 1.2 kb, the size of the acetamidase coding region. Since no promoter was identified immediately upstream of the coding region, this raises the possibility that a larger, primary transcript (possibly polycistronic) is cleaved to produce a stable form encoding the acetamidase protein.

Funding
This study was supported by the:
  • Postgraduate Research Studentship from the Medical Research Council of Great Britain
  • Canadian Diseases Network
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1997-07-01
2021-05-15
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References

  1. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for the screening of recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
    [Google Scholar]
  2. Bradley S.G. 1971; Criteria for the definition of Mycobacterium, Nocardia and the rhodocrous complex. Adv Front Plant Sci 28:349–362
    [Google Scholar]
  3. Brosius J. 1984; Plasmid vectors for the selection of promoters. Gene 27:151–160
    [Google Scholar]
  4. Chen Y. Y., Clancy K. A., Burne R. A. 1996; Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque streptococcus. Infect Immun 64:585–592
    [Google Scholar]
  5. Clark-Curtiss J. E., Jacobs W. R., Docherty M. A., Ritchie L. R., Curtiss R. , III 1985; Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae . J Bacteriol 161:1093–1102
    [Google Scholar]
  6. Clarke P. H., Meadow P. M. 1959; Evidence for the occurrence of permeases for tricarboxylic acid cycle intermediates in Pseudomonas aeruginosa . J Gen Microbiol 20:144–155
    [Google Scholar]
  7. Cohen S. N., Chang A., C.& Hsu L. 1972; Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114
    [Google Scholar]
  8. Cussac V., Ferrero R. L., Labigne A. 1992; Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions. J Bacteriol 174:2466–2473
    [Google Scholar]
  9. Das Gupta S. K., Bashyam M. D., Tyagi A. K. 1993; Cloning and assessment of mycobacterial promoters by using a plasmid shuttle vector. J Bacteriol 175:5186–5192
    [Google Scholar]
  10. Draper P. 1967; The aliphatic acylamide amidohydrolase of Mycobacterium smegmatis: its inducible nature and relation to acyl-transfer to hydroxylamine. J Gen Microbiol 46:111–123
    [Google Scholar]
  11. Dubos R. J., Davis B. D. 1946; Factors affecting the growth of tubercle bacilli in liquid media. J Exp Med 83:409–423
    [Google Scholar]
  12. Halpern Y. S., Grossowicz N. 1957; Hydrolysis of amides by extracts from mycobacteria. Biochem J 65:716–720
    [Google Scholar]
  13. Hopwood D. A., Wright H. M. 1978; Bacterial protoplast fusion; recombination in fused protoplasts of Streptomyces coelicolor . Mol Gen Genet 162:307–317
    [Google Scholar]
  14. Kohn H. I., Harris J. S. 1941; On the mode of action of sulphonamides. I. Action on E. coli . J Pharm Exp Ther 73:343–348
    [Google Scholar]
  15. Komeda H., Kobayashi M., Shimizu S. 1996a; Characterization of the gene cluster of high-molecular mass nitrile hydratase (H-NHase) induced by its own reaction product in Rhodococcus rhodochrous Jl. Proc Natl Acad Sci USA 93:4267–4272
    [Google Scholar]
  16. Komeda H., Kobayashi M., Shimizu S. 1996b; A novel gene cluster including the Rhodococcus rhodochrous J1nhlBA genes encoding a low-molecular mass nitrile hydratase (L-NHase) induced by its reaction product. J Biol Chem 271:15796–15802
    [Google Scholar]
  17. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  18. Mahenthiralingam E., Draper P., Davis E. O., Colston M. J. 1993; Cloning and sequencing of the gene which encodes the highly inducible acetamidase of Mycobacterium smegmatis . J Gen Microbiol 139:575–583
    [Google Scholar]
  19. Norrander J., Kempe T., Messing J. 1983; Construction of improved M13 vectors using oligodeoxynucleotide directed mutagenesis. Gene 26:101–106
    [Google Scholar]
  20. Oscarsson J., Mizunoe Y., Uhlin B. E., Haydon D. J. 1996; Induction of haemolytic activity in Escherichia coli by the slyA gene product. Mol Microbiol 20:191–199
    [Google Scholar]
  21. Padh H., Venkitasubramanian T. A. 1976; Adenosine 3ʹ,5ʹ-monophosphate in Mycobacterium phlei and Mycobacterium tuberculosis H37Ra. Microbios 16:183–189
    [Google Scholar]
  22. Ratledge C. 1982 Nutrition, growth and metabolism. . In The Biology of the Mycobacteria , vol. 1 , pp. 185–271 . Edited by Ratledge C., Stanford J. London: Academic Press;
    [Google Scholar]
  23. Rauzier J., Moniz-Pereira J., Gicquel-Sanzey B. 1988; Complete nucleotide sequence of pAL5000, a plasmid from Mycobacterium fortuitum . Gene 71:315–321
    [Google Scholar]
  24. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor; NY: Cold Spring Harbor Laboratory:
    [Google Scholar]
  25. Sanger F., Coulsen A. R., Barrell B. G., Smith A. J., Roe B. A. 1980; Cloning in single stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol 143:161–178
    [Google Scholar]
  26. Snapper S. B., Lugosi L., Jekkel A., Melton R. E., Kieser T., Bloom B. R., Jacobs W. R. J. 1988; Lysogeny and stable transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci USA 85:6987–6991
    [Google Scholar]
  27. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R. 1990; Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis . Mol Microbiol 4:1911–1919
    [Google Scholar]
  28. Wilson S., Drew R. 1991; Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product. J Bacteriol 173:4914–4921
    [Google Scholar]
  29. Wilson S. A., Wachira S. J., Drew R. E., Jones D., Pearl L. H. 1993; Antitermination of amidase expression in Pseudomonas aeruginosa is controlled by a novel cytoplasmic amide-binding protein. EMBO J 12:3637–3642
    [Google Scholar]
  30. Wilson S. A., Williams R. J., Pearl L. H., Drew R. E. 1995; Identification of two new genes in the Pseudomonas aeruginosa amidase operon, encoding an ATPase (AmiB) and a putative membrane protein (AmiS). J Biol Chem 270:18818–18824
    [Google Scholar]
  31. Wyborn N. R., Mills J., Williams S. G., Jones C. W. 1996; Molecular characterisation of formamidase from Methylophilus methylotrophus . Eur J Biochem 240:314–322
    [Google Scholar]
  32. Young R. A., Mehra V., Sweetser D., Buchanan T., Clark-Curtiss J., Davis R. W., Bloom B. R. 1985a; Genes for the major protein antigens of the leprosy parasite Mycobacterium leprae . Nature 316:450–452
    [Google Scholar]
  33. Young R. A., Bloom B. R., Grosskinsky C. M., Ivanyi J., Thomas D., Davis R.W. 1985b; Dissection of Mycobacterium tuberculosis antigens using recombinant DNA. Proc Natl Acad Sci USA 82:2583–2587
    [Google Scholar]
  34. Young D. B., Kaufmann S. H. E., Hermans P. W. M., Thole J. E. R. 1992; Mycobacterial protein antigens – a compilation. Mol Microbiol 6:133–145
    [Google Scholar]
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