1887

Abstract

mutants of Gram-negative and some Gram-positive bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, for example mammalian tissues, they will undergo lysis. This was previously exploited to develop vaccine strains of and cloning vectors containing as an selectable marker. As a first step for development of such systems for , the gene from wild-type strain PAO1 was cloned by a combined approach of PCR amplification from chromosomal DNA, construction of mini-libraries and by complementation of an mutant. The nucleotide sequence of a 2433 bp fragment was determined. This fragment contained the C-terminal 47 nucleotides of , encoding 3-isopropylmalate dehydrogenase; , encoding aspartate-β-semialdehyde dehydrogenase (Asd); and , whose product showed similarity to the Asd proteins from spp. By subcloning, was localized to a 1.24 kb DNA fragment which in an T7 expression system strongly expressed a 40000 Da protein. The amino acid sequence was deduced from the DNA sequence. A comparison of the Asd proteins from and revealed greater than 63% identity, demonstrating the conserved nature of Asd in Gram-negative bacteria, and defined the active-site-containing consensus sequence GGNCTVXMLMXXXLGLF as a possible signature motif. Chromosomal δ mutants were isolated. They were auxotrophic for DAP, lysine, methionine and threonine, and lysed in the absence of DAP. Genetic analyses indicated that probably is naturally frame-shifted and does not contribute to the Asd phenotype. By PFGE, the gene was mapped to between coordinates 1.89 and 2.15 Mbp, or 37-40 min, on the 5.9 Mbp chromosome.

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1997-03-01
2024-12-08
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References

  1. Biellmann J.P., Eid P., Hirth C., Joernvall H. 1980; Aspartate- β-semialdehyde dehydrogenase from Escherichia coli. Affinity labeling with the substrate analogue L-2-amino-4-oxo-5-chloro- pentanoic acid: an example of half-site reactivity.. Eur J Biochem 104:59–64
    [Google Scholar]
  2. Bodey G.P., Bolivar R., Fainstein V., Jadeja L. 1983; Infections caused by Pseudomonas aeruginosa. . Rev Infect Dis 5:279–313
    [Google Scholar]
  3. Cardineau G.A., Curtiss R. 1987; Nucleotide sequence of the asd gene of Streptococcus mutans. . J Biol Chem 262:3344–3353
    [Google Scholar]
  4. Chamberlain J.P. 1979; Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate.. Anal Biochem 98:132–135
    [Google Scholar]
  5. Cherepanov P.P., Wackernagel W. 1995; Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp- catalyzed excision of the antibiotic-resistance determinant.. Gene 158:9–14
    [Google Scholar]
  6. Clepet C., Borne F., Krishnapillai V., Baird C., Patte J.C., Cami B. 1992; Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes.. Mol Microbiol 6:3109–3119
    [Google Scholar]
  7. Cryz S.J. JR Pitt T.L., Furer E., Germaier R. 1984; Role of lipopolysaccharide in virulence of Pseudomonas aeruginosa. . Infect lmmun 44:508–513
    [Google Scholar]
  8. Foglino M., Borne F., Bally M., Ball G., Patte J.C. 1995; A direct sulfhydrylation pathway is used for methionine biosynthesis in Pseudomonas aeruginosa. . Microbiology 141:431–439
    [Google Scholar]
  9. Govan J.R.W. 1988; Alginate biosynthesis and other unusual characteristics associated with the pathogenesis of Pseudomonas aeruginosa in cystic fibrosis.. In Bacterial Infections of Respiratory and Gastrointestinal Mucosa pp. 67–96 Donachie W., Griffiths E., Stephen J. Edited by Oxford: IRL Press;
    [Google Scholar]
  10. Greene R.C. 1996; Biosynthesis of methionine.. In Escherichia coli and Salmonella pp. 542–560 Neidhardt F.C., Curtiss R. III Ingraham J.L. others Edited by Washington, DC: American Society for Microbiology;
    [Google Scholar]
  11. Haziza C., Stragier P., Patte J.-C. 1982; Nucleotide sequence of the asd gene of Escherichia coli: absence of a typical attenuation signal.. EMBO J 1:379–384
    [Google Scholar]
  12. Hoang T.T., Schweizer H.P. 1997; Leucine biosynthesis in Pseudomonas aeruginosa: identification and characterization of leuB, encoding 3-isopropylmalate dehydrogenase.. Mol Gen Genet in press
    [Google Scholar]
  13. Hoiby N., Pedersen S.S., Shand G.H., Doering G., Holder I.A. 1989; Pseudomonas aeruginosa infection.. Antibiot Chemother 42:1–300
    [Google Scholar]
  14. Holder I.A., Neely A.N. 1991; The role of proteases in Pseudomonas infections in burns: a current hypothesis.. Antibiot Chemother 44:99–105
    [Google Scholar]
  15. Holloway B.W., Zhang C. 1990; Genetic maps.. In Locus Maps of Complex Genomes pp. 2.71–2.78 O’Brien S.J. Edited by Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  16. Holloway B.W., Römling U., Tümmler B. 1994; Genomic mapping of Pseudomonas aeruginosa PAO.. Microbiology 140:2907–2929
    [Google Scholar]
  17. Kureishi A., Bryan L.E. 1992; Pre-boiling high GC content, mixed primers with ʹ3 complementation allows the successful PCR amplification of Pseudomonas aeruginosa DNA.. Nucleic Acids Res 20:1155
    [Google Scholar]
  18. Lightfoot J., Lam J.S. 1993; Chromosomal mapping, expression and synthesis of lipopolysaccharide in Pseudomonas aeruginosa: a role for guanosine diphospho (GDP)-D-mannose.. Mol Microbiol 8:771–782
    [Google Scholar]
  19. Liss L. 1987; New M13 host: DH5aF competent cells.. Focus 9:13
    [Google Scholar]
  20. Makowski G.S., Ramsby M.L. 1993; pH modification to enhance the molecular sieving properties of sodium dodecyl sulfate-10 % polyacrylamide gel.. Anal Biochem 212:283–285
    [Google Scholar]
  21. Martin G, Cami B., Jeenes D.D., Haas D., Patte J.-C. 1986; Heterologous expression and regulation of the lysA gene of Pseudomonas aeruginosa and Escherichia coli. . Mol Gen Genet 203:430–134
    [Google Scholar]
  22. Miller J.H. 1992 A Short Course in Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  23. Nakayama K., Kelly S.M., Curtiss R. 1988; Construction of an Asd+ expression-cloning vector: stable maintenance and high level expression of cloned genes in a Salmonella vaccine strain.. BioTechnology 6:693–697
    [Google Scholar]
  24. Ochsner U.A., Koch A.K., Fiechter A., Reiser J. 1994; Isolation and characterization of a regulatory gene affecting rhamnolipid synthesis in Pseudomonas aeruginosa. . J Bacteriol 176:2044–2054
    [Google Scholar]
  25. Patte J.-C. 1996; Biosynthesis of threonine and lysine.. In Escherichia coli and Salmonella pp. 528–541 F.C. Neidhardt, Curtiss R. III Ingraham J.L. Edited by others Washington, DC: American Society for Microbiology;
    [Google Scholar]
  26. Rosenberg M.C., Court D. 1979; Regulatory sequences involved in the promotion and termination of RNA transcription.. Annu Rev Genet 13:319–353
    [Google Scholar]
  27. 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]
  28. Schleifer K.H., Kandier O. 1972; Peptidoglycan types of bacterial cell walls and their taxonomic implications.. Bacteriol Rev 36:407–177
    [Google Scholar]
  29. Schweizer H.P. 1991; The agmR gene, an environmentally responsive gene, complements defective glpR, which encodes the putative activator for glycerol metabolism in Pseudomonas aeruginosa. . J Bacteriol 173:6798–6806
    [Google Scholar]
  30. Schweizer H.P., Hoang T. 1995; An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. . Gene 158:15–22
    [Google Scholar]
  31. Schweizer H.P., Po C. 1994; Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase from Pseudomonas aeruginosa. . J Bacteriol 176:2184–2193
    [Google Scholar]
  32. Schweizer H., Sweet G., Larson T.J. 1986; Physical and genetic structure of the glpD-malT interval of the Escherichia coli K-12 chromosome.. Mol Gen Genet 202:488–492
    [Google Scholar]
  33. Schweizer H.P., Klassen T.R., Hoang T. 1996; Improved methods for gene analysis and expression in Pseudomonas. . In Molecular Biology of Pseudomonads pp. 229–237 Nakazawa T., Furukawa K., Haas D., Silver S. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  34. Slauch J.M., Mahan M.J., Mekalanos J.J. 1994; In vivo expression technology for selection of genes specifically induced in host tissues.. Methods Enzymol 235:481–492
    [Google Scholar]
  35. Studier F.W., Rosenberg A.H., Dunn J.J., Dubendorff J.W. 1990; Use of T7 RNA polymerase to direct expression of cloned genes.. Methods Enzymol 185:60–89
    [Google Scholar]
  36. Tabor S. 1994; Expression using the T7 RNA polymerase/ promoter system.. In Short Protocols in Molecular Biology pp. 16.1–16.10 Ausubel F.M., Brent R., Kingston R.E. Edited by others New York: John Wiley;
    [Google Scholar]
  37. Vasil M.L. 1986; Pseudomonas aeruginosa:biology,mechanisms of virulence, epidemiology.. J Pediatr 108:800–805
    [Google Scholar]
  38. Vasil M.L., Graham M.L., Ostroff R.M., Shortridge V.D., Vasil A.I. 1991; Phospholipase C: molecular biology and contribution to the pathogenesis of Pseudomonas aeruginosa. . Antibiot Chemother 44:34–47
    [Google Scholar]
  39. Wang J., Mushegian A., Lory S., Jin S. 1996; Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection.. Proc Natl Acad Sei USA 9310434–10439
    [Google Scholar]
  40. West S.E.H., Iglewski B.H. 1988; Codon usage in Pseudomonas aeruginosa. . Nucleic Acids Res 16:9323
    [Google Scholar]
  41. West S.E.H., Schweizer H.P., Dall C, Sample A.K., Runyen-Janecky L.J. 1994; Construction of improved Escherichia- Pseudomonas shuttle vectors derived from pUC18/19 and the sequence of the region required for their replication in Pseudomonas aeruginosa. . Gene 128:81–86
    [Google Scholar]
  42. Wieslander L. 1979; A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels.. Anal Biochem 98:305–309
    [Google Scholar]
  43. Xiang C, Wang H., Shiel P., Berger P., Guerra D.J. 1994; A modified alkaline lysis miniprep protocol using a single microcentrifuge tube.. BioTechniques 17:30–31
    [Google Scholar]
  44. Yanisch-Perron C, Vieira J., Messing J. 1985; Improved M13 cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors.. Gene 33:103–119
    [Google Scholar]
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