Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1982 Aug;151(2):729–736. doi: 10.1128/jb.151.2.729-736.1982

Isolation and characterization of a Pseudomonas aeruginosa PAO mutant defective in the structural gene for the LIVAT-binding protein.

T Hoshino, K Nishio
PMCID: PMC220315  PMID: 6807959

Abstract

A mutant of Pseudomonas aeruginosa PAO which has a defect in the structural gene for a binding protein for leucine, isoleucine, valine, alanine, and threonine (LIVAT-binding protein) was isolated and characterized. DL-4-azaleucine was taken up via the high-affinity branched-chain amino acid transport system (LIV-I), but not via the low affinity system (LIV-II), and then inhibited the growth of P. aeruginosa cells. This finding enabled us to select mutants defective in the LIV-I transport system alone. Among such mutants, strain PAO3530 was found to produce an altered LIVAT-binding protein. The shock fluid of this strain contained a normal level of the protein which corresponded to the wild-type LIVAT-binding protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by an immunological test. However, the shock fluid showed almost no binding activity for branched-chain amino acids, suggesting that strain PAO3530 has a defect in the structural gene for the LIVAT-binding protein. The mutation locus (bra-310) was mapped in a region between cnu-9001 and oru-325 on the chromosome of P. aeruginosa PAO by conjugation mediated by plasmid FP5 or R68.45.

Full text

PDF
736

Images in this article

Selected References

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

  1. Ames G. F., Lever J. Components of histidine transport: histidine-binding proteins and hisP protein. Proc Natl Acad Sci U S A. 1970 Aug;66(4):1096–1103. doi: 10.1073/pnas.66.4.1096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson J. J., Oxender D. L. Escherichia coli transport mutants lacking binding protein and other components of the branched-chain amino acid transport systems. J Bacteriol. 1977 Apr;130(1):384–392. doi: 10.1128/jb.130.1.384-392.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anraku Y. Transport of sugars and amino acids in bacteria. 3. Studies on the restoration of active transport. J Biol Chem. 1968 Jun 10;243(11):3128–3135. [PubMed] [Google Scholar]
  4. Anraku Y. Transport of sugars and amino acids in bacteria. I. Purification and specificity of the galactose- and leucine-binding proteins. J Biol Chem. 1968 Jun 10;243(11):3116–3122. [PubMed] [Google Scholar]
  5. Berger E. A. Different mechanisms of energy coupling for the active transport of proline and glutamine in Escherichia coli. Proc Natl Acad Sci U S A. 1973 May;70(5):1514–1518. doi: 10.1073/pnas.70.5.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bowen B., Steinberg J., Laemmli U. K., Weintraub H. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 1980 Jan 11;8(1):1–20. doi: 10.1093/nar/8.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. FARGIE B., HOLLOWAY B. W. ABSENCE OF CLUSTERING OF FUNCTIONALLY RELATED GENES IN PSEUDOMONAS AERUGINOSA. Genet Res. 1965 Jul;6:284–299. doi: 10.1017/s0016672300004158. [DOI] [PubMed] [Google Scholar]
  8. HOLLOWAY B. W., FARGIE B. Fertility factors and genetic linkage in Pseudomonas aeruginosa. J Bacteriol. 1960 Sep;80:362–368. doi: 10.1128/jb.80.3.362-367.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haas D., Holloway B. W. R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa. Mol Gen Genet. 1976 Mar 30;144(3):243–251. doi: 10.1007/BF00341722. [DOI] [PubMed] [Google Scholar]
  10. Hong J. S., Hunt A. G., Masters P. S., Lieberman M. A. Requirements of acetyl phosphate for the binding protein-dependent transport systems in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1213–1217. doi: 10.1073/pnas.76.3.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoshino T., Kageyama M. Mutational separation of transport systems for branched-chain amino acids in Pseudomonas aeruginosa. J Bacteriol. 1982 Aug;151(2):620–628. doi: 10.1128/jb.151.2.620-628.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hoshino T., Kageyama M. Purification and properties of a binding protein for branched-chain amino acids in Pseudomonas aeruginosa. J Bacteriol. 1980 Mar;141(3):1055–1063. doi: 10.1128/jb.141.3.1055-1063.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hoshino T., Kageyama M. Sodium-dependent transport of L-leucine in membrane vesicles prepared from Pseudomonas aeruginosa. J Bacteriol. 1979 Jan;137(1):73–81. doi: 10.1128/jb.137.1.73-81.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hoshino T. Transport systems for branched-chain amino acids in Pseudomonas aeruginosa. J Bacteriol. 1979 Sep;139(3):705–712. doi: 10.1128/jb.139.3.705-712.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. 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]
  17. Matsumoto H., Tazaki T. FP5 factor, an undescribed sex factor of Pseudomonas aeruginosa. Jpn J Microbiol. 1973 Sep;17(5):409–417. doi: 10.1111/j.1348-0421.1973.tb00792.x. [DOI] [PubMed] [Google Scholar]
  18. Mizuno T., Kageyama M. Separation and characterization of the outer membrane of Pseudomonas aeruginosa. J Biochem. 1978 Jul;84(1):179–191. doi: 10.1093/oxfordjournals.jbchem.a132106. [DOI] [PubMed] [Google Scholar]
  19. Neal J. L. Analysis of Michaelis kinetics for two independent, saturable membrane transport functions. J Theor Biol. 1972 Apr;35(1):113–118. doi: 10.1016/0022-5193(72)90196-8. [DOI] [PubMed] [Google Scholar]
  20. Oxender D. L., Anderson J. J., Daniels C. J., Landick R., Gunsalus R. P., Zurawski G., Selker E., Yanofsky C. Structural and functional analysis of cloned DNA containing genes responsible for branched-chain amino acid transport in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1412–1416. doi: 10.1073/pnas.77.3.1412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Oxender D. L., Anderson J. J., Daniels C. J., Landick R., Gunsalus R. P., Zurawski G., Yanofsky C. Amino-terminal sequence and processing of the precursor of the leucine-specific binding protein, and evidence for conformational differences between the precursor and the mature form. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2005–2009. doi: 10.1073/pnas.77.4.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Oxender D. L. Membrane transport. Annu Rev Biochem. 1972;41(10):777–814. doi: 10.1146/annurev.bi.41.070172.004021. [DOI] [PubMed] [Google Scholar]
  23. Pemberton J. M., Holloway B. W. Chromosome mapping in Pseudomonas aeruginosa. Genet Res. 1972 Jun;19(3):251–260. doi: 10.1017/s0016672300014518. [DOI] [PubMed] [Google Scholar]
  24. Stanisich V. A., Holloway B. W. A mutant sex factor of Pseudomonas aeruginosa. Genet Res. 1972 Feb;19(1):91–108. doi: 10.1017/s0016672300014294. [DOI] [PubMed] [Google Scholar]
  25. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES