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. 1991 Mar;173(6):1855–1861. doi: 10.1128/jb.173.6.1855-1861.1991

Isolation of the braZ gene encoding the carrier for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO.

T Hoshino 1, K Kose-Terai 1, Y Uratani 1
PMCID: PMC207713  PMID: 1900503

Abstract

The braZ gene for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO was isolated and characterized. Determination of the nucleotide sequence showed that the braZ gene comprises 1,311 nucleotides specifying a protein of 437 amino acids. Hydropathy analysis suggested that the product is an integral membrane protein with 12 membrane-spanning segments. The amino acid sequence showed extensive homology to those of the braB and brnQ gene products, branched-chain amino acid carriers of P. aeruginosa and Salmonella typhimurium, respectively. By using the T7 RNA polymerase-promoter system, the braZ gene product was identified as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Properties of the transport system encoded by braZ were studied by using P. aeruginosa PAO3537, defective in both the high- and low-affinity branched-chain amino acid transport systems (LIV-I and LIV-II, respectively). The transport system encoded by braZ was found to be another effective branched-chain amino acid transport system in P. aeruginosa PAO and was thus designated as LIV-III. This system is specific for isoleucine and valine, giving the same Km value of 12 microM for these amino acids. The system was found, however, to have a very low affinity for leucine, with a Km value of 150 microM, which contrasts with the substrate specificities of LIV-I and LIV-II.

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Selected References

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

  1. Bagdasarian M. M., Amann E., Lurz R., Rückert B., Bagdasarian M. Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors. Gene. 1983 Dec;26(2-3):273–282. doi: 10.1016/0378-1119(83)90197-x. [DOI] [PubMed] [Google Scholar]
  2. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  3. Büchel D. E., Gronenborn B., Müller-Hill B. Sequence of the lactose permease gene. Nature. 1980 Feb 7;283(5747):541–545. doi: 10.1038/283541a0. [DOI] [PubMed] [Google Scholar]
  4. Frantz B., Ngai K. L., Chatterjee D. K., Ornston L. N., Chakrabarty A. M. Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13. J Bacteriol. 1987 Feb;169(2):704–709. doi: 10.1128/jb.169.2.704-709.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Frey J., Bagdasarian M., Feiss D., Franklin F. C., Deshusses J. Stable cosmid vectors that enable the introduction of cloned fragments into a wide range of gram-negative bacteria. Gene. 1983 Oct;24(2-3):299–308. doi: 10.1016/0378-1119(83)90090-2. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Hoshino T., Kose K. Cloning and nucleotide sequence of braC, the structural gene for the leucine-, isoleucine-, and valine-binding protein of Pseudomonas aeruginosa PAO. J Bacteriol. 1989 Nov;171(11):6300–6306. doi: 10.1128/jb.171.11.6300-6306.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hoshino T., Kose K. Cloning, nucleotide sequences, and identification of products of the Pseudomonas aeruginosa PAO bra genes, which encode the high-affinity branched-chain amino acid transport system. J Bacteriol. 1990 Oct;172(10):5531–5539. doi: 10.1128/jb.172.10.5531-5539.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hoshino T., Kose K., Uratani Y. Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO. Mol Gen Genet. 1990 Feb;220(3):461–467. doi: 10.1007/BF00391754. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Hoshino T., Tsuda M., Iino T., Nishio K., Kageyama M. Genetic mapping of bra genes affecting branched-chain amino acid transport in Pseudomonas aeruginosa. J Bacteriol. 1983 Mar;153(3):1272–1281. doi: 10.1128/jb.153.3.1272-1281.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Miyata T., Miyazawa S., Yasunaga T. Two types of amino acid substitutions in protein evolution. J Mol Evol. 1979 Mar 15;12(3):219–236. doi: 10.1007/BF01732340. [DOI] [PubMed] [Google Scholar]
  16. Nakao T., Yamato I., Anraku Y. Nucleotide sequence of putP, the proline carrier gene of Escherichia coli K12. Mol Gen Genet. 1987 Jun;208(1-2):70–75. doi: 10.1007/BF00330424. [DOI] [PubMed] [Google Scholar]
  17. Ohnishi K., Hasegawa A., Matsubara K., Date T., Okada T., Kiritani K. Cloning and nucleotide sequence of the brnQ gene, the structural gene for a membrane-associated component of the LIV-II transport system for branched-chain amino acids in Salmonella typhimurium. Jpn J Genet. 1988 Aug;63(4):343–357. doi: 10.1266/jjg.63.343. [DOI] [PubMed] [Google Scholar]
  18. Perlman D., Halvorson H. O. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J Mol Biol. 1983 Jun 25;167(2):391–409. doi: 10.1016/s0022-2836(83)80341-6. [DOI] [PubMed] [Google Scholar]
  19. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  20. Russel M., Model P. Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J Bacteriol. 1984 Sep;159(3):1034–1039. doi: 10.1128/jb.159.3.1034-1039.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Shine J., Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. doi: 10.1038/254034a0. [DOI] [PubMed] [Google Scholar]
  23. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  25. Tsuda M., Iino T. Ordering of the flagellar genes in Pseudomonas aeruginosa by insertions of mercury transposon Tn501. J Bacteriol. 1983 Feb;153(2):1008–1017. doi: 10.1128/jb.153.2.1008-1017.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Uratani Y., Hoshino T. Difference in sodium requirement of branched chain amino acid carrier between Pseudomonas aeruginosa PAO and PML strains is due to substitution of an amino acid at position 292. J Biol Chem. 1989 Nov 15;264(32):18944–18950. [PubMed] [Google Scholar]
  27. Uratani Y., Tsuchiya T., Akamatsu Y., Hoshino T. Na+(Li+)/branched-chain amino acid cotransport in Pseudomonas aeruginosa. J Membr Biol. 1989 Jan;107(1):57–62. doi: 10.1007/BF01871083. [DOI] [PubMed] [Google Scholar]
  28. Yamato I., Anraku Y. Transport of sugars and amino acids in bacteria. XVIII. Properties of an isoleucine carrier in the cytoplasmic membrane vesicles of Escherichia coli. J Biochem. 1977 May;81(5):1517–1523. [PubMed] [Google Scholar]
  29. Yamato I., Ohki M., Anraku Y. Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli. J Bacteriol. 1979 Apr;138(1):24–32. doi: 10.1128/jb.138.1.24-32.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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