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. 1988 Jun;170(6):2725–2734. doi: 10.1128/jb.170.6.2725-2734.1988

Anabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa: nucleotide sequence and transcriptional control of the argF structural gene.

Y Itoh 1, L Soldati 1, V Stalon 1, P Falmagne 1, Y Terawaki 1, T Leisinger 1, D Haas 1
PMCID: PMC211195  PMID: 3131308

Abstract

In Pseudomonas aeruginosa PAO the anabolic ornithine carbamoyltransferase (OTCase, EC 2.1.3.3) is the product of the argF gene and the only arginine biosynthetic enzyme whose synthesis is repressible by arginine. We have determined the complete nucleotide sequence of the argF gene including its promoter-control region. The deduced amino acid sequence of the anabolic OTCase consists of 305 residues (Mr 33,924), and this was confirmed by the N-terminal amino acid sequence, the total amino acid composition, and the subunit Mr of the purified enzyme. The native anabolic OTCase (Mr 110,000 to 125,000) was found to be a trimer by cross-linking experiments. P. aeruginosa also has a catabolic OTCase (the arcB gene product), which catalyzes the reverse reaction of the anabolic conversion. At the nucleotide sequence level, the P. aeruginosa argF gene had 52.4% identity with the arcB gene. The Escherichia coli argF and argI genes, which code for anabolic OTCase isoenzymes, had 47.3 and 44.9% identity, respectively, with the P. aeruginosa argF sequence. This suggests that these four genes have evolved from a common ancestral gene. The arcB gene appears to be more closely related to the E. coli argF gene than to the P. aeruginosa argF gene. Two transcripts (mRNA-1, mRNA-2) of the P. aeruginosa argF gene were identified by S1 mapping. The transcription initiation site for mRNA-1 was preceded by sequences having partial homology with the E. coli -35 and -10 consensus promoter sequences. No sequence similar to consensus promoters of enteric bacteria was found upstream of the 5' end of mRNA-2. E. coli carrying a P. aeruginosa argF+ recombinant plasmid produced mRNA-1 with low efficiency but no (or very little) mRNA-2. Arginine repressed argF transcription in P. aeruginosa. In the argF promoter region no sequence homologous to the "arg box" (arginine operator module) of E. coli was found. The mechanism of arginine repression in P. aeruginosa thus appears to be different from that in E. coli.

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Selected References

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  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. Baur H., Stalon V., Falmagne P., Luethi E., Haas D. Primary and quaternary structure of the catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa. Extensive sequence homology with the anabolic ornithine carbamoyltransferases of Escherichia coli. Eur J Biochem. 1987 Jul 1;166(1):111–117. doi: 10.1111/j.1432-1033.1987.tb13489.x. [DOI] [PubMed] [Google Scholar]
  3. Bencini D. A., Houghton J. E., Hoover T. A., Foltermann K. F., Wild J. R., O'Donovan G. A. The DNA sequence of argI from Escherichia coli K12. Nucleic Acids Res. 1983 Dec 10;11(23):8509–8518. doi: 10.1093/nar/11.23.8509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Chattoraj D. K., Snyder K. M., Abeles A. L. P1 plasmid replication: multiple functions of RepA protein at the origin. Proc Natl Acad Sci U S A. 1985 May;82(9):2588–2592. doi: 10.1073/pnas.82.9.2588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clarke P. H., Laverack P. D. Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli. J Bacteriol. 1983 Apr;154(1):508–512. doi: 10.1128/jb.154.1.508-512.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cunin R., Eckhardt T., Piette J., Boyen A., Piérard A., Glansdorff N. Molecular basis for modulated regulation of gene expression in the arginine regulon of Escherichia coli K-12. Nucleic Acids Res. 1983 Aug 11;11(15):5007–5019. doi: 10.1093/nar/11.15.5007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cunin R., Glansdorff N., Piérard A., Stalon V. Biosynthesis and metabolism of arginine in bacteria. Microbiol Rev. 1986 Sep;50(3):314–352. doi: 10.1128/mr.50.3.314-352.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dixon R. The xylABC promoter from the Pseudomonas putida TOL plasmid is activated by nitrogen regulatory genes in Escherichia coli. Mol Gen Genet. 1986 Apr;203(1):129–136. doi: 10.1007/BF00330393. [DOI] [PubMed] [Google Scholar]
  10. Falmagne P., Portetelle D., Stalon V. Immunological and structural relatedness of catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria. J Bacteriol. 1985 Feb;161(2):714–719. doi: 10.1128/jb.161.2.714-719.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grant C. C., Vasil M. L. Analysis of transcription of the exotoxin A gene of Pseudomonas aeruginosa. J Bacteriol. 1986 Dec;168(3):1112–1119. doi: 10.1128/jb.168.3.1112-1119.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haas D., Holloway B. W., Schamböck A., Leisinger T. The genetic organization of arginine biosynthesis in Pseudomonas aeruginosa. Mol Gen Genet. 1977 Jul 7;154(1):7–22. doi: 10.1007/BF00265571. [DOI] [PubMed] [Google Scholar]
  13. Haas D., Jann A., Reimmann C., Lüthi E., Leisinger T. Chromosome organization in Pseudomonas aeruginosa. Clustering and scattering of genes specifying four arginine catabolic pathways. Antibiot Chemother (1971) 1987;39:256–263. [PubMed] [Google Scholar]
  14. Haas D., Leisinger T. Multiple control of N-acetylglutamate synthetase from Pseudomonas aeruginosa: synergistic inhibition by acetylglutamate and polyamines. Biochem Biophys Res Commun. 1974 Sep 9;60(1):42–47. doi: 10.1016/0006-291x(74)90169-7. [DOI] [PubMed] [Google Scholar]
  15. Haas D., Leisinger T. N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Catalytic and regulatory properties. Eur J Biochem. 1975 Mar 17;52(2):377–393. [PubMed] [Google Scholar]
  16. Hass D., Evans R., Mercenier A., Simon J. P., Stalon V. Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase. J Bacteriol. 1979 Sep;139(3):713–720. doi: 10.1128/jb.139.3.713-720.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  18. Honzatko R. B., Lipscomb W. N. Interactions of phosphate ligands with Escherichia coli aspartate carbamoyltransferase in the crystalline state. J Mol Biol. 1982 Sep 15;160(2):265–286. doi: 10.1016/0022-2836(82)90176-0. [DOI] [PubMed] [Google Scholar]
  19. Houghton J. E., Bencini D. A., O'Donovan G. A., Wild J. R. Protein differentiation: a comparison of aspartate transcarbamoylase and ornithine transcarbamoylase from Escherichia coli K-12. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4864–4868. doi: 10.1073/pnas.81.15.4864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Inouye S., Asai Y., Nakazawa A., Nakazawa T. Nucleotide sequence of a DNA segment promoting transcription in Pseudomonas putida. J Bacteriol. 1986 Jun;166(3):739–745. doi: 10.1128/jb.166.3.739-745.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Isaac J. H., Holloway B. W. Control of arginine biosynthesis in Pseudomonas aeruginosa. J Gen Microbiol. 1972 Dec;73(3):427–438. doi: 10.1099/00221287-73-3-427. [DOI] [PubMed] [Google Scholar]
  22. Itoh Y., Haas D. Cloning vectors derived from the Pseudomonas plasmid pVS1. Gene. 1985;36(1-2):27–36. doi: 10.1016/0378-1119(85)90066-6. [DOI] [PubMed] [Google Scholar]
  23. Itoh Y., Watson J. M., Haas D., Leisinger T. Genetic and molecular characterization of the Pseudomonas plasmid pVS1. Plasmid. 1984 May;11(3):206–220. doi: 10.1016/0147-619x(84)90027-1. [DOI] [PubMed] [Google Scholar]
  24. Jann A., Stalon V., Wauven C. V., Leisinger T., Haas D. N-Succinylated intermediates in an arginine catabolic pathway of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4937–4941. doi: 10.1073/pnas.83.13.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jeenes D. J., Soldati L., Baur H., Watson J. M., Mercenier A., Reimmann C., Leisinger T., Haas D. Expression of biosynthetic genes from Pseudomonas aeruginosa and Escherichia coli in the heterologous host. Mol Gen Genet. 1986 Jun;203(3):421–429. doi: 10.1007/BF00422066. [DOI] [PubMed] [Google Scholar]
  26. Johnson K., Parker M. L., Lory S. Nucleotide sequence and transcriptional initiation site of two Pseudomonas aeruginosa pilin genes. J Biol Chem. 1986 Nov 25;261(33):15703–15708. [PubMed] [Google Scholar]
  27. Legrain C., Stalon V., Noullez J. P., Mercenier A., Simon J. P., Broman K., Wiame J. M. Structure and function of ornithine carbamoyltransferases. Eur J Biochem. 1977 Nov 1;80(2):401–409. doi: 10.1111/j.1432-1033.1977.tb11895.x. [DOI] [PubMed] [Google Scholar]
  28. Legrain C., Stalon V. Ornithine carbamoyltransferase from Escherichia coli W. Purification, structure and steady-state kinetic analysis. Eur J Biochem. 1976 Mar 16;63(1):289–301. doi: 10.1111/j.1432-1033.1976.tb10230.x. [DOI] [PubMed] [Google Scholar]
  29. Lerner C. G., Switzer R. L. Cloning and structure of the Bacillus subtilis aspartate transcarbamylase gene (pyrB). J Biol Chem. 1986 Aug 25;261(24):11156–11165. [PubMed] [Google Scholar]
  30. Lusty C. J., Jilka R. L., Nietsch E. H. Ornithine transcarbamylase of rat liver. Kinetic, physical, and chemical properties. J Biol Chem. 1979 Oct 25;254(20):10030–10036. [PubMed] [Google Scholar]
  31. Lüthi E., Mercenier A., Haas D. The arcABC operon required for fermentative growth of Pseudomonas aeruginosa on arginine: Tn5-751-assisted cloning and localization of structural genes. J Gen Microbiol. 1986 Oct;132(10):2667–2675. doi: 10.1099/00221287-132-10-2667. [DOI] [PubMed] [Google Scholar]
  32. Marshall M., Cohen P. P. Ornithine transcarbamylases. Ordering of S-cyano peptides and location of characteristically reactive cysteinyl residues within the sequence. J Biol Chem. 1980 Aug 10;255(15):7287–7290. [PubMed] [Google Scholar]
  33. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  34. McClure W. R. Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem. 1985;54:171–204. doi: 10.1146/annurev.bi.54.070185.001131. [DOI] [PubMed] [Google Scholar]
  35. Minton N. P., Clarke L. E. Identification of the promoter of the Pseudomonas gene coding for carboxypeptidase G2. J Mol Appl Genet. 1985;3(1):26–35. [PubMed] [Google Scholar]
  36. Mizusawa S., Nishimura S., Seela F. Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP. Nucleic Acids Res. 1986 Feb 11;14(3):1319–1324. doi: 10.1093/nar/14.3.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rella M., Mercenier A., Haas D. Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster. Gene. 1985;33(3):293–303. doi: 10.1016/0378-1119(85)90237-9. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Upshall A., Gilbert T., Saari G., O'Hara P. J., Weglenski P., Berse B., Miller K., Timberlake W. E. Molecular analysis of the argB gene of Aspergillus nidulans. Mol Gen Genet. 1986 Aug;204(2):349–354. doi: 10.1007/BF00425521. [DOI] [PubMed] [Google Scholar]
  41. Van Vliet F., Cunin R., Jacobs A., Piette J., Gigot D., Lauwereys M., Piérard A., Glansdorff N. Evolutionary divergence of genes for ornithine and aspartate carbamoyl-transferases--complete sequence and mode of regulation of the Escherichia coli argF gene; comparison of argF with argI and pyrB. Nucleic Acids Res. 1984 Aug 10;12(15):6277–6289. doi: 10.1093/nar/12.15.6277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vander Wauven C., Piérard A., Kley-Raymann M., Haas D. Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway. J Bacteriol. 1984 Dec;160(3):928–934. doi: 10.1128/jb.160.3.928-934.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Voellmy R., Leisinger T. Regulation of enzyme synthesis in the arginine biosynthetic pathway of Pseudomonas aeruginosa. J Gen Microbiol. 1978 Nov;109(1):25–35. doi: 10.1099/00221287-109-1-25. [DOI] [PubMed] [Google Scholar]
  44. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]
  45. Wozniak D. J., Cram D. C., Daniels C. J., Galloway D. R. Nucleotide sequence and characterization of toxR: a gene involved in exotoxin A regulation in Pseudomonas aeruginosa. Nucleic Acids Res. 1987 Mar 11;15(5):2123–2135. doi: 10.1093/nar/15.5.2123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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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