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. 1997 Sep;179(17):5300–5308. doi: 10.1128/jb.179.17.5300-5308.1997

Cloning and characterization of argR, a gene that participates in regulation of arginine biosynthesis and catabolism in Pseudomonas aeruginosa PAO1.

S M Park 1, C D Lu 1, A T Abdelal 1
PMCID: PMC179396  PMID: 9286980

Abstract

Gel retardation experiments indicated the presence in Pseudomonas aeruginosa cell extracts of an arginine-inducible DNA-binding protein that interacts with the control regions for the car and argF operons, encoding carbamoylphosphate synthetase and anabolic ornithine carbamoyltransferase, respectively. Both enzymes are required for arginine biosynthesis. The use of a combination of transposon mutagenesis and arginine hydroxamate selection led to the isolation of a regulatory mutant that was impaired in the formation of the DNA-binding protein and in which the expression of an argF::lacZ fusion was not controlled by arginine. Experiments with various subclones led to the conclusion that the insertion affected the expression of an arginine regulatory gene, argR, that encodes a polypeptide with significant homology to the AraC/XylS family of regulatory proteins. Determination of the nucleotide sequence of the flanking regions showed that argR is the sixth and terminal gene of an operon for transport of arginine. The argR gene was inactivated by gene replacement, using a gentamicin cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes encoded by the car and argF operons. Furthermore, argR inactivation abolished the induction of several enzymes of the arginine succinyltransferase pathway, which is considered the major route for arginine catabolism under aerobic conditions. Consistent with this finding and unlike the parent strain, the argR::Gm derivative was unable to utilize arginine or ornithine as the sole carbon source. The combined data indicate a major role for ArgR in the control of arginine biosynthesis and aerobic catabolism.

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Selected References

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  1. Abdelal A. T., Bussey L., Vickers L. Carbamoylphosphate synthetase from Pseudomonas aeruginosa. Subunit composition, kinetic analysis and regulation. Eur J Biochem. 1983 Jan 1;129(3):697–702. [PubMed] [Google Scholar]
  2. Abdelal A. T., Ingraham J. L. Carbamylphosphate synthetase from Salmonella typhimurium. Regulations, subunit composition, and function of the subunits. J Biol Chem. 1975 Jun 25;250(12):4410–4417. [PubMed] [Google Scholar]
  3. Auerswald E. A., Ludwig G., Schaller H. Structural analysis of Tn5. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):107–113. doi: 10.1101/sqb.1981.045.01.019. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Brennan R. G., Matthews B. W. Structural basis of DNA-protein recognition. Trends Biochem Sci. 1989 Jul;14(7):286–290. doi: 10.1016/0968-0004(89)90066-2. [DOI] [PubMed] [Google Scholar]
  6. Calogero S., Gardan R., Glaser P., Schweizer J., Rapoport G., Debarbouille M. RocR, a novel regulatory protein controlling arginine utilization in Bacillus subtilis, belongs to the NtrC/NifA family of transcriptional activators. J Bacteriol. 1994 Mar;176(5):1234–1241. doi: 10.1128/jb.176.5.1234-1241.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chung C. T., Niemela S. L., Miller R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2172–2175. doi: 10.1073/pnas.86.7.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Craven R., Montie T. C. Regulation of Pseudomonas aeruginosa chemotaxis by the nitrogen source. J Bacteriol. 1985 Nov;164(2):544–549. doi: 10.1128/jb.164.2.544-549.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Czaplewski L. G., North A. K., Smith M. C., Baumberg S., Stockley P. G. Purification and initial characterization of AhrC: the regulator of arginine metabolism genes in Bacillus subtilis. Mol Microbiol. 1992 Jan;6(2):267–275. doi: 10.1111/j.1365-2958.1992.tb02008.x. [DOI] [PubMed] [Google Scholar]
  11. Farinha M. A., Kropinski A. M. Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters. J Bacteriol. 1990 Jun;172(6):3496–3499. doi: 10.1128/jb.172.6.3496-3499.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gallegos M. T., Michán C., Ramos J. L. The XylS/AraC family of regulators. Nucleic Acids Res. 1993 Feb 25;21(4):807–810. doi: 10.1093/nar/21.4.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gambello M. J., Iglewski B. H. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J Bacteriol. 1991 May;173(9):3000–3009. doi: 10.1128/jb.173.9.3000-3009.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gamper M., Zimmermann A., Haas D. Anaerobic regulation of transcription initiation in the arcDABC operon of Pseudomonas aeruginosa. J Bacteriol. 1991 Aug;173(15):4742–4750. doi: 10.1128/jb.173.15.4742-4750.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Heimberg H., Boyen A., Crabeel M., Glansdorff N. Escherichia coli and Saccharomyces cerevisiae acetylornithine aminotransferase: evolutionary relationship with ornithine aminotransferase. Gene. 1990 May 31;90(1):69–78. doi: 10.1016/0378-1119(90)90440-3. [DOI] [PubMed] [Google Scholar]
  17. Holloway B. W., Römling U., Tümmler B. Genomic mapping of Pseudomonas aeruginosa PAO. Microbiology. 1994 Nov;140(Pt 11):2907–2929. doi: 10.1099/13500872-140-11-2907. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Itoh Y., Matsumoto H. Mutations affecting regulation of the anabolic argF and the catabolic aru genes in Pseudomonas aeruginosa PAO. Mol Gen Genet. 1992 Feb;231(3):417–425. doi: 10.1007/BF00292711. [DOI] [PubMed] [Google Scholar]
  20. Itoh Y., Soldati L., Stalon V., Falmagne P., Terawaki Y., Leisinger T., Haas D. Anabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa: nucleotide sequence and transcriptional control of the argF structural gene. J Bacteriol. 1988 Jun;170(6):2725–2734. doi: 10.1128/jb.170.6.2725-2734.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jann A., Matsumoto H., Haas D. The fourth arginine catabolic pathway of Pseudomonas aeruginosa. J Gen Microbiol. 1988 Apr;134(4):1043–1053. doi: 10.1099/00221287-134-4-1043. [DOI] [PubMed] [Google Scholar]
  22. Joannou C. L., Brown P. R. NAD-dependent glutamate dehydrogenase from Pseudomonas aeruginosa is a membrane-bound enzyme. FEMS Microbiol Lett. 1992 Jan 1;69(2):205–209. doi: 10.1016/0378-1097(92)90630-7. [DOI] [PubMed] [Google Scholar]
  23. Klingel U., Miller C. M., North A. K., Stockley P. G., Baumberg S. A binding site for activation by the Bacillus subtilis AhrC protein, a repressor/activator of arginine metabolism. Mol Gen Genet. 1995 Aug 21;248(3):329–340. doi: 10.1007/BF02191600. [DOI] [PubMed] [Google Scholar]
  24. Kwon D. H., Lu C. D., Walthall D. A., Brown T. M., Houghton J. E., Abdelal A. T. Structure and regulation of the carAB operon in Pseudomonas aeruginosa and Pseudomonas stutzeri: no untranslated region exists. J Bacteriol. 1994 May;176(9):2532–2542. doi: 10.1128/jb.176.9.2532-2542.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Leisinger T., O'Sullivan C., Haas D. Arginine analogues: effect on growth and on the first two enzymes of the arginine pathway in Pseudomonas aeruginosa. J Gen Microbiol. 1974 Oct;84(2):253–260. doi: 10.1099/00221287-84-2-253. [DOI] [PubMed] [Google Scholar]
  26. Lu C. D., Houghton J. E., Abdelal A. T. Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator. J Mol Biol. 1992 May 5;225(1):11–24. doi: 10.1016/0022-2836(92)91022-h. [DOI] [PubMed] [Google Scholar]
  27. Lu C. D., Kwon D. H., Abdelal A. T. Identification of greA encoding a transcriptional elongation factor as a member of the carA-orf-carB-greA operon in Pseudomonas aeruginosa PAO1. J Bacteriol. 1997 May;179(9):3043–3046. doi: 10.1128/jb.179.9.3043-3046.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Maas W. K. The arginine repressor of Escherichia coli. Microbiol Rev. 1994 Dec;58(4):631–640. doi: 10.1128/mr.58.4.631-640.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mountain A., Baumberg S. Map locations of some mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168. Mol Gen Genet. 1980;178(3):691–701. doi: 10.1007/BF00337880. [DOI] [PubMed] [Google Scholar]
  30. Park S. M., Lu C. D., Abdelal A. T. Purification and characterization of an arginine regulatory protein, ArgR, from Pseudomonas aeruginosa and its interactions with the control regions for the car, argF, and aru operons. J Bacteriol. 1997 Sep;179(17):5309–5317. doi: 10.1128/jb.179.17.5309-5317.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Prescott L. M., Jones M. E. Modified methods for the determination of carbamyl aspartate. Anal Biochem. 1969 Dec;32(3):408–419. doi: 10.1016/s0003-2697(69)80008-4. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Schweizer H. D. Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. Biotechniques. 1993 Nov;15(5):831–834. [PubMed] [Google Scholar]
  34. Smith M. C., Mountain A., Baumberg S. Cloning in Escherichia coli of a Bacillus subtilis arginine repressor gene through its ability to confer structural stability on a fragment carrying genes of arginine biosynthesis. Mol Gen Genet. 1986 Oct;205(1):176–182. doi: 10.1007/BF02428049. [DOI] [PubMed] [Google Scholar]
  35. Stibitz S., Black W., Falkow S. The construction of a cloning vector designed for gene replacement in Bordetella pertussis. Gene. 1986;50(1-3):133–140. doi: 10.1016/0378-1119(86)90318-5. [DOI] [PubMed] [Google Scholar]
  36. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  37. Vander Wauven C., Jann A., Haas D., Leisinger T., Stalon V. N2-succinylornithine in ornithine catabolism of Pseudomonas aeruginosa. Arch Microbiol. 1988;150(4):400–404. doi: 10.1007/BF00408314. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Vander Wauven C., Stalon V. Occurrence of succinyl derivatives in the catabolism of arginine in Pseudomonas cepacia. J Bacteriol. 1985 Nov;164(2):882–886. doi: 10.1128/jb.164.2.882-886.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  41. Voellmy R., Leisinger T. Dual role for N-2-acetylornithine 5-aminotransferase from Pseudomonas aeruginosa in arginine biosynthesis and arginine catabolism. J Bacteriol. 1975 Jun;122(3):799–809. doi: 10.1128/jb.122.3.799-809.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Winteler H. V., Haas D. The homologous regulators ANR of Pseudomonas aeruginosa and FNR of Escherichia coli have overlapping but distinct specificities for anaerobically inducible promoters. Microbiology. 1996 Mar;142(Pt 3):685–693. doi: 10.1099/13500872-142-3-685. [DOI] [PubMed] [Google Scholar]

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