Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Sep;174(18):5895–5909. doi: 10.1128/jb.174.18.5895-5909.1992

Origin, structure, and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression of argK in transgenic tobacco.

E Hatziloukas 1, N J Panopoulos 1
PMCID: PMC207126  PMID: 1522066

Abstract

Pseudomonas syringae pv. phaseolicola produces the tripeptide N delta(N'-sulfo-diaminophosphinyl)-ornithylalanyl-homoarginin e (phaseolotoxin), which functions as a chlorosis-inducing toxin in the bean halo blight disease by inhibiting ornithine carbamoyltransferase (OCT). The bacterium possesses duplicate OCT genes, one of which, argK, encodes a toxin-resistant enzyme (ROCT) and imparts resistance to phaseolotoxin. We sequenced the argK gene from strain NPS3121, defined its promoter region, analyzed its regulation, and characterized its transcripts. The gene probably originated from another organism, since it is very distantly related to the argF gene encoding the housekeeping toxin-sensitive OCT and has low G+C content compared with the bacterial genome as a whole and with other protein-coding genes from P. syringae pv. phaseolicola. Optimized alignments of 13 OCT sequences allowed us to define key residues that may be responsible for toxin resistance and to identify a distinct prokaryotic amino acid signature, in ROCT, which argues for a prokaryotic origin of argK. An in-frame fusion of the argK coding region with the chloroplast transit peptide segment of the pea rbcS gene was introduced in Nicotiana tabacum by Agrobacterium-mediated transformation. The presence of an ROCT activity in transgenic plants was demonstrated by in vitro and in vivo assays. Some plants were toxin resistant, suggesting that pathogen-derived resistance to the toxin should be feasible in the pathogen's host.

Full text

PDF
5895

Images in this article

5899Image
on p.5899
5902Image
on p.5902
5904Image
on p.5904

Selected References

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

  1. An G. Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells. Plant Physiol. 1986 May;81(1):86–91. doi: 10.1104/pp.81.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baur H., Tricot C., Stalon V., Haas D. Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme. J Biol Chem. 1990 Sep 5;265(25):14728–14731. [PubMed] [Google Scholar]
  3. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Brickman E., Beckwith J. Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages. J Mol Biol. 1975 Aug 5;96(2):307–316. doi: 10.1016/0022-2836(75)90350-2. [DOI] [PubMed] [Google Scholar]
  5. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cundliffe E. How antibiotic-producing organisms avoid suicide. Annu Rev Microbiol. 1989;43:207–233. doi: 10.1146/annurev.mi.43.100189.001231. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Del Sal G., Manfioletti G., Schneider C. A one-tube plasmid DNA mini-preparation suitable for sequencing. Nucleic Acids Res. 1988 Oct 25;16(20):9878–9878. doi: 10.1093/nar/16.20.9878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gavel Y., von Heijne G. A conserved cleavage-site motif in chloroplast transit peptides. FEBS Lett. 1990 Feb 26;261(2):455–458. doi: 10.1016/0014-5793(90)80614-o. [DOI] [PubMed] [Google Scholar]
  10. Glenn E. Properties and subcellular distribution of two partially purified ornithine transcarbamoylases in cell suspensions of sugarcane. Plant Physiol. 1977 Jul;60(1):122–126. doi: 10.1104/pp.60.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gouaux J. E., Lipscomb W. N. Three-dimensional structure of carbamoyl phosphate and succinate bound to aspartate carbamoyltransferase. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4205–4208. doi: 10.1073/pnas.85.12.4205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guerineau F., Woolston S., Brooks L., Mullineaux P. An expression cassette for targeting foreign proteins into chloroplasts. Nucleic Acids Res. 1988 Dec 9;16(23):11380–11380. doi: 10.1093/nar/16.23.11380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Higgins D. G., Sharp P. M. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene. 1988 Dec 15;73(1):237–244. doi: 10.1016/0378-1119(88)90330-7. [DOI] [PubMed] [Google Scholar]
  14. Horwich A. L., Fenton W. A., Williams K. R., Kalousek F., Kraus J. P., Doolittle R. F., Konigsberg W., Rosenberg L. E. Structure and expression of a complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase. Science. 1984 Jun 8;224(4653):1068–1074. doi: 10.1126/science.6372096. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Huygen R., Crabeel M., Glansdorff N. Nucleotide sequence of the ARG3 gene of the yeast Saccharomyces cerevisiae encoding ornithine carbamoyltransferase. Comparison with other carbamoyltransferases. Eur J Biochem. 1987 Jul 15;166(2):371–377. doi: 10.1111/j.1432-1033.1987.tb13525.x. [DOI] [PubMed] [Google Scholar]
  17. Jahn O., Sauerstein J., Reuter G. Characterization of two ornithine carbamoyltransferases from Pseudomonas syringae pv. phaseolicola, the producer of phaseolotoxin. Arch Microbiol. 1987 Mar;147(2):174–178. doi: 10.1007/BF00415280. [DOI] [PubMed] [Google Scholar]
  18. Jahn O., Sauerstein J., Reuter G. Detection of two ornithine carbamoyltransferases in a phaseolotoxin-producing strain Pseudomonas syringae pv. phaseolicola. J Basic Microbiol. 1985;25(8):543–546. doi: 10.1002/jobm.3620250821. [DOI] [PubMed] [Google Scholar]
  19. KING E. O., WARD M. K., RANEY D. E. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med. 1954 Aug;44(2):301–307. [PubMed] [Google Scholar]
  20. Kantrowitz E. R., Lipscomb W. N. Escherichia coli aspartate transcarbamylase: the relation between structure and function. Science. 1988 Aug 5;241(4866):669–674. doi: 10.1126/science.3041592. [DOI] [PubMed] [Google Scholar]
  21. Ke H. M., Honzatko R. B., Lipscomb W. N. Structure of unligated aspartate carbamoyltransferase of Escherichia coli at 2.6-A resolution. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4037–4040. doi: 10.1073/pnas.81.13.4037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kinney D. M., Lusty C. J. Arginine restriction induced by delta-N-(phosphonacetyl)-L-ornithine signals increased expression of HIS3, TRP5, CPA1, and CPA2 in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Nov;9(11):4882–4888. doi: 10.1128/mcb.9.11.4882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Krause K. L., Volz K. W., Lipscomb W. N. Structure at 2.9-A resolution of aspartate carbamoyltransferase complexed with the bisubstrate analogue N-(phosphonacetyl)-L-aspartate. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1643–1647. doi: 10.1073/pnas.82.6.1643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kuo L. C., Caron C., Lee S., Herzberg W. Zn2+ regulation of ornithine transcarbamoylase. II. Metal binding site. J Mol Biol. 1990 Jan 5;211(1):271–280. doi: 10.1016/0022-2836(90)90026-I. [DOI] [PubMed] [Google Scholar]
  25. Kuo L. C., Herzberg W., Lipscomb W. N. Substrate specificity and protonation state of ornithine transcarbamoylase as determined by pH studies. Biochemistry. 1985 Aug 27;24(18):4754–4761. doi: 10.1021/bi00339a007. [DOI] [PubMed] [Google Scholar]
  26. Kuo L. C., Miller A. W., Lee S., Kozuma C. Site-directed mutagenesis of Escherichia coli ornithine transcarbamoylase: role of arginine-57 in substrate binding and catalysis. Biochemistry. 1988 Nov 29;27(24):8823–8832. doi: 10.1021/bi00424a021. [DOI] [PubMed] [Google Scholar]
  27. Kuo L. C., Seaton B. A. X-ray diffraction analysis on single crystals of recombinant Escherichia coli ornithine transcarbamoylase. J Biol Chem. 1989 Sep 25;264(27):16246–16248. [PubMed] [Google Scholar]
  28. 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]
  29. Major J. G., Jr, Wales M. E., Houghton J. E., Maley J. A., Davidson J. N., Wild J. R. Molecular evolution of enzyme structure: construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase. J Mol Evol. 1989 May;28(5):442–450. doi: 10.1007/BF02603079. [DOI] [PubMed] [Google Scholar]
  30. Martin P. R., Cooperider J. W., Mulks M. H. Sequence of the argF gene encoding ornithine transcarbamoylase from Neisseria gonorrhoeae. Gene. 1990 Sep 28;94(1):139–140. doi: 10.1016/0378-1119(90)90482-7. [DOI] [PubMed] [Google Scholar]
  31. Melchers L. S., Regensburg-Tuïnk T. J., Bourret R. B., Sedee N. J., Schilperoort R. A., Hooykaas P. J. Membrane topology and functional analysis of the sensory protein VirA of Agrobacterium tumefaciens. EMBO J. 1989 Jul;8(7):1919–1925. doi: 10.1002/j.1460-2075.1989.tb03595.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mosqueda G., Van den Broeck G., Saucedo O., Bailey A. M., Alvarez-Morales A., Herrera-Estrella L. Isolation and characterization of the gene from Pseudomonas syringae pv. phaseolicola encoding the phaseolotoxin-insensitive ornithine carbamoyltransferase. Mol Gen Genet. 1990 Jul;222(2-3):461–466. doi: 10.1007/BF00633857. [DOI] [PubMed] [Google Scholar]
  33. Mountain A., Smith M. C., Baumberg S. Nucleotide sequence of the Bacillus subtilis argF gene encoding ornithine carbamoyltransferase. Nucleic Acids Res. 1990 Aug 11;18(15):4594–4594. doi: 10.1093/nar/18.15.4594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Muto A., Osawa S. The guanine and cytosine content of genomic DNA and bacterial evolution. Proc Natl Acad Sci U S A. 1987 Jan;84(1):166–169. doi: 10.1073/pnas.84.1.166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pastra-Landis S. C., Foote J., Kantrowitz E. R. An improved colorimetric assay for aspartate and ornithine transcarbamylases. Anal Biochem. 1981 Dec;118(2):358–363. doi: 10.1016/0003-2697(81)90594-7. [DOI] [PubMed] [Google Scholar]
  36. Peet R. C., Lindgren P. B., Willis D. K., Panopoulos N. J. Identification and cloning of genes involved in phaseolotoxin production by Pseudomonas syringae pv. "phaseolicola". J Bacteriol. 1986 Jun;166(3):1096–1105. doi: 10.1128/jb.166.3.1096-1105.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Peet R. C., Panopoulos N. J. Ornithine carbamoyltransferase genes and phaseolotoxin immunity in Pseudomonas syringae pv. phaseolicola. EMBO J. 1987 Dec 1;6(12):3585–3591. doi: 10.1002/j.1460-2075.1987.tb02689.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sanger F., Coulson A. R. The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 1978 Mar 1;87(1):107–110. doi: 10.1016/0014-5793(78)80145-8. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Savioz A., Jeenes D. J., Kocher H. P., Haas D. Comparison of proC and other housekeeping genes of Pseudomonas aeruginosa with their counterparts in Escherichia coli. Gene. 1990 Jan 31;86(1):107–111. doi: 10.1016/0378-1119(90)90121-7. [DOI] [PubMed] [Google Scholar]
  41. Scherer S. E., Veres G., Caskey C. T. The genetic structure of mouse ornithine transcarbamylase. Nucleic Acids Res. 1988 Feb 25;16(4):1593–1601. doi: 10.1093/nar/16.4.1593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schneider K., Beck C. F. New expression vectors for identifying and testing signal structures for initiation and termination of transcription. Methods Enzymol. 1987;153:452–461. doi: 10.1016/0076-6879(87)53071-3. [DOI] [PubMed] [Google Scholar]
  43. Slocum R. D., Richardson D. P. Purification and characterization of ornithine transcarbamylase from pea (Pisum sativum L.). Plant Physiol. 1991;96:262–268. doi: 10.1104/pp.96.1.262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Staskawicz B. J., Panopoulos N. J., Hoogenraad N. J. Phaseolotoxin-insensitive ornithine carbamoyltransferase of Pseudomonas syringae pv. phaseolicola: basis for immunity to phaseolotoxin. J Bacteriol. 1980 May;142(2):720–723. doi: 10.1128/jb.142.2.720-723.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Staskawicz B. J., Panopoulos N. J. Phaseolotoxin transport in Escherichia coli and Salmonella typhimurium via the oligopeptide permease. J Bacteriol. 1980 May;142(2):474–479. doi: 10.1128/jb.142.2.474-479.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stebbins J. W., Xu W., Kantrowitz E. R. Three residues involved in binding and catalysis in the carbamyl phosphate binding site of Escherichia coli aspartate transcarbamylase. Biochemistry. 1989 Mar 21;28(6):2592–2600. doi: 10.1021/bi00432a037. [DOI] [PubMed] [Google Scholar]
  47. Takiguchi M., Miura S., Mori M., Tatibana M., Nagata S., Kaziro Y. Molecular cloning and nucleotide sequence of cDNA for rat ornithine carbamoyltransferase precursor. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7412–7416. doi: 10.1073/pnas.81.23.7412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Templeton M. D., Mitchell R. E., Sullivan P. A., Shepherd M. G. The inactivation of ornithine transcarbamoylase by N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine. Biochem J. 1985 Jun 1;228(2):347–352. doi: 10.1042/bj2280347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tricot C., De Coen J. L., Momin P., Falmagne P., Stalon V. Evolutionary relationships among bacterial carbamoyltransferases. J Gen Microbiol. 1989 Sep;135(9):2453–2464. doi: 10.1099/00221287-135-9-2453. [DOI] [PubMed] [Google Scholar]
  50. Turner J. G., Mitchell R. E. Association between Symptom Development and Inhibition of Ornithine Carbamoyltransferase in Bean Leaves Treated with Phaseolotoxin. Plant Physiol. 1985 Oct;79(2):468–473. doi: 10.1104/pp.79.2.468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Van Vliet F., Boyen A., Glansdorff N. On interspecies gene transfer: the case of the argF gene of Escherichia coli. Ann Inst Pasteur Microbiol. 1988 Jul-Aug;139(4):493–496. doi: 10.1016/0769-2609(88)90111-1. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Veres G., Gibbs R. A., Scherer S. E., Caskey C. T. The molecular basis of the sparse fur mouse mutation. Science. 1987 Jul 24;237(4813):415–417. doi: 10.1126/science.3603027. [DOI] [PubMed] [Google Scholar]
  55. Verwoerd T. C., Dekker B. M., Hoekema A. A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res. 1989 Mar 25;17(6):2362–2362. doi: 10.1093/nar/17.6.2362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Volź K. W., Krause K. L., Lipscomb W. N. The binding of N-(phosphonacetyl)-L-aspartate to aspartate carbamoyltransferase of Escherichia coli. Biochem Biophys Res Commun. 1986 Apr 29;136(2):822–826. doi: 10.1016/0006-291x(86)90514-0. [DOI] [PubMed] [Google Scholar]
  57. Wild J. R., Wales M. E. Molecular evolution and genetic engineering of protein domains involving aspartate transcarbamoylase. Annu Rev Microbiol. 1990;44:193–218. doi: 10.1146/annurev.mi.44.100190.001205. [DOI] [PubMed] [Google Scholar]
  58. Xu W., Kantrowitz E. R. Function of threonine-55 in the carbamoyl phosphate binding site of Escherichia coli aspartate transcarbamoylase. Biochemistry. 1989 Dec 26;28(26):9937–9943. doi: 10.1021/bi00452a010. [DOI] [PubMed] [Google Scholar]
  59. de Ruiter H., Kollöffel C. Properties of Ornithine Carbamoyltransferase from Pisum sativum L. Plant Physiol. 1985 Mar;77(3):695–699. doi: 10.1104/pp.77.3.695. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES