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. 1995 Jul;177(14):4009–4020. doi: 10.1128/jb.177.14.4009-4020.1995

Analysis of the syrB and syrC genes of Pseudomonas syringae pv. syringae indicates that syringomycin is synthesized by a thiotemplate mechanism.

J H Zhang 1, N B Quigley 1, D C Gross 1
PMCID: PMC177131  PMID: 7608074

Abstract

The syrB and syrC genes are required for synthesis of syringomycin, a lipodepsipeptide phytotoxin produced by Pseudomonas syringae pv. syringae, and are induced by plant-derived signal molecules. A 4,842-bp chromosomal region containing the syrB and syrC genes of strain B301D was sequenced and characterized. The open reading frame (ORF) of syrB was 2,847 bp in length and was predicted to encode an approximately 105-kDa protein, SyrB, with 949 amino acids. Searches of databases revealed that SyrB shares homology with members of a superfamily of adenylate-forming enzymes involved in peptide antibiotic and siderophore synthesis in a diverse spectrum of microorganisms. SyrB exhibited the highest degree of overall similarity (56.4%) and identity (33.8%) with the first amino acid-activating domain of pyoverdin synthetase, PvdD, of Pseudomonas aeruginosa. The N-terminal portion of SyrB contained a domain of approximately 600 amino acids that resembles the amino acid-activating domains of thiotemplate-employing peptide synthetases. The SyrB domain contained six signature core sequences with the same order and spacing as observed in all known amino acid-activating domains involved in nonribosomal peptide synthesis. Core sequence 6 of SyrB, for example, was similar to the binding site for 4'-phosphopantetheine, a cofactor required for thioester formation. The syrC ORF (1,299 bp) was located 175 bp downstream of the syrB ORF. Analysis of the transcriptional and translational relationship between the syrB and syrC genes demonstrated that they are expressed independently. The syrC ORF was predicted to encode an approximately 48-kDa protein product of 433 amino acids which is 42 to 48% similar to a number of thioesterases, including fatty acid thioesterases, haloperoxidases, and acyltransferases, that contain a characteristic GXS (C) XG motif. In addition, a zinc-binding motif was found near the C terminus of SyrC. The data suggest that SyrB and SyrC function as peptide synthetases in a thiotemplate mechanism of syringomycin biosynthesis.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Cosmina P., Rodriguez F., de Ferra F., Grandi G., Perego M., Venema G., van Sinderen D. Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis. Mol Microbiol. 1993 May;8(5):821–831. doi: 10.1111/j.1365-2958.1993.tb01629.x. [DOI] [PubMed] [Google Scholar]
  3. D'Souza C., Nakano M. M., Corbell N., Zuber P. Amino-acylation site mutations in amino acid-activating domains of surfactin synthetase: effects on surfactin production and competence development in Bacillus subtilis. J Bacteriol. 1993 Jun;175(11):3502–3510. doi: 10.1128/jb.175.11.3502-3510.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fernández-Moreno M. A., Martínez E., Boto L., Hopwood D. A., Malpartida F. Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin. J Biol Chem. 1992 Sep 25;267(27):19278–19290. [PubMed] [Google Scholar]
  7. Gocht M., Marahiel M. A. Analysis of core sequences in the D-Phe activating domain of the multifunctional peptide synthetase TycA by site-directed mutagenesis. J Bacteriol. 1994 May;176(9):2654–2662. doi: 10.1128/jb.176.9.2654-2662.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grant S. G., Jessee J., Bloom F. R., Hanahan D. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4645–4649. doi: 10.1073/pnas.87.12.4645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gross D. C. Regulation of syringomycin synthesis in Pseudomonas syringae pv. syringae and defined conditions for its production. J Appl Bacteriol. 1985 Feb;58(2):167–174. doi: 10.1111/j.1365-2672.1985.tb01444.x. [DOI] [PubMed] [Google Scholar]
  10. Haese A., Schubert M., Herrmann M., Zocher R. Molecular characterization of the enniatin synthetase gene encoding a multifunctional enzyme catalysing N-methyldepsipeptide formation in Fusarium scirpi. Mol Microbiol. 1993 Mar;7(6):905–914. doi: 10.1111/j.1365-2958.1993.tb01181.x. [DOI] [PubMed] [Google Scholar]
  11. Hori K., Yamamoto Y., Minetoki T., Kurotsu T., Kanda M., Miura S., Okamura K., Furuyama J., Saito Y. Molecular cloning and nucleotide sequence of the gramicidin S synthetase 1 gene. J Biochem. 1989 Oct;106(4):639–645. doi: 10.1093/oxfordjournals.jbchem.a122909. [DOI] [PubMed] [Google Scholar]
  12. Itoh N., Morinaga N., Kouzai T. Purification and characterization of a novel metal-containing nonheme bromoperoxidase from Pseudomonas putida. Biochim Biophys Acta. 1994 Aug 17;1207(2):208–216. doi: 10.1016/0167-4838(94)00053-0. [DOI] [PubMed] [Google Scholar]
  13. Kleinkauf H., von Döhren H. Nonribosomal biosynthesis of peptide antibiotics. Eur J Biochem. 1990 Aug 28;192(1):1–15. doi: 10.1111/j.1432-1033.1990.tb19188.x. [DOI] [PubMed] [Google Scholar]
  14. Krätzschmar J., Krause M., Marahiel M. A. Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases. J Bacteriol. 1989 Oct;171(10):5422–5429. doi: 10.1128/jb.171.10.5422-5429.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Ladin B. F., Accuroso E. M., Mielenz J. R., Wilson C. R. A rapid procedure for isolating RNA from small-scale Bacillus cultures. Biotechniques. 1992 May;12(5):672–676. [PubMed] [Google Scholar]
  17. 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]
  18. Libertini L. J., Smith S. Purification and properties of a thioesterase from lactating rat mammary gland which modifies the product specificity of fatty acid synthetase. J Biol Chem. 1978 Mar 10;253(5):1393–1401. [PubMed] [Google Scholar]
  19. Liu T. N., M'Timkulu T., Geigert J., Wolf B., Neidleman S. L., Silva D., Hunter-Cevera J. C. Isolation and characterization of a novel nonheme chloroperoxidase. Biochem Biophys Res Commun. 1987 Jan 30;142(2):329–333. doi: 10.1016/0006-291x(87)90277-4. [DOI] [PubMed] [Google Scholar]
  20. Marahiel M. A. Multidomain enzymes involved in peptide synthesis. FEBS Lett. 1992 Jul 27;307(1):40–43. doi: 10.1016/0014-5793(92)80898-q. [DOI] [PubMed] [Google Scholar]
  21. Menkhaus M., Ullrich C., Kluge B., Vater J., Vollenbroich D., Kamp R. M. Structural and functional organization of the surfactin synthetase multienzyme system. J Biol Chem. 1993 Apr 15;268(11):7678–7684. [PubMed] [Google Scholar]
  22. Merriman T. R., Merriman M. E., Lamont I. L. Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases. J Bacteriol. 1995 Jan;177(1):252–258. doi: 10.1128/jb.177.1.252-258.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mittenhuber G., Weckermann R., Marahiel M. A. Gene cluster containing the genes for tyrocidine synthetases 1 and 2 from Bacillus brevis: evidence for an operon. J Bacteriol. 1989 Sep;171(9):4881–4887. doi: 10.1128/jb.171.9.4881-4887.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mo Y. Y., Geibel M., Bonsall R. F., Gross D. C. Analysis of Sweet cherry (Prunus avium L.) Leaves for Plant Signal Molecules That Activate the syrB Gene Required for Synthesis of the Phytotoxin, Syringomycin, by Pseudomonas syringae pv syringae. Plant Physiol. 1995 Feb;107(2):603–612. doi: 10.1104/pp.107.2.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mo Y. Y., Gross D. C. Plant signal molecules activate the syrB gene, which is required for syringomycin production by Pseudomonas syringae pv. syringae. J Bacteriol. 1991 Sep;173(18):5784–5792. doi: 10.1128/jb.173.18.5784-5792.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Narahashi Y. The amino acid sequence of zinc-carboxypeptidase from Streptomyces griseus. J Biochem. 1990 Jun;107(6):879–886. doi: 10.1093/oxfordjournals.jbchem.a123142. [DOI] [PubMed] [Google Scholar]
  27. Pfeifer O., Pelletier I., Altenbuchner J., van Pée K. H. Molecular cloning and sequencing of a non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762. J Gen Microbiol. 1992 Jun;138(6):1123–1131. doi: 10.1099/00221287-138-6-1123. [DOI] [PubMed] [Google Scholar]
  28. Poulose A. J., Rogers L., Cheesbrough T. M., Kolattukudy P. E. Cloning and sequencing of the cDNA for S-acyl fatty acid synthase thioesterase from the uropygial gland of mallard duck. J Biol Chem. 1985 Dec 15;260(29):15953–15958. [PubMed] [Google Scholar]
  29. Quigley N. B., Gross D. C. Syringomycin production among strains of Pseudomonas syringae pv. syringae: conservation of the syrB and syrD genes and activation of phytotoxin production by plant signal molecules. Mol Plant Microbe Interact. 1994 Jan-Feb;7(1):78–90. doi: 10.1094/mpmi-7-0078. [DOI] [PubMed] [Google Scholar]
  30. Quigley N. B., Mo Y. Y., Gross D. C. SyrD is required for syringomycin production by Pseudomonas syringae pathovar syringae and is related to a family of ATP-binding secretion proteins. Mol Microbiol. 1993 Aug;9(4):787–801. doi: 10.1111/j.1365-2958.1993.tb01738.x. [DOI] [PubMed] [Google Scholar]
  31. Quigley N. B., Reeves P. R. Chloramphenicol resistance cloning vector based on pUC9. Plasmid. 1987 Jan;17(1):54–57. doi: 10.1016/0147-619x(87)90008-4. [DOI] [PubMed] [Google Scholar]
  32. Randhawa Z. I., Smith S. Complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase from rat mammary gland. Biochemistry. 1987 Mar 10;26(5):1365–1373. doi: 10.1021/bi00379a024. [DOI] [PubMed] [Google Scholar]
  33. Rusnak F., Sakaitani M., Drueckhammer D., Reichert J., Walsh C. T. Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine. Biochemistry. 1991 Mar 19;30(11):2916–2927. doi: 10.1021/bi00225a027. [DOI] [PubMed] [Google Scholar]
  34. Saraste M., Sibbald P. R., Wittinghofer A. The P-loop--a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci. 1990 Nov;15(11):430–434. doi: 10.1016/0968-0004(90)90281-f. [DOI] [PubMed] [Google Scholar]
  35. Schlumbohm W., Stein T., Ullrich C., Vater J., Krause M., Marahiel M. A., Kruft V., Wittmann-Liebold B. An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase. J Biol Chem. 1991 Dec 5;266(34):23135–23141. [PubMed] [Google Scholar]
  36. Scott-Craig J. S., Panaccione D. G., Pocard J. A., Walton J. D. The cyclic peptide synthetase catalyzing HC-toxin production in the filamentous fungus Cochliobolus carbonum is encoded by a 15.7-kilobase open reading frame. J Biol Chem. 1992 Dec 25;267(36):26044–26049. [PubMed] [Google Scholar]
  37. Segre A., Bachmann R. C., Ballio A., Bossa F., Grgurina I., Iacobellis N. S., Marino G., Pucci P., Simmaco M., Takemoto J. Y. The structure of syringomycins A1, E and G. FEBS Lett. 1989 Sep 11;255(1):27–31. doi: 10.1016/0014-5793(89)81054-3. [DOI] [PubMed] [Google Scholar]
  38. Smith D. J., Earl A. J., Turner G. The multifunctional peptide synthetase performing the first step of penicillin biosynthesis in Penicillium chrysogenum is a 421,073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases. EMBO J. 1990 Sep;9(9):2743–2750. doi: 10.1002/j.1460-2075.1990.tb07461.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stachelhaus T., Marahiel M. A. Modular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis. FEMS Microbiol Lett. 1995 Jan 1;125(1):3–14. doi: 10.1111/j.1574-6968.1995.tb07328.x. [DOI] [PubMed] [Google Scholar]
  40. Stein T., Vater J., Kruft V., Wittmann-Liebold B., Franke P., Panico M., Mc Dowell R., Morris H. R. Detection of 4'-phosphopantetheine at the thioester binding site for L-valine of gramicidinS synthetase 2. FEBS Lett. 1994 Feb 28;340(1-2):39–44. doi: 10.1016/0014-5793(94)80169-x. [DOI] [PubMed] [Google Scholar]
  41. Tan F., Chan S. J., Steiner D. F., Schilling J. W., Skidgel R. A. Molecular cloning and sequencing of the cDNA for human membrane-bound carboxypeptidase M. Comparison with carboxypeptidases A, B, H, and N. J Biol Chem. 1989 Aug 5;264(22):13165–13170. [PubMed] [Google Scholar]
  42. Turgay K., Krause M., Marahiel M. A. Four homologous domains in the primary structure of GrsB are related to domains in a superfamily of adenylate-forming enzymes. Mol Microbiol. 1992 Feb;6(4):529–546. doi: 10.1111/j.1365-2958.1992.tb01498.x. [DOI] [PubMed] [Google Scholar]
  43. Ullrich M., Bender C. L. The biosynthetic gene cluster for coronamic acid, an ethylcyclopropyl amino acid, contains genes homologous to amino acid-activating enzymes and thioesterases. J Bacteriol. 1994 Dec;176(24):7574–7586. doi: 10.1128/jb.176.24.7574-7586.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vollenbroich D., Mehta N., Zuber P., Vater J., Kamp R. M. Analysis of surfactin synthetase subunits in srfA mutants of Bacillus subtilis OKB105. J Bacteriol. 1994 Jan;176(2):395–400. doi: 10.1128/jb.176.2.395-400.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Witkowski A., Naggert J., Witkowska H. E., Randhawa Z. I., Smith S. Utilization of an active serine 101----cysteine mutant to demonstrate the proximity of the catalytic serine 101 and histidine 237 residues in thioesterase II. J Biol Chem. 1992 Sep 15;267(26):18488–18492. [PubMed] [Google Scholar]
  46. Wolfframm C., Lingens F., Mutzel R., van Pée K. H. Chloroperoxidase-encoding gene from Pseudomonas pyrrocinia: sequence, expression in heterologous hosts, and purification of the enzyme. Gene. 1993 Aug 16;130(1):131–135. doi: 10.1016/0378-1119(93)90356-8. [DOI] [PubMed] [Google Scholar]
  47. Xu G. W., Gross D. C. Physical and functional analyses of the syrA and syrB genes involved in syringomycin production by Pseudomonas syringae pv. syringae. J Bacteriol. 1988 Dec;170(12):5680–5688. doi: 10.1128/jb.170.12.5680-5688.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. van Schijndel J. W., Simons L. H., Vollenbroek E. G., Wever R. The vanadium chloroperoxidase from the fungus, Curvularia inaequalis. Evidence for the involvement of a histidine residue in the binding of vanadate. FEBS Lett. 1993 Dec 27;336(2):239–242. doi: 10.1016/0014-5793(93)80811-8. [DOI] [PubMed] [Google Scholar]

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