1887

Abstract

Phosphopantetheinyltransferase (PPTase) enzymes fulfil essential roles in primary and secondary metabolism in prokaryotes, archaea and eukaryotes. PPTase enzymes catalyse the essential modification of the carrier protein domain of fatty acid synthases, polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs). In bacteria and fungi, NRPS and PKS enzymes are often responsible for the biosynthesis of secondary metabolites with clinically relevant properties; these secondary metabolites include a variety of antimicrobial peptides. We have previously shown that in the Gram-negative bacterium Db10, the PPTase enzyme PswP is essential for the biosynthesis of an NRPS-PKS dependent antibiotic called althiomycin. In this work we utilize bioinformatic analyses to classify PswP as belonging to the F/KES subfamily of Sfp type PPTases and to putatively identify additional NRPS substrates of PswP, in addition to the althiomycin NRPS-PKS, in Db10. We show that PswP is required for the production of three diffusible metabolites by this organism, each possessing antimicrobial activity against . Genetic analyses identify the three metabolites as althiomycin, serrawettin W2 and an as-yet-uncharacterized siderophore, which may be related to enterobactin. Our results highlight the use of an individual PPTase enzyme in multiple biosynthetic pathways, each contributing to the ability of to inhibit competitor bacteria by the production of antimicrobial secondary metabolites.

Funding
This study was supported by the:
  • the Wellcome Trust (Award 093711/B/10/Z)
  • Royal Society of Edinburgh/Scottish Government
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.078576-0
2014-08-01
2022-01-27
Loading full text...

Full text loading...

/deliver/fulltext/micro/160/8/1609.html?itemId=/content/journal/micro/10.1099/mic.0.078576-0&mimeType=html&fmt=ahah

References

  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. ( 1997). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [View Article][PubMed]
    [Google Scholar]
  2. Beld J., Sonnenschein E. C., Vickery C. R., Noel J. P., Burkart M. D. ( 2014). The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life. Nat Prod Rep 31:61–108 [View Article][PubMed]
    [Google Scholar]
  3. Blin K., Medema M. H., Kazempour D., Fischbach M. A., Breitling R., Takano E., Weber T. ( 2013). antiSMASH 2.0–a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res 41:Web Server issueW204–W212 [View Article][PubMed]
    [Google Scholar]
  4. Chirgadze N. Y., Briggs S. L., McAllister K. A., Fischl A. S., Zhao G. ( 2000). Crystal structure of Streptococcus pneumoniae acyl carrier protein synthase: an essential enzyme in bacterial fatty acid biosynthesis. EMBO J 19:5281–5287 [View Article][PubMed]
    [Google Scholar]
  5. Copp J. N., Neilan B. A. ( 2006). The phosphopantetheinyl transferase superfamily: phylogenetic analysis and functional implications in cyanobacteria. Appl Environ Microbiol 72:2298–2305 [View Article][PubMed]
    [Google Scholar]
  6. Corsini G., Karahanian E., Tello M., Fernandez K., Rivero D., Saavedra J. M., Ferrer A. ( 2010). Purification and characterization of the antimicrobial peptide microcin N. FEMS Microbiol Lett 312:119–125 [View Article][PubMed]
    [Google Scholar]
  7. Coulthurst S. J., Williamson N. R., Harris A. K., Spring D. R., Salmond G. P. ( 2006). Metabolic and regulatory engineering of Serratia marcescens: mimicking phage-mediated horizontal acquisition of antibiotic biosynthesis and quorum-sensing capacities. Microbiology 152:1899–1911 [View Article][PubMed]
    [Google Scholar]
  8. Crosa J. H., Walsh C. T. ( 2002). Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev 66:223–249 [View Article][PubMed]
    [Google Scholar]
  9. Elovson J., Vagelos P. R. ( 1968). Acyl carrier protein. X. Acyl carrier protein synthetase. J Biol Chem 243:3603–3611[PubMed]
    [Google Scholar]
  10. Fichtlscherer F., Wellein C., Mittag M., Schweizer E. ( 2000). A novel function of yeast fatty acid synthase. Subunit alpha is capable of self-pantetheinylation. Eur J Biochem 267:2666–2671 [View Article][PubMed]
    [Google Scholar]
  11. Fineran P. C., Slater H., Everson L., Hughes K., Salmond G. P. ( 2005). Biosynthesis of tripyrrole and beta-lactam secondary metabolites in Serratia: integration of quorum sensing with multiple new regulatory components in the control of prodigiosin and carbapenem antibiotic production. Mol Microbiol 56:1495–1517 [View Article][PubMed]
    [Google Scholar]
  12. Fischbach M. A., Walsh C. T. ( 2006). Assembly-line enzymology for polyketide and nonribosomal Peptide antibiotics: logic, machinery, and mechanisms. Chem Rev 106:3468–3496 [View Article][PubMed]
    [Google Scholar]
  13. Flyg C., Kenne K., Boman H. G. ( 1980). Insect pathogenic properties of Serratia marcescens: phage-resistant mutants with a decreased resistance to Cecropia immunity and a decreased virulence to Drosophila . J Gen Microbiol 120:173–181[PubMed]
    [Google Scholar]
  14. Gerc A. J., Song L., Challis G. L., Stanley-Wall N. R., Coulthurst S. J. ( 2012). The insect pathogen Serratia marcescens Db10 uses a hybrid non-ribosomal peptide synthetase-polyketide synthase to produce the antibiotic althiomycin. PLoS ONE 7:e44673 [View Article][PubMed]
    [Google Scholar]
  15. Grinter N. J. ( 1983). A broad-host-range cloning vector transposable to various replicons. Gene 21:133–143 [View Article][PubMed]
    [Google Scholar]
  16. Herrero M., de Lorenzo V., Timmis K. N. ( 1990). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 172:6557–6567[PubMed]
    [Google Scholar]
  17. Jack R. L., Buchanan G., Dubini A., Hatzixanthis K., Palmer T., Sargent F. ( 2004). Coordinating assembly and export of complex bacterial proteins. EMBO J 23:3962–3972 [View Article][PubMed]
    [Google Scholar]
  18. Kadouri D. E., Shanks R. M. ( 2013). Identification of a methicillin-resistant Staphylococcus aureus inhibitory compound isolated from Serratia marcescens . Res Microbiol 164:821–826 [View Article][PubMed]
    [Google Scholar]
  19. Kaniga K., Delor I., Cornelis G. R. ( 1991). A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica . Gene 109:137–141 [View Article][PubMed]
    [Google Scholar]
  20. Lambalot R. H., Walsh C. T. ( 1995). Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase. J Biol Chem 270:24658–24661 [View Article][PubMed]
    [Google Scholar]
  21. Lambalot R. H., Gehring A. M., Flugel R. S., Zuber P., LaCelle M., Marahiel M. A., Reid R., Khosla C., Walsh C. T. ( 1996). A new enzyme superfamily - the phosphopantetheinyl transferases. Chem Biol 3:923–936 [View Article][PubMed]
    [Google Scholar]
  22. Li H., Tanikawa T., Sato Y., Nakagawa Y., Matsuyama T. ( 2005). Serratia marcescens gene required for surfactant serrawettin W1 production encodes putative aminolipid synthetase belonging to nonribosomal peptide synthetase family. Microbiol Immunol 49:303–310 [View Article][PubMed]
    [Google Scholar]
  23. Matsuyama T., Kaneda K., Nakagawa Y., Isa K., Hara-Hotta H., Yano I. ( 1992). A novel extracellular cyclic lipopeptide which promotes flagellum-dependent and -independent spreading growth of Serratia marcescens . J Bacteriol 174:1769–1776[PubMed]
    [Google Scholar]
  24. Matsuyama T., Tanikawa T., Nakagawa T. ( 2011). Serrawettins and other surfactants produced by Serratia . Biosurfactants (Microbiology Monographs vol. 20)93–120 Soberón-Chávez G. Heidelberg: Springer; [View Article]
    [Google Scholar]
  25. Miethke M., Marahiel M. A. ( 2007). Siderophore-based iron acquisition and pathogen control. Microbiol Mol Biol Rev 71:413–451 [View Article][PubMed]
    [Google Scholar]
  26. Mootz H. D., Finking R., Marahiel M. A. ( 2001). 4′-phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis . J Biol Chem 276:37289–37298 [View Article][PubMed]
    [Google Scholar]
  27. Murugan E., Liang Z. X. ( 2008). Evidence for a novel phosphopantetheinyl transferase domain in the polyketide synthase for enediyne biosynthesis. FEBS Lett 582:1097–1103 [View Article][PubMed]
    [Google Scholar]
  28. Parris K. D., Lin L., Tam A., Mathew R., Hixon J., Stahl M., Fritz C. C., Seehra J., Somers W. S. ( 2000). Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites. Structure 8:883–895 [View Article][PubMed]
    [Google Scholar]
  29. Perego M., Hoch J. A. ( 1988). Sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in Bacillus subtilis . J Bacteriol 170:2560–2567[PubMed]
    [Google Scholar]
  30. Persmark M., Expert D., Neilands J. B. ( 1989). Isolation, characterization, and synthesis of chrysobactin, a compound with siderophore activity from Erwinia chrysanthemi . J Biol Chem 264:3187–3193[PubMed]
    [Google Scholar]
  31. Petty N. K., Foulds I. J., Pradel E., Ewbank J. J., Salmond G. P. ( 2006). A generalized transducing phage (phiIF3) for the genomically sequenced Serratia marcescens strain Db11: a tool for functional genomics of an opportunistic human pathogen. Microbiology 152:1701–1708 [View Article][PubMed]
    [Google Scholar]
  32. Pradel E., Zhang Y., Pujol N., Matsuyama T., Bargmann C. I., Ewbank J. J. ( 2007). Detection and avoidance of a natural product from the pathogenic bacterium Serratia marcescens by Caenorhabditis elegans . Proc Natl Acad Sci U S A 104:2295–2300 [View Article][PubMed]
    [Google Scholar]
  33. Quadri L. E., Weinreb P. H., Lei M., Nakano M. M., Zuber P., Walsh C. T. ( 1998). Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37:1585–1595 [View Article][PubMed]
    [Google Scholar]
  34. Raymond K. N., Dertz E. A., Kim S. S. ( 2003). Enterobactin: an archetype for microbial iron transport. Proc Natl Acad Sci U S A 100:3584–3588 [View Article][PubMed]
    [Google Scholar]
  35. Rebuffat S. ( 2012). Microcins in action: amazing defence strategies of Enterobacteria . Biochem Soc Trans 40:1456–1462 [View Article][PubMed]
    [Google Scholar]
  36. Reuter K., Mofid M. R., Marahiel M. A., Ficner R. ( 1999). Crystal structure of the surfactin synthetase-activating enzyme sfp: a prototype of the 4′-phosphopantetheinyl transferase superfamily. EMBO J 18:6823–6831 [View Article][PubMed]
    [Google Scholar]
  37. Schwyn B., Neilands J. B. ( 1987). Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56 [View Article][PubMed]
    [Google Scholar]
  38. Seyedsayamdost M. R., Cleto S., Carr G., Vlamakis H., João Vieira M., Kolter R., Clardy J. ( 2012). Mixing and Matching Siderophore Clusters: Structure and Biosynthesis of Serratiochelins from Serratia sp. V4. J Am Chem Soc 134:13550–13553 [CrossRef]
    [Google Scholar]
  39. Sibbald M. J., Winter T., van der Kooi-Pol M. M., Buist G., Tsompanidou E., Bosma T., Schäfer T., Ohlsen K., Hecker M. & other authors ( 2010). Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in Staphylococcus aureus . J Bacteriol 192:3788–3800 [View Article][PubMed]
    [Google Scholar]
  40. Singh P., Cameotra S. S. ( 2004). Potential applications of microbial surfactants in biomedical sciences. Trends Biotechnol 22:142–146 [View Article][PubMed]
    [Google Scholar]
  41. Sunaga S., Li H., Sato Y., Nakagawa Y., Matsuyama T. ( 2004). Identification and characterization of the pswP gene required for the parallel production of prodigiosin and serrawettin W1 in Serratia marcescens . Microbiol Immunol 48:723–728 [View Article][PubMed]
    [Google Scholar]
  42. Thomson N. R., Crow M. A., McGowan S. J., Cox A., Salmond G. P. ( 2000). Biosynthesis of carbapenem antibiotic and prodigiosin pigment in Serratia is under quorum sensing control. Mol Microbiol 36:539–556 [View Article][PubMed]
    [Google Scholar]
  43. Walsh C. T., Gehring A. M., Weinreb P. H., Quadri L. E., Flugel R. S. ( 1997). Post-translational modification of polyketide and nonribosomal peptide synthases. Curr Opin Chem Biol 1:309–315 [View Article][PubMed]
    [Google Scholar]
  44. Wasserman H. H., Keggi J. J., McKeon J. E. ( 1962). The structure of serratamolide1–3 . J Am Chem Soc 84:2978–2982 [View Article]
    [Google Scholar]
  45. Weissman K. J., Hong H., Oliynyk M., Siskos A. P., Leadlay P. F. ( 2004). Identification of a phosphopantetheinyl transferase for erythromycin biosynthesis in Saccharopolyspora erythraea . ChemBioChem 5:116–125 [View Article][PubMed]
    [Google Scholar]
  46. Williamson N. R., Simonsen H. T., Harris A. K., Leeper F. J., Salmond G. P. ( 2006). Disruption of the copper efflux pump (CopA) of Serratia marcescens ATCC 274 pleiotropically affects copper sensitivity and production of the tripyrrole secondary metabolite, prodigiosin. J Ind Microbiol Biotechnol 33:151–158 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.078576-0
Loading
/content/journal/micro/10.1099/mic.0.078576-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error