Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.
Marahiel MA (2009) Working outside the protein-synthesis rules: insights into nonribosomal peptide synthesis. J Pept Sci 15: 799–807.
[3]
Hertweck C, Luzhetskyy A, Rebets Y, Bechthold A (2007) Type II polyketide synthases: gaining a deeper insight into enzymatic teamwork. Nat Prod Rep 24: 162–190.
[4]
Liu F, Garneau S, Walsh CT (2004) Hybrid nonribosomal peptide-polyketide interfaces in epothilone biosynthesis. Chem Biol 11: 1533–1542.
[5]
Park SR, Yoo YJ, Ban YH, Yoon YJ (2010) Biosynthesis of rapamycin and its regulation: past achievements and recent progress. J Antibiot 63: 434–441.
[6]
Miller DA, Luo L, Hillson N, Keating TA, Walsh CT (2002) Yersiniabactin synthetase: a four-protein assembly line producing the nonribosomal peptide/polyketide hybrid siderophore of Yersinia pestis. Chem Biol 9: 333–344.
[7]
Mattheus W, Gao LJ, Herdewijn P, Landuyt B, Verhaegen J, et al. (2010) Isolation and purification of a new kalimantacin/batumin-related polyketide antibiotic and elucidation of its biosynthesis gene cluster. Chem Biol 17: 149–159.
[8]
Shulse CN, Allen EE (2011) Widespread occurrence of secondary lipid biosynthesis potential in microbial lineages. PLoS One 6: e20146.
[9]
Okuyama H, Orikasa Y, Nishida T, Morita N (2007) Bacterial genes responsible for the biosynthesis of eicosapentaenoic and docosahexaenoic acids and their heterologous expression. Appl Environ Microbiol 73: 665–670.
[10]
Van Houdt R, Aertsen A, Jansen A, Quintana AL, Michiels CW (2004) Biofilm formation and cell-to-cell signaling in Gram-negative bacteria isolated from a food processing environment. J Appl Microbiol 96: 177–184.
[11]
Van Houdt R, Moons P, Aertsen A, Jansen A, Vanoirbeek K, et al. (2007) Characterization of a luxI/luxR-type quorum sensing system and N-acyl-homoserine lactone-dependent regulation of exo-enzyme and antibacterial component production in Serratia plymuthica RVH1. Res Microbiol 158: 150–158.
[12]
Van Houdt R, Moons P, Buj MH, Michiels CW (2006) N-acyl-L-homoserine lactone quorum sensing controls butanediol fermentation in Serratia plymuthica RVH1 and Serratia marcescens MG1. J Bacteriol 188: 4570–4572.
[13]
Moons P, Van Houdt R, Vivijs B, Michiels CW, Aertsen A (2011) Integrated regulation of acetoin fermentation by quorum sensing and pH in Serratia plymuthica RVH1. Appl Environ Microbiol 77: 3422–3427.
[14]
Moons P (2008) Influence of the Serratia plymuthica RVH1 quorum sensing system on biofilm formation and interspecies interactions. PhD Thesis, Katholieke Universiteit Leuven (Belgium).
[15]
Van Houdt R, Moons P, Jansen A, Vanoirbeek K, Michiels CW (2005) Genotypic and phenotypic characterization of a biofilm-forming Serratia plymuthica isolate from a raw vegetable processing line. FEMS Microbiol Lett 246: 265–272.
[16]
Moons P, Van Houdt R, Aertsen A, Vanoirbeek K, Engelborghs Y, et al. (2006) Role of quorum sensing and antimicrobial component production by Serratia plymuthica in formation of biofilms, including mixed biofilms with Escherichia coli. Appl Environ Microbiol 72: 7294–7300.
[17]
Wu J, Zhang H-B, Xu J-L, Cox RJ, Simpson TJ, et al. (2010) 13C labeling reveals multiple amination reactions in the biosynthesis of a novel polyketide polyamine antibiotic zeamine from Dickeya zeae. Chem Comm 46: 333–335.
[18]
Zhou J, Zhang H, Wu J, Liu Q, Xi P, et al. (2011) A novel multidomain polyketide synthase is essential for zeamine production and the virulence of Dickeya zeae. Mol Plant-Microbe Interact 24: 1156–1164.
[19]
Mahlen SD (2011) Serratia infections, from military experiments to current practice. Clin Microbiol Rev 24: 755–791.
[20]
Neupane S, Finlay RD, Alstr？m S, Goodwin L, Kyrpides NC, et al. (2012) Complete genome sequence of Serratia plymuthica strain AS12. Stand Genomic Sci 6: 165–173.
[21]
Neupane S, H？gberg N, Alstr？m S, Lucas S, Han J, et al. (2012) Complete genome sequence of the rapeseed plant-growth promoting Serratia plymuthica strain AS9. Stand Genomic Sci 6: 54–62.
[22]
Yazawa K (1996) Production of eicosapentaenoic acid from marine bacteria. Lipids S297–S300.
[23]
Hirota K, Nodasaka Y, Orikasa Y, Okuyama H, Yumoto I (2005) Shewanella pneumatophori sp. nov., an eicosapentaenoic acid-producing marine bacterium isolated from the intestines of Pacific mackerel (Pneumatophorus japonicus). Int J Syst Evol Microbiol 55: 2355–2359.
[24]
Tanaka M, Ueno A, Kawasaki K, Yumoto I, Ohgiya S, et al. (1999) Isolation of clustered genes that are notably homologous to the eicosapentaenoic acid biosynthesis gene cluster from the docosahexaenoic acid-producing bacterium Vibrio marinus strain MP-1. Biotechnol Lett 21: 939–945.
[25]
Allen EE, Bartlett DH (2002) Structure and regulation of the omega-3 polyunsaturated fatty acid synthase genes from the deep-sea bacterium Photobacterium profundum strain SS9. Microbiology 148: 1903–1913.
[26]
Orikasa Y, Nishida T, Hase A, Watanabe K, Morita N, et al. (2006) A phosphopantetheinyl transferase gene essential for biosynthesis of n-3 polyunsaturated fatty acids from Moritella marina strain MP-1. FEBS Lett 580: 4423–4429.
[27]
Sugihara S, Orikasa Y, Okuyama H (2008) An EntD-like phosphopantetheinyl transferase gene from Photobacterium profundum SS9 complements pfa genes of Moritella marina strain MP-1 involved in biosynthesis of docosahexaenoic acid. Biotechnol Lett 30: 411–414.
[28]
Sugihara S, Orikasa Y, Okuyama H (2010) The Escherichia coli highly expressed entD gene complements the pfaE deficiency in a pfa gene clone responsible for the biosynthesis of long-chain n-3 polyunsaturated fatty acids. FEMS Microbiol Lett 307: 207–211.
[29]
Gaitatzis N, Kunze B, Müller R (2001) In vitro reconstitution of the myxochelin biosynthetic machinery of Stigmatella aurantiaca Sg a15: Biochemical characterization of a reductive release mechanism from nonribosomal peptide synthetases. Proc Nat Acad Sci USA 98: 11136–11141.
[30]
Aparicio JF, Molnár I, Schwecke T, K？nig A, Haydock SF, et al. (1996) Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase. Gene 169: 9–16.
[31]
Haydock SF, Aparicio JF, Molnar I, Schwecke T, Khaw LE, et al. (1995) Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl carrier protein transacylase domains in modular polyketide synthases. FEBS Lett 374: 246–248.
[32]
Yadav G, Gokhale RS, Mohanty D (2003) Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases. J Mol Biol 328: 335–363.
[33]
Reid R, Piagentini M, Rodriguez E, Ashley G, Viswanathan N, et al. (2003) A model of structure and catalysis for ketoreductase domains in modular polyketide synthases. Biochemistry 42: 72–79.
[34]
Caffrey P (2003) Conserved amino acid residues correlating with ketoreductase stereospecificity in modular polyketide synthases. ChemBioChem 4: 649–662.
[35]
De Crécy-Lagard V, Marlière P, Saurin W (1995) Multienzymatic nonribosomal peptide biosynthesis: identification of the functional domains catalysing peptide elongation and epimerisation. C R Acad Sci Ser III 318: 927–936.
[36]
Rausch C, Weber T, Kohlbacher O, Wohlleben W, Huson DH (2005) Specificity prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using transductive support vector machines (TSVMs). Nucleic Acids Res 33: 5799–5808.
[37]
R？ttig M, Medema MH, Blin K, Weber T, Rausch C, et al. (2011) NRPSpredictor2 - a web server for predicting NRPS adenylation domain specificity. Nucleic Acids Res 39: W362–367.
[38]
Schlumbohm W, Stein T, Ullrich C, Vater J, Krause M, et al. (1991) An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase. J Biol Chem 266: 23135–23141.
[39]
Pace HC, Brenner C (2001) The nitrilase superfamily: classification, structure and function. Genome Biol 2: reviews0001.1–reviews0001.9.
[40]
Brenner C (2002) Catalysis in the nitrilase superfamily. Curr Opin Struct Biol 12: 775–782.
[41]
Blight MA, Chervaux C, Holland IB (1994) Protein secretion pathway in Escherichia coli. Curr Opin Biotechnol 5: 468–474.
[42]
Barrett AJ, Rawlings ND (1992) Oligopeptidases, and the emergence of the prolyl oligopeptidase family. Biol Chem Hoppe Seyler 373: 353–360.
[43]
Metz JG, Roessler P, Facciotti D, Levering C, Dittrich F, et al. (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293: 290–293.
[44]
Duitman EH, Hamoen LW, Rembold M, Venema G, Seitz H, et al. (1999) The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proc Nat Acad Sci USA 96: 13294–13299.
[45]
Aron ZD, Dorrestein PC, Blackhall JR, Kelleher NL, Walsh CT (2005) Characterization of a new tailoring domain in polyketide biogenesis: the amine transferase domain of MycA in the mycosubtilin gene cluster. J Am Chem Soc 127: 14986–14987.
[46]
Silakowski B, Kunze B, Nordsiek G, Bl？cker H, H？fle G, et al. (2000) The myxochelin iron transport regulon of the myxobacterium Stigmatella aurantiaca Sg a15. Eur J Biochem 267: 6476–6485.
[47]
Keating TA, Marshall CG, Walsh CT (2000) Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains. Biochemistry 39: 15513–15521.
[48]
Keating TA, Marshall CG, Walsh CT (2000) Reconstitution and characterization of the Vibrio cholerae vibriobactin synthetase from VibB, VibE, VibF, and VibH. Biochemistry 39: 15520–15530.
[49]
Wilkinson B, Foster G, Rudd BA, Taylor NL, Blackaby AP, et al. (2000) Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase. Chem Biol 7: 111–117.
[50]
Moss SJ, Martin CJ, Wilkinson B (2004) Loss of co-linearity by modular polyketide synthases: a mechanism for the evolution of chemical diversity. Nat Prod Rep 21: 575–593.
[51]
Reimer D, Pos KM, Thines M, Grün P, Bode HB (2011) A natural prodrug activation mechanism in nonribosomal peptide synthesis. Nat Chem Biol 7: 888–890.
[52]
Fujii I, Yasuoka Y, Tsai HF, Chang YC, Kwon-Chung KJ, et al. (2004) Hydrolytic polyketide shortening by ayg1p, a novel enzyme involved in fungal melaninbiosynthesis. J Biol Chem 279: 44613–44620.
[53]
Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Nat Biotechnol 1: 784–791.
[54]
Van Houdt R, Aertsen A, Moons P, Vanoirbeek K, Michiels C (2006) N-acyl-L homoserine lactone signal interception by Escherichia coli. FEMS Microbiol Lett 256: 83–89.
[55]
Davis AL, Laue ED, Keeler J, Moskau D, Lohman J (1991) Absortion-mode 2-dimensional NMR-spectra recorded using pulsed field gradients. J Magn Reson 94: 637–644.
[56]
Sattler M, Schwendinger MG, Schleucher J, Griesinger C (1995) Novel strategies of sensitivity enhancement in heteronuclear multi-dimensional NMR experiments employing pulsed field gradients. J Biomol NMR 6: 11–22.
[57]
Davis AL, Keeler J, Laue ED, Moskau D (1992) Experiments for recording pure –absorption heteronuclear correlation spectra using pulsed field gradients. J Magn Reson 98: 207–216.
[58]
Griesinger C, Otting G, Wüthrich K, Ernst RR (1988) Clean TOCSY for 1H spin system identification in macromolecules. J Am Chem Soc 110: 7870–7872.
[59]
Shaka AJ, Barker PB, Freeman R (1985) Computer-optimized decoupling scheme for wideband applications and low-level operation. J Magn Reson 64: 574.
[60]
De Lorenzo V, Timmis K (1994) Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10- derived minitransposons. Methods Enzymol 235: 386–405.
[61]
Osoegawa K, Woon PY, Zhao B, Frengen E, Tateno M, et al. (1998) An improved method for construction of bacterial artificial chromosome libraries. Genomics 52: 1–8.
[62]
Borodovsky M, Mills R, Besemer J, Lomsadze A (2003) Prokaryotic gene prediction using GeneMark and GeneMark.hmm. Curr Protoc Bioinformatics Chapter 4, Unit 4.5.
