RESUMEN
Syringomycin is a lipodepsinonapeptide phytotoxin synthesized by Pseudomonas syringae pv. syringae on multienzymatic peptide synthetases. Sequence analysis of the interval between the syrB and syrD genes of P. syringae pv. syringae strain B301D revealed a 1,059-bp open reading frame (ORF), designated syrP. The predicted product of this ORF was a 39.6-kDa protein consisting of 353 amino acid residues. Searches of protein sequence databases demonstrated that SyrP was most similar to histidine kinases such as the CheA regulatory protein of Escherichia coli. The predicted SyrP sequence was aligned with the N terminus of CheA, a region corresponding to the phosphotransfer and acceptor domains of CheA. The SyrP region that aligns with the phosphotransfer domain of CheA contained a His at position 101 which is flanked by a weak consensus sequence of the unorthodox sensory kinase subfamily of two-component regulatory systems. Strain B301D-31, obtained by site-directed insertional mutagenesis of the syrP gene, exhibited an unusual pleiotropic phenotype including a failure to produce syringomycin in liquid media in contrast to production of elevated levels of the toxin on agar media. The syrP mutant was relieved of the suppression of toxin production that accompanies inorganic phosphate concentrations of > 1 mM on agar media. Nevertheless, the syrP mutant was substantially less virulent than the wild-type strain in pathogenicity assays in cherry fruits. These results suggest that the syrP gene encodes a regulatory protein that participates in a phosphorylation cascade controlling syringomycin production and virulence in P. syringae pv. syringae.
Asunto(s)
Proteínas Bacterianas/genética , Péptido Sintasas , Pseudomonas/genética , Transportadoras de Casetes de Unión a ATP , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteínas Portadoras/genética , Mapeo Cromosómico , Escherichia coli/genética , Proteínas de Escherichia coli , Frutas/microbiología , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Histidina/genética , Histidina Quinasa , Proteínas de la Membrana/genética , Proteínas Quimiotácticas Aceptoras de Metilo , Datos de Secuencia Molecular , Mutagénesis Insercional , Sistemas de Lectura Abierta , Proteínas Quinasas/genética , Pseudomonas/patogenicidad , Recombinación Genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Virulencia/genéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Genes Bacterianos/genética , Pseudomonas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Expresión Génica , Datos de Secuencia Molecular , Péptido Sintasas/genética , Filogenia , Biosíntesis de Proteínas , Pseudomonas/enzimología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Tioléster Hidrolasas/genética , Transcripción GenéticaRESUMEN
The syrB and syrD genes of Pseudomonas syringae pv. syringae are predicted to encode proteins that function in the synthesis and export of syringomycin, respectively. Using portions of the syr genes as DNA probes, both genes were shown to be conserved as single copies within a 15-kb or smaller DNA region among a broad spectrum of P. s. pv. syringae strains that produce syringomycin or one of its amino acid analogs, syringotoxin and syringostatin. Strains representative of P. viridiflava and six pathovars of P. syringae failed to hybridize with the gene probes, demonstrating that syr sequences are highly specific to P. s. pv. syringae and related nonpathogenic strains. Maximum parsimony analysis of restriction fragment length polymorphism profiles was used to evaluate relatedness among strains within the syrB and syrD gene region. A tree, conveying the smallest number of evolutionary changes among strains, revealed considerable diversity within the syr gene region; subclusters of strains were identified that appear to share specific qualities relevant to the plant-pathogen interaction. Because both the syrB gene and syringomycin production can be induced in response to plant signal molecules, 42 strains containing homologous syr sequences were tested for signal-mediated induction of toxin production. Over 90% of the toxigenic strains produced larger quantities of toxin when the plant signal molecules, arbutin and D-fructose, were added to syringomycin-minimal medium; 13 of the strains produced > or = 10-fold higher toxin levels. Some strains, such as 5D428, produced toxin only in the presence of these signals.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Portadoras/biosíntesis , Genes Bacterianos , Fenómenos Fisiológicos de las Plantas , Polimorfismo de Longitud del Fragmento de Restricción , Pseudomonas/genética , Pseudomonas/metabolismo , Toxinas Biológicas/biosíntesis , Transportadoras de Casetes de Unión a ATP , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Southern Blotting , Proteínas Portadoras/genética , ADN Bacteriano/análisis , ADN Bacteriano/metabolismo , Datos de Secuencia Molecular , Plantas/microbiología , Plásmidos , Pseudomonas/patogenicidad , Mapeo Restrictivo , Virulencia/genéticaRESUMEN
The syrD gene of Pseudomonas syringae pathovar syringae strain B301D-R was characterized and sequenced. The syrD open reading frame is 1695 bp long and encodes a predicted protein, SyrD, of approximately 63 kDa. Database searches revealed that SyrD shares a high degree of similarity with the ATP-binding cassette (ABC) superfamily of transporter proteins which are responsible for specific nutrient uptake and for secretion of certain cellular products in prokaryotes, and for multiple drug resistance in mammals. The amino acid sequence homology between SyrD and the ABC proteins was greatest at the conserved residues which constitute the ATP-binding cassette of these proteins; these residues lie in the hydrophilic C-terminal half of SyrD. The N-terminus of SyrD is predicted to be hydrophobic and to contain six membrane-spanning alpha-helices. syrD mutants of strain B301D-R were significantly less virulent than other syr mutants, were deficient in four large polypeptides thought to be components of a syringomycin synthetase complex, and showed reduced expression of a syrB-lacZ reporter gene fusion in trans. It is proposed that SyrD is a cytoplasmic membrane protein that functions as an ATP-driven efflux pump for the secretion of syringomycin.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Familia de Multigenes , Pseudomonas/genética , Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/metabolismo , Secuencia de Consenso , Datos de Secuencia Molecular , Mutagénesis Insercional , Sistemas de Lectura Abierta , Enfermedades de las Plantas/microbiología , Pseudomonas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , VirulenciaRESUMEN
The O antigens of Salmonella serogroups A, B, and D differ structurally in their side-chain sugar residue. These genes encoding O-antigen biosynthesis are clustered in the rfb operon. We report here the molecular cloning and analysis of the rfb operons of Salmonella paratyphi A (serogroup A) and S. typhi (serogroup D). The regions of DNA nonhomology between the rfb operons of these serogroup A, B, and D representatives are identified, and the evolutionary derivation of serogroup A from a serogroup D progenitor is discussed.
Asunto(s)
Antígenos Bacterianos/genética , Genes Bacterianos , Operón , Salmonella/genética , Variación Antigénica , Mapeo Cromosómico , Clonación Molecular , Cósmidos , Enzimas de Restricción del ADN , ADN Bacteriano/genética , ADN Bacteriano/ultraestructura , Microscopía Electrónica , Hibridación de Ácido Nucleico , Antígenos O , Salmonella/inmunología , Salmonella paratyphi A/genética , Salmonella paratyphi A/inmunología , Salmonella typhi/genética , Salmonella typhi/inmunología , Salmonella typhimurium/genética , Salmonella typhimurium/inmunología , Homología de Secuencia de Ácido NucleicoRESUMEN
Biosynthesis of the Salmonella typhimurium LT2 O antigen is encoded by genes which map in the rfb cluster. The cloning and restriction enzyme analysis of part of this cluster have been described previously (H. N. Brahmbhatt, N. B. Quigley, and P. R. Reeves, Mol. Gen. Genet. 203:172-176, 1986). The entire rfb gene cluster has now been cloned, and a detailed restriction enzyme map has been constructed which has enabled us to map the approximate positions of individual rfb genes.
Asunto(s)
Antígenos Bacterianos/genética , Genes Bacterianos , Salmonella typhimurium/genética , Mapeo Cromosómico , Clonación Molecular , Enzimas de Restricción del ADN , ADN Bacteriano/genética , Hibridación de Ácido Nucleico , Antígenos O , Plásmidos , Salmonella typhimurium/enzimología , Salmonella typhimurium/inmunologíaRESUMEN
A low copy number cosmid was constructed by subcloning the pair of cos sites and the kanamycin resistance gene of pcos2EMBL into pGB2. The resulting cosmid, pPR691, has the pSC101 replicon and specifies resistance to kanamycin, spectinomycin, and streptomycin. pPR691 also carries restriction sites suitable for cloning partial Sau3A digests using the strategy of Bates and Swift (P. F. Bates and R. A. Swift, 1983, Gene 26, 137-146). A library of Salmonella typhimurium chromosomal DNA was made using this cosmid and the rfb gene cluster (map position 42) was isolated from this library.
Asunto(s)
Clonación Molecular , Cósmidos , Escherichia coli/genética , Enzimas de Restricción del ADN , Farmacorresistencia Microbiana , Kanamicina/farmacología , Factores RRESUMEN
Plasmid pPR328 has been constructed for use as a chloramphenicol-resistant, intermediate cloning vector when manipulating DNA fragments cloned in vectors of the pUC family. The method used in its construction may be of general use for the replacement of the ampicillin resistance marker of pBR322-based plasmids with other drug resistance cassettes.
Asunto(s)
Cloranfenicol , Clonación Molecular/métodos , Vectores Genéticos , ADN Bacteriano/genética , Factores R , beta-Galactosidasa/genéticaRESUMEN
The rfb gene cluster of Salmonella typhimurium encodes the enzymes required for the biosynthesis of the O-Antigen. A part of it has been cloned in plasmid vectors pBR322 and pUC9 using an adjacent, previously cloned, part of the his operon (Barnes 1981) as a molecular probe for the first clone. A detailed restriction enzyme map of 7.57 kb of rfb DNA is presented and the approximate locations of two of the genes, rfbK and rfbM have been defined.
