RESUMEN
Syringopeptin is a necrosis-inducing phytotoxin, composed of 22 amino acids attached to a 3-hydroxy fatty acid tail. Syringopeptin, produced by Pseudomonas syringae pv. syringae, functions as a virulence determinant in the plant-pathogen interaction. A 73,800-bp DNA region was sequenced, and analysis identified three large open reading frames, sypA, sypB, and sypC, that are 16.1, 16.3, and 40.6 kb in size. Sequence analysis of the putative SypA, SypB, and SypC sequences determined that they are homologous to peptide synthetases, containing five, five, and twelve amino acid activation modules, respectively. Each module exhibited characteristic domains for condensation, aminoacyl adenylation, and thiolation. Within the aminoacyl adenylation domain is a region responsible for substrate specificity. Phylogenetic analysis of the substrate-binding pockets resulted in clustering of the 22 syringopeptin modules into nine groups. This clustering reflects the substrate amino acids predicted to be recognized by each of the respective modules based on placement of the syringopeptin NRPS (nonribosomal peptide synthetase) system in the linear (type A) group. Finally, SypC contains two C-terminal thioesterase domains predicted to catalyze the release of syringopeptin from the synthetase and peptide cyclization to form the lactone ring. The syringopeptin synthetases, which carry 22 NRPS modules, represent the largest linear NRPS system described for a prokaryote.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Lipoproteínas/biosíntesis , Péptido Sintasas/genética , Péptidos Cíclicos/biosíntesis , Pseudomonas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Clonación Molecular , Escherichia coli/genética , Datos de Secuencia Molecular , Péptidos Cíclicos/genética , Filogenia , Plásmidos , Pseudomonas/enzimología , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Characterization of the biological roles of proteins is essential for functional genomics of pseudomonads. Heterologous proteins overproduced in Escherichia coli frequently fail to exhibit biological function. To circumvent this problem, vector pMEKm12 was constructed and used to overexpress proteins in Pseudomonas. The vector contains the pRO1600 replication origin, the maltose-binding protein (MBP) fusion system, and an inducible tac promoter. The pMEKm12 was successfully used to overexpress the syringomycin synthetase SyrB1 protein fused to MBP in Pseudomonas syringae pv. syringae. Furthermore, expression of the MBP-SyrB1 protein in the syrB1 mutant BR132A1 resulted in the restoration of syringomycin production. This vector will facilitate confirmation of the biochemical roles of nonribosomal peptide synthetase genes in Pseudomonas syringae, and studies of gene function from a wide spectrum of pseudomonads.
Asunto(s)
Proteínas Bacterianas/genética , Vectores Genéticos/biosíntesis , Pseudomonas/genética , Proteínas Bacterianas/biosíntesis , ADN Bacteriano/genética , Escherichia coli/genética , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Origen de Réplica , Mapeo RestrictivoRESUMEN
Sequence analysis of the right border of the syr gene cluster of Pseudomonas syringae pv. syringae strain B301D revealed the presence of the salA gene 8,113 bp downstream of syrE. The predicted SalA protein of strain B301D differs by one amino acid from that of strain B728a. Two homologs of salA, designated syrF and syrG, were identified between syrE and salA. All three proteins contain helix-turn-helix DNA-binding motifs at their C termini and exhibit homology to regulatory proteins of the LuxR family. A salA mutant failed to produce syringomycin, whereas syrF and syrG mutants produced 12 and 50%, respectively, of syringomycin relative to the wild-type strain. The salA, syrF, and syrG mutants were significantly reduced in virulence, forming small, nonspreading lesions in immature cherry fruits. Translational fusions to the uidA gene were constructed to evaluate expression of syrB1 in regulatory mutant backgrounds and to determine the relationship among the three regulatory loci. Expression of a syrB1::uidA fusion required functional salA and syrF genes and, in series, the expression of a syrF::uidA fusion required a functional salA gene. These results demonstrate that salA is located upstream of syrF in the regulatory hierarchy controlling syringomycin production and virulence in P. syringae pv. syringae.