RESUMEN
Genes coding for the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the plant cell wall-degrading enzymes, are under the coordinate control of global regulator systems including both positive and negative factors. In addition to this global control, some virulence determinants are subject to specific regulation. We have previously shown that mutations in the pehR locus result in reduced virulence and impaired production of one of these enzymes, an endopolygalacturonase (PehA). In contrast, these pehR strains produce essentially wild-type levels of other extracellular enzymes including pectate lyases and cellulases. In this work, we characterized the pehR locus and showed that the DNA sequence is composed of two genes, designated pehR and pehS, present in an operon. Mutations in either pehR or pehS caused a Peh-negative phenotype and resulted in reduced virulence on tobacco seedlings. Complementation experiments indicated that both genes are required for transcriptional activation of the endopolygalacturonase gene, pehA, as well as restoration of virulence. Structural characterization of the pehR-pehS operon demonstrated that the corresponding polypeptides are highly similar to the two-component transcriptional regulators PhoP-PhoQ of both Escherichia coli and Salmonella typhimurium. Functional similarity of PehR-PehS with PhoP-PhoQ of E. coli and S. typhimurium was demonstrated by genetic complementation.
Asunto(s)
Proteínas Bacterianas/genética , Pectobacterium carotovorum/genética , Poligalacturonasa/genética , Proteínas Quinasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Mapeo Cromosómico , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Prueba de Complementación Genética , Datos de Secuencia Molecular , Pectobacterium carotovorum/enzimología , Pectobacterium carotovorum/patogenicidad , Poligalacturonasa/biosíntesis , Proteínas Quinasas/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Alineación de Secuencia , VirulenciaRESUMEN
Chlorocatechol 1,2-dioxygenase (CC12O) and 1,2-dichlorophenol hydroxylase (DCPH) encoding genes tfdC and tfdB are located on a 4.2-kb DNA fragment cloned from the 2,4-dichlorophenoxyacetic acid (2,4D) degrading plasmid pEST4011. The nucleotide sequences of tfdC and tfdB were determined. The DCPH is coded by a 1758-bp gene and CC12O is coded by a 762-bp gene. The deduced M(r) of these proteins are 64.09 kDa and 28.2 kDa, respectively. Expression analysis of tfdB and tfdC in Escherichia coli suggested that these genes form one operon, tfdCB.