RESUMEN
SsrA and SmpB are known to play important roles in translational quality control and are essential for virulence in many human and animal pathogenic bacteria. The physiological roles and contribution of SsrA and SmpB to plant pathogen are unclear. Here, we present evidence to show that ssrA and smpB are involved in pathogenesis of Xanthomonas campestris pathovar campestris, the cause of black rot diseases in crucifers. The ssrA and smpB mutants exhibited defects in bacterial attachment, cell motility, and extracellular enzyme activity. The mutation of ssrA and smpB also resulted in a reduction in temperature tolerance. These altered phenotypes of the ssrA and smpB mutants could be complemented to wild-type levels by the intact ssrA and smpB genes. This is the first demonstration of the roles of SsrA and SmpB in phytopathogen.
Asunto(s)
Xanthomonas campestris , Animales , Humanos , Xanthomonas campestris/genética , Proteínas Bacterianas/genética , Virulencia/genética , Factores de Virulencia/genética , Proteómica , Enfermedades de las Plantas/microbiologíaRESUMEN
BACKGROUND: Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium that can cause black rot disease in crucifers. The lipoprotein outer membrane localization (Lol) system is involved in the lipoprotein sorting to the outer membrane. Although Xcc has a set of annotated lol genes, there is still little known about the physiological role in this phytopathogen. In this study, we aimed to characterize the role of LolB of Xcc in bacterial attachment, stress tolerance, and virulence. RESULTS: To characterize the role of LolB, lolB mutant was constructed and phenotypic evaluation was performed. The lolB mutant revealed reductions in bacterial attachment, extracellular enzyme production, and virulence. Mutation of lolB also resulted in reduced tolerance to a myriad of stresses, including heat and a range of membrane-perturbing agents. Trans-complementation of lolB mutant with intact lolB gene reverted these altered phenotypes to the wild-type levels. From subsequent reporter assay and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, the expression of genes that encode the major extracellular enzymes and the stress-related proteins was reduced after lolB mutation. CONCLUSIONS: The results in this work contribute to the functional understanding of lolB in Xanthomonas for the first time, and provide new insights into the function of lolB in bacteria.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Xanthomonas campestris/fisiología , Xanthomonas campestris/patogenicidad , Adaptación Fisiológica/genética , Adhesión Bacteriana/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Mutación , Enfermedades de las Plantas/microbiología , Virulencia/genética , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismoRESUMEN
The caseinolytic protease (Clp) system is essential for survival under stress conditions and for virulence in several pathogenic bacteria. Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen which causes black rot disease in crucifers. In this study, the Xcc clpP gene which is annotated to encode the proteolytic core of Clp was characterized. Mutation of clpP resulted in susceptibility to high temperature and puromycin stresses. Site-directed mutagenesis revealed that S105, H130, and D179 are critical amino acid residues for ClpP function in puromycin tolerance. Inactivation of clpP also revealed an attenuation of virulence on the host plant and a reduction in the production of extracellular cellulase, mannanase, pectinase, and protease. The affected phenotypes of the clpP mutant could be complemented to wild-type levels by the intact clpP gene. Transcriptional analysis revealed that expression of clpP is induced under heat shock condition.
Asunto(s)
Endopeptidasas , Regulación Bacteriana de la Expresión Génica , Xanthomonas campestris , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Mutagénesis Sitio-Dirigida , Enfermedades de las Plantas/microbiología , Factores de Virulencia/genética , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidadRESUMEN
Xanthomonas campestris pv. campestris is a bacterial pathogen and the causal agent of black rot in crucifers. In this study, a clpX mutant was obtained by EZ-Tn5 transposon mutagenesis of the X. campestris pv. campestris. The clpX gene was annotated to encode ClpX, the ATP-binding subunit of ATP-dependent Clp protease. The clpX mutant exhibited reduced bacterial attachment, extracellular enzyme production and virulence. Mutation of clpX also resulted in increased sensitivity to a myriad of stresses, including heat, puromycin, and sodium dodecyl sulfate. These altered phenotypes of the clpX mutant could be restored to wild-type levels by in trans expression of the intact clpX gene. Proteomic analysis revealed that the expression of 211 proteins differed not less than twofold between the wild-type and mutant strains. Cluster of orthologous group analysis revealed that these proteins are mainly involved in metabolism, cell wall biogenesis, chaperone, and signal transduction. The reverse transcription quantitative real-time polymerase chain reaction analysis demonstrated that the expression of genes encoding attachment-related proteins, extracellular enzymes, and virulence-associated proteins was reduced after clpX mutation. The results in this study contribute to the functional understanding of the role of clpX in Xanthomonas for the first time, and extend new insights into the function of clpX in bacteria.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Endopeptidasa Clp/metabolismo , Xanthomonas campestris/enzimología , Xanthomonas campestris/patogenicidad , Proteínas Bacterianas/genética , Endopeptidasa Clp/genética , Regulación Bacteriana de la Expresión Génica , Mutación , Enfermedades de las Plantas/microbiología , Proteómica , Virulencia , Xanthomonas campestris/genética , Xanthomonas campestris/fisiologíaRESUMEN
Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. Here, one EZ-Tn5 transposon mutant of Xcc, altered in bacterial attachment, was isolated. Further analysis revealed that the transposon was inserted in the wxcX gene (encodes a hypothetical protein) of the transposon mutant. Sequence analysis revealed that WxcX is highly conserved in Xanthomonas, but none has been characterized. In this study, it was indicated that mutation of wxcX resulted in enhanced bacterial attachment, reduced virulence on the host cabbage, and increased sensitivity to sodium dodecyl sulfate. The affected phenotypes of the wxcX mutant could be complemented to wild-type levels by the intact wxcX gene. Site-directed mutagenesis revealed that E408 and E411 are critical amino acid residues for WxcX function in bacterial attachment. Taken together, our results demonstrate the roles of wxcX in attachment, virulence, and tolerance to sodium dodecyl sulfate in Xanthomonas for the first time.