Project description:The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and â??tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26Â°C and 37Â°C. The most significant changes in the transcriptome of the â??tatC mutant were seen at 26Â°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. Y. pseudotuberculosis IP32953 was grown at 26Â°C in LB medium under aeration on a rotary shaker. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.

Project description:The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and âtatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26Â°C and 37Â°C. The most significant changes in the transcriptome of the âtatC mutant were seen at 26Â°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. Y. pseudotuberculosis IP32953 was grown at 26Â°C in LB medium under aeration. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.

Project description:The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and âtatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26Â°C and 37Â°C. The most significant changes in the transcriptome of the âtatC mutant were seen at 26Â°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. Y. pseudotuberculosis IP32953 was grown in LB medium supplemented with 2.5 mM CaCl2 under aeration for one hour at 26Â°C and then shifted to 37Â°C. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.

Project description:Colonization of the intestinal tract and dissemination into deeper tissues by the enteric pathoÂgen Yersinia pseudotuberculosis demands expression of a special set of virulence factors important for the initiation and the persistence of the infection. In this study we demonstrate that many virulence-associated functions are coregulated with the carbohydrate metabolism. This link is mediated by the carbon storage regulator (Csr) system, including the regulatory RNAs CsrB and CsrC, and the cAMP receptor protein (Crp), which both control virulence gene expression in response to the nutrient composition of the medium. Here, we show that Crp regulates the synthesis of both Csr RNAs in an opposite manner. A loss of the crp gene resulted in a strong upregulation of CsrB synthesis, whereas CsrC levels were strongly reduced leading to downregulation of the viruÂlence regulator RovA. Switching of the Csr RNA involves Crp-mediated reÂpression of the response regulator UvrY which activates csrB transcription. To elucidate the regulatory links between virulence and carbon metabolism, we performed comparative metabolome, transÂÂÂcripÂtome and phenotypic microarray analyses and found that Crp promotes oxidative catabolism of many different carbon sources, whereas fermentative patterns of metabolism are favoured when crp is deleted. Mouse infection experiments further demonstrated that Crp is pivotal for a successÂful Y. pseudoÂtuberÂculosis infection. In summary, placement of the Csr system and important virulence factors under control of Crp enables this pathogen to link its nutritional status to virulence in order to optimize bioÂlogical fitness and infection efficiency through the infecÂtious life cycle. Y. pseudotuberculosis YPIII or the isogenic crp mutant strain were grown to late stationary phase at 25Â°C. Four biological replicates were employed for each experiment consisting of two pooled individual cultures and two pooled RNA preparation samples, respectively. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.

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Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis

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