Project description:Gene expression analysis of wild type Pectobacteriumwasabie SCC3193 wildtype compared to a SCC3193 inactivation mutant of RsmA, inactivation mutant of ExpA and an ExpA RsmA double inactivation mutant.
Project description:Posttranscriptional regulator RsmA is part of a regulatory system that controls production of plant cell wall degrading enzymes (PCWDEs), motility and other virulence factors among the Pectobacterium spp of plant pathogens. We constructed the knockout mutation of rsmA gene in Pectobacterium wasabiae strain SCC3193. The rsmA- strain has three times lower growth rate but increased virulence when compared to wild type. To explain this phenotype we compared wild type and rsmA- mRNA levels using genome wide microarray. We found that the synthesis of all known virulence factors and flagella is upregulated, while cell division and peptidoglycan synthesis is down regulated in rsmA- strain. The TCA cycle is directed fully to energy production, indicating low energetic status of the cell that is a probable reason for the low growth rate of the mutant. Growth experiments showed that the enzymatic activities of extracellular virulence factors polygalacturonase and pectate lyase are very high throughout the growth curve even without induction by plant component. Wild type cells are rod shaped in liquid media, but elongated and hyperflagellated in swarm plate containing potato tuber extract and in potato tuber. The rsmA- strain exhibits the same conditional phenotype, but differentiation into swarmer cells is faster and cell elongation more pronounced. Virulence experiments in potato tuber showed that although rsmA- strain causes bigger tissue maceration, its ability to compete with the natural bacterial community in the host is decreased. We conclude that the lack of RsmA switches the cells to infective phenotype characterized by expression of virulence factors and swarming. The lack of control over these energy costly processes is probably the reason for decreased growth rate and fitness.
Project description:Posttranscriptional regulator RsmA is part of a regulatory system that controls production of plant cell wall degrading enzymes (PCWDEs), motility and other virulence factors among the Pectobacterium spp of plant pathogens. We constructed the knockout mutation of rsmA gene in Pectobacterium wasabiae strain SCC3193. The rsmA- strain has three times lower growth rate but increased virulence when compared to wild type. To explain this phenotype we compared wild type and rsmA- mRNA levels using genome wide microarray. We found that the synthesis of all known virulence factors and flagella is upregulated, while cell division and peptidoglycan synthesis is down regulated in rsmA- strain. The TCA cycle is directed fully to energy production, indicating low energetic status of the cell that is a probable reason for the low growth rate of the mutant. Growth experiments showed that the enzymatic activities of extracellular virulence factors polygalacturonase and pectate lyase are very high throughout the growth curve even without induction by plant component. Wild type cells are rod shaped in liquid media, but elongated and hyperflagellated in swarm plate containing potato tuber extract and in potato tuber. The rsmA- strain exhibits the same conditional phenotype, but differentiation into swarmer cells is faster and cell elongation more pronounced. Virulence experiments in potato tuber showed that although rsmA- strain causes bigger tissue maceration, its ability to compete with the natural bacterial community in the host is decreased. We conclude that the lack of RsmA switches the cells to infective phenotype characterized by expression of virulence factors and swarming. The lack of control over these energy costly processes is probably the reason for decreased growth rate and fitness. Gene expression analysis from P. wasabie SCC3193 wildtype and SCC3193 rsmA mutant was performed on RNA samples extraxcted from the rsmA mutant (at 40 and 48 hours) and wildtype (at 24 hours). The strain were grown on 1.5% agar M9 minimal media plates supplemented with 10% potato extract. Three biological replicates were used from each strain at each time point.
Project description:The phytopathogen Pectobacterium wasabiae SCC3193 is a model for research on virulence regulation in soft rot pathogens. Two proteins acting as global regulators of the expression on genes involved in virulence in SCC3193 are ExpA, a DNA binding transcriptional regulator, and RsmA, an RNA binding post transcriptional regulator. Studies of related enterobacterial phytopathogens suggest that homologues of ExpA (GacA) affect gene expression partly by modulating RsmA-like protein activity. However, the exact extent of this regulon overlap has not been mapped. To elucidate the role of RsmA in ExpA controlled gene expression goes through RsmA in P. wasabiae we performed a gene expression microarray analysis of the wild type strain, an expA mutant, an rsmA mutant, and an expA rsmA double mutant. The microarray-based data of various virulence related genes was confirmed through quantitative RT-PCR. Subsequently, assays were made to link observed transcriptomic differences to changes in growth rate, PCWDE production, and virulence in planta between the four strains. We could conclude that the majority of ExpA regulation seems routed via RsmA modulation. However we could identify genes involved in electron transport, oligogalacturonide transport and metabolism, which ExpA seems to influence independently of RsmA, directly or indirectly.
Project description:HexA is a global transcriptional regulator involved in the modulation of virulence and environmental adaptation in Pectobacterium carotovorum. HexA strongly represses expression of ipdC gene, which encodes indole-3-pyruvic acid decarboxylase. To identify the flux-associated genes regulated by HexA, we performed RNA-seq analysis comparing the wild-type strain PCC27 with the hexA mutant, the ipdC mutant, and hexA/ipdC mutant. This study provides comprehensive transcriptomic profiles that reveal how HexA influences gene expression in connection with indole metabolism.
Project description:HexA is a global transcriptional regulator involved in the modulation of virulence and environmental adaptation in Pectobacterium carotovorum. To identify the genes regulated by HexA, we performed RNA-seq analysis comparing the wild-type strain PCC27 and a hexA deletion mutant at mid-log and early stationary growth phases. This study provides comprehensive transcriptomic profiles that reveal the influence of HexA on gene expression dynamics across growth stages.
Project description:The phytopathogen Pectobacterium wasabiae SCC3193 is a model for research on virulence regulation in soft rot pathogens. Two proteins acting as global regulators of the expression on genes involved in virulence in SCC3193 are ExpA, a DNA binding transcriptional regulator, and RsmA, an RNA binding post transcriptional regulator. Studies of related enterobacterial phytopathogens suggest that homologues of ExpA (GacA) affect gene expression partly by modulating RsmA-like protein activity. However, the exact extent of this regulon overlap has not been mapped. To elucidate the role of RsmA in ExpA controlled gene expression goes through RsmA in P. wasabiae we performed a gene expression microarray analysis of the wild type strain, an expA mutant, an rsmA mutant, and an expA rsmA double mutant. The microarray-based data of various virulence related genes was confirmed through quantitative RT-PCR. Subsequently, assays were made to link observed transcriptomic differences to changes in growth rate, PCWDE production, and virulence in planta between the four strains. We could conclude that the majority of ExpA regulation seems routed via RsmA modulation. However we could identify genes involved in electron transport, oligogalacturonide transport and metabolism, which ExpA seems to influence independently of RsmA, directly or indirectly. Gene expression analysis from P. wasabie SCC3193 wildtype, SCC3193 rsmA mutant, SCC3193 expA mutant and SCC3193 expA rsmA double mutant was performed on RNA samples extraxcted from the strains at late-logarithmic (late-log) and early stationary (stat) growth phase. Strains were grown in liquid hrp inducing minimal medium cultures supplemented with 0.4% polygalacturonic acid at 200 rpm shaking and 28 degrees celsius. Three biological replicates were used from each strain at each growth phase point.