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:Whole genome analysis of gene expression by Pectobacterium atrosepticum strain SCRI1043 wildtype and its relA, expI and rpoS deletion mutants when grown to exponential and stationary phase in PMB media. The data is further described in Bowden et al (2013) Virulence in Pectobacterium atrosepticum is regulated by a coincidence circuit involving quorum sensing and the stress alarmone, (p)ppGpp. Molecular Microbiology. DOI: 10.1111/mmi.12369
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:Whole genome analysis of gene expression by Pectobacterium atrosepticum strain SCRI1043 wildtype and its relA, expI and rpoS deletion mutants when grown to exponential and stationary phase in PMB media. The data is further described in Bowden et al (2013) Virulence in Pectobacterium atrosepticum is regulated by a coincidence circuit involving quorum sensing and the stress alarmone, (p)ppGpp. Molecular Microbiology. DOI: 10.1111/mmi.12369 A 24 chip study using total RNA recovered from three separate wild-type cultures of Pectobacterium atrosepticum SCRI1043 and three separate cultures from three single mutant strains of SCRI1043 possessing deletions within relA (ECA3569), expI (ECA0105) or rpoS (ECA3530) when grown in Pel Minimal Broth (PMB) media to log-phase (6h) or early stationary phase (14h) growth. Each chip measures the expression level of 4,472 genes from Pectobacterium atrosepticum SCRI1043 with eight 60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.