Regulatory networks controlling virulence in the plant pathogen Erwinia carotovora ssp. carotovora

Erwinia carotovora ssp. carotovora (Ecc) is a Gram-negative enterobacterium that causes soft rot in potato and other plants. The main virulence factors, secreted extracellular enzymes, degrade the plant cell wall, leading to plant tissue maceration. Extracellular enzymes, cellulases, proteases and pectinolytic enzymes are subject to complex regulatory networks including quorum sensing, two-component systems, sigma factors and regulatory RNAs. One of the most important regulatory systems activating the production of extracellular enzymes and virulence in Ecc is the two-component system ExpS-ExpA. ExpS is a cytoplasmic membrane-associated protein suggested to activate its cognate partner ExpA, a response regulator protein. However, the signal activating this two-component system is unknown, and the target genes of the DNA-binding protein ExpA remain to be unravelled. ExpA and its orthologues are currently thought to mediate their effects through other regulatory components in diverse bacterial species. The aim here was to characterize regulatory components acting in concert with ExpA or serving as downstream regulators of ExpA in the control of extracellular enzyme genes. We applied a second-site transposon mutagenesis to an avirulent expA mutant to identify genes whose inactivation results in suppression of an extracellular enzyme-negative phenotype of the expA mutant. We identified an insertion in the kdgR gene, which was earlier characterized to repress expression of extracellular enzyme genes and a small regulatory RsmB RNA. Our results indicated that both regulatory proteins ExpA and KdgR affect expression of the RsmA-RsmB RNA regulatory system and that activation of extracellular enzyme genes by ExpA is partially mediated through this system. However, other mechanisms are likely also involved in activation of virulence factors by ExpA. In a second study, we identified a novel two-component system for plant pathogenic bacteria, PmrA-PmrB, which affects production of extracellular enzymes, virulence and bacterial survival in planta. The PmrA-PmrB regulatory system was suggested to modify bacterial lipopolysaccharide, thus promoting bacterial resistance to plant-derived antimicrobial peptides. Furthermore, a functional PmrA was necessary for survival excess of iron in conditions where pH was mildly acidic. However, the putative interaction between the two-component systems ExpS-ExpA and PmrA-PmrB requires further clarification. We also identified a novel small functional RNA RyhB, which affects expression of the celV1 gene encoding a cellulase. Expression of RyhB RNA was induced in iron-limiting conditions, suggesting its role in iron homeostasis. However, Ecc RyhB RNA was not involved in regulation of the sdhCDAB operon encoding succinate dehydrogenase enzyme, which requires Fe ions for activity. Strikingly, ExpA was found to regulate the sdhCDAB operon, and inactivation of expA resulted in inability to utilize C4-dicarboxylates succinate or fumarate, which act as chemoattractants for some plant-associated bacteria. In conclusion, our findings indicate that response regulator ExpA mediates its effects through another system, RsmA-RsmB RNA, and that the complex regulatory network includes novel mechanisms controlling virulence such as PmrA-PmrB and RyhB RNA. Furthermore, although ExpA is a central activator for expression of extracellular enzyme genes, it also has a role in another regulatory pathway, carbon metabolism.