Effects of the Two-Component System Comprising GacA and GacS of Erwinia carotovora subsp. carotovora on the Production of Global Regulatory rsmB RNA, Extracellular Enzymes, and HarpinEcc

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Abstract
Posttranscriptional regulation mediated by the regulator of secondary metabolites (RSM) RsmA-rsmB pair is the most important factor in the expression of genes for extracellular enzymes and HarpinEcc in Erwinia carotovora subsp. carotovora. RsmA is a small RNA-binding protein, which acts by lowering the half-life of a mRNA species. rsmB specifies an untranslated regulatory RNA and neutralizes the RsmA effect. It has been speculated that GacA-GacS, members of a two-component system, may affect gene expression via RsmA. Because expA, a gacA homolog, and expS (or rpfA), a gacS homolog, have been identified in E. carotovora subsp. carotovora, we examined the effects of these gacA and gacS homologs on the expression of rsmA, rsmB, and an assortment of exoprotein genes. The gacA gene of E. carotovora subsp. carotovora strain 71 stimulated transcription of genes for several extracellular enzymes (pel-1, a pectate lyase gene; peh-1, a polygalacturonase gene; and celV, a cellulase gene), hrpNEcc (an E. carotovora subsp. carotovora gene specifying the elicitor of hypersensitive reaction), and rsmB in GacA+ and GacS+ E. carotovora subsp. carotovora strains. Similarly, the E. carotovora subsp. carotovora gacA gene stimulated csrB (rsmB) transcription in Escherichia coli. A GacS¯ mutant of E. carotovora subsp. carotovora strain AH2 and a GacA¯ mutant of E. carotovora subsp. carotovora strain Ecc71 compared with their parent strains produced very low levels of rsmB, pel-1, peh-1, celV, and hrpNEcc transcripts but produced similar levels of rsmA RNA and RsmA protein as well as transcripts of hyper-production of extracellular enzymes (Hex) hexA, kdgR (repressor of genes for uronate and pectate catabolism), rsmC, and rpoS (gene for Sigma-S, an alternate Sigma factor). The levels of rsmB, pel-1, peh-1, celV, and hrpNEcc transcripts as well as production of pectate lyase, polygalacturonase, cellulase, protease, and HarpinEcc proteins were stimulated in GacS¯ and GacA¯ mutants by GacS+ or GacA+ plasmids, respectively. The GacA effect on exoenzyme genes and hrpNEcc was abrogated in E. carotovora subsp. carotovora mutants deficient in RsmA and RsmC or RsmA, RsmC, and rsmB RNA. The expression of lacZ transcriptional fusions of rsmB of Erwinia amylovora and Erwinia herbicola pv. gypsophilae was markedly reduced in a GacA¯ and a GacS¯ mutant of Pseudomonas syringae pv. syringae. Southern blot hybridization revealed the presence of gacA and gacS homologs in all tested strains of soft-rotting Erwinia spp. and several nonsoft-rotting Erwinia species such as E. amylovora, E. rhapontici, E. herbicola, E. stewartii, and E. herbicola pv. gypsophilae. These findings establish that the GacA-GacS system controls transcription of rsmB of E. carotovora subsp. carotovora, E. amylovora, and E. herbicola pv. gypsophilae and support the hypothesis that the effects of this two-component system on extracellular protein production in E. carotovora subsp. carotovora is mediated, at least in part, via the levels of rsmB transcripts.

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