kdgREcc negatively regulates genes for pectinases, cellulase, protease, HarpinEcc, and a global RNA regulator in Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp, carotovora produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium. E. carotovora subsp. carotovora also produces Harpin(Ecc), the elicitor of the hypersensitive reaction. We document here that KdgR(Ecc) (Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the E. chrysanthemi repressor, KdgR(Ech) and the Escherichia coli repressor, KdgR(Eco), negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and Harpin(Ecc) production in E. carotovora subsp. carotovora. The levels of pel-1, peh-1, celV, and hrpN(Ecc) transcripts are markedly affected by KdgR(Ecc). The KdgR(Ecc)(-) mutant is more virulent than the KdgR(Ecc)(+) parent. Thus, our data for the first time establish a global regulatory role for KdgR(Ecc) in E. carotovora subsp. carotovora. Another novel observation is the negative effect of KdgR(Ecc) on the transcription of rsmB (previously aepH), which specifies an RNA regulator controlling exoenzyme and Harpin(Ecc) production. The levels of rsmB RNA are higher in the KdgR(Ecc)(-) mutant than in the KdgR(Ecc)(+) parent. Moreover. by DNase I protection assays we determined that purified KdgR(Ecc) protected three 25-bp regions within the transcriptional unit of rsmB. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgR(Ecc)-binding site as 5'-G/AA/TA/TGAAA [N-6]TTTCAG/TG/TA-3'. Two such KdgR(Ecc)-binding sites occur in rsmB DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgR(Ecc)-binding site within nucleotides +207 and +231, Analysis of lacZ transcriptional fusions shows that the KdgR binding sites negatively affect the expression of rsmB. KdgR(Ecc) also binds the operator DNAs of pel-1 and peh-1 genes and represses expression of a pel1-lacZ and a peh1-lacZ transcriptional fusions. We conclude that KdgR(Ecc) affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgR(Ecc) affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription.