CHARACTERIZATION OF A NOVEL RNA REGULATOR OF ERWINIA-CAROTOVORA SSP. CAROTOVORA THAT CONTROLS PRODUCTION OF EXTRACELLULAR ENZYMES AND SECONDARY METABOLITES

The enterobacterium Erwinia carotovora ssp. carotovora strain 71 (here after Ecc71) produces extracellular enzymes such as pectate lyase isoz ymes (Pels), cellulase (Cel), polygalacturonase (Peh) and protease (Pr t). These enzymes degrade plant cell wall components and are largely r esponsible for the elicitation of softrot diseases in plants and plant products, Ecc71 also produces Harpin(Ecc), the elicitor of hypersensi tive reaction (HR) and the quorum-sensing signal, N-(3-oxo-hexanoyl)-L -homoserine lactone (OHL). OHL controls extracellular enzyme and Harpi n(Ecc) production. The revels of these enzymes, as well as the express ion of hrpN(Ecc), the structural gene for Harpin(Ecc) and ohll, the ge ne specifying OHL synthesis, are negatively regulated by RsmA, rsmB, f ormerly aepH, on the other hand, positively regulates extracellular en zyme production. 6His-RsmA recombinant protein purified from E. coil b inds rsmB RNA as indicated by gel mobility shift assays. rsmB comprise s 547 bp DNA, which is transcribed from a single start site immediatel y after a sigma(70)-like promoter, In Ecc71, two rsmB RNA species are detected: a full-length 479 base rsmB RNA and a 259 base rsmB' RNA, rs mB' DNA hybridizes with the 259 base and the 479 base transcripts, A 3 ' RNase protection assay revealed that the 259 base and the 479 base R NA species end at the same position immediately after the putative rho -independent terminator. The expression of rsmB-lacZ transcriptional f usions established that the rsmB' RNA is not produced because of the a ctivation of an internal promoter. These data strongly suggest that th e 259 base rsmB' RNA is derived by processing of the primary rsmB RNA, In Ecc71, rsmB' expression driven by the lac promoter causes overprod uction of Pel, Peh, Cel and Prt, and accumulation of pel-1, peh-1, hrp N(Ecc) and ohll transcripts. By contrast, a plasmid with the rsmS' DNA sequence deleted fails to cause overproduction of the extracellular e nzymes in Ecc71, The rsmB' effect also occurs in Escherichia coil as g lycogen accumulation is stimulated in the presence of rsmB'. In vivo a nd in vitro translation as well as mutational analysis of rsmB' have e stablished that rsmB' RNA does not yield a translational product. Ther efore, we concluded that the rsmS' RNA itself functions as the regulat or, Indeed, the expression of rsmS' DNA leads to neutralization of the negative effects of the RNA-binding protein, RsmA, in Ecc71 and Serra tia marcescens strain SM274. We propose a model that explains how RsmA and rsmB control the expression of genes for extracellular enzymes.