hexA of Erwinia carotovora ssp carotovora strain Ecc71 negatively regulates production of RpoS and rsmB RNA, a global regulator of extracellular proteins, plant virulence and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone

The soft-rotting bacterium, Erwinia carotovora ssp. carotovora (E. c. carotovora), produces an array of extracellular enzymes (= exoenzymes), including pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel) and protease (Prt), as well as Harpin(Ecc), the elicitor of hypersensitive reaction (HR). The production of these exoenzymes and Harpin(Ecc),, responds to plant products and the quorum-sensing signal [N-(3-oxohexanoyl)-L-homoserine lactone; OHL] and is subject to both transcriptional and post-transcriptional regulation. hexA of E. c. carotovora strain Ecc71 (hereafter hexA(71),), like that of another E. c. carotovora strain, negatively controls the production of exoenzymes, OHL and virulence in E. c. carotovora strain Ecc71. In addition to exoenzymes, HexA(71) negatively regulates the expression of hrpN(Ecc),,, the structural gene for Harpin(Ecc). Exoenzyme overproduction is abolished by OHL deficiency in a HexA(-) and OhII(-) double mutant, indicating that HexA and OHL are components of a common regulatory pathway controlling exoenzyme production. HexA(71) negatively affects RpoS, as the levels of this alternative sigma factor are higher in the HexA- mutant than in the HexA(71) strain. However, a HexA- and RpoS(-) double mutant produces higher levels of exoenzymes and transcripts of pel-l, peh-1 and ceIV genes than the HexA(-) and RpoS(+) parent. Thus, the elevated levels of RpoS protein in the HexA- mutant do not account for exoenzyme overproduction. The following evidence associates for the first time the phenotypic changes in the HexA(-) mutant to overproduction of rsmB RNA, a global regulator of exoenzymes, Harpin(Ecc),,, OHL and secondary metabolites. Analyses of rsmB transcripts and expression of an rsmB-lacZ operon fusion in E. c. carotovora strain Ecc71 revealed that HexA(71), negatively regulates transcription of rsmB. Multiple copies of hexA(71)(+) RNA suppress various phenotypes, including exoenzyme production in E. c. carotovora strain Ecc71, and concomitantly inhibit the production of rsmB, pel-1, peh-1, ceIV and hrpN(Ecc) transcripts. Multiple copies of rsmB(+) DNA, on the other hand, stimulate exoenzyme production by relieving the negative effects of a chromosomal copy of hexA(+). The occurrence of hexA homologues and the negative effect of the dosage of hexA,, DNA on rsmB transcripts were also detected in other E. c. carotovora strains as well as Erwinia carotovora atroseptica and Erwinia carotovora betavasculorum. Extrapolating from the findings with LrhA, the Escherichia coli homologue of HexA, and the presence of sprE homologues in E. carotovora subspecies, we propose that HexA,, controls several regulatory pathways in E. caratovora including rsmB transcription and the production of SprE(Ecc),, which, in turn, affects RpoS levels. A model is presented that integrates the findings presented here and our current knowledge of the major regulatory network that controls exoprotein production in soft-potting Erwinia carotovora subspecies.