1.5.6S
TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL REGULATION OF SECRETED PROTEINS IN ERWINIA CAROTOVORA SUBSP. CAROTOVORA

AK CHATTERJEE, YANG LIU, Y CUI, C KORSI DUMENYO and A MUKHERJEE

Department of Plant Pathology, University of Missouri, Columbia, MO 65211, USA

Erwinia carotovora subsp. carotovora (Ecc) produces extracellular enzymes (exoenzymes), such as pectate lyase, isozymes (Pel), cellulase (Cel), polygalacturonase (Peh) and protease (Prt), as well as the quorum-sensing signal, N-acyl homoserine lactone (AHL), and HarpinEcc, the elicitor of a hypersensitive reaction (HR). While exoenzymes, especially the pectinases, are known to be required for tissue maceration, the role of HarpinEcc in disease development awaits clarification. However, the production of exoenzymes and HarpinEcc occurs in a growth-phase-dependent manner and responds to AHL. Studies in several laboratories, including our own, have led to the identification of several additional factors that control exoenzyme production. For example, we have documented that transcription in Ecc strain 71 is affected by aepA and aepB genes (activators of extracellular protein production), as well as plant signals. Inactivation of aepA and aepB abolishes exoenzyme production. Another set of genes, designated rsm for regulator of secondary metabolites, also controls exoenzyme production in Ecc. We have thus far identified four such genes: rsmA which encodes a 6.8-kDa RNA-binding protein, rsmB which specifies an RNA regulator; rsmC which controls rsmC expression; and rsmD which specifies a LysR-type regulator.

We have determined that the genes for those exoenzymes, HarpinEcc and AHL, are also subject to post-transcriptional regulation. For example, the levels of mRNAs of pel and peh, hrpNEcc (the structural gene for HarpinEcc) and ahl1, the gene specifying AHL synthesis, are negatively affected by RsmA. The accumulation of transcripts of those and other genes in RsmA- mutants causes a profound change in their phenotype which is characterized by over-production of Pel, Pch, Cel, Prt and HarpinEcc; hypervirulence; and the ability to elicit the HR. Moreover, RsmA- mutants do not require the quorum-sensing signal, as the RsmA- and Ahl- double mutants produce exoenzymes and HarpinEcc like their Ahl+ counterparts. The available evidence suggests that RsmA controls gene expression by affecting mRNA stability. RsmB, formerly aepH [1], stimulates the production of pel, peh and hrpNEcc transcripts as well as Cel and Prt activities. The RsmB gene specifies two RNA species: a full-length 479-base rsmB RNA, and a 259-base rsmB RNA; the latter apparently through processing of the primary rsmB RNA. rsmB RNA causes over-production of Pel, Peh, Cel, Prt, HarpinEcc and AHL, primarily by neutralizing the negative effect of RsmA. Since structural or functional homologues of rsmA and rsmB occur in various Erwinia and enterobacterial species including Escherichia coli, we predict that the post-transcriptional regulatory system consisting of RsmA and rsmB has been conserved in plant and animal pathogenic bacteria.

The regulation of pectin lyase (Pnl) production in Ecc and most other soft-rotting Erwinia occurs by a very different pathway in sponse to signals generated upon DNA damage [2]. It entails the following sequential events: activation of RecA; RecA*-promoted cleavage of RdgA, which functions as an auto-repressor and a repressor of rdgB; synthesis of RdgB; and then the activation of transcription of pnlA, the structural gene for Pnl by RdgB. We propose that Pnl is an ancillary virulence factor whose production is activated only upon stress inflicted by DNA-damaging agents. It is now apparent that Ecc and other soft-rotting Erwinia have developed highly sophisticated and novel regulatory systems that control the production of pathogenicity/virulence factors.

References
1. Murata H, Chatterjee A, Liu Y, Chatterjee AK, 1994. Applied and Environmental Microbiology 60, 3150-3159.
2. Liu Y, Wang X, Mukherjee A, Chatterjee AK, 1996. Mol. Microbiol. 22, 909-918.