rsmC of the Soft-Rotting Bacterium Erwinia carotovora subsp. carotovora Negatively Controls Extracellular Enzyme and Harpin Ecc Production and Virulence by Modulating Levels of Regulatory RNA ( rsmB ) and RNA-Binding Protein (RsmA)

Abstract

ABSTRACT Previous studies have shown that the production of extracellular enzymes (pectate lyase [Pel], polygalacturonase [Peh], cellulase [Cel], and protease [Prt]) and harpin Ecc (the elicitor of hypersensitive reaction) in Erwinia carotovora subsp. carotovora is regulated by RsmA, an RNA-binding protein, and rsmB , a regulatory RNA (Rsm stands for regulator of secondary metabolites) (Y. Liu et al., Mol. Microbiol. 29:219–234, 1998). We have cloned and characterized a novel regulatory gene, rsmC , that activates RsmA production and represses extracellular enzyme and harpin Ecc production, rsmB transcription, and virulence in E. carotovora subsp. carotovora . In an rsmC knockout mutant of E. carotovora subsp. carotovora Ecc71 carrying the chromosomal copy of the wild-type rsmA + allele, the basal levels of Pel, Peh, Cel, Prt, and harpin Ecc as well as the amounts of rsmB , pel-1 , peh-1 , celV , and hrpN Ecc transcripts are high, whereas the levels of rsmA transcripts and RsmA protein are low. Furthermore, the expression of an rsmA-lacZ gene fusion is lower in the RsmC − mutant than in the RsmC + parent. Conversely, the expression of an rsmB-lacZ operon fusion is higher in the RsmC − mutant than in the RsmC + parent. These observations establish that RsmC negatively regulates rsmB transcription but positively affects RsmA production. Indeed, comparative studies with an RsmC − mutant, an RsmA − mutant, and an RsmA − RsmC − double mutant have revealed that the negative effects on exoprotein production and virulence are due to the cumulative regulatory effects of RsmC on rsmA and rsmB . Exoprotein production by the RsmC − mutant is partially dependent on the quorum sensing signal, N -(3-oxohexanoyl)- l -homoserine lactone. Southern blot data and analysis of PCR products disclosed the presence of rsmC sequences in E. carotovora subsp. atroseptica , E. carotovora subsp. betavasculorum , and E. carotovora subsp. carotovora . These findings collectively support the idea that rsmA and rsmB expression in these plant pathogenic Erwinia species is controlled by RsmC or a functional homolog of RsmC.