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)

Previous studies have shown that the production of extracellular enzymes (p ectate 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 metaboli tes) (Y. Liu et al., Mel. 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 transc ription, and virulence in E. carotavora subsp. carotovora. In an rsmC knock out mutant of E. carotovora subsp. carotovora Ecc71 carrying the chromosoma l copy of the wild-type rsmA(+) allele, the basal levels of Pel, Feb, Gel, Prt, and harpin(Ecc) as well as the amounts of rsmB, pel-1, peh-1, celV, an d hrpN(Ecc) transcripts are high, whereas the levels of rsmA transcripts an d RsmA protein are low. Furthermore, the expression of an rsmA-lacZ gene fu sion 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(-) muta nt than in the RsmC(+) parent. These observations establish that RsmC negat ively regulates rsmB transcription but positively affects RsmA production. Indeed, comparative studies with an RsmC(-) mutant, an RsmC(-) mutant, and an RsmA(-) RsmC(-) double mutant have revealed that the negative effects on exoprotein production and virulence are due to the cumulative regulatory e ffects of RsmC on rsmA and rsmB. Exoprotein production by the RsmC(-) mutan t is partially dependent on the quorum sensing signal, N-(3-oxohexanoyl) -L -homoserine lactone. Southern blot data and analysis of PCR products disclo sed the presence of rsmC sequences in E. carotovora subsp. atroseptica, E, carotovora subsp. hetavasculorum, and E. carotovora a subsp. carotovora. Th ese findings collectively support the idea that rsmA and rsmB expression in these plant pathogenic Erwinia species is controlled by RsmC or a function al homolog of RsmC.