Inactivation of rsmA leads to overproduction of extracellular pectinases, cellulases, and proteases in Erwinia carotovora subsp. carotovora in the absence of the starvation/cell density-sensing signal, N-(3-oxohexanoyl)-L- homoserine lactone

Abstract

The soft-rotting bacterium, Erwinia carotovora subsp. carotovora 71, produces extracellular enzymes such as pectate lyase isozymes (Pels), cellulase (Cel), polygalacturonase (Peh), and protease (Prt). While the extracellular levels of these enzymes are extremely low when the bacterium is grown in salts-yeast extract-glycerol (SYG) medium, the enzymatic activities are highly induced in SYG medium supplemented with celery extract. By transposon (mini-Tn5) mutagenesis, we isolated a RsmA- mutant, AC5070, which overproduces extracellular enzymes; the basal levels of Pel, Peh, and Cel in AC5070 are higher than the induced levels in the RsmA+ parent, AC5047. While Peh production is mostly constitutive in AC5070, Pel, Cel, and Prt production is still inducible with celery extract. The high basal levels of pel-1, pel- 3, and peh-1 mRNAs in AC5070 demonstrate that overproduction of the pectolytic enzymes is due to the stimulation of transcription. Using chromosomal DNA flanking mini-Tn5 as a probe, we cloned the wild-type rsmA+ allele, which suppresses Pel, Peh, Cel, and Prt production in both RsmA+ and RsmA- strains. The RsmA- mutant, like its parent, produces N-(3- oxohexanoyl)-L-homoserine lactone (HSL), a starvation/cell density-sensing signal required for extracellular enzyme production. To examine the role of HSL, we constructed HSL-deficient strains by replacing hslI, a locus required for HSL production, with hslI::Tn3HoHo1-Spc. While the basal levels of Pel, Peh, Cel, and Prt are comparable in the RsmA- mutant and its HSL- derivative, these enzymes are barely detectable in the Hsl- derivative of the RsmA+ parent strain. The Hsl- RsmA+ strain fails to elicit soft rot, whereas the Hsl- RsmA- strain, like its Hsl+ RsmA- parent, remains hypervirulent. These findings demonstrate that the RsmA-mutant produces extracellular enzymes and macerates plant tissue in the absence of HSL. We conclude that overproduction of extracellular enzymes in an HSL-independent manner occurs because of the inactivation of a global repressor locus, rsmA.