Regulatory Network Controlling Extracellular Proteins inErwinia carotovorasubsp.carotovora: FlhDC, the Master Regulator of Flagellar Genes, ActivatesrsmBRegulatory RNA Production by AffectinggacAandhexA(lrhA) Expression

Erwinia carotovora subsp.carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal,N -acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA,rsmB RNA).rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT inErwinia chrysanthemi ; LrhA [LysR homolog A] inEscherichia coli ) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA withrsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to theflhDC operon ofE. coli also controls extracellular enzyme production. A transposon insertion FlhDC− mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, andE. carotovora subsp.carotovora Harpin (HarpinEcc ) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC+ plasmid. Sequence analysis and transcript assays disclosed that theflhD operon ofE. carotovora subsp.carotovora , like those of other enterobacteria, consists offlhD andflhC . Complementation analysis revealed that the regulatory effect requires functions of bothflhD andflhC products. The data presented here show that FlhDC positively regulatesgacA ,rsmC , andfliA and negatively regulateshexA (lrhA ). Evidence shows that FlhDC controls extracellular protein production through cumulative effects onhexA andgacA . Reduced levels of GacA and elevated levels of HexA in the FlhDC− mutant are responsible for the inhibition ofrsmB RNA production, a condition conducive to the accumulation of free RsmA. Indeed, studies with an RsmA− FlhDC− double mutant and multiple copies ofrsmB + DNA establish that the negative effect of FlhDC deficiency is exerted via RsmA. The FlhDC-mediated regulation offliA has no bearing on exoprotein production inE. carotovora subsp.carotovora . Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA, andrsmB RNA in the context of the exoprotein production and virulence ofE. carotovora subsp.carotovora .