Regulatory Network Controlling Extracellular Proteins in Erwinia carotovora subsp. carotovora : FlhDC, the Master Regulator of Flagellar Genes, Activates rsmB Regulatory RNA Production by Affecting gacA and hexA ( lrhA ) Expression

Abstract

ABSTRACT 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 in Erwinia chrysanthemi ; LrhA [LysR homolog A] in Escherichia coli ) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC − mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin Ecc ) 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 the flhD operon of E. carotovora subsp. carotovora , like those of other enterobacteria, consists of flhD and flhC . Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA , rsmC , and fliA and negatively regulates hexA ( lrhA ). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA . Reduced levels of GacA and elevated levels of HexA in the FlhDC − mutant are responsible for the inhibition of rsmB RNA production, a condition conducive to the accumulation of free RsmA. Indeed, studies with an RsmA − FlhDC − double mutant and multiple copies of rsmB + DNA establish that the negative effect of FlhDC deficiency is exerted via RsmA. The FlhDC-mediated regulation of fliA has no bearing on exoprotein production in E. carotovora subsp. carotovora . Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA, and rsmB RNA in the context of the exoprotein production and virulence of E. carotovora subsp. carotovora .