Regulatory network controlling extracellular proteins in Erwinia carotovora ssp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory sRNA production by affecting gacA and hexA (lrhA) expression

Division of Plant Sciences, 108 Waters Hall, University of Missouri, Columbia, MO 65211, U. S. A.

^* To whom correspondence should be addressed. Email: chatterjeeas{at}missouri.edu.

	Abstract

Erwinia carotovora ssp. carotovora (Ecc) produces an array of extracellular proteins (= exoproteins) including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are co-regulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, RsmC) and post-transcriptional 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 E. 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 Tn-insertion FlhDC^- mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease and 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 Ecc, 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 inhibition of rsmB RNA production, a condition conducive to 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 Ecc. Our observations for the first time establish a regulatory connection between FlhDC, HexA, GacA and rsmB RNA in the context of exoprotein production and virulence of Ecc.