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

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.Erwinia carotovora subsp. carotovora (Ecc), a member of the Enterobacteriaceae family, causes soft-rotting disease on a wide variety of plants worldwide. A consortium of plant cell walldegrading extracellular enzymes comprising pectate lyase (Pel), polygalacturonase (Peh), protease (Prt), and cellulase (Cel) contribute to its plant virulence (4,15,19,61,71). Among them, extracellular pectinases, including Pel and Peh, play a crucial role in tissue maceration and cell death. In addition, motility and some effectors secreted th...

A role for the Rcs phosphorelay in regulating expression of plant cell wall degrading enzymes in Pectobacterium carotovorum subsp. carotovorum

“…butes to the virulence of Pcc (Cui et al, 2008). The results of this study demonstrate that inactivation of rcsB in an flhDC-negative mutant does not restore expression of PCWDE.…”

“…2a), demonstrating that direct transcriptional regulation of rprA and flhDC expression by the Rcs phosphorelay is not exclusive to E. coli and Salmonella (Francez-Charlot et al, 2003;Majdalani et al, 2002;Wang et al, 2007). The flagella master regulator, FlhDC, has been previously shown to positively regulate RsmB RNA levels in Pcc (Cui et al, 2008;Fig. 2b); thus RcsB may regulate transcription of rsmB both directly and indirectly through modulating the expression of flhDC.…”

“…Since the flhDC mutant and the flhDC/rcsB double mutant exhibited the same phenotype, a model where the Rcs phosphorelay targets PCWDE genes indirectly via FlhDC is supported. Cui et al (2008) reported that FlhDC activates expression of PCWDE indirectly by reducing the activity of the Rsm regulatory system. Based on these data and our results, we hypothesize that the Rcs phosphorelay regulates the expression of PCWDE by modulating the activity of the Rsm system directly (only in certain conditions) by regulating rsmB expression and indirectly through flhDC (Fig.…”

“…Recently, the Rsm system was shown to be dependent on expression of a global regulator of flagellar genes, FlhDC. FlhDC activates the expression of PCWDE in Pcc strain 71 by modulating transcription of RsmB (Cui et al, 2008).…”

“…Recently, the Rsm system was shown to be dependent on expression of a global regulator of flagellar genes, FlhDC. FlhDC activates the expression of PCWDE in Pcc strain 71 by modulating transcription of RsmB (Cui et al, 2008).Motility is a key feature of many microbes' physiology, and flagella provide the necessary propulsion to the microbes in a liquid environment. FlhDC, which regulates the expression of flagellar genes (for a review, see Prüß, 2000), also regulates the consumption of glucose (Entner-Doudoroff pathway) and anaerobic respiration in Escherichia coli, as well as induction of purine and pyrimidine synthesis and repression of the urea cycle in Yersinia enterocolitica (Kapatral et al, 2004; Prüß et al, 2001 Prüß et al, , 2003 motility is also a pathogenicity determinant in pectobacteria, and appears to be necessary for the successful invasion and infection of a plant host (Pirhonen et al, 1991; Pérombelon, 2002).…”