PehN, a Polygalacturonase Homologue with a Low Hydrolase Activity, Is Coregulated with the Other Erwinia chrysanthemi Polygalacturonases

Abstract

Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-␣-D-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi. Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN. The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent pehN transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in peh regulation.The enterobacterium Erwinia chrysanthemi is a well-characterized phytopathogen which causes soft-rot disease of various plants. The typical soft-rot maceration of plant tissues involves the depolymerization of the pectin in plant cell walls. The cleavage of this polysaccharide, an ␣-1,4-galacturonan (or polygalacturonate), is accomplished by a variety of microbial or fungal enzymes produced, in particular, by plant pathogens and by saprophytic microorganisms. Variations in the structure of the galacturonan chain occur in the extent of esterifications by O-acetyl and methoxyl groups, as well as in some modifications, such as xylose substitution or insertion in the polysaccharide backbone of single 1,2-linked ␣-L-rhamnose (27,37,46). Two type...

Molecular Cloning and Expression of Cellulase and Polygalacturonase Genes in E. coli as a Promising Application for Biofuel Production

“…Comparison of these results with those of others [58] reveals replacement of a histidine residue with an arginine residue in their identified pehN gene of E. chrysanthemi 3937. The possible replacement of the pehN gene was inferred to suggest a mechanism adapted to different substrate specificities [58].…”

Definition of a Consensus DNA-binding Site for PecS, a Global Regulator of Virulence Gene Expression in Erwinia chrysanthemi and Identification of New Members of the PecS Regulon

“…The PecS Consensus Is Present in the Regulatory Regions of the Target Genes-By using in vitro DNase I footprinting and missing contact experiments, the presence and position of PecS-binding sites has been identified on various controlled genes: celZ, pecS, pelD, pehX, pehV, pehW, and outC (22,23,25,26). Sequences displaying significant homology with the defined consensus were identified in each of the regions protected by PecS in DNase I footprinting experiments on these target genes (Fig.…”

“…DNA Sequences-Traditional in vitro methods for identifying protein binding sites, such as DNase I footprinting or missing contact experiments, failed to reveal a DNA-binding consensus for PecS (22,23,(25)(26)(27). Therefore, a SELEX strategy for selecting targets from random DNA sequences was adopted.…”

“…In E. chrysanthemi, PecS acts as a repressor on the production of the degradative enzymes, i.e. pectate lyases and cellulases (21), and up-regulates the synthesis of polygalacturonase enzymes (22,23). Furthermore, pecS mutants produce an extracellular blue pigment called indigoidine, which is involved in the resistance to the products of oxidative burst, including hydrogen peroxide (24).…”

“…Previous experiments carried out on 11 natural PecS target genes have suggested that PecS binds to DNA as a dimer and acts by means of a direct mechanism by interacting with the regulatory regions of the controlled genes (22)(23)(24)(25)(26)(27). Despite these works, relatively little is known about how PecS regulates gene expression.…”