Characterization of the pecT control region from Erwinia chrysanthemi 3937

Abstract

Erwinia chrysanthemi synthesizes and secretes pectate lyases that attack components of the plant cell wall and, therefore, play a major role in the pathogenesis of soft rot disease. We isolated a new mutant (designated pec-1), by Tn5 mutagenesis, that displays weak pectate lyase production and decreased motility and mucoidicity. Maceration and pathogenicity tests done on different plant organs showed that the pec-1 strain displays a reduced virulence compared to that of the parental strain. The Tn5 insertion was localized between the pelL and the out loci and defines a new regulatory region. Sequencing of the pec-1::Tn5 insertion revealed that pec-1 is tightly linked to the pecT regulatory gene that also controls pectate lyase synthesis. Moreover, the pecT mutation is dominant over the pec-1 mutation, suggesting that these two loci are involved in the same regulatory network. We demonstrated, by Northern blot analysis, that the pec-1::Tn5 insertion provokes derepression of pecT transcription and defines a cis-acting element. Introduction of the pecT gene in trans of a pecT::uidA fusion induced a decrease of pecT::uidA transcription, indicating a negative autoregulation. Band shift experiments confirmed that the PecT repressor specifically interacts with the pecT regulatory region. We also demonstrated that the PecT protein interacts with the regulatory region of the pelD gene encoding a pectate lyase. Therefore, the abolition of the pecT autoregulation in the pec-1 mutant provokes an overproduction of the PecT repressor that is responsible for the decrease of pectate lyase synthesis. Mutagenesis of the pecT regulatory region revealed the presence of two sites in which insertions reproduced the pec-1 phenotype. This result suggests that pecT autoregulation requires the presence of two functional operator sites. From this study, we propose that the PecT repressor binds to these two sites, generating a loop that blocks pecT transcription.

Erwinia carotovora has two KdgR-like proteins belonging to the IcIR family of transcriptional regulators: identification and characterization of the RexZ activator and the KdgR repressor of pathogenesis

“…Analysis of the distribution of these different regulators in bacteria of the genus Erwinia has revealed the presence of pecT/hexA (Castillo & Reverchon, 1997;Harris et al, 1998), expllcarl Reverchon et al, 1998;Jones et al, 1993;Pirhonen et al, 1993), and rexZ and kdgR homologues (this work) in both E. chrysantherni and E. carotovora. However, in vivo comparative studies for many of these proteins, such as PecT/HexA and proteins encoded by the quorum-sensing locus, suggest that the mechanism by which they are involved in pel gene expression is different in these two Erwinia species Reverchon et al, 1998 ;Harris et al, 1998).…”

“…In addition to kdgR, two other loci that negatively regulate the expression of the pectinase genes, pecS-pecM and pecT, have also been characterized in E. chrysanthemi. However, the signal to which they respond remains unknown Praillet et al, 1994;Surgey at al., 1996;Castillo & Reverchon, 1997).…”

“…In addition to kdgR, two other loci that negatively regulate the expression of the pectinase genes, pecS-pecM and pecT, have also been characterized in E. chrysanthemi. However, the signal to which they respond remains unknown Praillet et al, 1994;Surgey at al., 1996;Castillo & Reverchon, 1997).In the related soft-rot species, Erwinia carotovora subsp. caiotovoru, analysis of regulatory mutants has allowed the identification of several loci involved in the regulation of exoenzyme genes : expl/carI /hsll, hor, aepA, aepH (rsmB), rsmA and hexA.…”

Characterization of the Erwinia chrysanthemi expI–expR locus directing the synthesis of two N‐acyl‐homoserine lactone signal molecules

“…In E. chrysanthemi, it has been demonstrated that the full expression of the pectin catabolism genes requires the presence of the cAMP-CRP complex Reverchon et al, 1997) and that the KdgR repressor essentially mediates the induction of these catabolic pathway genes by pectic compounds (Reverchon et al, 1991;Nasser et al, 1994). In addition to kdgR, two other loci involved in negative regulation of the pectinase gene expression, pecS-pecM and pecT, were also characterized in E. chrysanthemi, but the signal to which they respond still remains unknown (Praillet et al, 1996;Reverchon et al, 1994;Surgey et al, 1996;Castillo and Reverchon, 1997).…”

Integration of the quorum‐sensing system in the regulatory networks controlling virulence factor synthesis in Erwinia chrysanthemi

“…Very few mutants with decreased pectinase production were obtained in E. chrysanthemi. Only the crp and pec-1 mutants are deficient in pectinase synthesis Castillo and Reverchon, 1997). While CRP, the global regulator of sugar catabolism, acts by a direct mechanism involving specific interactions with pel gene regulatory regions , the effect of the pec-1 mutation is indirect and results in an increased production of the PecT repressor (Castillo and Reverchon, 1997).…”