Characterization of the pecT control region from Erwinia chrysanthemi 3937.

AUTOR(ES)

Castillo, A

RESUMO

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.

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