The Erwinia chrysanthemi pecS gene encodes a repressor that negatively regulates the expression of virulence factors such as pectinases or cellulases. The cloned pecS gene was overexpressed using a phage T7 system. The purification of PecS involved DEAE-anion exchange and TSK-heparin columns and delivered the PecS protein that was purified to homogeneity. The purified repressor displayed an 18 kDa apparent molecular mass and an isoelectric point near to neutrality (pl = 6.5). Gel-filtration experiments revealed that the PecS protein is a dimer. Bandshift assays demonstrated that the PecS protein could specifically bind in vitro to the regulatory sites of the in vivo PecS-regulated genes. The interaction between the PecS protein and its DNA-binding site was characterized by a relatively low affinity (about 10(-8) M). DNase I footprintings revealed short protected sequences only with the most in vivo PecS-regulated genes. Alignment of these PecS-binding sites did not show a well-conserved consensus sequence. Immunoblotting demonstrated that the copy number of the PecS protein was approximately 50 dimers per cell. The low affinity of the PecS repressor for its DNA targets and the low cellular PecS content suggest the existence of E. chrysanthemi-specific factors able to potentiate PecS protein activity in vivo.

SummaryThe Erwinia chrysanthemi pecS mutant displays constitutive production of virulence factors, such as pectinases or cellulases. Complementation of the pecS mutation can be obtained in the presence of the pecS wild-type gene on a low-copy-number plasmid. Moreover, the resulting plasmid decreases the expression of a pecS::uidA chromosomal fusion, indicating the existence of an autoregulation mechanism. This negative autoregulation was confirmed and quantified by analysis of the pecS transcripts using primer-extension experiments. Band-shift assays and DNase I footprinting experiments demonstrated that the PecS protein could bind to the intergenic regulatory region, located between the pecS and pecM genes, with a relatively high affinity (apparent dissociation constant (K Ј d ) close to 4 nM). These PecS-binding sites overlap the pecS and pecM promoters. The comparison of these new PecS-binding sites with those previously characterized on the target genes confirms the absence of a consensus. This observation was in accordance with the results of the missing-contact experiments performed on the pecS-pecM intergenic regulatory region and the celZ operator. Concurrently, we demonstrated that the PecS protein negatively controls the expression of the divergently transcribed pecM gene located 400 bp upstream from the pecS gene. By following the efficiency of pecS autoregulation in a double E. chrysanthemi pecM-pecS mutant, we established that the PecM protein potentiates PecS activity in vivo.

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The pecS regulatory locus is responsible for the down-expression of many virulence genes in Erwinia chrysanthemi. This locus consists of two genes, pecS and pecM, divergently transcribed. Genetic evidence indicates that the PecM protein modulates the regulatory activity of PecS. Purification and characterization of PecS, expressed either from E. coli, from the wild-type E. chrysanthemi strain or from a pecM mutant, showed that the PecS protein produced in these three genetic backgrounds displays the same biochemical properties. Band-shift assay analysis with the three PecS isoforms confirmed the involvement of the PecM protein in modulating the PecS DNA-binding capacity. Moreover, determination of the Kdapp for operator regions of the PecS protein, produced either by the wild-type E. chrysanthemi or by E. coli, reveals similar affinities. Thus, in E. coli, there is likely to be at least one other PecM-like protein able to cross-react with the E. chrysanthemi PecS protein.