1.5.4S
COMPLEX REGULATORY NETWORK DIRECTING THE SYNTHESIS OF PECTINASES, THE MAIN VIRULENCE FACTORS OF ERWINIA CHRYSANTHEMI

W NASSER, G CONDEMINE, N HUGOUVIEUX-COTTE-PATTAT, J ROBERT-BAUDOUY and S REVERCHON

Laboratoire de Genetique Moleculaire des Microorganismes et des Interactions Cellulaires, CNRS UMR 5577, INSA Batiment 406, 20 Avenue Albert Einstein, 69621 Villeurbanne, France

Background and objectives
The phytopathogenicity of the plant pathogenic soft-rot enterobacteria Erwinia is correlated with their ability to produce and secrete plant cell wall-degrading enzymes such as pectinases, cellulases and proteases. Of these enzymes, pectinases have attracted most attention because of their capacity to macerate plant tissue. Erwinia chrysanthemi strain 3937 synthesizes multiple isoforms of pectinases, including the product of pelA, B, C ,D, E genes, that cleave pectin and generate unsaturated digalacturonate. These compounds are transported into the bacterium, where they are catabolized by the product of the genes ogl, kdul, kduD, kdgK and kdgA [1). Expression of the pectinase genes responds to several environmental and metabolic stimuli, including pectin degradation products, temperature, catabolic repression, presence of plant extracts and growth phase [1]. The major goal of our laboratory is to elucidate the molecular mechanisms directing pectinase gene expression.

Results and conclusions
ln vivo mutagenesis experiments have led to the identification of three regulatory loci kdgR, pecs-pecM and pecT [1]. The KdgR protein negatively regulates all of the genes involved in pectin degradation. kdgR interacts with the target DNA via a 17-bp motif that is highly conserved in the regulatory regions of the majority of the pectinolysis genes. Moreover, the presence of 2-keto-3-deoxygluconate (KDG), an inducer of pectinolysis, prevents the binding of the KdgR repressor to its operators. The pecS-pecM locus controls the synthesis of the pectinases, some proteins of the secretion machinery, the cellulase EGZ and the production of a blue pigment. According to the results of subcellular fractionation, a transmembrane sensor function and a cytoplasmic regulator role were assigned to PecM and PecS, respectively. In vitro experiments have demonstrated that the PecS repressor acts by specific interaction with the regulatory regions of the controlled genes. PecS also controls its own gene expression and that of the adjacent gene pecM. The PecM protein modulates the regulatory activity of PecS. Indeed, the DNA-binding capacity of PecS is strongly reduced in a pecM mutant. The pecT gene encodes a protein of the LysR family which represses the expression of some pel genes. PecT acts by direct binding to the regulatory region of the controlled genes. In addition to these three negative regulators, recent work allowed the identification of two activator loci: crp and expl-expR. The E. chrysanthemi crp mutant is deficient in pectate lyase production and severely affected in its virulence on saintpaulia plants.

The position of the CRP-binding sites relative to the transcription start sites suggests that CRP acts as a primary activator at the peIB, peIC, pelE, pelD, kdgTpi and oglP promoters. On the other hand, transcription at the kdul and oglPi promoters seems to require another transcriptional activator acting in synergy with CRP [2]. Investigation of the simultaneous binding of CRP and KdgR to the regulatory regions of the pectinolysis genes showed that binding of KdgR is preferential and exclusive in the case of ogl and kdgT, whereas the binding of these two regulators is independent in the case of pelB, C, D, E and kdul. These data indicate that the antagonistic effects of CRP and KdgR on the expression of the pectinolysis genes occur by different mechanisms, including direct competition between the two regulators or between the repressor and RNA polymerase for the occupation of a common DNA region on the target genes [2]. The expl-expR locus directs the synthesis of two acyl-homoserine lactones and partially ensure the growth phase regulation of the pectinase gene expression. CRP acts as an activator of expR expression but as a repressor of expl transcription. A second level of control of expR and expl occurs through the PecS repressor. PecS represses expl expression while ExpR activates pecS transcription. Regulation of pectinase synthesis in soft rot Erwinia then appears as a complex network of multiple cross-acting regulatory elements.

References
1. Hugouvieux-Cotte-Pattat N, Condemine G, Nasser WI, Reverchon S, 1998. Annual Review of Microbiology 50, 213-257.
2. Nasser W, Robert-Baudouy J, Reverchon S, 1997. Microbiology 26,1071-1082.