Purification and functional characterization of PecS, a regulator of virulence-factor synthesis in Erwinia chrysanthemi.
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.
Praillet
T
T
Laboratoire de Génétique Moléculaire des Microorganismes, CNRS-UMR 5577, Villeurbanne, France.
Nasser
W
W
Robert-Baudouy
J
J
Reverchon
S
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D016974:Dickeya chrysanthemi / Q000378:metabolism* / Q000472:pathogenicity; D004885:Erwinia / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014774:Virulence
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C088080:pecS protein, Erwinia chrysanthemi; D003850:Deoxyribonuclease I