The PecT repressor interacts with regulatory regions of pectate lyase genes in Erwinia chrysanthemi.
Erwinia chrysanthemi is a broad host range phytopathogenic enterobacterium responsible for soft-rot disease of many plant species. The pecT gene encodes a repressor that negatively regulates the expression of virulence factors, such as pectinases, motility or exopolysaccharide synthesis. The cloned pecT gene was overexpressed using a phage T7 system. The purification of PecT involved the use of a TSK-heparin column and delivered the PecT protein that was purified to near homogeneity. The purified repressor displayed a 34 kDa apparent molecular mass. Gel-filtration experiments revealed that the PecT protein is a dimer. Band-shift assays demonstrated that the tetramer of the PecT protein could specifically bind in vitro to the regulatory regions of the pectate lyase genes with variable affinities. In addition, we demonstrated that PecT represses its own synthesis by interacting independently with two 200 bp regions, R1 and R2, located from -382 to -632 and -17 to -234, respectively, from the distal P1 promoter and from -465 to -715 and -100 to -317 from the P2 proximal promoter. We propose a model that explains the regulation exerted by PecT on its target genes and that integrates the phenotype obtained with a PecT overproducing pec-1 mutant or a pecT mutant.
Castillo
A
A
Laboratoire de Génétique Moléculaire des Microorganismes, CNRS UMR 5577 INSA, Bat 406, 20 Avenue Albert Einstein, 69621 Villeurbanne, France.
Nasser
W
W
Condemine
G
G
Reverchon
S
S
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D016974:Dickeya chrysanthemi / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology; D010944:Plants / Q000382:microbiology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C098627:PecT protein, Erwinia chrysanthemi; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D011133:Polysaccharide-Lyases; C023911:pectate lyase