Definition of a consensus DNA-binding site for PecS, a global regulator of virulence gene expression in Erwinia chrysanthemi and identification of new members of the PecS regulon.
In Erwinia chrysanthemi, production of pectic enzymes is modulated by a complex network involving several regulators. One of them, PecS, which belongs to the MarR family, also controls the synthesis of various other virulence factors, such as cellulases and indigoidine. Here, the PecS consensus-binding site is defined by combining a systematic evolution of ligands by an exponential enrichment approach and mutational analyses. The consensus consists of a 23-base pair palindromic-like sequence (C(-11)G(-10)A(-9)N(-8)W(-7)T(-6)C(-5)G(-4)T(-3)A(-2))T(-1)A(0)T(1)(T(2)A(3)C(4)G(5)A(6)N(7)N(8)N(9)C(10)G(11)). Mutational experiments revealed that (i) the palindromic organization is required for the binding of PecS, (ii) the very conserved part of the consensus (-6 to 6) allows for a specific interaction with PecS, but the presence of the relatively degenerated bases located apart significantly increases PecS affinity, (iii) the four bases G, A, T, and C are required for efficient binding of PecS, and (iv) the presence of several binding sites on the same promoter increases the affinity of PecS. This consensus is detected in the regions involved in PecS binding on the previously characterized target genes. This variable consensus is in agreement with the observation that the members of the MarR family are able to bind various DNA targets as dimers by means of a winged helix DNA-binding motif. Binding of PecS on a promoter region containing the defined consensus results in a repression of gene transcription in vitro. Preliminary scanning of the E. chrysanthemi genome sequence with the consensus revealed the presence of strong PecS-binding sites in the intergenic region between fliE and fliFGHIJKLMNOPQR which encode proteins involved in the biogenesis of flagellum. Accordingly, PecS directly represses fliE expression. Thus, PecS seems to control the synthesis of virulence factors required for the key steps of plant infection.
Rouanet
Carine
C
Unité de Microbiologie et Génétique CNRS-Institut National des Sciences Appliquées-Université Claude Bernard Lyon, Unité Mixte de Recherche 5122 Université Claude Bernard Lyon, 69622 Villeurbanne, France.
Reverchon
Sylvie
S
Rodionov
Dmitry A
DA
Nasser
William
W
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D016974:Dickeya chrysanthemi / Q000472:pathogenicity*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011196:Potassium Permanganate / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014774:Virulence
D001426:Bacterial Proteins; D017931:DNA Primers; D008024:Ligands; D009841:Oligonucleotides; D012097:Repressor Proteins; C088080:pecS protein, Erwinia chrysanthemi; D011196:Potassium Permanganate; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases