Competition between VanU G Repressor and VanR G Activator Leads to Rheostatic Control of vanG Vancomycin Resistance Operon Expression

International audienceEnterococcus faecalis BM4518 is resistant to vancomycin by synthesis of peptidoglycan precursors ending in D-alanyl-D-serine. In the chromosomal vanG locus, transcription of the resistance genes from the P-YG resistance promoter is inducible and, upstream from these genes, there is an unusual three-component regulatory system encoded by the vanURS(G) operon from the P-UG regulatory promoter. In contrast to the other van operons in enterococci, the vanG operon possesses the additional vanU(G) gene which encodes a transcriptional regulator whose role remains unknown. We show by DNase I footprinting, RT-qPCR, and reporter proteins activities that VanU(G), but not VanR(G), binds to P-UG and negatively autoregulates the vanURSG operon and that it also represses PYG where it overlaps with VanR(G) for binding. In clinical isolate BM4518, the transcription level of the resistance genes was dependent on vancomycin concentration whereas, in a Delta vanUG mutant, resistance was expressed at a maximum level even at low concentrations of the inducer. The binding competition between VanU(G) and VanR(G) on the P-YG resistance promoter allowed rheostatic activation of the resistance operon depending likely on the level of VanR(G) phosphorylation by the VanS(G) sensor. In addition, there was cross-talk between VanS(G) and VanR'(G), a VanR(G) homolog, encoded elsewhere in the chromosome indicating a sophisticated and subtle regulation of vancomycin resistance expression by a complex two-component system

Two-Component Regulatory Systems : the moment of truth

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Régulation multifactorielle du système respiratoire triméthylamine N-oxyde (TMAO) réductase chez Escherichia coli

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Fonctionnement et régulation des systèmes respiratoires triméthylamine N-oxyde (TMAO) réductase chez Escherichia coli et Shewanella oneidensis

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Etude de la réponse adaptative à l'oxyde de triméthylamine et de son mécanisme de détection chez Escherichia coli et Shewanella oneidensis

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Caracterisation of the KplE1 prophage site-specific recombination module in Escherichia coli (from intasome assembly to genetics regulation)

KplE1 est l un des dix prophages présents sur le chromosome de la souche Escherichia coli K12. Nous avons montré in vivo que ce prophage est compétant pour s exciser du chromosome bactérien bien qu il soit incapable de former des particules virales et de lyser son hôte. Au laboratoire, nous avons identifié les protéines IntS (intégrase) et TorI (RDF), codées sur le prophage KplE1, et la protéine IHF (NBP) de l hôte comme seules impliquées dans le mécanisme de recombinaison spécifique de site (RSS). Nous avons cartographié sur les régions attL et attR, les sites de fixations des protéines de recombinaison permettant l assemblage de l intasome, le complexe nucléoprotéique compétant pour la RSS. L ensemble de ces sites ainsi que les gènes intS et torI qui chevauchent respectivement les régions attL et attR, ont permis de définir un module de recombinaison de type KplE1. Ce module est très conservé et se retrouve chez des phages infectant différentes souches d E. coli et de shigella. Le modèle en terme de RSS est celui décrit pour les bactériophages de type . Cependant, le nombre et l organisation des sites de recombinaison suggèrent que l architecture de l intasome de type KplE1 diffère de celle de . Nos résultats renforcent ainsi l idée que l assemblage de l intasome est spécifique du module de RSS considéré même si, in fine, la réaction catalysée demeure similaire.En ce qui concerne l expression des gènes intS et torI, le fait que ces gènes soient localisés à chacune des extrémités du prophage, rend ainsi impossible leur couplage transcriptionnel à partir d un promoteur commun au moment de la commutation lyse/lysogénie, tel qu il est connu pour les phages lambdoïdes. De part son orientation atypique sur attL, la présence de sites de fixations des protéines IntS et TorI au niveau du promoteur du gène intS, nous ont logiquement amené à étudier sa régulation. Nous avons ainsi montré que le gène intS est négativement régulé par son propre produit ainsi que par la protéine RDF TorI. Nos résultats in vivo et in vitro indiquent que l efficacité de la réaction de recombinaison excisive est intimement liée à la quantité d intégrase présente, pouvant alors justifier la raison d être de ce contrôle strict de l expression du gène intS. En parallèle, une approche in silico a révélé que cette orientation atypique du gène codant pour l intégrase est largement répandue sur les génomes des prophages, nous amenant à généraliser ce mécanisme atypique de régulation négative de l intégrase.KplE1 is one of the 10 prophage region present on the Escherichia coli K12 chromosome. We showed in vivo that this prophage is fully competent to excise from the bacterial chromosome, although it is unable to form viral particles and lyse its host. In the laboratory, we have identified Ints (integrase) and TorI (RDF) proteins, encoded on the KplE1 prophage, and the host protein IHF (NBP) only involved in the mechanism of site-specific recombination (SSR). We have mapped on attL and attR regions, the binding sites of recombinant proteins for the assembly of the intasome, the nucleoprotein complex competent for SSR. All of these sites as well as intS and torI genes that overlap respectively attL and attR regions, have permit to define a KplE1 recombination module. This module is highly conserved and is found among phages infecting different E. coli and shigella strains. The model in terms of RSS is that described for bacteriophage. However, the number and organization of recombination sites suggests that the architecture of the KplE1 intasome differs from that of . Our findings reinforce the idea that the intasome assembly is specific to the SSR module considered even if ultimately the catalyzed reaction is similar.Regarding the intS and torI gene expressions, the fact that these genes are located at each end of the prophage, prevented the transcriptional coupling of these genes from a common promoter when the lysis/lysogeny switch occurs. Because of its atypical orientation on attL, and the presence of IntS and TorI protein binding sites that overlap its promoter region, we have logically studied the regulation of the intS gene. We have shown that intS is negatively regulated by both IntS and TorI proteins. Our in vivo and in vitro results suggest that the efficiency of the excision recombination reaction is closely related to the amount of this integrase, which can justify the strict control of the intS gene expression. In parallel, an in silico approach has revealed that the atypical orientation of the integrase gene is widespread in prophage genomes, leading us to generalize this atypical mechanism of negative regulation of integraseAIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Identification par approche génomique des gènes contrôlés par le système de régulation TorS/TorR en réponse au triméthylamine N-oxyde (TMAO) chez Escherichia coli et Shewanella oneidensis

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Competition between VanU_G and VanR_G for binding to the <i>P_YG</i> resistance promoter.

<p>(A) DNase I footprinting analysis. A 233-bp DNA fragment was amplified from the <i>PYG</i> region using a labeled reverse primer (YG10) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005170#pgen.1005170.s006" target="_blank">S2 Table</a>) to radiolabel the template strand. Increasing amounts of VanR_G and two fixed amounts of VanU_G, indicated at the top, were incubated with the DNA probe. The bracket indicates the region protected from DNase I cleavage by VanU_G and/or VanR_G and the co-ordinates of protection relative to the transcriptional start site are indicated on the left. M is the A+G Maxam and Gilbert sequencing reaction lane of the probes used as a size marker and the nucleotide positions are indicated at the right. (B) Model for the binding competition between VanU_G and VanR_G-P in the absence or in the presence of various concentrations of vancomycin (Vm).</p

Schematic representation of the <i>vanG</i> operon.

<p>Open arrows represent coding sequences and indicate direction of transcription. The regulatory genes are in red, the resistance genes in blue and accessory genes in green. The additional regulatory gene, <i>vanUG</i>, is in yellow. The vertical bar in <i>vanYG</i> indicates a frameshift mutation leading to a truncated protein.</p

Growth rate.

<p>^a Exponential growth rate measured in the absence of antibiotic or in the presence of vancomycin (1μg/ml) (Vm1); average of at least four independent experiments ± standard deviations.</p><p>^b Relative growth rate was calculated as the ratio of the growth rate of the strain induced by 1μg/ml of vancomycin versus the non induced strain.</p><p>Growth rate.</p