Protein binding sites involved in the assembly of the KplE1 prophage intasome

AbstractThe organization of the recombination regions of the KplE1 prophage in Escherichia coli K12 differs from that observed in the λ prophage. Indeed, the binding sites characterized for the IntS integrase, the TorI recombination directionality factor (RDF) and the integration host factor (IHF) vary in number, spacing and orientation on the attL and attR regions. In this paper, we performed site-directed mutagenesis of the recombination sites to decipher if all sites are essential for the site-specific recombination reaction and how the KplE1 intasome is assembled. We also show that TorI and IntS form oligomers that are stabilized in the presence of their target DNA. Moreover, we found that IHF is the only nucleoid associated protein (NAP) involved in KplE1 recombination, although it is dispensable. This is consistent with the presence of only one functional IHF site on attR and none on attL

P2CS: a two-component system resource for prokaryotic signal transduction research

BACKGROUND: With the escalation of high throughput prokaryotic genome sequencing, there is an ever-increasing need for databases that characterise, catalogue and present data relating to particular gene sets and genomes/metagenomes. Two-component system (TCS) signal transduction pathways are the dominant mechanisms by which micro-organisms sense and respond to external as well as internal environmental changes. These systems respond to a wide range of stimuli by triggering diverse physiological adjustments, including alterations in gene expression, enzymatic reactions, or protein-protein interactions. DESCRIPTION: We present P2CS (Prokaryotic 2-Component Systems), an integrated and comprehensive database of TCS signal transduction proteins, which contains a compilation of the TCS genes within 755 completely sequenced prokaryotic genomes and 39 metagenomes. P2CS provides detailed annotation of each TCS gene including family classification, sequence features, functional domains, as well as genomic context visualization. To bypass the generic problem of gene underestimation during genome annotation, we also constituted and searched an ORFeome, which improves the recovery of TCS proteins compared to searches on the equivalent proteomes. CONCLUSION: P2CS has been developed for computational analysis of the modular TCSs of prokaryotic genomes and metagenomes. It provides a complete overview of information on TCSs, including predicted candidate proteins and probable proteins, which need further curation/validation. The database can be browsed and queried with a user-friendly web interface at

Tight Regulation of the intS Gene of the KplE1 Prophage: A New Paradigm for Integrase Gene Regulation

Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host's chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excisive recombination are discussed

The Cyst-Dividing Bacterium Ramlibacter tataouinensis TTB310 Genome Reveals a Well-Stocked Toolbox for Adaptation to a Desert Environment

Ramlibacter tataouinensis TTB310T (strain TTB310), a betaproteobacterium isolated from a semi-arid region of South Tunisia (Tataouine), is characterized by the presence of both spherical and rod-shaped cells in pure culture. Cell division of strain TTB310 occurs by the binary fission of spherical “cyst-like” cells (“cyst-cyst” division). The rod-shaped cells formed at the periphery of a colony (consisting mainly of cysts) are highly motile and colonize a new environment, where they form a new colony by reversion to cyst-like cells. This unique cell cycle of strain TTB310, with desiccation tolerant cyst-like cells capable of division and desiccation sensitive motile rods capable of dissemination, appears to be a novel adaptation for life in a hot and dry desert environment. In order to gain insights into strain TTB310's underlying genetic repertoire and possible mechanisms responsible for its unusual lifestyle, the genome of strain TTB310 was completely sequenced and subsequently annotated. The complete genome consists of a single circular chromosome of 4,070,194 bp with an average G+C content of 70.0%, the highest among the Betaproteobacteria sequenced to date, with total of 3,899 predicted coding sequences covering 92% of the genome. We found that strain TTB310 has developed a highly complex network of two-component systems, which may utilize responses to light and perhaps a rudimentary circadian hourglass to anticipate water availability at the dew time in the middle/end of the desert winter nights and thus direct the growth window to cyclic water availability times. Other interesting features of the strain TTB310 genome that appear to be important for desiccation tolerance, including intermediary metabolism compounds such as trehalose or polyhydroxyalkanoate, and signal transduction pathways, are presented and discussed

Burden of community-acquired and nosocomial rotavirus gastroenteritis in the pediatric population of Western Europe: a scoping review

<p>Abstract</p> <p>Background</p> <p>Rotavirus affects 95% of children worldwide by age 5 years and is the leading cause of severe dehydrating diarrhea. The objective of this review was to estimate the burden of rotavirus gastroenteritis (RVGE) in the Western European pediatric population.</p> <p>Methods</p> <p>A comprehensive literature search (1999-2010) was conducted in PubMed and other sources (CDC; WHO, others). Data on the epidemiology and burden of RVGE among children < 5 years-old in Western Europe --including hospital-acquired disease--were extracted.</p> <p>Results</p> <p>76 studies from 16 countries were identified. The mean percentage of acute gastroenteritis (AGE) cases caused by rotavirus ranged from 25.3%-63.5% in children < 5 years of age, peaking during winter. Incidence rates of RVGE ranged from 1.33-4.96 cases/100 person- years. Hospitalization rates for RVGE ranged from 7% to 81% among infected children, depending on the country. Nosocomial RVGE accounted for 47%-69% of all hospital-acquired AGE and prolonged hospital stays by 4-12 days. Each year, RVGE incurred $0.54-$53.6 million in direct medical costs and $1.7-$22.4 million in indirect costs in the 16 countries studied. Full serotyping data was available for 8 countries. G1P[8], G2P[4], G9P[8], and G3P[8] were the most prevalent serotypes (cumulative frequency: 57.2%- 98.7%). Serotype distribution in nosocomial RVGE was similar.</p> <p>Conclusions</p> <p>This review confirms that RVGE is a common disease associated with significant morbidity and costs across Western Europe. A vaccine protecting against multiple serotypes may decrease the epidemiological and cost burden of RVGE in Western Europe.</p

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

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

Prophages in Salmonella enterica : a driving force in reshaping the genome and physiology of their bacterial host?

International audienceThanks to the exponentially increasing number of publicly available bacterial genome sequences, one can now estimate the important contribution of integrated viral sequences to the diversity of bacterial genomes. Indeed, temperate bacteriophages are able to stably integrate the genome of their host through site-specific recombination and transmit vertically to the host siblings. Lysogenic conversion has been long acknowledged to provide additional functions to the host, and particularly to bacterial pathogen genomes where prophages contribute important virulence factors. This review aims particularly at highlighting the current knowledge and questions about lysogeny in Salmonella genomes where functional prophages are abundant, and where genetic interactions between host and prophages are of particular importance for human health considerations

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

TorI, a response regulator inhibitor of phage origin in Escherichia coli

The torI gene has been identified by using a genetic multicopy approach as a negative regulator of the torCAD operon that encodes the trimethylamine N-oxide reductase respiratory system in Escherichia coli. The negative effect was due to a previously unidentified small ORF (66 aa) of phage origin that we called torI for Tor inhibition. Overexpression of torI led to an 8-fold decrease of the torCAD operon transcription. This operon is positively regulated, in the presence of trimethylamine N-oxide, by a four-step phosphorelay involving the TorS sensor and the TorR response regulator. Epistatic experiments showed that TorI acts downstream of TorS and needs the presence of TorR. In vitro experiments showed that it is neither a TorR phosphatase nor a histidine kinase inhibitor and that it binds to the effector domain of TorR. Unexpectedly, TorI did not impede TorR DNA binding, and we propose that it may prevent RNA polymerase recruitment to the torC promoter. This study thus reveals a previously uncharacterized class of response regulator inhibitors