Yves Quentin

Cell signalling by oligosaccharides. Two-component systems in Pseudomonas aeruginosa: why so many?

Screening the Pseudomonas aeruginosa genome has led to the identification of the highest number of putative genes encoding two-component regulatory systems of all bacterial genomes sequenced to date (64 and 63 encoding response regulators and histidine kinases, respectively). Sixteen atypical kinases, among them 11 devoid of an Hpt domain, and three independent Hpt modules were retrieved. These data suggest that P. aeruginosa possesses complex control strategies with which to respond to environmental challenges.

Genes but Not Genomes Reveal Bacterial Domestication of Lactococcus Lactis

Background The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST) scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE). Methodology/Principal Findings The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content) did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST) differing by up to 230 kb in genome size. Conclusion/Significance The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between “environmental” strains, the main contributors to the genetic diversity within the subspecies, and “domesticated” strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the “domesticated” strains essentially arose through substantial genomic flux within the dispensable genome.

Molecular Evidence of Male-to-Female Sexual Transmission of Hepatitis C Virus after Vaginal and Anal Intercourse

ABSTRACT Hepatitis C virus (HCV) was transmitted from a chronic carrier to his female partner during unprotected anal and vaginal intercourse. Based on HVR1 and phylogenetic tree analysis, the couple had closely related isolates. These findings confirm sexual transmission of HCV without other risk factors.

Direct involvement of DprA, the transformation-dedicated RecA loader, in the shut-off of pneumococcal competence

Significance This article concerns the control of competence for bacterial genetic transformation. Competence is transient in the human pathogen Streptococcus pneumoniae, involving the specific expression of ∼100 genes that are turned ON suddenly and OFF almost as abruptly. Although the mechanism rendering all cells in a culture simultaneously competent is well understood, how competence stops has remained unknown. Here, we unravel the mechanism of shut-off, describing the discovery that a key recombination protein, DprA, exerts a negative control on competence through direct, physical interaction with the master regulator of competence, the response regulator ComE, to abolish transcription from ComE-activated promoters. Natural bacterial transformation is a genetically programmed process allowing genotype alterations that involves the internalization of DNA and its chromosomal integration catalyzed by the universal recombinase RecA, assisted by its transformation-dedicated loader, DNA processing protein A (DprA). In Streptococcus pneumoniae, the ability to internalize DNA, known as competence, is transient, developing suddenly and stopping as quickly. Competence is induced by the comC-encoded peptide, competence stimulating peptide (CSP), via a classic two-component regulatory system ComDE. Upon CSP binding, ComD phosphorylates the ComE response-regulator, which then activates transcription of comCDE and the competence-specific σX, leading to a sudden rise in CSP levels and rendering all cells in a culture competent. However, how competence stops has remained unknown. We report that DprA, under σX control, interacts with ComE∼P to block ComE-driven transcription, chiefly impacting σX production. Mutations of dprA specifically disrupting interaction with ComE were isolated and shown to map mainly to the N-terminal domain of DprA. Wild-type DprA but not ComE interaction mutants affected in vitro binding of ComE to its promoter targets. Once introduced at the dprA chromosomal locus, mutations disrupting DprA interaction with ComE altered competence shut-off. The absence of DprA was found to negatively impact growth following competence induction, highlighting the importance of DprA for pneumococcal physiology. DprA has thus two key roles: ensuring production of transformants via interaction with RecA and competence shut-off via interaction with ComE, avoiding physiologically detrimental consequences of prolonged competence. Finally, phylogenetic analyses revealed that the acquisition of a new function by DprA impacted its evolution in streptococci relying on ComE to regulate comX expression.

Emergence of master sequences in families of retroposons derived from 7sl RNA.

The past few years have brought new insight into the evolution of families of retroposons. These are composed of a very small number of master sequences able to duplicate, and a large majority of copies that are inactive for retroposition. During the course of time, successive replacements of master sequences have produced waves of amplification that are recognizable as subfamilies. In the Alu and the B1 families, one can distinguish two evolutionary periods. The first involves only monomeric elements that are now extinguished (fossil elements) and is characterized by deep remodeling of the sequences. This period ends, in primates, with the fusion of a free left and a free right Alu monomer, producing the first modern Alu dimeric element; in rodents it ends with a tandem duplication of 29 bp to create the first modern B1 element. The second period is characterized by a great stability of the master sequences. The observed turn-over of master sequences is still an enigma. However, analysis of the contemporary master sequences and of the oldest master sequences provide some clues. Here, we review the very first stages of the appearance of the Alu and the B1 families in mammalian genomes.

Use of Phylogenetic Analysis of Hepatitis C Virus (HCV) Hypervariable Region 1 Sequences To Trace an Outbreak of HCV in an Autodialysis Unit

ABSTRACT Hemodialysis patients are at high risk of infection by hepatitis C virus (HCV). The aim of this study was to investigate an HCV outbreak that occurred in an autodialysis unit by using epidemiological and molecular methods. Seroconversion to HCV antibody (anti-HCV) was observed in two patients over an 18-month period; two other patients had previously been recorded as anti-HCV positive. All four patients involved in the outbreak were tested for HCV RNA, and hepatitis C genotype determination was accomplished by a reverse hybridization assay. Furthermore, part of hypervariable region 1 (HVR1) of the hepatitis C genome was amplified and sequenced in samples from all HCV RNA-positive patients. Phylogenetic analysis of the nucleotide sequences obtained was carried out in order to investigate any possible epidemiological linkages among patients. The nucleotide sequences of the HVR1 regions of both newly infected patients were found to be identical to sequences of samples from previously recorded anti-HCV-positive original patients, suggesting that they were infected by the same isolate. Molecular and epidemiological analysis suggested that nosocomial patient-to-patient transmission was the most likely explanation for the virus spread in the autodialysis unit under study.

Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences

REPs are highly repeated intergenic palindromic sequences often clustered into structures called BIMEs including two individual REPs separated by short linker of variable length. They play a variety of key roles in the cell. REPs also resemble the sub-terminal hairpins of the atypical IS200/605 family of insertion sequences which encode Y1 transposases (TnpAIS200/IS605). These belong to the HUH endonuclease family, carry a single catalytic tyrosine (Y) and promote single strand transposition. Recently, a new clade of Y1 transposases (TnpAREP) was found associated with REP/BIME in structures called REPtrons. It has been suggested that TnpAREP is responsible for REP/BIME proliferation over genomes. We analysed and compared REP distribution and REPtron structure in numerous available E. coli and Shigella strains. Phylogenetic analysis clearly indicated that tnpAREP was acquired early in the species radiation and was lost later in some strains. To understand REP/BIME behaviour within the host genome, we also studied E. coli K12 TnpAREP activity in vitro and demonstrated that it catalyses cleavage and recombination of BIMEs. While TnpAREP shared the same general organization and similar catalytic characteristics with TnpAIS200/IS605 transposases, it exhibited distinct properties potentially important in the creation of BIME variability and in their amplification. TnpAREP may therefore be one of the first examples of transposase domestication in prokaryotes.

Archaeal β-CASP ribonucleases of the aCPSF1 family are orthologs of the eukaryal CPSF-73 factor

Bacterial RNase J and eukaryal cleavage and polyadenylation specificity factor (CPSF-73) are members of the β-CASP family of ribonucleases involved in mRNA processing and degradation. Here we report an in-depth phylogenomic analysis that delineates aRNase J and archaeal CPSF (aCPSF) as distinct orthologous groups and establishes their repartition in 110 archaeal genomes. The aCPSF1 subgroup, which has been inherited vertically and is strictly conserved, is characterized by an N-terminal extension with two K homology (KH) domains and a C-terminal motif involved in dimerization of the holoenzyme. Pab-aCPSF1 (Pyrococcus abyssi homolog) has an endoribonucleolytic activity that preferentially cleaves at single-stranded CA dinucleotides and a 5′–3′ exoribonucleolytic activity that acts on 5′ monophosphate substrates. These activities are the same as described for the eukaryotic cleavage and polyadenylation factor, CPSF-73, when engaged in the CPSF complex. The N-terminal KH domains are important for endoribonucleolytic cleavage at certain specific sites and the formation of stable high molecular weight ribonucleoprotein complexes. Dimerization of Pab-aCPSF is important for exoribonucleolytic activity and RNA binding. Altogether, our results suggest that aCPSF1 performs an essential function and that an enzyme with similar activities was present in the last common ancestor of Archaea and Eukarya.

The Q15H mutation enables Crh, a Bacillus subtilis HPr-like protein, to carry out some regulatory HPr functions, but does not make it an effective phosphocarrier for sugar transport.

Crh of Bacillus subtilis exhibits 45% sequence identity when compared to histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS). Crh can be phosphorylated by ATP at the regulatory Ser-46 and similar to P-Ser-HPr, P-Ser-Crh plays a role in carbon-catabolite repression. The sequence around the phosphorylatable Ser-46 in Crh exhibits strong similarity to the corresponding sequence of HPr of Gram-positive and a few Gram-negative bacteria. In contrast, the catalytic His-15, the site of PEP-dependent phosphorylation in HPr, is replaced with a glutamine in Crh. When Gln-15 was exchanged for a histidyl residue, in vitro PEP-dependent enzyme I-catalysed phosphorylation of the mutant Crh was observed. However, expression of the crhQ15H mutant allele did not restore growth of a ptsH deletion strain on the PTS sugars glucose, fructose or mannitol or on the non-PTS sugar glycerol. In contrast, Q15H mutant Crh could phosphorylate the transcriptional activator LevR as well as LevD, the enzyme IIA of the fructose-specific lev-PTS, which together with enzyme I, HPr and LevE forms the phosphorylation cascade regulating induction of the lev operon via LevR. As a consequence, the constitutive expression from the lev promoter observed in a (delta)ptsH strain became inducible with fructose when the crhQ15H allele was expressed in this strain.

RNase E in the γ-Proteobacteria: conservation of intrinsically disordered noncatalytic region and molecular evolution of microdomains

RNase E of Escherichia coli is a membrane-associated endoribonuclease that has a major role in mRNA degradation. The enzyme has a large C-terminal noncatalytic region that is mostly intrinsically disordered (ID). Under standard growth conditions, RhlB, enolase and PNPase associate with the noncatalytic region to form the multienzyme RNA degradosome. To elucidate the origin and evolution of the RNA degradosome, we have identified and characterized orthologs of RNase E in the γ-Proteobacteria, a phylum of bacteria with diverse ecological niches and metabolic phenotypes and an ancient origin contemporary with the radiation of animals, plants and fungi. Intrinsic disorder, composition bias and tandem sequence repeats are conserved features of the noncatalytic region. Composition bias is bipartite with a catalytic domain proximal ANR-rich region and distal AEPV-rich region. Embedded in the noncatalytic region are microdomains (also known as MoRFs, MoREs or SLiMs), which are motifs that interact with protein and other ligands. Our results suggest that tandem repeat sequences are the progenitors of microdomains. We have identified 24 microdomains with phylogenetic signals that were acquired once with few losses. Microdomains involved in membrane association and RNA binding are universally conserved suggesting that they were present in ancestral RNase E. The RNA degradosome of E. coli arose in two steps with RhlB and PNPase acquisition early in a major subtree of the γ-Proteobacteria and enolase acquisition later. We propose a mechanism of microdomain acquisition and evolution and discuss implications of these results for the structure and function of the multienzyme RNA degradosome.