Publications by authors named "Marie-Agnès Petit"

38 Publications

Bacterial Consumption of T4 Phages.

Microorganisms 2021 Aug 31;9(9). Epub 2021 Aug 31.

GenPhySE, INRAE, Université de Toulouse, 31320 Castanet-Tolosan, France.

The bacterial consumption of viruses not been reported on as of yet even though bacteria feed on almost anything. Viruses are widely distributed but have no acknowledged active biocontrol. Viral biomass undoubtedly reintegrates trophic cycles; however, the mechanisms of this phase still remain unknown. C-labelled T4 phages monitor the increase of the density of the bacterial DNA concomitant with the decrease of plaque forming units. We used C T4 phages as a control. T4 phage disappearance in wastewater sludge was found to occur mainly through predation by . Phage consumption also favours significant in situ bacterial growth. Furthermore, an isolated strain of was observed to grow on T4 phages as sole the source of carbon, nitrogen, and phosphorus. Bacterial species are capable of consuming bacteriophages in situ, which is likely a widespread and underestimated type of biocontrol. This assay is anticipated as a starting point for harnessing the bacterial potential in limiting the diffusion of harmful viruses within environments such as in the gut or in water.
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http://dx.doi.org/10.3390/microorganisms9091852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8472381PMC
August 2021

PHROG: families of prokaryotic virus proteins clustered using remote homology.

NAR Genom Bioinform 2021 Sep 5;3(3):lqab067. Epub 2021 Aug 5.

Université Clermont Auvergne, CNRS, LMGE, F-63000 Clermont-Ferrand, France.

Viruses are abundant, diverse and ancestral biological entities. Their diversity is high, both in terms of the number of different protein families encountered and in the sequence heterogeneity of each protein family. The recent increase in sequenced viral genomes constitutes a great opportunity to gain new insights into this diversity and consequently urges the development of annotation resources to help functional and comparative analysis. Here, we introduce PHROG (Prokaryotic Virus Remote Homologous Groups), a library of viral protein families generated using a new clustering approach based on remote homology detection by HMM profile-profile comparisons. Considering 17 473 reference (pro)viruses of prokaryotes, 868 340 of the total 938 864 proteins were grouped into 38 880 clusters that proved to be a 2-fold deeper clustering than using a classical strategy based on BLAST-like similarity searches, and yet to remain homogeneous. Manual inspection of similarities to various reference sequence databases led to the annotation of 5108 clusters (containing 50.6 % of the total protein dataset) with 705 different annotation terms, included in 9 functional categories, specifically designed for viruses. Hopefully, PHROG will be a useful tool to better annotate future prokaryotic viral sequences thus helping the scientific community to better understand the evolution and ecology of these entities.
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http://dx.doi.org/10.1093/nargab/lqab067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341000PMC
September 2021

Virulent coliphages in 1-year-old children fecal samples are fewer, but more infectious than temperate coliphages.

Nat Commun 2020 01 17;11(1):378. Epub 2020 Jan 17.

Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350, Jouy-en-Josas, France.

Bacteriophages constitute an important part of the human gut microbiota, but their impact on this community is largely unknown. Here, we cultivate temperate phages produced by 900 E. coli strains isolated from 648 fecal samples from 1-year-old children and obtain coliphages directly from the viral fraction of the same fecal samples. We find that 63% of strains hosted phages, while 24% of the viromes contain phages targeting E. coli. 150 of these phages, half recovered from strain supernatants, half from virome (73% temperate and 27% virulent) were tested for their host range on 75 E. coli strains isolated from the same cohort. Temperate phages barely infected the gut strains, whereas virulent phages killed up to 68% of them. We conclude that in fecal samples from children, temperate coliphages dominate, while virulent ones have greater infectivity and broader host range, likely playing a role in gut microbiota dynamics.
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http://dx.doi.org/10.1038/s41467-019-14042-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969025PMC
January 2020

The enemy from within: a prophage of Roseburia intestinalis systematically turns lytic in the mouse gut, driving bacterial adaptation by CRISPR spacer acquisition.

ISME J 2020 03 11;14(3):771-787. Epub 2019 Dec 11.

Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350, Jouy-en-Josas, France.

Despite an overall temporal stability in time of the human gut microbiota at the phylum level, strong variations in species abundance have been observed. We are far from a clear understanding of what promotes or disrupts the stability of microbiome communities. Environmental factors, like food or antibiotic use, modify the gut microbiota composition, but their overall impacts remain relatively low. Phages, the viruses that infect bacteria, might constitute important factors explaining temporal variations in species abundance. Gut bacteria harbour numerous prophages, or dormant viruses, which can evolve to become ultravirulent phage mutants, potentially leading to important bacterial death. Whether such phenomenon occurs in the mammal's microbiota has been largely unexplored. Here we studied temperate phage-bacteria coevolution in gnotoxenic mice colonised with Roseburia intestinalis, a dominant symbiont of the human gut microbiota, and Escherichia coli, a sub-dominant member of the same microbiota. We show that R. intestinalis L1-82 harbours two active prophages, Jekyll and Shimadzu. We observed the systematic evolution in mice of ultravirulent Shimadzu phage mutants, which led to a collapse of R. intestinalis population. In a second step, phage infection drove the fast counter-evolution of host phage resistance mainly through phage-derived spacer acquisition in a clustered regularly interspaced short palindromic repeats array. Alternatively, phage resistance was conferred by a prophage originating from an ultravirulent phage with a restored ability to lysogenize. Our results demonstrate that prophages are a potential source of ultravirulent phages that can successfully infect most of the susceptible bacteria. This suggests that prophages can play important roles in the short-term temporal variations observed in the composition of the gut microbiota.
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http://dx.doi.org/10.1038/s41396-019-0566-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031369PMC
March 2020

Viral metagenomic analysis of the cheese surface: A comparative study of rapid procedures for extracting viral particles.

Food Microbiol 2020 Feb 24;85:103278. Epub 2019 Jul 24.

UMR1319 MICALIS, INRA, AgroParisTech, Université Paris-Saclay, Domaine de Vilvert, 78350, Jouy-en-Josas, France.

The structure and functioning of microbial communities from fermented foods, including cheese, have been extensively studied during the past decade. However, there is still a lack of information about both the occurrence and the role of viruses in modulating the function of this type of spatially structured and solid ecosystems. Viral metagenomics was recently applied to a wide variety of environmental samples and standardized procedures for recovering viral particles from different type of materials has emerged. In this study, we adapted a procedure originally developed to extract viruses from fecal samples, in order to enable efficient virome analysis of cheese surface. We tested and validated the positive impact of both addition of a filtration step prior to virus concentration and substitution of purification by density gradient ultracentrifugation by a simple chloroform treatment to eliminate membrane vesicles. Viral DNA extracted from the several procedures, as well as a vesicle sample, were sequenced using Illumina paired-end MiSeq technology and the subsequent clusters assembled from the virome were analyzed to assess those belonging to putative phages, plasmid-derived DNA, or even from bacterial chromosomal DNA. The best procedure was then chosen, and used to describe the first cheese surface virome, using Epoisses cheese as example. This study provides the basis of future investigations regarding the ecological importance of viruses in cheese microbial ecosystems.
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http://dx.doi.org/10.1016/j.fm.2019.103278DOI Listing
February 2020

Countermeasures Defeat a Virulent Bacteriophage.

Viruses 2019 01 10;11(1). Epub 2019 Jan 10.

Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France.

is an opportunistic pathogen that has emerged as a major cause of nosocomial infections worldwide. Many clinical strains are indeed resistant to last resort antibiotics and there is consequently a reawakening of interest in exploiting virulent phages to combat them. However, little is still known about phage receptors and phage resistance mechanisms in enterococci. We made use of a prophageless derivative of the well-known clinical strain V583 to isolate a virulent phage belonging to the subfamily and to the P68 genus that we named Idefix. Interestingly, most isolates of tested-including V583-were resistant to this phage and we investigated more deeply into phage resistance mechanisms. We found that V583 prophage 6 was particularly efficient in resisting Idefix infection thanks to a new abortive infection (Abi) mechanism, which we designated Abiα. It corresponded to the Pfam domain family with unknown function DUF4393 and conferred a typical Abi phenotype by causing a premature lysis of infected . The gene is widespread among prophages of enterococci and other Gram-positive bacteria. Furthermore, we identified two genes involved in the synthesis of the side chains of the surface rhamnopolysaccharide that are important for Idefix adsorption. Interestingly, mutants in these genes arose at a frequency of ~10 resistant mutants per generation, conferring a supplemental bacterial line of defense against Idefix.
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http://dx.doi.org/10.3390/v11010048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356687PMC
January 2019

Minimum Information about an Uncultivated Virus Genome (MIUViG).

Nat Biotechnol 2019 01 17;37(1):29-37. Epub 2018 Dec 17.

Department of Physiology, Genetics and Microbiology, University of Alicante, Alicante, Spain.

We present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere.
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http://dx.doi.org/10.1038/nbt.4306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6871006PMC
January 2019

Sak4 of Phage HK620 Is a RecA Remote Homolog With Single-Strand Annealing Activity Stimulated by Its Cognate SSB Protein.

Front Microbiol 2018 24;9:743. Epub 2018 Apr 24.

Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France.

Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing , but only weakly compared to the phage λ Redβ protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the gene in various species of phages, highly stimulates its recombineering activity , Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of and from HK620 can promote low-level of recombination , though Sak4 and its SSB are unable to promote strand exchange . Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves.
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http://dx.doi.org/10.3389/fmicb.2018.00743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928155PMC
April 2018

Phages infecting Faecalibacterium prausnitzii belong to novel viral genera that help to decipher intestinal viromes.

Microbiome 2018 04 3;6(1):65. Epub 2018 Apr 3.

Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350, Jouy-en-Josas, France.

Background: Viral metagenomic studies have suggested a role for bacteriophages in intestinal dysbiosis associated with several human diseases. However, interpretation of viral metagenomic studies is limited by the lack of knowledge of phages infecting major human gut commensal bacteria, such as Faecalibacterium prausnitzii, a bacterial symbiont repeatedly found depleted in inflammatory bowel disease (IBD) patients. In particular, no complete genomes of phages infecting F. prausnitzii are present in viral databases.

Methods: We identified 18 prophages in 15 genomes of F. prausnitzii, used comparative genomics to define eight phage clades, and annotated the genome of the type phage of each clade. For two of the phages, we studied prophage induction in vitro and in vivo in mice. Finally, we aligned reads from already published viral metagenomic data onto the newly identified phages.

Results: We show that each phage clade represents a novel viral genus and that a surprisingly large fraction of them (10 of the 18 phages) codes for a diversity-generating retroelement, which could contribute to their adaptation to the digestive tract environment. We obtained either experimental or in silico evidence of activity for at least one member of each genus. In addition, four of these phages are either significantly more prevalent or more abundant in stools of IBD patients than in those of healthy controls.

Conclusion: Since IBD patients generally have less F. prausnitzii in their microbiota than healthy controls, the higher prevalence or abundance of some of its phages may indicate that they are activated during disease. This in turn suggests that phages could trigger or aggravate F. prausnitzii depletion in patients. Our results show that prophage detection in sequenced strains of the microbiota can usefully complement viral metagenomic studies.
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http://dx.doi.org/10.1186/s40168-018-0452-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5883640PMC
April 2018

"French Phage Network"-Third Meeting Report.

Viruses 2018 03 10;10(3). Epub 2018 Mar 10.

Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91190 Gif-sur-Yvette CEDEX, France.

In its third year of existence, the French Phage Network (Phages.fr) is pursuing its expansion. With more than 25 groups, mostly based in France, working on the various aspects of phage research, the network has increased its visibility, interactivity, and activity. The third meeting of the Phages.fr network, held on November 2017 at the Gif-sur-Yvette Centre National de la Recherche Scientifique (CNRS) campus, was a great opportunity for many young scientists to present their work and interact with more senior scientists, amongst which several were invited from abroad. Here we provide a summary of the work presented at this occasion during the oral presentations and poster sessions.
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http://dx.doi.org/10.3390/v10030123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869516PMC
March 2018

[A century of research on bacteriophages].

Virologie (Montrouge) 2020 02;24(1):9-22

Unité de Recherches de Pathologie Végétale, UR407, Inrae PACA, 67 allée des Chênes CS 60094, Domaine Saint-Maurice, F84143 Montfavet cedex, France.

Bacteriophages have a prominent place in the living world. They participate to our understanding of the living world through three main aspects : (i) the dissection of the most intimist aspects of viral infection molecular mechanisms (molecular biology), (ii) the description and functioning mechanisms of ecosystems (ecology), and (iii) the adaptive dynamics of integrated viral and host-cell populations (evolution). This review looks back at the genesis of these fundamental findings and draws a picture of the most active fields of current research.
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http://dx.doi.org/10.1684/vir.2020.0809DOI Listing
February 2020

[Antibacterial applications of bacteriophages].

Virologie (Montrouge) 2020 02;24(1):23-36

Unité de Recherches de Pathologie Végétale, UR407, Inrae PACA, 67 allée des Chênes CS 60094, Domaine Saint-Maurice, F84143 Montfavet cedex, France.

In the 1917 article in which Félix d'Hérelle describes his first observations and proposes the name of bacteriophage, he also reports the first use of these viruses to treat bacterial infections, thus giving birth to phage therapy. Soon after antibiotics supplanted bacteriophages. Today, bacteria resistant to multiple antibiotics become a growing public health issue worldwide. This situation has revived research aiming at developing the antibacterial activity of bacteriophages to treat patients as well as diseases in animals and plants. In fact, the areas of applications of bacteriophages as antibacterial are widening as current solutions of chemical nature are questioned. This review summarizes the basic principles of therapeutic applications of bacteriophages and presents recent data in areas where commercial exploitation is occurring or about to emerge.
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http://dx.doi.org/10.1684/vir.2019.0805DOI Listing
February 2020

Domestication of Lambda Phage Genes into a Putative Third Type of Replicative Helicase Matchmaker.

Genome Biol Evol 2017 06;9(6):1561-1566

Universite de Versailles Saint-Quentin en Yvelines UFR des Sciences, France.

At the onset of the initiation of chromosome replication, bacterial replicative helicases are recruited and loaded on the DnaA-oriC nucleoprotein platform, assisted by proteins like DnaC/DnaI or DciA. Two orders of bacteria appear, however, to lack either of these factors, raising the question of the essentiality of these factors in bacteria. Through a phylogenomic approach, we identified a pair of genes that could have substituted for dciA. The two domesticated genes are specific of the dnaC/dnaI- and dciA-lacking organisms and apparently domesticated from lambdoid phage genes. They derive from λO and λP and were renamed dopC and dopE, respectively. DopE is expected to bring the replicative helicase to the bacterial origin of replication, while DopC might assist DopE in this function. The confirmation of the implication of DopCE in the handling of the replicative helicase at the onset of replication in these organisms would generalize to all bacteria and therefore to all living organisms the need for specific factors dedicated to this function.
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http://dx.doi.org/10.1093/gbe/evx111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509034PMC
June 2017

Complete Genome Sequences of Pseudomonas aeruginosa Phages vB_PaeP_PcyII-10_P3P1 and vB_PaeM_PcyII-10_PII10A.

Genome Announc 2016 Nov 17;4(6). Epub 2016 Nov 17.

Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France.

vB_PaeP_PcyII-10_P3P1 and vB_PaeM_PcyII-10_PII10A are Pseudomonas aeruginosa bacteriophages belonging, respectively, to the Lit1virus genus of the Podoviridae family and the Pbunavirus genus of the Myoviridae family. Their genomes are 72,778 bp and 65,712 bp long, containing 94 and 93 predicted open reading frames, respectively.
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http://dx.doi.org/10.1128/genomeA.00916-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114362PMC
November 2016

Phages rarely encode antibiotic resistance genes: a cautionary tale for virome analyses.

ISME J 2017 01 21;11(1):237-247. Epub 2016 Jun 21.

Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France.

Antibiotic resistance genes (ARGs) are pervasive in gut microbiota, but it remains unclear how often ARGs are transferred, particularly to pathogens. Traditionally, ARG spread is attributed to horizontal transfer mediated either by DNA transformation, bacterial conjugation or generalized transduction. However, recent viral metagenome (virome) analyses suggest that ARGs are frequently carried by phages, which is inconsistent with the traditional view that phage genomes rarely encode ARGs. Here we used exploratory and conservative bioinformatic strategies found in the literature to detect ARGs in phage genomes, and experimentally assessed a subset of ARG predicted using exploratory thresholds. ARG abundances in 1181 phage genomes were vastly overestimated using exploratory thresholds (421 predicted vs 2 known), due to low similarities and matches to protein unrelated to antibiotic resistance. Consistent with this, four ARGs predicted using exploratory thresholds were experimentally evaluated and failed to confer antibiotic resistance in Escherichia coli. Reanalysis of available human- or mouse-associated viromes for ARGs and their genomic context suggested that bona fide ARG attributed to phages in viromes were previously overestimated. These findings provide guidance for documentation of ARG in viromes, and reassert that ARGs are rarely encoded in phages.
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http://dx.doi.org/10.1038/ismej.2016.90DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315482PMC
January 2017

Phagonaute: A web-based interface for phage synteny browsing and protein function prediction.

Virology 2016 09 30;496:42-50. Epub 2016 May 30.

Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France. Electronic address:

Distant homology search tools are of great help to predict viral protein functions. However, due to the lack of profile databases dedicated to viruses, they can lack sensitivity. We constructed HMM profiles for more than 80,000 proteins from both phages and archaeal viruses, and performed all pairwise comparisons with HHsearch program. The whole resulting database can be explored through a user-friendly "Phagonaute" interface to help predict functions. Results are displayed together with their genetic context, to strengthen inferences based on remote homology. Beyond function prediction, this tool permits detections of co-occurrences, often indicative of proteins completing a task together, and observation of conserved patterns across large evolutionary distances. As a test, Herpes simplex virus I was added to Phagonaute, and 25% of its proteome matched to bacterial or archaeal viral protein counterparts. Phagonaute should therefore help virologists in their quest for protein functions and evolutionary relationships.
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http://dx.doi.org/10.1016/j.virol.2016.05.007DOI Listing
September 2016

Carriage of λ Latent Virus Is Costly for Its Bacterial Host due to Frequent Reactivation in Monoxenic Mouse Intestine.

PLoS Genet 2016 Feb 12;12(2):e1005861. Epub 2016 Feb 12.

Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France.

Temperate phages, the bacterial viruses able to enter in a dormant prophage state in bacterial genomes, are present in the majority of bacterial strains for which the genome sequence is available. Although these prophages are generally considered to increase their hosts' fitness by bringing beneficial genes, studies demonstrating such effects in ecologically relevant environments are relatively limited to few bacterial species. Here, we investigated the impact of prophage carriage in the gastrointestinal tract of monoxenic mice. Combined with mathematical modelling, these experimental results provided a quantitative estimation of key parameters governing phage-bacteria interactions within this model ecosystem. We used wild-type and mutant strains of the best known host/phage pair, Escherichia coli and phage λ. Unexpectedly, λ prophage caused a significant fitness cost for its carrier, due to an induction rate 50-fold higher than in vitro, with 1 to 2% of the prophage being induced. However, when prophage carriers were in competition with isogenic phage susceptible bacteria, the prophage indirectly benefited its carrier by killing competitors: infection of susceptible bacteria led to phage lytic development in about 80% of cases. The remaining infected bacteria were lysogenized, resulting overall in the rapid lysogenization of the susceptible lineage. Moreover, our setup enabled to demonstrate that rare events of phage gene capture by homologous recombination occurred in the intestine of monoxenic mice. To our knowledge, this study constitutes the first quantitative characterization of temperate phage-bacteria interactions in a simplified gut environment. The high prophage induction rate detected reveals DNA damage-mediated SOS response in monoxenic mouse intestine. We propose that the mammalian gut, the most densely populated bacterial ecosystem on earth, might foster bacterial evolution through high temperate phage activity.
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http://dx.doi.org/10.1371/journal.pgen.1005861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4752277PMC
February 2016

Heterogeneity in Induction Level, Infection Ability, and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates.

Appl Environ Microbiol 2016 Jan 29;82(7):2177-2186. Epub 2016 Jan 29.

Université Paris-Est, Anses, Laboratory for Food Safety, Maisons-Alfort, France

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event.
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http://dx.doi.org/10.1128/AEM.03463-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807521PMC
January 2016

Bacterial genome remodeling through bacteriophage recombination.

FEMS Microbiol Lett 2015 Jan 4;362(1):1-10. Epub 2014 Dec 4.

Laboratoire de Chimie Bactérienne, UMR7283, Centre National de la Recherche Scientifique, Aix Marseille Université, 13402 Marseille Cedex 20, France

Bacteriophages co-exist and co-evolve with their hosts in natural environments. Virulent phages lyse infected cells through lytic cycles, whereas temperate phages often remain dormant and can undergo lysogenic or lytic cycles. In their lysogenic state, prophages are actually part of the host genome and replicate passively in rhythm with host division. However, prophages are far from being passive residents: they can modify or bring new properties to their host. In this review, we focus on two important phage-encoded recombination mechanisms, i.e. site-specific recombination and homologous recombination, and how they remodel bacterial genomes.
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http://dx.doi.org/10.1093/femsle/fnu022DOI Listing
January 2015

Automated classification of tailed bacteriophages according to their neck organization.

BMC Genomics 2014 Nov 27;15:1027. Epub 2014 Nov 27.

CEA, iBiTecS, Gif-sur-Yvette, F-91191 Paris, France.

Background: The genetic diversity observed among bacteriophages remains a major obstacle for the identification of homologs and the comparison of their functional modules. In the structural module, although several classes of homologous proteins contributing to the head and tail structure can be detected, proteins of the head-to-tail connection (or neck) are generally more divergent. Yet, molecular analyses of a few tailed phages belonging to different morphological classes suggested that only a limited number of structural solutions are used in order to produce a functional virion. To challenge this hypothesis and analyze proteins diversity at the virion neck, we developed a specific computational strategy to cope with sequence divergence in phage proteins. We searched for homologs of a set of proteins encoded in the structural module using a phage learning database.

Results: We show that using a combination of iterative profile-profile comparison and gene context analyses, we can identify a set of head, neck and tail proteins in most tailed bacteriophages of our database. Classification of phages based on neck protein sequences delineates 4 Types corresponding to known morphological subfamilies. Further analysis of the most abundant Type 1 yields 10 Clusters characterized by consistent sets of head, neck and tail proteins. We developed Virfam, a webserver that automatically identifies proteins of the phage head-neck-tail module and assign phages to the most closely related cluster of phages. This server was tested against 624 new phages from the NCBI database. 93% of the tailed and unclassified phages could be assigned to our head-neck-tail based categories, thus highlighting the large representativeness of the identified virion architectures. Types and Clusters delineate consistent subgroups of Caudovirales, which correlate with several virion properties.

Conclusions: Our method and webserver have the capacity to automatically classify most tailed phages, detect their structural module, assign a function to a set of their head, neck and tail genes, provide their morphologic subtype and localize these phages within a "head-neck-tail" based classification. It should enable analysis of large sets of phage genomes. In particular, it should contribute to the classification of the abundant unknown viruses found on assembled contigs of metagenomic samples.
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http://dx.doi.org/10.1186/1471-2164-15-1027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362835PMC
November 2014

Killing the killers.

Elife 2014 Sep 2;3:e04168. Epub 2014 Sep 2.

Marie-Agnès Petit is in the French National Institute for Agricultural Research and AgroParisTech, Micalis Institute, Jouy-en-Josas, France

The bacteria that infect humans and cause cholera are themselves infected by viruses, which have the potential to influence the course of a cholera infection.
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http://dx.doi.org/10.7554/eLife.04168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151084PMC
September 2014

Bacteriophages: an underestimated role in human and animal health?

Front Cell Infect Microbiol 2014 28;4:39. Epub 2014 Mar 28.

Institut National de la Recherche Agronomique, Micalis, UMR 1319 Jouy en Josas, France ; Agroparistech, Micalis, UMR 1319 Jouy en Josas, France.

Metagenomic approaches applied to viruses have highlighted their prevalence in almost all microbial ecosystems investigated. In all ecosystems, notably those associated with humans or animals, the viral fraction is dominated by bacteriophages. Whether they contribute to dysbiosis, i.e., the departure from microbiota composition in symbiosis at equilibrium and entry into a state favoring human or animal disease is unknown at present. This review summarizes what has been learnt on phages associated with human and animal microbiota, and focuses on examples illustrating the several ways by which phages may contribute to a shift to pathogenesis, either by modifying population equilibrium, by horizontal transfer, or by modulating immunity.
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http://dx.doi.org/10.3389/fcimb.2014.00039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3975094PMC
September 2014

Shifting the paradigm from pathogens to pathobiome: new concepts in the light of meta-omics.

Front Cell Infect Microbiol 2014 5;4:29. Epub 2014 Mar 5.

UMR 1332 Biologie du Fruit et Pathologie, Université de Bordeaux Villenave d'Ornon Cedex, France.

The concept of pathogenesis has evolved considerably over recent years, and the scenario "a microbe + virulence factors = disease" is probably far from reality in a number of cases. Actual pathogens have extremely broad biological diversity and are found in all major groups of microorganisms (viruses, bacteria, fungi, protozoa…). Their pathogenicity results from strong and often highly specific interactions they have with either their microbial environment, hosts and/or arthropod vectors. In this review, we explore the contribution of metagenomic approaches toward understanding pathogens within the context of microbial communities. With this broader view, we discussed the concept of "pathobiome" and the research questions that this raises.
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http://dx.doi.org/10.3389/fcimb.2014.00029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942874PMC
September 2014

Temperate phages acquire DNA from defective prophages by relaxed homologous recombination: the role of Rad52-like recombinases.

PLoS Genet 2014 Mar 6;10(3):e1004181. Epub 2014 Mar 6.

INRA, UMR1319, Micalis, domaine de Vilvert, Jouy en Josas, France; AgroParisTech, UMR1319, Micalis, domaine de Vilvert, Jouy en Josas, France.

Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like functions, and play an important role in bacterial evolution itself.
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http://dx.doi.org/10.1371/journal.pgen.1004181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945230PMC
March 2014

Aerobic respiration metabolism in lactic acid bacteria and uses in biotechnology.

Annu Rev Food Sci Technol 2012 7;3:37-58. Epub 2011 Nov 7.

Department of Physiology, Cultures & Enzymes Division, Chr. Hansen A/S, DK-2970 Hørsholm, Denmark.

The lactic acid bacteria (LAB) are essential for food fermentations and their impact on gut physiology and health is under active exploration. In addition to their well-studied fermentation metabolism, many species belonging to this heterogeneous group are genetically equipped for respiration metabolism. In LAB, respiration is activated by exogenous heme, and for some species, heme and menaquinone. Respiration metabolism increases growth yield and improves fitness. In this review, we aim to present the basics of respiration metabolism in LAB, its genetic requirements, and the dramatic physiological changes it engenders. We address the question of how LAB acquired the genetic equipment for respiration. We present at length how respiration can be used advantageously in an industrial setting, both in the context of food-related technologies and in novel potential applications.
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http://dx.doi.org/10.1146/annurev-food-022811-101255DOI Listing
June 2012

Low efficiency of homology-facilitated illegitimate recombination during conjugation in Escherichia coli.

PLoS One 2011 15;6(12):e28876. Epub 2011 Dec 15.

INRA, UMR 1319, Micalis, Jouy en Josas, France.

Homology-facilitated illegitimate recombination has been described in three naturally competent bacterial species. It permits integration of small linear DNA molecules into the chromosome by homologous recombination at one end of the linear DNA substrate, and illegitimate recombination at the other end. We report that homology-facilitated illegitimate recombination also occurs in Escherichia coli during conjugation with small non-replicative plasmids, but at a low frequency of 3×10(-10) per recipient cell. The fate of linear DNA in E. coli is either RecBCD-dependent degradation, or circularisation by ligation, and integration into the chromosome by single crossing-over. We also report that the observed single crossing-overs are recA-dependent, but essentially recBCD, and recFOR independent. This suggests that other, still unknown, proteins may act as mediator for the loading of RecA on DNA during single crossing-over recombination in E. coli.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028876PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3240628PMC
August 2012

DNA motifs that sculpt the bacterial chromosome.

Nat Rev Microbiol 2011 Jan;9(1):15-26

INRA, UMR 1319, Institut Micalis, FR-78352, Jouy-en-Josas, France.

During the bacterial cell cycle, the processes of chromosome replication, DNA segregation, DNA repair and cell division are coordinated by precisely defined events. Tremendous progress has been made in recent years in identifying the mechanisms that underlie these processes. A striking feature common to these processes is that non-coding DNA motifs play a central part, thus 'sculpting' the bacterial chromosome. Here, we review the roles of these motifs in the mechanisms that ensure faithful transmission of genetic information to daughter cells. We show how their chromosomal distribution is crucial for their function and how it can be analysed quantitatively. Finally, the potential roles of these motifs in bacterial chromosome evolution are discussed.
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http://dx.doi.org/10.1038/nrmicro2477DOI Listing
January 2011

Small variable segments constitute a major type of diversity of bacterial genomes at the species level.

Genome Biol 2010 30;11(4):R45. Epub 2010 Apr 30.

INRA, UMR1319, Micalis, Bat 222, Jouy en Josas, 78350, France.

Background: Analysis of large scale diversity in bacterial genomes has mainly focused on elements such as pathogenicity islands, or more generally, genomic islands. These comprise numerous genes and confer important phenotypes, which are present or absent depending on strains. We report that despite this widely accepted notion, most diversity at the species level is composed of much smaller DNA segments, 20 to 500 bp in size, which we call microdiversity.

Results: We performed a systematic analysis of the variable segments detected by multiple whole genome alignments at the DNA level on three species for which the greatest number of genomes have been sequenced: Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes. Among the numerous sites of variability, 62 to 73% were loci of microdiversity, many of which were located within genes. They contribute to phenotypic variations, as 3 to 6% of all genes harbor microdiversity, and 1 to 9% of total genes are located downstream from a microdiversity locus. Microdiversity loci are particularly abundant in genes encoding membrane proteins. In-depth analysis of the E. coli alignments shows that most of the diversity does not correspond to known mobile or repeated elements, and it is likely that they were generated by illegitimate recombination. An intriguing class of microdiversity includes small blocks of highly diverged sequences, whose origin is discussed.

Conclusions: This analysis uncovers the importance of this small-sized genome diversity, which we expect to be present in a wide range of bacteria, and possibly also in many eukaryotic genomes.
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http://dx.doi.org/10.1186/gb-2010-11-4-r45DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2884548PMC
October 2010

Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs.

Nucleic Acids Res 2010 Jul 1;38(12):3952-62. Epub 2010 Mar 1.

CEA, iBiTecS, F-91191 Gif sur Yvette, France.

Homologous recombination is a key in contributing to bacteriophages genome repair, circularization and replication. No less than six kinds of recombinase genes have been reported so far in bacteriophage genomes, two (UvsX and Gp2.5) from virulent, and four (Sak, Red beta, Erf and Sak4) from temperate phages. Using profile-profile comparisons, structure-based modelling and gene-context analyses, we provide new views on the global landscape of recombinases in 465 bacteriophages. We show that Sak, Red beta and Erf belong to a common large superfamily adopting a shortcut Rad52-like fold. Remote homologs of Sak4 are predicted to adopt a shortcut Rad51/RecA fold and are discovered widespread among phage genomes. Unexpectedly, within temperate phages, gene-context analyses also pinpointed the presence of distant Gp2.5 homologs, believed to be restricted to virulent phages. All in all, three major superfamilies of phage recombinases emerged either related to Rad52-like, Rad51-like or Gp2.5-like proteins. For two newly detected recombinases belonging to the Sak4 and Gp2.5 families, we provide experimental evidence of their recombination activity in vivo. Temperate versus virulent lifestyle together with the importance of genome mosaicism is discussed in the light of these novel recombinases. Screening for these recombinases in genomes can be performed at http://biodev.extra.cea.fr/virfam.
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http://dx.doi.org/10.1093/nar/gkq096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2896510PMC
July 2010

Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths.

PLoS Genet 2009 Jan 23;5(1):e1000344. Epub 2009 Jan 23.

Atelier de BioInformatique, Université Pierre et Marie Curie--Paris 6, Paris, France.

The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the approximately 18,000 families of orthologous genes, we found approximately 2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome's long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome.
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http://dx.doi.org/10.1371/journal.pgen.1000344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2617782PMC
January 2009
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