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1.  Clonogrouping, a Rapid Multiplex PCR Method for Identification of Major Clones of Listeria monocytogenes 
Journal of Clinical Microbiology  2015;53(10):3355-3358.
Three multiplex PCR assays were developed to identify the 11 most common Listeria monocytogenes clones in clinical and food samples; 270 (95.7%) of 282 strains of serogroups IVb, IIb, IIa, and IIc were identified accurately. This novel tool is a rapid and efficient alternative to multilocus sequence typing for identification of L. monocytogenes clones.
PMCID: PMC4572523  PMID: 26224848
2.  Identification of protein secretion systems in bacterial genomes 
Scientific Reports  2016;6:23080.
Bacteria with two cell membranes (diderms) have evolved complex systems for protein secretion. These systems were extensively studied in some model bacteria, but the characterisation of their diversity has lagged behind due to lack of standard annotation tools. We built online and standalone computational tools to accurately predict protein secretion systems and related appendages in bacteria with LPS-containing outer membranes. They consist of models describing the systems’ components and genetic organization to be used with MacSyFinder to search for T1SS-T6SS, T9SS, flagella, Type IV pili and Tad pili. We identified ~10,000 candidate systems in bacterial genomes, where T1SS and T5SS were by far the most abundant and widespread. All these data are made available in a public database. The recently described T6SSiii and T9SS were restricted to Bacteroidetes, and T6SSii to Francisella. The T2SS, T3SS, and T4SS were frequently encoded in single-copy in one locus, whereas most T1SS were encoded in two loci. The secretion systems of diderm Firmicutes were similar to those found in other diderms. Novel systems may remain to be discovered, since some clades of environmental bacteria lacked all known protein secretion systems. Our models can be fully customized, which should facilitate the identification of novel systems.
PMCID: PMC4793230  PMID: 26979785
3.  Bacteria in Ostreococcus tauri cultures – friends, foes or hitchhikers? 
Marine phytoplankton produce half of the oxygen we breathe and their astounding diversity is just starting to be unraveled. Many microbial phytoplankton are thought to be phototrophic, depending solely on inorganic sources of carbon and minerals for growth rather than preying on other planktonic cells. However, there is increasing evidence that symbiotic associations, to a large extent with bacteria, are required for vitamin or nutrient uptake for many eukaryotic microalgae. Here, we use in silico approaches to look for putative symbiotic interactions by analysing the gene content of microbial communities associated with 13 different Ostreococcus tauri (Chlorophyta, Mamilleophyceae) cultures sampled from the Mediterranean Sea. While we find evidence for bacteria in all cultures, there is no ubiquitous bacterial group, and the most prevalent group, Flavobacteria, is present in 10 out of 13 cultures. Among seven of the microbiomes, we detected genes predicted to encode type 3 secretion systems (T3SS, in 6/7 microbiomes) and/or putative type 6 secretion systems (T6SS, in 4/7 microbiomes). Phylogenetic analyses show that the corresponding genes are closely related to genes of systems identified in bacterial-plant interactions, suggesting that these T3SS might be involved in cell-to-cell interactions with O. tauri.
PMCID: PMC4224133  PMID: 25426102
phytoplankton; bacterial symbiosis; secretion system; illumina sequencing; bacterial diversity; microbiome; phycosphere; Ostreococcus tauri
4.  Origin in Acinetobacter guillouiae and Dissemination of the Aminoglycoside-Modifying Enzyme Aph(3′)-VI 
mBio  2014;5(5):e01972-14.
The amikacin resistance gene aphA6 was first detected in the nosocomial pathogen Acinetobacter baumannii and subsequently in other genera. Analysis of 133 whole-genome sequences covering the taxonomic diversity of Acinetobacter spp. detected aphA6 in the chromosome of 2 isolates of A. guillouiae, which is an environmental species, 1 of 8 A. parvus isolates, and 5 of 34 A. baumannii isolates. The gene was also present in 29 out of 36 A. guillouiae isolates screened by PCR, indicating that it is ancestral to this species. The Pnative promoter for aphA6 in A. guillouiae and A. parvus was replaced in A. baumannii by PaphA6, which was generated by use of the insertion sequence ISAba125, which brought a −35 sequence. Study of promoter strength in Escherichia coli and A. baumannii indicated that PaphA6 was four times more potent than Pnative. There was a good correlation between aminoglycoside MICs and aphA6 transcription in A. guillouiae isolates that remained susceptible to amikacin. The marked topology differences of the phylogenetic trees of aphA6 and of the hosts strongly support its recent direct transfer within Acinetobacter spp. and also to evolutionarily remote bacterial genera. Concomitant expression of aphA6 must have occurred because, contrary to the donors, it can confer resistance to the new hosts. Mobilization and expression of aphA6 via composite transposons and the upstream IS-generating hybrid PaphA6, followed by conjugation, seems the most plausible mechanism. This is in agreement with the observation that, in the recipients, aphA6 is carried by conjugative plasmids and flanked by IS that are common in Acinetobacter spp. Our data indicate that resistance genes can also be found in susceptible environmental bacteria.
We speculated that the aphA6 gene for an enzyme that confers resistance to amikacin, the most active aminoglycoside for the treatment of nosocomial infections due to Acinetobacter spp., originated in this genus before disseminating to phylogenetically distant genera pathogenic for humans. Using a combination of whole-genome sequencing of a collection of Acinetobacter spp. covering the breadth of the known taxonomic diversity of the genus, gene cloning, detailed promoter analysis, study of heterologous gene expression, and comparative analysis of the phylogenetic trees of aphA6 and of the bacterial hosts, we found that aphA6 originated in Acinetobacter guillouiae, an amikacin-susceptible environmental species. The gene conferred, upon mobilization, high-level resistance to the new hosts. This work stresses that nonpathogenic bacteria can act as reservoirs of resistance determinants, and it provides an example of the use of a genomic library to study the origin and dissemination of an antibiotic resistance gene to human pathogens.
PMCID: PMC4212838  PMID: 25336457
5.  MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems 
PLoS ONE  2014;9(10):e110726.
Biologists often wish to use their knowledge on a few experimental models of a given molecular system to identify homologs in genomic data. We developed a generic tool for this purpose.
Macromolecular System Finder (MacSyFinder) provides a flexible framework to model the properties of molecular systems (cellular machinery or pathway) including their components, evolutionary associations with other systems and genetic architecture. Modelled features also include functional analogs, and the multiple uses of a same component by different systems. Models are used to search for molecular systems in complete genomes or in unstructured data like metagenomes. The components of the systems are searched by sequence similarity using Hidden Markov model (HMM) protein profiles. The assignment of hits to a given system is decided based on compliance with the content and organization of the system model. A graphical interface, MacSyView, facilitates the analysis of the results by showing overviews of component content and genomic context. To exemplify the use of MacSyFinder we built models to detect and class CRISPR-Cas systems following a previously established classification. We show that MacSyFinder allows to easily define an accurate “Cas-finder” using publicly available protein profiles.
Availability and Implementation
MacSyFinder is a standalone application implemented in Python. It requires Python 2.7, Hmmer and makeblastdb (version 2.2.28 or higher). It is freely available with its source code under a GPLv3 license at It is compatible with all platforms supporting Python and Hmmer/makeblastdb. The “Cas-finder” (models and HMM profiles) is distributed as a compressed tarball archive as Supporting Information.
PMCID: PMC4201578  PMID: 25330359
6.  The Genomic Diversification of the Whole Acinetobacter Genus: Origins, Mechanisms, and Consequences 
Genome Biology and Evolution  2014;6(10):2866-2882.
Bacterial genomics has greatly expanded our understanding of microdiversification patterns within a species, but analyses at higher taxonomical levels are necessary to understand and predict the independent rise of pathogens in a genus. We have sampled, sequenced, and assessed the diversity of genomes of validly named and tentative species of the Acinetobacter genus, a clade including major nosocomial pathogens and biotechnologically important species. We inferred a robust global phylogeny and delimited several new putative species. The genus is very ancient and extremely diverse: Genomes of highly divergent species share more orthologs than certain strains within a species. We systematically characterized elements and mechanisms driving genome diversification, such as conjugative elements, insertion sequences, and natural transformation. We found many error-prone polymerases that may play a role in resistance to toxins, antibiotics, and in the generation of genetic variation. Surprisingly, temperate phages, poorly studied in Acinetobacter, were found to account for a significant fraction of most genomes. Accordingly, many genomes encode clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems with some of the largest CRISPR-arrays found so far in bacteria. Integrons are strongly overrepresented in Acinetobacter baumannii, which correlates with its frequent resistance to antibiotics. Our data suggest that A. baumannii arose from an ancient population bottleneck followed by population expansion under strong purifying selection. The outstanding diversification of the species occurred largely by horizontal transfer, including some allelic recombination, at specific hotspots preferentially located close to the replication terminus. Our work sets a quantitative basis to understand the diversification of Acinetobacter into emerging resistant and versatile pathogens.
PMCID: PMC4224351  PMID: 25313016
comparative genomics; bacterial genus; evolution; mobile genetic elements; nosocomial pathogens
7.  The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts 
Nucleic Acids Research  2014;42(16):10618-10631.
The roles of restriction-modification (R-M) systems in providing immunity against horizontal gene transfer (HGT) and in stabilizing mobile genetic elements (MGEs) have been much debated. However, few studies have precisely addressed the distribution of these systems in light of HGT, its mechanisms and its vectors. We analyzed the distribution of R-M systems in 2261 prokaryote genomes and found their frequency to be strongly dependent on the presence of MGEs, CRISPR-Cas systems, integrons and natural transformation. Yet R-M systems are rare in plasmids, in prophages and nearly absent from other phages. Their abundance depends on genome size for small genomes where it relates with HGT but saturates at two occurrences per genome. Chromosomal R-M systems might evolve under cycles of purifying and relaxed selection, where sequence conservation depends on the biochemical activity and complexity of the system and total gene loss is frequent. Surprisingly, analysis of 43 pan-genomes suggests that solitary R-M genes rarely arise from the degradation of R-M systems. Solitary genes are transferred by large MGEs, whereas complete systems are more frequently transferred autonomously or in small MGEs. Our results suggest means of testing the roles for R-M systems and their associations with MGEs.
PMCID: PMC4176335  PMID: 25120263
8.  A PNPase Dependent CRISPR System in Listeria 
PLoS Genetics  2014;10(1):e1004065.
The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in “CRISPRology”.
Author Summary
CRISPR-Cas systems confer to bacteria and archaea an adaptive immunity that protects them against invading bacteriophages and plasmids. In this study, we characterize a CRISPR (RliB-CRISPR) that is present in all L. monocytogenes strains at the same genomic locus but is never associated with a cas operon. It is an unusual CRISPR that, as we demonstrate, has a secondary structure consisting of basepair interactions between the repeat sequence and the adjacent spacer. We show that the RliB-CRISPR is processed by the endogenously encoded polynucleotide phosphorylase enzyme (PNPase). In addition, we show that the RliB-CRISPR system requires PNPase and presence of trans encoded cas genes of a second CRISPR-Cas system, to mediate DNA interference directed against a plasmid carrying a matching protospacer. Altogether, our data reveal a novel type of CRISPR system in bacteria that requires endogenously encoded PNPase enzyme for its processing and interference activity.
PMCID: PMC3886909  PMID: 24415952
9.  Manipulating or Superseding Host Recombination Functions: A Dilemma That Shapes Phage Evolvability 
PLoS Genetics  2013;9(9):e1003825.
Phages, like many parasites, tend to have small genomes and may encode autonomous functions or manipulate those of their hosts'. Recombination functions are essential for phage replication and diversification. They are also nearly ubiquitous in bacteria. The E. coli genome encodes many copies of an octamer (Chi) motif that upon recognition by RecBCD favors repair of double strand breaks by homologous recombination. This might allow self from non-self discrimination because RecBCD degrades DNA lacking Chi. Bacteriophage Lambda, an E. coli parasite, lacks Chi motifs, but escapes degradation by inhibiting RecBCD and encoding its own autonomous recombination machinery. We found that only half of 275 lambdoid genomes encode recombinases, the remaining relying on the host's machinery. Unexpectedly, we found that some lambdoid phages contain extremely high numbers of Chi motifs concentrated between the phage origin of replication and the packaging site. This suggests a tight association between replication, packaging and RecBCD-mediated recombination in these phages. Indeed, phages lacking recombinases strongly over-represent Chi motifs. Conversely, phages encoding recombinases and inhibiting host recombination machinery select for the absence of Chi motifs. Host and phage recombinases use different mechanisms and the latter are more tolerant to sequence divergence. Accordingly, we show that phages encoding their own recombination machinery have more mosaic genomes resulting from recent recombination events and have more diverse gene repertoires, i.e. larger pan genomes. We discuss the costs and benefits of superseding or manipulating host recombination functions and how this decision shapes phage genome structure and evolvability.
Author Summary
Bacterial viruses, called bacteriophages, are extremely abundant in the biosphere. They have key roles in the regulation of bacterial populations and in the diversification of bacterial genomes. Among these viruses, lambdoid phages are very abundant in enterobacteria and exchange genetic material very frequently. This latter process is thought to increase phage diversity and therefore facilitate adaptation to hosts. Recombination is also essential for the replication of many lambdoid phages. Lambdoids have been described to encode their own recombination genes and inhibit their hosts'. In this study, we show that lambdoids are split regarding their capacity to encode autonomous recombination functions and that this affects the abundance of recombination-related sequence motifs. Half of the phages encode an autonomous system and inhibit their hosts'. The trade-off between superseding and manipulating the hosts' recombination functions has important consequences. The phages encoding autonomous recombination functions have more diverse gene repertoires and recombine more frequently. Viruses, as many other parasites, have small genomes and depend on their hosts for several housekeeping functions. Hence, they often face trade-offs between supersession and manipulation of molecular machineries. Our results suggest these trade-offs may shape viral gene repertoires, their sequence composition and even influence their evolvability.
PMCID: PMC3784561  PMID: 24086157
10.  Characterization of Novel Phages Isolated in Coagulase-Negative Staphylococci Reveals Evolutionary Relationships with Staphylococcus aureus Phages 
Journal of Bacteriology  2012;194(21):5829-5839.
Despite increasing interest in coagulase-negative staphylococci (CoNS), little information is available about their bacteriophages. We isolated and sequenced three novel temperate Siphoviridae phages (StB12, StB27, and StB20) from the CoNS Staphylococcus hominis and S. capitis species. The genome sizes are around 40 kb, and open reading frames (ORFs) are arranged in functional modules encoding lysogeny, DNA metabolism, morphology, and cell lysis. Bioinformatics analysis allowed us to assign a potential function to half of the predicted proteins. Structural elements were further identified by proteomic analysis of phage particles, and DNA-packaging mechanisms were determined. Interestingly, the three phages show identical integration sites within their host genomes. In addition to this experimental characterization, we propose a novel classification based on the analysis of 85 phage and prophage genomes, including 15 originating from CoNS. Our analysis established 9 distinct clusters and revealed close relationships between S. aureus and CoNS phages. Genes involved in DNA metabolism and lysis and potentially in phage-host interaction appear to be widespread, while structural genes tend to be cluster specific. Our findings support the notion of a possible reciprocal exchange of genes between phages originating from S. aureus and CoNS, which may be of crucial importance for pathogenesis in staphylococci.
PMCID: PMC3486100  PMID: 22923589
11.  The Adaptation of Temperate Bacteriophages to Their Host Genomes 
Molecular Biology and Evolution  2012;30(4):737-751.
Rapid turnover of mobile elements drives the plasticity of bacterial genomes. Integrated bacteriophages (prophages) encode host-adaptive traits and represent a sizable fraction of bacterial chromosomes. We hypothesized that natural selection shapes prophage integration patterns relative to the host genome organization. We tested this idea by detecting and studying 500 prophages of 69 strains of Escherichia and Salmonella. Phage integrases often target not only conserved genes but also intergenic positions, suggesting purifying selection for integration sites. Furthermore, most integration hotspots are conserved between the two host genera. Integration sites seem also selected at the large chromosomal scale, as they are nonrandomly organized in terms of the origin–terminus axis and the macrodomain structure. The genes of lambdoid prophages are systematically co-oriented with the bacterial replication fork and display the host high frequency of polarized FtsK-orienting polar sequences motifs required for chromosome segregation. matS motifs are strongly avoided by prophages suggesting counter selection of motifs disrupting macrodomains. These results show how natural selection for seamless integration of prophages in the chromosome shapes the evolution of the bacterium and the phage. First, integration sites are highly conserved for many millions of years favoring lysogeny over the lytic cycle for temperate phages. Second, the global distribution of prophages is intimately associated with the chromosome structure and the patterns of gene expression. Third, the phage endures selection for DNA motifs that pertain exclusively to the biology of the prophage in the bacterial chromosome. Understanding prophage genetic adaptation sheds new lights on the coexistence of horizontal transfer and organized bacterial genomes.
PMCID: PMC3603311  PMID: 23243039
12.  Complete Genome Sequence of Flavobacterium indicum GPSTA100-9T, Isolated from Warm Spring Water 
Journal of Bacteriology  2012;194(11):3024-3025.
We report here the complete annotated genome sequence of Flavobacterium indicum CIP 109464T (= GPTSA100-9T), isolated from warm spring water in Assam, India. The genome sequence of F. indicum revealed a number of interesting features and genes in relation to its environmental lifestyle.
PMCID: PMC3370609  PMID: 22582381
13.  Rapid Evolution of the Sequences and Gene Repertoires of Secreted Proteins in Bacteria 
PLoS ONE  2012;7(11):e49403.
Proteins secreted to the extracellular environment or to the periphery of the cell envelope, the secretome, play essential roles in foraging, antagonistic and mutualistic interactions. We hypothesize that arms races, genetic conflicts and varying selective pressures should lead to the rapid change of sequences and gene repertoires of the secretome. The analysis of 42 bacterial pan-genomes shows that secreted, and especially extracellular proteins, are predominantly encoded in the accessory genome, i.e. among genes not ubiquitous within the clade. Genes encoding outer membrane proteins might engage more frequently in intra-chromosomal gene conversion because they are more often in multi-genic families. The gene sequences encoding the secretome evolve faster than the rest of the genome and in particular at non-synonymous positions. Cell wall proteins in Firmicutes evolve particularly fast when compared with outer membrane proteins of Proteobacteria. Virulence factors are over-represented in the secretome, notably in outer membrane proteins, but cell localization explains more of the variance in substitution rates and gene repertoires than sequence homology to known virulence factors. Accordingly, the repertoires and sequences of the genes encoding the secretome change fast in the clades of obligatory and facultative pathogens and also in the clades of mutualists and free-living bacteria. Our study shows that cell localization shapes genome evolution. In agreement with our hypothesis, the repertoires and the sequences of genes encoding secreted proteins evolve fast. The particularly rapid change of extracellular proteins suggests that these public goods are key players in bacterial adaptation.
PMCID: PMC3506625  PMID: 23189144
14.  Complete Genome Sequence of the Fish Pathogen Flavobacterium branchiophilum▿† 
Applied and Environmental Microbiology  2011;77(21):7656-7662.
Members of the genus Flavobacterium occur in a variety of ecological niches and represent an interesting diversity of lifestyles. Flavobacterium branchiophilum is the main causative agent of bacterial gill disease, a severe condition affecting various cultured freshwater fish species worldwide, in particular salmonids in Canada and Japan. We report here the complete genome sequence of strain FL-15 isolated from a diseased sheatfish (Silurus glanis) in Hungary. The analysis of the F. branchiophilum genome revealed putative mechanisms of pathogenicity strikingly different from those of the other, closely related fish pathogen Flavobacterium psychrophilum, including the first cholera-like toxin in a non-Proteobacteria and a wealth of adhesins. The comparison with available genomes of other Flavobacterium species revealed a small genome size, large differences in chromosome organization, and fewer rRNA and tRNA genes, in line with its more fastidious growth. In addition, horizontal gene transfer shaped the evolution of F. branchiophilum, as evidenced by its virulence factors, genomic islands, and CRISPR (clustered regularly interspaced short palindromic repeats) systems. Further functional analysis should help in the understanding of host-pathogen interactions and in the development of rational diagnostic tools and control strategies in fish farms.
PMCID: PMC3209149  PMID: 21926215
15.  CRISPR Distribution within the Escherichia coli Species Is Not Suggestive of Immunity-Associated Diversifying Selection ▿ †  
Journal of Bacteriology  2011;193(10):2460-2467.
In order to get further insights into the role of the clustered, regularly interspaced, short palindromic repeats (CRISPRs) in Escherichia coli, we analyzed the CRISPR diversity in a collection of 290 strains, in the phylogenetic framework of the strains represented by multilocus sequence typing (MLST). The set included 263 natural E. coli isolates exposed to various environments and isolated over a 20-year period from humans and animals, as well as 27 fully sequenced strains. Our analyses confirm that there are two largely independent pairs of CRISPR loci (CRISPR1 and -2 and CRISPR3 and -4), each associated with a different type of cas genes (Ecoli and Ypest, respectively), but that each pair of CRISPRs has similar dynamics. Strikingly, the major phylogenetic group B2 is almost devoid of CRISPRs. The majority of genomes analyzed lack Ypest cas genes and contain CRISPR3 with spacers matching Ypest cas genes. The analysis of relatedness between strains in terms of spacer repertoire and the MLST tree shows a pattern where closely related strains (MLST phylogenetic distance of <0.005 corresponding to at least hundreds of thousands of years) often exhibit identical CRISPRs while more distantly related strains (MLST distance of >0.01) exhibit completely different CRISPRs. This suggests rare but radical turnover of spacers in CRISPRs rather than CRISPR gradual change. We found no link between the presence, size, or content of CRISPRs and the lifestyle of the strains. Our data suggest that, within the E. coli species, CRISPRs do not have the expected characteristics of a classical immune system.
PMCID: PMC3133152  PMID: 21421763
16.  The Small, Slow and Specialized CRISPR and Anti-CRISPR of Escherichia and Salmonella 
PLoS ONE  2010;5(6):e11126.
Prokaryotes thrive in spite of the vast number and diversity of their viruses. This partly results from the evolution of mechanisms to inactivate or silence the action of exogenous DNA. Among these, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are unique in providing adaptive immunity against elements with high local resemblance to genomes of previously infecting agents. Here, we analyze the CRISPR loci of 51 complete genomes of Escherichia and Salmonella. CRISPR are in two pairs of loci in Escherichia, one single pair in Salmonella, each pair showing a similar turnover rate, repeat sequence and putative linkage to a common set of cas genes. Yet, phylogeny shows that CRISPR and associated cas genes have different evolutionary histories, the latter being frequently exchanged or lost. In our set, one CRISPR pair seems specialized in plasmids often matching genes coding for the replication, conjugation and antirestriction machinery. Strikingly, this pair also matches the cognate cas genes in which case these genes are absent. The unexpectedly high conservation of this anti-CRISPR suggests selection to counteract the invasion of mobile elements containing functional CRISPR/cas systems. There are few spacers in most CRISPR, which rarely match genomes of known phages. Furthermore, we found that strains divergent less than 250 thousand years ago show virtually identical CRISPR. The lack of congruence between cas, CRISPR and the species phylogeny and the slow pace of CRISPR change make CRISPR poor epidemiological markers in enterobacteria. All these observations are at odds with the expectedly abundant and dynamic repertoire of spacers in an immune system aiming at protecting bacteria from phages. Since we observe purifying selection for the maintenance of CRISPR these results suggest that alternative evolutionary roles for CRISPR remain to be uncovered.
PMCID: PMC2886076  PMID: 20559554
17.  A Module Located at a Chromosomal Integration Hot Spot Is Responsible for the Multidrug Resistance of a Reference Strain from Escherichia coli Clonal Group A▿ †  
Escherichia coli clonal group A (CGA) commonly exhibits a distinctive multidrug antimicrobial resistance phenotype—i.e., resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and trimethoprim (ACSSuTTp)—and has accounted for up to 50% of trimethoprim-sulfamethoxazole-resistant E. coli urinary tract infections in some locales. Annotation of the whole-genome sequencing of UMN026, a reference CGA strain, clarified the genetic basis for this strain's ACSSuTTp antimicrobial resistance phenotype. Most of the responsible genes were clustered in a unique 23-kbp chromosomal region, designated the genomic resistance module (GRM), which occurred within a 105-kbp genomic island situated at the leuX tRNA. The GRM is characterized by numerous remnants of mobilization and rearrangement events suggesting multiple horizontal transfers. Additionally, comparative genomic analysis of the leuX tRNA genomic island in 14 sequenced E. coli genomes showed that this region is a hot spot of integration, with the presence/absence of specific subregions being uncorrelated with either the phylogenetic group or the pathotype. Our data illustrate the importance of whole-genome sequencing in the detection of genetic elements involved in antimicrobial resistance. Additionally, this is the first documentation of the blaTEM and dhfrVII genes in a chromosomal location in E. coli strains.
PMCID: PMC2687200  PMID: 19364861
18.  Horizontal Gene Transfer of the Secretome Drives the Evolution of Bacterial Cooperation and Virulence 
Current Biology  2009;19(20):1683-1691.
Microbes engage in a remarkable array of cooperative behaviors, secreting shared proteins that are essential for foraging, shelter, microbial warfare, and virulence. These proteins are costly, rendering populations of cooperators vulnerable to exploitation by nonproducing cheaters arising by gene loss or migration. In such conditions, how can cooperation persist?
Our model predicts that differential gene mobility drives intragenomic variation in investment in cooperative traits. More mobile loci generate stronger among-individual genetic correlations at these loci (higher relatedness) and thereby allow the maintenance of more cooperative traits via kin selection. By analyzing 21 Escherichia genomes, we confirm that genes coding for secreted proteins—the secretome—are very frequently lost and gained and are associated with mobile elements. We show that homologs of the secretome are overrepresented among human gut metagenomics samples, consistent with increased relatedness at secretome loci across multiple species. The biosynthetic cost of secreted proteins is shown to be under intense selective pressure, even more than for highly expressed proteins, consistent with a cost of cooperation driving social dilemmas. Finally, we demonstrate that mobile elements are in conflict with their chromosomal hosts over the chimeric ensemble's social strategy, with mobile elements enforcing cooperation on their otherwise selfish hosts via the cotransfer of secretome genes with “mafia strategy” addictive systems (toxin-antitoxin and restriction-modification).
Our analysis matches the predictions of our model suggesting that horizontal transfer promotes cooperation, as transmission increases local genetic relatedness at mobile loci and enforces cooperation on the resident genes. As a consequence, horizontal transfer promoted by agents such as plasmids, phages, or integrons drives microbial cooperation.
PMCID: PMC2773837  PMID: 19800234
19.  Organised Genome Dynamics in the Escherichia coli Species Results in Highly Diverse Adaptive Paths 
PLoS Genetics  2009;5(1):e1000344.
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 ∼18,000 families of orthologous genes, we found ∼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.
Author Summary
Although abundant knowledge has been accumulated regarding the E. coli laboratory strain K-12, little is known about the evolutionary trajectories that have driven the high diversity observed among natural isolates of the species, which encompass both commensal and highly virulent intestinal and extraintestinal pathogenic strains. We have annotated or 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. Although recombination rates are much higher than mutation rates, we were able to reconstruct a robust phylogeny based on the ∼2,000 genes common to all strains. Based on this phylogeny, we established the evolutionary scenario of gains and losses of thousands of specific genes, identifying functional classes under opposite selection pressures. This genome flux is confined to very few positions in the chromosome, which are the same for every genome. Notably, we identified few or no extraintestinal virulence-specific genes. We also defined a long-scale structure of recombination in the genome with lower recombination rates at the terminus of replication. These findings demonstrate that, despite a very high gene flow, genes can co-exist in an organised genome.
PMCID: PMC2617782  PMID: 19165319
20.  Transcription-coupled and splicing-coupled strand asymmetries in eukaryotic genomes 
Nucleic Acids Research  2004;32(17):4969-4978.
Under no-strand bias conditions, each genomic DNA strand should present equimolarities of A and T and of G and C. Deviations from these rules are attributed to asymmetric properties intrinsic to DNA mutation–repair processes. In bacteria, strand biases are associated with replication or transcription. In eukaryotes, recent studies demonstrate that human genes present transcription-coupled biases that might reflect transcription-coupled repair processes. Here, we study strand asymmetries in intron sequences of evolutionarily distant eukaryotes, and show that two superimposed intron biases can be distinguished. (i) Biases that are maximum at intron extremities and decrease over large distances to zero values in internal regions, possibly reflecting interactions between pre-mRNA and splicing machinery; these extend over ∼0.5 kb in mammals and Arabidopsis thaliana, and over 1 kb in Caenorhabditis elegans and Drosophila melanogaster. (ii) Biases that are constant along introns, possibly associated with transcription. Strikingly, in C.elegans, these latter biases extend over intergenic regions that separate co-oriented genes. When appropriately examined, all genomes present transcription-coupled excess of T over A in the coding strand. On the opposite, GC skews are either positive (mammals, plants) or negative (invertebrates). These results suggest that transcription-coupled asymmetries result from mutation–repair mechanisms that differ between vertebrates and invertebrates.
PMCID: PMC521644  PMID: 15388799

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