[Show abstract][Hide abstract] ABSTRACT: Comparative genomics is the cornerstone of identification of gene functions. The immense number of living organisms precludes experimental identification of functions except in a handful of model organisms. The bacterial domain is split into large branches, among which the Firmicutes occupy a considerable space. Bacillus subtilis has been the model of Firmicutes for decades and its genome has been a reference for more than 10 years. Sequencing the genome involved more than 30 laboratories, with different expertises, in a attempt to make the most of the experimental information that could be associated with the sequence. This had the expected drawback that the sequencing expertise was quite varied among the groups involved, especially at a time when sequencing genomes was extremely hard work. The recent development of very efficient, fast and accurate sequencing techniques, in parallel with the development of high-level annotation platforms, motivated the present resequencing work. The updated sequence has been reannotated in agreement with the UniProt protein knowledge base, keeping in perspective the split between the paleome (genes necessary for sustaining and perpetuating life) and the cenome (genes required for occupation of a niche, suggesting here that B. subtilis is an epiphyte). This should permit investigators to make reliable inferences to prepare validation experiments in a variety of domains of bacterial growth and development as well as build up accurate phylogenies.
[Show abstract][Hide abstract] ABSTRACT: Bacterial populations are subject to complex processes of diversification that involve mutation and horizontal DNA transfer mediated by transformation, transduction, or conjugation. Tracing the evolutionary events leading to genetic changes allows us to infer the history of a microbe. Here, we combine experimental and in silico approaches to explore the forces that drive the genome dynamics of Streptococcus agalactiae, the leading cause of neonatal infections. We demonstrate that large DNA segments of up to 334 kb of the chromosome of S. agalactiae can be transferred through conjugation from multiple initiation sites. Consistently, a genome-wide map analysis of nucleotide polymorphisms among eight human isolates demonstrated that each chromosome is a mosaic of large chromosomal fragments from different ancestors suggesting that large DNA exchanges have contributed to the genome dynamics in the natural population. The analysis of the resulting genetic flux led us to propose a model for the evolutionary history of this species in which clonal complexes of clinical importance derived from a single clone that evolved by exchanging large chromosomal regions with more distantly related strains. The emergence of this clone could be linked to selective sweeps associated with the reduction of genetic diversity in three regions within a large panel of human isolates. Up to now sex in bacteria has been assumed to involve mainly small regions; our results define S. agalactiae as an alternative paradigm in the study of bacterial evolution.
Full-text · Article · Nov 2008 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Genomics can provide the basis for understanding the evolution of emerging, lethal human pathogens such as Legionella pneumophila, the causative agent of Legionnaires' disease. This bacterium replicates within amoebae and persists in the environment as a free-living microbe. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease and within the 15 serogroups (Sg), L. pneumophila Sg1 causes over 84% of Legionnaires' disease worldwide. Why L. pneumophila Sg1 is so predominant is unknown. Here, we report the first comprehensive screen of the gene content of 217 L. pneumophila and 32 non-L. pneumophila strains isolated from humans and the environment using a Legionella DNA-array. Strikingly, we uncovered a high conservation of virulence- and eukaryotic-like genes, indicating strong environmental selection pressures for their preservation. No specific hybridization profile differentiated clinical and environmental strains or strains of different serogroups. Surprisingly, the gene cluster coding the determinants of the core and the O side-chain synthesis of the lipopolysaccaride (LPS cluster) determining Sg1 was present in diverse genomic backgrounds, strongly implicating the LPS of Sg1 itself as a principal cause of the high prevalence of Sg1 strains in human disease and suggesting that the LPS cluster can be transferred horizontally. Genomic analysis also revealed that L. pneumophila is a genetically diverse species, in part due to horizontal gene transfer of mobile genetic elements among L. pneumophila strains, but also between different Legionella species. However, the genomic background also plays a role in disease causation as demonstrated by the identification of a globally distributed epidemic strain exhibiting the genotype of the sequenced L. pneumophila strain Paris.
[Show abstract][Hide abstract] ABSTRACT: Listeria monocytogenes is a food-borne, opportunistic, bacterial pathogen causing a wide spectrum of diseases, including meningitis, septicemia,
abortion, and gastroenteritis, in humans and animals. Among the 13 L. monocytogenes serovars described, human listeriosis is mostly associated with strains of serovars 4b, 1/2b, and 1/2a. Within the species
L. monocytogenes, three phylogenetic lineages are described. Serovar 1/2a belongs to phylogenetic lineage I, while serovars 4b and 1/2b group
in phylogenetic lineage II. To explore the role of gene expression in the adaptation of L. monocytogenes strains of these two major lineages to different environments, as well as in virulence, we performed whole-genome expression
profiling of six L. monocytogenes isolates of serovars 4b, 1/2b, and 1/2a of distinct origins, using a newly constructed Listeria multigenome DNA array. Comparison of the global gene expression profiles revealed differences among strains. The expression
profiles of two strains having distinct 50% lethal doses, as assessed in the mouse model, were further analyzed. Gene ontology
term enrichment analysis of the differentially expressed genes identified differences in protein-, nucleic acid-, carbon metabolism-,
and virulence-related gene expression. Comparison of the expression profiles of the core genomes of all strains revealed differences
between the two lineages with respect to cell wall synthesis, the stress-related sigma B regulon and virulence-related genes.
These findings suggest different patterns of interaction with host cells and the environment, key factors for host colonization
and survival in the environment.
Full-text · Article · Nov 2007 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Adaptation to the host environment and exploitation of host cell functions are critical to the success of intracellular pathogens. Here, insight to these virulence mechanisms was obtained for the first time from the transcriptional program of the human pathogen Legionella pneumophila during infection of its natural host, Acanthamoeba castellanii. The biphasic life cycle of L. pneumophila was reflected by a major shift in gene expression from replicative to transmissive phase, concerning nearly half of the genes predicted in the genome. However, three different L. pneumophila strains showed similar in vivo gene expression patterns, indicating that common regulatory mechanisms govern the Legionella life cycle, despite the plasticity of its genome. During the replicative phase, in addition to components of aerobic metabolism and amino acid catabolism, the Entner-Doudoroff pathway, a NADPH producing mechanism used for sugar and/or gluconate assimilation, was expressed, suggesting for the first time that intracellular L. pneumophila may also scavenge host carbohydrates as nutrients and not only proteins. Identification of genes only upregulated in vivo but not in vitro, may explain higher virulence of in vivo grown L. pneumophila. Late in the life cycle, L. pneumophila upregulates genes predicted to promote transmission and manipulation of a new host cell, therewith priming it for the next attack. These including substrates of the Dot/Icm secretion system, other factors associated previously with invasion and virulence, the motility and the type IV pilus machineries, and > 90 proteins not characterized so far. Analysis of a fliA (sigma28) deletion mutant identified genes coregulated with the flagellar regulon, including GGDEF/EAL regulators and factors that promote host cell entry and survival.
Full-text · Article · Aug 2006 · Cellular Microbiology
[Show abstract][Hide abstract] ABSTRACT: A chikungunya virus outbreak of unprecedented magnitude is currently ongoing in Indian Ocean territories. In Réunion Island, this alphavirus has already infected about one-third of the human population. The main clinical symptom of the disease is a painful and invalidating poly-arthralgia. Besides the arthralgic form, 123 patients with a confirmed chikungunya infection have developed severe clinical signs, i.e., neurological signs or fulminant hepatitis.
We report the nearly complete genome sequence of six selected viral isolates (isolated from five sera and one cerebrospinal fluid), along with partial sequences of glycoprotein E1 from a total of 127 patients from Réunion, Seychelles, Mauritius, Madagascar, and Mayotte islands. Our results indicate that the outbreak was initiated by a strain related to East-African isolates, from which viral variants have evolved following a traceable microevolution history. Unique molecular features of the outbreak isolates were identified. Notably, in the region coding for the non-structural proteins, ten amino acid changes were found, four of which were located in alphavirus-conserved positions of nsP2 (which contains helicase, protease, and RNA triphosphatase activities) and of the polymerase nsP4. The sole isolate obtained from the cerebrospinal fluid showed unique changes in nsP1 (T301I), nsP2 (Y642N), and nsP3 (E460 deletion), not obtained from isolates from sera. In the structural proteins region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Homology 3D modelling allowed mapping of these two changes to regions that are important for membrane fusion and virion assembly. Change E1-A226V was absent in the initial strains but was observed in >90% of subsequent viral sequences from Réunion, denoting evolutionary success possibly due to adaptation to the mosquito vector.
The unique molecular features of the analyzed Indian Ocean isolates of chikungunya virus demonstrate their high evolutionary potential and suggest possible clues for understanding the atypical magnitude and virulence of this outbreak.
[Show abstract][Hide abstract] ABSTRACT: Group B streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. Multilocus sequence typing (MLST) revealed that the sequence type ST-17 defines a "highly virulent" serotype III clone strongly associated with neonatal invasive infections. Our aim was to identify a target sequence enabling rapid, simple, and specific detection of this clone by a real-time PCR assay. Conventional methods for DNA manipulation and gene analyses were used to characterize the gbs2018 gene variant specific for ST-17 clone and to design ST-17- and GBS-specific primers. Conventional and real-time PCR assays were developed to detect GBS and ST-17 clones in bacterial cultures and directly on clinical samples. One hundred and fifty-six French GBS strains from various geographical areas in France isolated between 1990 and 2005 were screened by PCR with ST-17-specific primers. Forty strains were positive, and all were validated by MLST as ST-17. A representative sampling of 49 ST-17-PCR-negative strains was confirmed by MLST as non-ST-17. Real-time PCR was further used to directly test 85 vaginal samples. Among these, 13 were GBS-positive, and one was identified as ST-17. The association between strain invasiveness and ST-17 lineage in neonates with late onset disease was highly significant: 78% (P<0.0001) of strains isolated were ST-17. In conclusion, an ST-17-specific gbs2018 allele was identified and used to develop a sensitive and specific rapid-screening molecular assay for identifying ST-17 "highly virulent" GBS. Using this technique, accurate identification of women and neonates colonized by ST-17 can be readily achieved within less than 2 h.
No preview · Article · Jul 2006 · Microbes and Infection
[Show abstract][Hide abstract] ABSTRACT: Streptococcus agalactiae is a leading cause of invasive infections in neonates, and responsible for bovine mastitis. It is also a commensal bacterium adapted to asymptomatic colonization of the mammalian gut and of the genitourinary tract. Here, we report the analysis of a collection of 75 strains of human and animal origin by using serotyping, multilocus sequence typing, whole genome DNA-array hybridizations and sequence comparison of putatively virulence-associated loci. Although the most variable parts of the genome are the previously predicted genomic islands, significant genetic variations were present in the genome backbone. Evolution within genes encoding surface and secreted proteins and those involved in the biosynthesis of different capsular types is mainly due to recombination events leading to the replacement of a locus of several genes or to the allelic exchange of the internal part of a gene. These two processes, which led to a broad diversity of surface protein patterns, are probably involved in the diversity of interactions with the host and its immune system. According to gene content comparisons and phylogeny, recent gene replacements by horizontal gene transfer may occur but are rare events. Although specific gene patterns, with respect to the origin of the strains and the epidemiological characteristics, were not identified, we show that the recently described hypervirulent ST-17 lineage is a homogeneous group. The study highlights for the first time that this lineage contains a specific and conserved set of surface proteins, probably accounting for its high capacity to cause infections in newborns.
Full-text · Article · May 2006 · Microbes and Infection
[Show abstract][Hide abstract] ABSTRACT: Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal pneumonia, sepsis, and meningitis. An in silico genome analysis
indicated that GBS strain NEM316 encodes 35 proteins containing an LPXTG motif which are thought to be covalently linked to
the peptidoglycan by an enzyme called sortase. The role of these cell wall-anchored proteins in GBS pathogenesis was evaluated
on a global level by inactivating the srtA gene. This gene encodes the major sortase SrtA that anchors most of the LPXTG-containing proteins. We chose the C5a peptidase
(ScpB) and Alp2, an abundant immunogenic protein, as prototypical LPXTG-containing proteins. As expected, the SrtA knockout
mutant was unable to anchor the C5a peptidase (ScpB) and Alp2 to the cell wall. Complementation with plasmid-borne srtA inserted into the chromosome restored the correct surface localization of both ScpB and Alp2. Interestingly, the SrtA mutant
was impaired for binding to the major extracellular matrix components fibronectin and fibrinogen and displayed a significant
reduction in adherence to human (A549, HeLa, and Caco-2) and murine (L2) epithelial cells compared to the parental wild-type
strain. Surprisingly, the inactivation of srtA had no effect on the virulence of the type III strain of GBS in a neonatal rat model (measured by the 50% lethal dose and
lung colonization) but strongly impaired the capacity of the strain to colonize the intestines of gnotobiotic mice in a competition
assay. These results demonstrate that LPXTG-containing proteins are involved in cell adhesion and GBS persistence in vivo.
Full-text · Article · Jul 2005 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: Photorhabdus is an entomopathogenic bacterium belonging to the Enterobacteriaceae. The genome of the TT01 strain of Photorhabdus luminescens was recently sequenced and a large number of toxin-encoding genes were found. Genomic analysis predicted the presence on the chromosome of genes encoding a type three secretion system (TTSS), the main role of which is the delivery of effector proteins directly into eukaryotic host cells. We report here the functional characterization of the TTSS. The locus identified encodes the secretion/translocation apparatus, gene expression regulators and an effector protein - LopT - homologous to the Yersinia cysteine protease cytotoxin YopT. Heterologous expression in Yersinia demonstrated that LopT was translocated into mammal cells in an active form, as shown by the appearance of a form of the RhoA GTPase with modified electrophoretic mobility. In vitro study showed that recombinant LopT was able to release RhoA and Rac from human and insect cell membrane. In vivo assays of infection of the cutworm Spodoptera littoralis and the locust Locusta migratoria with a TT01 strain carrying a translational fusion of the lopT gene with the gfp reporter gene revealed that the lopT gene was switched on only at sites of cellular defence reactions, such as nodulation, in insects. TTSS-mutant did not induce nodule formation and underwent phagocytosis by insect macrophage cells, suggesting that the LopT effector plays an essential role in preventing phagocytosis and indicating an unexpected link between TTSS expression and the nodule reaction in insects.
[Show abstract][Hide abstract] ABSTRACT: In this study, we carried out a detailed structural and functional analysis of a Streptococcus agalactiae (GBS) two-component system which is orthologous to the CovS/CovR (CsrS/CsrR) regulatory system of Streptococcus pyogenes. In GBS, covR and covS are part of a seven gene operon transcribed from two promoters that are not regulated by CovR. A DeltacovSR mutant was found to display dramatic phenotypic changes such as increased haemolytic activity and reduced CAMP activity on blood agar. Adherence of the DeltacovSR mutant to epithelial cells was greatly increased and analysis by transmission electron microscopy revealed the presence at its surface of a fibrous extracellular matrix that might be involved in these intercellular interactions. However, the DeltacovSR mutant was unable to initiate growth in RPMI and its viability in human normal serum was greatly impaired. A major finding of this phenotypic analysis was that the CovS/CovR system is important for GBS virulence, as a 3 log increase of the LD(50) of the mutant strain was observed in the neonate rat sepsis model. The pleiotropic phenotype of the DeltacovSR mutant is in full agreement with the large number of genes controlled by CovS/CovR as seen by expression profiling analysis, many of which encode potentially secreted or cell surface-associated proteins: 76 genes are repressed whereas 63 were positively regulated. CovR was shown to bind directly to the regulatory regions of several of these genes and a consensus CovR recognition sequence was proposed using both DNase I footprinting and computational analyses.
Full-text · Article · Jan 2005 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, replicates as an intracellular parasite of amoebae and persists in the environment as a free-living microbe. Here we have analyzed the complete genome sequences of L. pneumophila Paris (3,503,610 bp, 3,077 genes), an endemic strain that is predominant in France, and Lens (3,345,687 bp, 2,932 genes), an epidemic strain responsible for a major outbreak of disease in France. The L. pneumophila genomes show marked plasticity, with three different plasmids and with about 13% of the sequence differing between the two strains. Only strain Paris contains a type V secretion system, and its Lvh type IV secretion system is encoded by a 36-kb region that is either carried on a multicopy plasmid or integrated into the chromosome. Genetic mobility may enhance the versatility of L. pneumophila. Numerous genes encode eukaryotic-like proteins or motifs that are predicted to modulate host cell functions to the pathogen's advantage. The genome thus reflects the history and lifestyle of L. pneumophila, a human pathogen of macrophages that coevolved with fresh-water amoebae.
[Show abstract][Hide abstract] ABSTRACT: Entomopathogenic bacteria of the genus Photorhabdus harbor a type III secretion system. This system was probably acquired prior to the separation of the species within this
genus. Furthermore, the core components of the secretion machinery are highly conserved but the predicted effectors differ
between Photorhabdus luminescens and P. asymbiotica, two highly related species with different hosts.
Full-text · Article · Aug 2004 · Journal of Bacteriology
[Show abstract][Hide abstract] ABSTRACT: Photorhabdus luminescens is an insect-pathogenic bacterium that forms a symbiosis with specific entomopathogenic nematodes. In this bacterium, a symbiosis-'deficient' phenotypic variant (known as the secondary variant or form II) arises at a low frequency during prolonged incubation. A knock-out mutant was generated of the regulator of a newly identified two-component regulatory system, designated AstR-AstS. Interestingly, this mutation altered the timing of phenotypic switching. Variant cells arose in the mutant strain several days before they did in the wild-type population, suggesting that AstRS is directly or indirectly involved in the genetic mechanism underlying variant cell formation. This mutation also affected motility and antibiotic synthesis. To identify AstRS-regulated genes, a comparative analysis using two-dimensional gel electrophoresis was performed. Seventeen proteins with modified synthesis in stationary phase were identified by mass spectrometry and shown to be involved in electron-transport systems, energy metabolism, iron acquisition and stress responses. The results imply that AstRS is involved in the adaptation of cells to the stationary phase, whilst negatively affecting the competitive advantage of form I cells. The link between AstRS-dependent stationary-phase adaptation and phenotypic variation is discussed.
[Show abstract][Hide abstract] ABSTRACT: Analysis of the Photorhabdus luminescens genome sequence revealed that the pts region is related to the tail synthesis gene core of the P2 phage. The pts locus encodes a DNA invertase homologue. PCR-RFLP analysis showed the two potential tail fiber regions of the pts locus present DNA inversions. Electron microscopy revealed a phage tail-like particle, related to the R-type family and named R-photorhabdicin, in the culture supernatant of P. luminescens. Mass spectrometry analysis of two sub-units of R-photorhabdicin revealed that they are encoded by the pts locus. The role of this P2-related prophage remnant in the Photorhabdus genome is discussed.
Full-text · Article · May 2004 · FEMS Microbiology Letters
[Show abstract][Hide abstract] ABSTRACT: Contigs-Assembly and Annotation Tool-Box (CAAT-Box) is a software package developed for the computational part of a genome project where the sequence is obtained by a shotgun strategy. CAAT-Box contains new tools to predict links between contigs by using similarity searches with other whole genome sequences. Most importantly, it allows annotation of a genome to commence during the finishing phase using a gene-oriented strategy. For this purpose, CAAT-Box creates an Individual Protein file (IPF) for each ORF of an assembly. The nucleotide sequence reported in an IPF corresponds to the sequence of the ORF with 500 additional bases before the ORF and 200 bases after. For annotation, additional information like Blast results can be added or linked to the IPFs as well as automatic and/or manual annotations. When a new assembly is performed, CAAT-Box creates new IPFs according to the old IPF panel. CAAT-Box recognizes the modified IPFs which are the only ones used for a new automatic analysis after each assembly. Using this strategy, the user works with a group of IPFs independently of the closure phase progression. The IPFs are accessible by a web server and can therefore be modified and commented by different groups.
CAAT-Box was used to obtain and to annotate several complete genomes like Listeria monocytogenes or Streptococcus agalactiae.
The program may be obtained from the authors and is freely available to non-profit organisations.
[Show abstract][Hide abstract] ABSTRACT: Photorhabdus luminescens is a symbiont of entomopathogenic nematodes. Analysis of the genome sequence of this organism revealed a homologue of PhoP-PhoQ,
a two-component system associated with virulence in intracellular bacterial pathogens. This organism was shown to respond
to the availability of environmental magnesium. A mutant with a knockout mutation in the regulatory component of this system
(phoP) had no obvious growth defect. It was, however, more motile and more sensitive to antimicrobial peptides than its wild-type
parent. Remarkably, the mutation eliminated virulence in an insect model. No insect mortality was observed after injection
of a large number of the phoP bacteria, while very small amounts of parental cells killed insect larvae in less than 48 h. At the molecular level, the
PhoPQ system mediated Mg2+-dependent modifications in lipopolysaccharides and controlled a locus (pbgPE) required for incorporation of 4-aminoarabinose into lipid A. Mg2+-regulated gene expression of pbgP1 was absent in the mutant and was restored when phoPQ was complemented in trans. This finding highlights the essential role played by PhoPQ in the virulence of an entomopathogen.
Full-text · Article · Apr 2004 · Journal of Bacteriology