Sequencing and analysis of bacterial genomes:

Biodiversity and evolution - Comparative genomics :

Transcriptome studies - Regulatory networks :


Streptococcus agalactiae CovS/CovR regulon


Mol Microbiol. 2004 Dec;54(5):1250-68

CovS/CovR of group B streptococcus: a two-component global regulatory system involved in virulence.

Lamy MC, Zouine M, Fert J, Vergassola M, Couve E, Pellegrini E, Glaser P, Kunst F, Msadek T, Trieu-Cuot P, Poyart C

Summary 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 Delta covSR mutant was found to display dramatic phenotypic changes such as increased haemolytic activity and reduced CAMP activity on blood agar. Adherence of the Delta covSR 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.


Listeria monocytogenes serovar 4b Genome

Description:

L. monocytogenes, an intracellular pathogen, is the causative agent of serious epidemic and sporadic food-borne listeriosis. The clinical features of listeriosis include meningitis, meningo-encephalitis, septicemia, abortion, perinatal infections and gastroenterits. Although rare when compared to other food-borne diseases, a significant feature of listeriosis is the high lethality rate (about 30%), which makes L. monocytogenes an important human pathogen.

However, not all strains of L. monocytogenes seem to be equally capable of causing disease in humans. Isolates from four (1/2a; 1/2c; 1/2b; 4b) of the 13 serovars identified within this species are responsible for over 98% of the human listeriosis cases reported. Furthermore, all major food-borne outbreaks of listeriosis, as well as the majority of sporadic cases, have been caused by serovar 4b strains suggesting that strains of this sv may possess unique virulence properties.

We have sequenced the genome of the epidemic L. monocytogenes serotype 4b strain CLIP 80459. Its size is 2906 kb and annotation and analysis of its genome is under way. We have recently determined the complete genome sequences of L. monocytogenes strain EGDe (serovar 1/2a) and Listeria innocua strain CLIP11262 (Glaser et al. 2001). The sequence of L. monocytogenes serotype 4b will help to asses genetic diversity among the different L. monocytogenes serotypes and to determine whether genetic loci exist that are specific to epidemic L. monocytogenes.


For inquiries on Listeria monocytogenes serotype 4b sequences, please contact Carmen Buchrieser or Philippe Glaser.



Listeria grayi Genome

Description:


Listeria grayi ("L. murrayi") is an environmental bacterium belonging to the genus Listeria. L. grayi is the most distantly related species within the genus, a fact, which lead to a long controversy about the taxonomic position of L. grayi ("L. murrayi"). However, L. grayi ("L. murrayi") and L. monocytogenes share a number of similarities: they cluster in all numerical taxonomic studies, possess lipoteichoic acid, teichoic acid of the polyribitol phosphate type, peptidoglycan of the A1 gamma variation, nonhydrogenated menaquinones of the MK-7 type, and the same cytochromes. In contrast L. grayi shows a slight difference in G+C % content (42%), a small number of biochemical reactions, differences in the nature of substitution of the lipoteichoic acids, slight differences of protein electrophoregrams, cellular fatty acids composition, antigenic structure and low DNA homology values. Finally, all data together with 16S rRNA gene sequencing defined L. grayi , which is divided into two subspecies L. grayi subsp. grayi and L. grayi subsp. murrayi as belonging to the genus Listeria.

We are determining the complete genome sequence of L. grayi subsp. murrayi strain CLIP12515. The genome has an estimated size of 2.6 Mb. The whole-genome random sequencing strategy is followed. A 8x coverage of the genome is foreseen in the shotgun phase and a BAC library was constructed and end sequenced to help in the finishing phase. Furthermore for the finishing phase the complete genome sequences of different Listeria monocytogenes and a Listeria innocua strains available should help. The availability of the complete genome sequence of L. grayi, which constitutes an "outgroup" in the genus Listeria and its comparison with Listeria monocytogenes, Listeria innocua and the remaining three Listeria species, Listeria ivanovii, Listeria seeligeri and Listeria welshimeri, being sequenced in collaboration with the German Pathogenomik Network, will allow comparative genomic analyses among all Listeria species. The results from these comparisons should help to uncover evolution of virulence determinants in the genus Listeria and help to better understand phylogenetic relationships and evolution within the genus Listeria.

Collaboations:

This project is carried out in collaboration with the German Pathogenomik Network (Werner Goebel and Juergen Kreft, Universitaet Wuerzburg, Gerhard Gottschalk, and Axel Strittmatter University Goettingen)

For inquiries on Listeria grayi sequences, please contact Philippe Glaser.


Listeria ivanovii Genome

Description:

Listeria ivanovii, primarily a ruminant pathogen, is a gram-positive facultative intracellular parasite. Human cases of Listeria ivanovii infections are rare, the vast majority of reported isolations of this species being from abortions, stillbirths, and neonatal septicemia in sheep and cattle.

We are determining the complete genome sequence of Listeria ivanovii strain PAM 55 (serotype 5). The strain was isolated from an abortion during an outbreak in ewe in 1997 in Spain. The LD50 in the mouse model is a log lower than that for the type strain (ATCC 19119). The size is estimated between 3 to 3.2 Mb. Sequencing and the informatics part are done in collaboration by all partners.

The availability of the sequences of Listeria monocytogenes EGD and Listeria innocua will allow comparative genomic analyses between three Listeria species. The results from this comparison should help to uncover new putative virulence determinants, aid the study of the exact mode of action of known virulence genes of L. monocytogenes as well as of L. invanovii and help to better understand phylogenetic relationships and evolution within the genus Listeria.

Collaborations:

This project is carried out in collaboration with Pascale Cossart, Unité des Interactions Bactéries-Cellules, Institut Pasteur, José-Antonio Vazquez-Boland, Universidad de Leon, Spain, and the Competence Centre Pathogenomik Wuerzburg (Werner Goebel and Juergen Kreft, Universitaet Wuerzburg, Trinad Chakraborty, Universitaet Giessen, Juergen Wehland, Braunschweig, Germany).

Sequencing strategy:

The whole-genome random sequencing strategy is followed. A 6x coverage of the genome is foreseen in the shotgun phase. For the finishing phase the completed genome sequence of Listeria monocytogenes and Listeria innocua should help.

Statistics:

The progress of this genome project is summarized in the section Listeria ivanovii statistics.

Contact:

For inquiries on Listeria ivanovii sequences, please contact Philippe Glaser.

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Listeria monocytogenes sigma 54 regulon


Microbiology. 2004 May;150(Pt 5):1581-90.

Global analysis of gene expression in an rpoN mutant of Listeria monocytogenes

Arous S, Buchrieser C, Folio P, Glaser P, Namane A, Hebraud M, Hechard Y

The role of the alternative sigma(54) factor, encoded by the rpoN gene, was investigated in Listeria monocytogenes by comparing the global gene expression of the wild-type EGDe strain and an rpoN mutant. Gene expression, using whole-genome macroarrays, and protein content, using two-dimensional gel electrophoresis, were analysed. Seventy-seven genes and nine proteins, whose expression was modulated in the rpoN mutant as compared to the wild-type strain, were identified. Most of the modifications were related to carbohydrate metabolism and in particular to pyruvate metabolism. However, under the conditions studied, only the mptACD operon was shown to be directly controlled by sigma(54). Therefore, the remaining modifications seem to be due to indirect effects. In parallel, an in silico analysis suggests that sigma(54) may directly control the expression of four different phosphotransferase system (PTS) operons, including mptACD. PTS activity is known to have a direct effect on the pyruvate pool and on catabolite regulation. These results suggest that sigma(54) is mainly involved in the control of carbohydrate metabolism in L. monocytogenes via direct regulation of PTS activity, alteration of the pyruvate pool and modulation of carbon catabolite regulation


This project was carried out in collaboration with Yann Hechard (Equipe de Microbiologie Fondamentale et Appliquee, Universite de Poitiers)and the Plate-forme Proteome, Pasteur Genopole. Ile de France (directed by Abdelkade Namane)


Legionella pneumophila Genome


Nature Genetics, 2004 Nov;36(11):1165-73. Epub 2004 Oct 03, Pub Med Supplemental Information


Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity

Christel Cazalet, Christophe Rusniok, Holger Brueggemann, Nora Zidane, Arnaud Magnier, Laurence Ma, Magalie Tichit, Sophie Jarraud, Christiane Bouchier, Frangois Vandenesch, Frank Kunst, Jerome Etienne, Philippe Glaser & Carmen Buchrieser

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.


This project was carried out in collaboration with the NRC for Legionella (directed by Prof. J. Etienne), the Plate-forme GCenomique, Pasteur Genopole. Ile de France (directed by C. Bouchier) and Veolia Water

The complete annotated sequences of Legionella pneumophila strain Paris and strain Lens are available at an interactive web server at the Institut Pasteur LegioList

Press release

For inquiries on L pneumophila sequences, please contact Carmen Buchrieser or Christel Cazalet.

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Photorhabdus luminescens Genome


Nat Biotechnol. 2003 Nov;21(11):1307-13.

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens.

Duchaud E, Rusniok C, Frangeul L, Buchrieser C, Givaudan A, Taourit S, Bocs S, Boursaux-Eude C, Chandler M, Charles JF, Dassa E, Derose R, Derzelle S, Freyssinet G, Gaudriault S, Medigue C, Lanois A, Powell K, Siguier P, Vincent R, Wingate V, Zouine M, Glaser P, Boemare N, Danchin A and F Kunst.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.


Collaborations:

Functional analysis is performed at the Unité de Régulation de l'Expression Génétique, Institut Pasteur (Responsible scientist : Antoine Danchin).

The interactions of Photorhabdus with both hosts, the nematode and the insect, is studied in collaboration with the Laboratoire de Pathologie Comparée, INRA, Montpellier.

Contact:

For scientific correspondance, please contact Dr. Eric Duchaud.


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Streptococcus agalactiae Genome

Mol Microbiol. 2002 Sep;45(6):1499-513


Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease.

Glaser P, Rusniok C, Buchrieser C, Chevalier F, Frangeul L, Msadek T, Zouine M, Couve E, Lalioui L, Poyart C, Trieu-Cuot P, Kunst F.

Streptococcus agalactiae is a commensal bacterium colonizing the intestinal tract of a significant proportion of the human population. However, it is also a pathogen which is the leading cause of invasive infections in neonates and causes septicaemia, meningitis and pneumonia. We sequenced the genome of the serogroup III strain NEM316, responsible for a fatal case of septicaemia. The genome is 2 211 485 base pairs long and contains 2118 protein coding genes. Fifty-five per cent of the predicted genes have an ortholog in the Streptococcus pyogenes genome, representing a conserved backbone between these two streptococci. Among the genes in S. agalactiae that lack an ortholog in S. pyogenes, 50% are clustered within 14 islands. These islands contain known and putative virulence genes, mostly encoding surface proteins as well as a number of genes related to mobile elements. Some of these islands could therefore be considered as pathogenicity islands. Compared with other pathogenic streptococci, S. agalactiae shows the unique feature that pathogenicity islands may have an important role in virulence acquisition and in genetic diversity.

Collaborations:

The projects on Streptococcus agalactaie are carried out in collaboration with Claire POYART and Patrick TRIEU-CUOT (Laboratoire Mixte Pasteur-Necker de Recherche sur les Streptocoques) and with Tarek MSADEK (Unité de Biochimie Microbienne, Institut Pasteur).

For more information on S. agalactiae genome go to the SagaList WWW server

For inquiries on the Streptococcus agalactiae projcts, please contact P. Glaser or F. Kunst.


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Listeria monocytogenes and Listeria innocua genomes

Science 294 (5543) 849-852, supplementary information (pdf)

Comparative genomics of Listeria species

Glaser P, Frangeul L, Buchrieser C, Rusniok C, Amend A, Baquero F, Berche P, Bloecker H, Brandt P, Chakraborty T, Charbit A, Chetouani F, Couve E, de Daruvar A, Dehoux P, Domann E, Dominguez-Bernal G, Duchaud E, Durant L, Dussurget O, Entian KD, Fsihi H, Portillo FG, Garrido P, Gautier L, Goebel W, Gomez-Lopez N, Hain T, Hauf J, Jackson D, Jones LM, Kaerst U, Kreft J, Kuhn M, Kunst F, Kurapkat G, Madueno E, Maitournam A, Vicente JM, Ng E, Nedjari H, Nordsiek G, Novella S, de Pablos B, Perez-Diaz JC, Purcell R, Remmel B, Rose M, Schlueter T, Simoes N, Tierrez A, Vazquez-Boland JA, Voss H, Wehland J, Cossart P
Listeria monocytogenes european consortium

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

For more information on Listeria genomes go to the ListiList WWW server


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Listeria monocytogenes PrfA regulon

Mol Microbiol. 2003 Mar;47(6):1613-25.


Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA.

Milohanic E, Glaser P, Coppee J Y, Frangeul L, Vega Y, Vazquez-Boland J A, Kunst F, Cossart P and C. Buchrieser.


PrfA is the major regulator of Listeria virulence gene expression. This protein is a member of the Crp/Fnr family of transcription regulators. To gain a deeper understanding of the PrfA regulon we constructed a whole-genome array based on the complete genome sequence of Listeria monocytogenes strain EGDe and evaluated the expression profiles of the wild-type EGDe and a prfA-deleted mutant (EGDe Delta prfA). Both strains were grown at 37 degrees C in brain-heart infusion broth (BHI) and BHI supplemented with either activated charcoal, a compound known to enhance virulence gene expression, or cellobiose, a sugar reported to downregulate virulence gene expression in spite of full expression of PrfA. We identified three groups of genes that are regulated differently. Group I comprises, in addition tothe 10 already known genes, two new genes, lmo2219 and lmo0788, both positively regulated and preceded by a putative PrfA box. Group II comprises eight negatively regulated genes: lmo0278 is preceded by a putative PrfA box, and the remaining seven genes (lmo0178-lmo0184) are organized in an operon. Group III comprises 53 genes, of which only two (lmo0596 and lmo2067) are preceded by a putative PrfA box. Charcoal addition induced upregulation of group I genes but abolished regulation by PrfA of most group III genes. In the presence of cellobiose, allthe group I genes were downregulated, whereas group III genes remained fully activated. Group II genes were repressed in all conditions tested. A comparison of the expression profiles between a second L. monocytogenes strain (P14), its spontaneous mutant expressing a constitutively active PrfA variant (P14prfA*) and its corresponding prfA-deleted mutant (P14 Delta prfA) and the EGDe strain revealed interesting strain-specific differences. Sequences strongly similar to a sigma B-dependent promoter were identified upstream of 22 group III genes. These resultssuggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box and probablyexpressed from a sigmaA-dependent promoter. In contrast, a second set of PrfA-regulated genes lack a PrfA box and are expressed from a sigma B-dependent promoter. This study reveals that PrfA can act as an activator or a repressor and suggests that PrfA may directly or indirectly activate different sets of genes in association with different sigma factors.



Shigella flexneri virulence plasmid

Mol Microbiol 38 (4) 760-771

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri.

Buchrieser C, Glaser P, Rusniok C, Nedjari H, d'Hauteville H, Kunst F, Sansonetti P and Parsot C

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.



Biodiversity within the species Strepotcoccus agalactiae


Microbes Infect. 2006, 8(5):1227-43, Supplemental Table S1 , Supplemental Table S2


Genomic diversity and evolution within the species Streptococcus agalactiae

Brochet M, Couve E, Zouine M, Vallaeys T, Rusniok C, Lamy MC, Buchrieser C, Trieu-Cuot P, Kunst F, Poyart C, Glaser P.

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.



In vivo gene expression of Legionella pneumophila in Acanthamoeba castellani and the FliA regulon


Cellular Microbiology, 2006, Online publication date: 3-Mar-2006 doi: 10.1111/j.1462-5822.2006.00703.x, Pub Med Supplemental Information


Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila

Holger Brueggemann, Arne Hagman, Matthieu Jules, Odile Sismeiro, Marie-Agnes Dillies, Catherine Gouyette, Frank Kunst, Michael Steinert, Klaus Heuner, Jean-Yves Coppie, Carmen Buchrieser

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.



Biodiversity within the species Listeria monocytogens and the genus Listeria

Infect Immun 72 (4) 1072-1083, supplemental material

New Aspects Regarding Evolution and Virulence of Listeria monocytogenes Revealed by Comparative Genomics and DNA Arrays

M Doumith, C Cazalet, N Simoes, L Frangeul, C Jacquet, F Kunst, P Martin, P Cossart, P Glaser, and C Buchrieser

Listeria monocytogenes is a food-borne bacterial pathogen that causes a wide spectrum of diseases, such as meningitis,septicemia, abortion, and gastroenteritis, in humans and animals. Among the 13 L. monocytogenes serovars described, invasive disease is mostly associated with serovar 4b strains. To investigate the genetic diversity of L. monocytogenes strains with different virulence potentials, we partially sequenced an epidemic serovar 4b strain and compared it with the complete sequence of the nonepidemic L. monocytogenes EGDe serovar 1/2a strain. We identified an unexpected genetic divergence between the two strains, as about 8% of the sequences were serovar 4b specific. These sequences included seven genes coding for surface proteins, two of which belong to the internalin family, and three genes coding for transcriptional regulators, all of which might be important in different steps of the infectious process. Based on the sequence information, we then characterized the gene content of 113 Listeria strains by using a newly designed Listeria array containing the "flexible" part of the sequenced Listeria genomes. Hybridization results showed that all of the previously identified virulence factors of L. monocytogenes were present in the 93 L. monocytogenes strains tested. However, distinct patterns of the presence or absence of other genes were identified among the different L. monocytogenes serovars and Listeria species. These results allow new insights into the evolution of L. monocytogenes, suggesting that early divergence of the ancestral L. monocytogenes serovar 1/2c strains from the serovar 1/2b strains led to two major phylogenetic lineages, one of them including the serogroup 4 strains, which branched off the serovar 1/2b ancestral lineage, leading (mostly by gene loss) to the species Listeria innocua. The identification of 30 L. monocytogenes-specific and several serovar-specific marker genes, such as three L. monocytogenes serovar 4b-specific surface protein-coding genes, should prove powerful for the rapid tracing of listeriosis outbreaks, but it also represents a fundamental basis for the functional study of virulence differences between L. monocytogenes strains.



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Pathogenicity island of Yersinia pestis

Infect Immun 1999 Sep;67(9):4851-61

The 102-kilobase pgm locus of Yersinia pestis: sequence analysis and comparison of selected regions among different Yersinia pestis and Yersinia pseudotuberculosis strains.

Buchrieser C, Rusniok C, Frangeul L, Couve E, Billault A, Kunst F, Carniel E, Glaser P

We report the complete 119,443-bp sequence of the pgm locus from Yersinia pestis and its flanking regions. Sequence analysis confirms that the 102-kb unstable pgm locus is composed of two distinct parts: the pigmentation segment and a high-pathogenicity island (HPI) which carries virulence genes involved in iron acquisition (yersiniabactin biosynthetic gene cluster). Within the HPI, three genes coding for proteins related to phage proteins were uncovered. They are located at both extremities indicating that the entire HPI was acquired en bloc by phage-mediated horizontal transfer. We identified, within the pigmentation segment, two novel loci that may be involved in virulence: a fimbriae gene cluster and a locus probably encoding a two component regulatory system similar to the BvgAS regulatory system of Bordetella pertussis. Three genes containing frameshift mutations and two genes interrupted by insertion element insertion were found within this region. To investigate diversity among different Y. pestis and Yersinia pseudotuberculosis strains, the sequence of selected regions of the pgm locus and flanking regions were compared from 20 different Y. pestis and 10 Y. pseudotuberculosis strains. The results showed that the genes interrupted in Y. pestis are intact in Y. pseudotuberculosis. However, one of these mutations, in the bvgS homologue, is only present in Y. pestis strains of biovar Orientalis and not in those of the biovars Antiqua and Medievalis. The results obtained by analysis of variable positions in the sequence are in accordance with historical records, confirming that biovar Orientalis is the most recent lineage. Furthermore, sequence comparisons among 29 Yersinia strains suggest that Y. pestis is a recently emerged pathogen that is probably entering the initial phase of reductive evolution.



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Genome comparison of Escherichia coli

Microbiology 2005 Feb;151(Pt 2):385-98 supplemental material

Characterization of the flexible genome complement of the commensal Escherichia coli strain A0 34/86 (O83 : K24 : H31).

Hejnova J, Dobrindt U, Nemcova R, Rusniok C, Bomba A, Frangeul L, Hacker J, Glaser P, Sebo P, Buchrieser C.

Colonization by the commensal Escherichia coli strain A0 34/86 (O83 : K24 : H31) has proved to be safe and efficient in the prophylaxis and treatment of nosocomial infections and diarrhoea of preterm and newborn infants in Czech paediatric clinics over the past three decades. In searching for traits contributing to this beneficial effect related to the gut colonization capacity of the strain, the authors have analysed its genome by DNA-DNA hybridization to E. coli K-12 (MG1655) genomic DNA arrays and to 'Pathoarrays', as well as by multiplex PCR, bacterial artificial chromosome (BAC) library cloning and shotgun sequencing. Four hundred and ten E. coli K-12 ORFs were absent from A0 34/86, while 72 out of 456 genes associated with pathogenicity islands of E. coli and Shigella were also detected in E. coli A0 34/86. Furthermore, extraintestinal pathogenic E. coli-related genes involved in iron uptake and adhesion were detected by multiplex PCR, and genes encoding the HlyA and cytotoxic necrotizing factor toxins, together with 21 genes of the uropathogenic E. coli 536 pathogenicity island II, were identified by analysis of 2304 shotgun and 1344 BAC clone sequences of A0 34/86 DNA. Multiple sequence comparisons identified 31 kb of DNA specific for E. coli A0 34/86; some of the genes carried by this DNA may prove to be implicated in the colonization capacity of the strain, enabling it to outcompete pathogens. Among 100 examined BAC clones roughly covering the A0 34/86 genome, one reproducibly conferred on the laboratory strain DH10B an enhanced capacity to persist in the intestine of newborn piglets. Sequencing revealed that this BAC clone carried gene clusters encoding gluconate and mannonate metabolism, adhesion (fim), invasion (ibe) and restriction/modification functions. Hence, the genome of this clinically safe and highly efficient colonizer strain appears to harbour many 'virulence-associated' genes. These results highlight the thin line between bacterial 'virulence' and 'fitness' or 'colonization' factors, and question the definition of enterobacterial virulence factors.

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