Genomic island excisions in Bordetella petrii

<p>Abstract</p> <p>Background</p> <p>Among the members of the genus <it>Bordetella B. petrii </it>is unique, since it is the only species isolated from the environment, while the pathogenic Bordetellae are obligately associated with host organisms. Another feature distinguishing <it>B. petrii </it>from the other sequenced Bordetellae is the presence of a large number of mobile genetic elements including several large genomic regions with typical characteristics of genomic islands collectively known as integrative and conjugative elements (ICEs). These elements mainly encode accessory metabolic factors enabling this bacterium to grow on a large repertoire of aromatic compounds.</p> <p>Results</p> <p>During <it>in vitro </it>culture of <it>Bordetella petrii </it>colony variants appear frequently. We show that this variability can be attributed to the presence of a large number of metastable mobile genetic elements on its chromosome. In fact, the genome sequence of <it>B. petrii </it>revealed the presence of at least seven large genomic islands mostly encoding accessory metabolic functions involved in the degradation of aromatic compounds and detoxification of heavy metals. Four of these islands (termed GI1 to GI3 and GI6) are highly related to ICE<it>clc </it>of <it>Pseudomonas knackmussii </it>sp. strain B13. Here we present first data about the molecular characterization of these islands. We defined the exact borders of each island and we show that during standard culture of the bacteria these islands get excised from the chromosome. For all but one of these islands (GI5) we could detect circular intermediates. For the <it>clc</it>-like elements GI1 to GI3 of <it>B. petrii </it>we provide evidence that tandem insertion of these islands which all encode highly related integrases and attachment sites may also lead to incorporation of genomic DNA which originally was not part of the island and to the formation of huge composite islands. By integration of a tetracycline resistance cassette into GI3 we found this island to be rather unstable and to be lost from the bacterial population within about 100 consecutive generations. Furthermore, we show that GI3 is self transmissible and by conjugation can be transferred to <it>B. bronchiseptica </it>thus proving it to be an active integrative and conjugative element</p> <p>Conclusion</p> <p>The results show that phenotypic variation of <it>B. petrii </it>is correlated with the presence of genomic islands. Tandem integration of related islands may contribute to island evolution by the acquisition of genes originally belonging to the bacterial core genome. In conclusion, <it>B. petrii </it>appears to be the first member of the genus in which horizontal gene transfer events have massively shaped its genome structure.</p

Detection of small RNAs in Bordetella pertussis and identification of a novel repeated genetic element

Background: Small bacterial RNAs (sRNAs) have been shown to participate in the regulation of gene expression and have been identified in numerous prokaryotic species. Some of them are involved in the regulation of virulence in pathogenic bacteria. So far, little is known about sRNAs in Bordetella, and only very few sRNAs have been identified in the genome of Bordetella pertussis, the causative agent of whooping cough. Results: An in silico approach was used to predict sRNAs genes in intergenic regions of the B. pertussis genome. The genome sequences of B. pertussis, Bordetella parapertussis, Bordetella bronchiseptica and Bordetella avium were compared using a Blast, and significant hits were analyzed using RNAz. Twenty-three candidate regions were obtained, including regions encoding the already documented 6S RNA, and the GCVT and FMN riboswitches. The existence of sRNAs was verified by Northern blot analyses, and transcripts were detected for 13 out of the 20 additional candidates. These new sRNAs were named Bordetella pertussis RNAs, bpr. The expression of 4 of them differed between the early, exponential and late growth phases, and one of them, bprJ2, was found to be under the control of BvgA/BvgS two-component regulatory system of Bordetella virulence. A phylogenetic study of the bprJ sequence revealed a novel, so far undocumented repeat of ~90 bp, found in numerous copies in the Bordetella genomes and in that of other Betaproteobacteria. This repeat exhibits certain features of mobil

The Airway Microbiota in Cystic Fibrosis: A Complex Fungal and Bacterial Community—Implications for Therapeutic Management

International audienceBackground Given the polymicrobial nature of pulmonary infections in patients with cystic fibrosis (CF), it is essential to enhance our knowledge on the composition of the microbial community to improve patient management. In this study, we developed a pyrosequencing approach to extensively explore the diversity and dynamics of fungal and prokaryotic populations in CF lower airways. Methodology and Principal Findings Fungi and bacteria diversity in eight sputum samples collected from four adult CF patients was investigated using conventional microbiological culturing and high-throughput pyrosequencing approach targeting the ITS2 locus and the 16S rDNA gene. The unveiled microbial community structure was compared to the clinical profile of the CF patients. Pyrosequencing confirmed recently reported bacterial diversity and observed complex fungal communities, in which more than 60% of the species or genera were not detected by cultures. Strikingly, the diversity and species richness of fungal and bacterial communities was significantly lower in patients with decreased lung function and poor clinical status. Values of Chao1 richness estimator were statistically correlated with values of the Shwachman-Kulczycki score, body mass index, forced vital capacity, and forced expiratory volume in 1 s (p = 0.046, 0.047, 0.004, and 0.001, respectively for fungal Chao1 indices, and p = 0.010, 0.047, 0.002, and 0.0003, respectively for bacterial Chao1 values). Phylogenetic analysis showed high molecular diversities at the sub-species level for the main fungal and bacterial taxa identified in the present study. Anaerobes were isolated with Pseudomonas aeruginosa, which was more likely to be observed in association with Candida albicans than with Aspergillus fumigatus

Méthode ab initio de prédiction d'opérons chez les procaryotes et validations biologiques chez les Bordetelles

Aujourd'hui comprendre les mécanismes moléculaires qui régissent la vie est un challenge de la biologie. C'est avec l'objectif de comprendre les voies métaboliques et leur régulation que de nombreuses méthodes in silico ont été développées. La conservation de l'organisation des gènes sur les génomes procaryotes reflète des interactions fonctionnelles, c'est pourquoi annoter ces structures est primordial. Au cours de nos travaux, nous avons développé un algorithme de prédiction d'opérons travaillant uniquement sur base d'une séquence génomique. Notre méthode combine la prédiction de plusieurs éléments génomiques ainsi qu'une analyse de la fréquence de distribution des nucléotides et dinucléotides afin de discriminer les séquences intergéniques transcrites des non transcrites. La robustesse de notre algorithme a été éprouvée sur deux jeux de données validés d'E coli et de B. subtilis et atteint une sensibilité de 88% et une spécificité de 89%. Afin d'annoter de nouvelles structures opéroniques chez E coli, nous avons biologiquement validé certains opérons prédits. Quatre nouveaux opérons ont été expérimentalement confinnés. De plus, nous avons vérifié biologiquement deux opérons, prédits par notre logiciel comme unités polycistroniques mais précédemment décrits dans la littérature comme juxtapositions d'unités monogéniques. Dans une dernIère partie, nous avons appliqué notre méthode au niveau de trois génomes de la famille des Bordetelles. Par une comparaison des annotations opéroniques, nous cherchons à mettre en évidence des différences d'organisation génomique pouvant entraîner des changements dans les spécificités d'hôte et pathogénicités de trois espèces très proches des Bordetelles, à savoir pertussis, parapertussis et bronchiseptica.LILLE1-BU (590092102) / SudocSudocFranceF

Hybrid approach based on a combination of methods for operon prediction

International audienc

Multilocus Variable-Number Tandem Repeat Typing of Mycobacterium ulcerans

The apparent genetic homogeneity of Mycobacterium ulcerans contributes to the poorly understood epidemiology of M. ulcerans infection. Here, we report the identification of variable number tandem repeat (VNTR) sequences as novel polymorphic elements in the genome of this species. A total of 19 potential VNTR loci identified in the closely related M. marinum genome sequence were screened in a collection of 23 M. ulcerans isolates, one Mycobacterium species referred to here as an intermediate species, and five M. marinum strains. Nine of the 19 loci were polymorphic in the three species (including the intermediate species) and revealed eight M. ulcerans and five M. marinum genotypes. The results from the VNTR analysis corroborated the genetic relationships of M. ulcerans isolates from various geographical origins, as defined by independent molecular markers. Although these results further highlight the extremely high clonal homogeneity within certain geographic regions, we report for the first time the discrimination of the two South American strains from Surinam and French Guyana. These findings support the potential of a VNTR-based genotyping method for strain discrimination within M. ulcerans and M. marinum

Temporal analysis of French Bordetella pertussis isolates by comparative whole-genome hybridization

Bordetella pertussis, a gram-negative beta-proteobacterium, is the agent of whooping cough in humans. Whooping cough remains a public health problem worldwide, despite well-implemented infant/child vaccination programs. It continues to be endemic and is observed cyclically in vaccinated populations. Classical molecular subtyping methods indicate that genome diversity among B. pertussis isolates is limited. Although the whole bacterial genome has been studied by pulsed-field gel electrophoresis, the genes implicated in the diversity have not been identified. We developed a B. pertussis whole-genome DNA microarray representing over 91% of the predicted coding sequences of the sequenced strain Tohama I. Genomic DNA from clinical isolates with various pulsed-field gel electrophoresis profile patterns was competitively hybridized with the DNA microarray and coding sequences were classified as present, absent or duplicated. Our data strongly suggest that the B. pertussis population is dynamic. In France, with highly vaccinated population, the genetic diversity is low and decreasing with time, and clonal expansion correlates with cycles of the disease. This decrease in diversity is essentially due to loss of genes and pseudogenes. The genes deleted are most of the time flanked by insertion sequences

Population Snapshot of Streptococcus pneumoniae Serotype 19A Isolates before and after Introduction of Seven-Valent Pneumococcal Vaccination for French Children▿

Serotype 19A Streptococcus pneumoniae strains are now more frequent in French children than before the introduction of a seven-valent conjugate vaccine (PCV7). By applying multilocus sequence typing to 144 serotype 19A isolates collected before and after beginning PCV7 vaccination, we detected clonal expansion of the preexisting penicillin-intermediate sequence type 276

Draft genome sequence of the intestinal parasite Blastocystis subtype 4-isolate WR1

The intestinal protistan parasite Blastocystis is characterized by an extensive genetic variability with 17 subtypes (ST1–ST17) described to date. Only the whole genome of a human ST7 isolate was previously sequenced. Here we report the draft genome sequence of Blastocystis ST4-WR1 isolated from a laboratory rodent at Singapore