Architecture of Burkholderia cepacia complex σ70 gene family: evidence of alternative primary and clade-specific factors, and genomic instability

<p>Abstract</p> <p>Background</p> <p>The <it>Burkholderia cepacia </it>complex (Bcc) groups bacterial species with beneficial properties that can improve crop yields or remediate polluted sites but can also lead to dramatic human clinical outcomes among cystic fibrosis (CF) or immuno-compromised individuals. Genome-wide regulatory processes of gene expression could explain parts of this bacterial duality. Transcriptional σ⁷⁰factors are components of these processes. They allow the reversible binding of the DNA-dependent RNA polymerase to form the holoenzyme that will lead to mRNA synthesis from a DNA promoter region. Bcc genome-wide analyses were performed to investigate the major evolutionary trends taking place in the σ⁷⁰family of these bacteria.</p> <p>Results</p> <p>Twenty σ⁷⁰paralogous genes were detected in the <it>Burkholderia cenocepacia </it>strain J2315 (<it>Bcen</it>-J2315) genome, of which 14 were of the ECF (extracytoplasmic function) group. Non-ECF paralogs were related to primary (<it>rpoD</it>), alternative primary, stationary phase (<it>rpoS</it>), flagellin biosynthesis (<it>fliA</it>), and heat shock (<it>rpoH</it>) factors. The number of σ⁷⁰genetic determinants among this genome was of 2,86 per Mb. This number is lower than the one of <it>Pseudomonas aeruginosa</it>, a species found in similar habitats including CF lungs. These two bacterial groups showed strikingly different σ⁷⁰family architectures, with only three ECF paralogs in common (<it>fecI</it>-like, <it>pvdS </it>and <it>algU</it>). <it>Bcen</it>-J2315 σ⁷⁰paralogs showed clade-specific distributions. Some paralogs appeared limited to the ET12 epidemic clone (<it>ecfA2</it>), particular Bcc species (<it>sigI</it>), the <it>Burkholderia </it>genus (<it>ecfJ</it>, <it>ecfF</it>, and <it>sigJ</it>), certain proteobacterial groups (<it>ecfA1</it>, <it>ecfC</it>, <it>ecfD</it>, <it>ecfE</it>, <it>ecfG</it>, <it>ecfL</it>, <it>ecfM </it>and <it>rpoS</it>), or were broadly distributed in the eubacteria (<it>ecfI</it>, <it>ecfK</it>, <it>ecfH</it>, <it>ecfB</it>, and <it>rpoD</it>-, <it>rpoH</it>-, <it>fliA</it>-like genes). Genomic instability of this gene family was driven by chromosomal inversion (<it>ecfA2</it>), recent duplication events (<it>ecfA </it>and <it>RpoD</it>), localized (<it>ecfG</it>) and large scale deletions (<it>sigI</it>, <it>sigJ</it>, <it>ecfC</it>, <it>ecfH</it>, and <it>ecfK</it>), and a phage integration event (<it>ecfE</it>).</p> <p>Conclusion</p> <p>The Bcc σ⁷⁰gene family was found to be under strong selective pressures that could lead to acquisition/deletion, and duplication events modifying its architecture. Comparative analysis of Bcc and <it>Pseudomonas aeruginosa </it>σ⁷⁰gene families revealed distinct evolutionary strategies, with the Bcc having selected several alternative primary factors, something not recorded among <it>P. aeruginosa </it>and only previously reported to occur among the actinobacteria.</p

Development and Application of a Simple Plaque Assay for the Human Malaria Parasite Plasmodium falciparum.

Malaria is caused by an obligate intracellular protozoan parasite that replicates within and destroys erythrocytes. Asexual blood stages of the causative agent of the most virulent form of human malaria, Plasmodium falciparum, can be cultivated indefinitely in vitro in human erythrocytes, facilitating experimental analysis of parasite cell biology, biochemistry and genetics. However, efforts to improve understanding of the basic biology of this important pathogen and to develop urgently required new antimalarial drugs and vaccines, suffer from a paucity of basic research tools. This includes a simple means of quantifying the effects of drugs, antibodies and gene modifications on parasite fitness and replication rates. Here we describe the development and validation of an extremely simple, robust plaque assay that can be used to visualise parasite replication and resulting host erythrocyte destruction at the level of clonal parasite populations. We demonstrate applications of the plaque assay by using it for the phenotypic characterisation of two P. falciparum conditional mutants displaying reduced fitness in vitro

Enkele proeven over de invloed van de stikstofvorm op het optreden van stip bij paprika

<p><b>Copyright information:</b></p><p>Taken from "Architecture of complex σgene family: evidence of alternative primary and clade-specific factors, and genomic instability"</p><p>http://www.biomedcentral.com/1471-2164/8/308</p><p>BMC Genomics 2007;8():308-308.</p><p>Published online 4 Sep 2007</p><p>PMCID:PMC2194791.</p><p></p>mes using ACT [36]. -J2315 ORF annotations were assigned using other annotated genomes. ORF numbers assigned by the Sanger Institute are indicated. Dashes indicate missing regions. Phage DNA in (c) indicates a potential phage insertion. Arrows indicating ORF of sigma factors are filled with dots, and of anti-sigma factors are filled with vertical bars

The "Bourg-en-Bresse" epidemic clone of Burkholderia cenocepacia : origin, phylogenetic position and genetic events associated with its emergence

The Burkholderia cepacia complex (Bcc) comprises 17 species found in lung infections of individuals with cystic fibrosis. The bacteria of this complex are present in the soil, the rhizosphere of field crops, wastewater and may also be encountered in nosocomial infections. In France, the B. multivorans and B. cenocepacia species are the major species in infections of cystic fibrosis patients. Various epidemic clones have been described within the B. cenocepacia species whose ET12 clone associated with "cepacia syndrome". In 2004, a nosocomial outbreak involving a clone of Bcc occurred in a French hospital. During this outbreak, origin of this clone (B&B clone), its classification within the Bcc and several genetic events associated with its emergence have been studied. These investigations have identified this clone as belonging to the species B. cenocepacia with a strong proximity with the ET12 lineage. The study of transcriptional factors of σ70 family within the Bcc has revealed a similar genetic structure between the ET12 lineage and this clone, but different from that observed in other species of Bcc. Analysis of genetic elements repeated family of insertion sequences (IS), however, allowed to observe a distinct genomic organization of the ET12 lineage. It has been linked to phenomen of genetic instability including acquisition of mobile genetic elements like genomic island (GI). All of this work has helped to characterize a set of genetic events may explain the emergence of epidemic clones such as clone B&B.Le complexe Burkholderia cepacia (Bcc) englobe 17 espèces retrouvées dans les infections pulmonaires d'individus atteints de mucoviscidose. Les bactéries de ce complexe sont présentes dans les sols, la rhizosphère de grandes cultures, les eaux usées et peuvent également être rencontrées dans le cadre d'infections nosocomiales. En France, les espèces B. multivorans et B. cenocepacia (Bcen) sont les espèces majoritaires au niveau des infections de patients atteints de mucoviscidose. Divers clones épidémiques ont été décrits au sein de l'espèce Bcen dont le clone ET12 associé au "syndrome cepacia". En 2004, une épidémie nosocomiale impliquant un clone du Bcc est survenue dans un hôpital de l'Ain. Durant ce travail, l'origine de ce clone (B&B), sa classification au sein du Bcc et certains phénomènes génétiques liés à son émergence ont été étudiés. Cela a permis d'identifier ce clone comme appartenant à l'espèce Bcen et une forte proximité de celui-ci avec la lignée ET12. L'étude des facteurs transcriptionnels de la famille σ70 au sein du Bcc a mis en évidence une structure génétique similaire entre la lignée ET12 et ce clone, mais différente de celle observée chez les autres espèces du Bcc. L'analyse d'éléments génétiques répétés de la famille des séquences d'insertion (IS) a cependant permis d'observer une organisation génomique distincte de la lignée ET12. Celle-ci a été reliée à des phénomènes d'instabilité génétique notamment à des phénomènes d'acquisition d'éléments génétiques mobiles de type îlot génomique. L'ensemble de ce travail a permis de caractériser un ensemble de phénomènes génétiques pouvant expliquer l'émergence de clones épidémiques tels que le clone B&B

Le clone épidémique "Bourg-en-Bresse" de l’espèce Burkholderia cenocepacia : origine, positionnement phylétique et phénomènes génétiques liés à son émergence

The Burkholderia cepacia complex (Bcc) comprises 17 species found in lung infections of individuals with cystic fibrosis. The bacteria of this complex are present in the soil, the rhizosphere of field crops, wastewater and may also be encountered in nosocomial infections. In France, the B. multivorans and B. cenocepacia species are the major species in infections of cystic fibrosis patients. Various epidemic clones have been described within the B. cenocepacia species whose ET12 clone associated with "cepacia syndrome". In 2004, a nosocomial outbreak involving a clone of Bcc occurred in a French hospital. During this outbreak, origin of this clone (B&B clone), its classification within the Bcc and several genetic events associated with its emergence have been studied. These investigations have identified this clone as belonging to the species B. cenocepacia with a strong proximity with the ET12 lineage. The study of transcriptional factors of σ70 family within the Bcc has revealed a similar genetic structure between the ET12 lineage and this clone, but different from that observed in other species of Bcc. Analysis of genetic elements repeated family of insertion sequences (IS), however, allowed to observe a distinct genomic organization of the ET12 lineage. It has been linked to phenomen of genetic instability including acquisition of mobile genetic elements like genomic island (GI). All of this work has helped to characterize a set of genetic events may explain the emergence of epidemic clones such as clone B&B.Le complexe Burkholderia cepacia (Bcc) englobe 17 espèces retrouvées dans les infections pulmonaires d'individus atteints de mucoviscidose. Les bactéries de ce complexe sont présentes dans les sols, la rhizosphère de grandes cultures, les eaux usées et peuvent également être rencontrées dans le cadre d'infections nosocomiales. En France, les espèces B. multivorans et B. cenocepacia (Bcen) sont les espèces majoritaires au niveau des infections de patients atteints de mucoviscidose. Divers clones épidémiques ont été décrits au sein de l’espèce Bcen dont le clone ET12 associé au "syndrome cepacia". En 2004, une épidémie nosocomiale impliquant un clone du Bcc est survenue dans un hôpital de l’Ain. Durant ce travail, l’origine de ce clone (B&B), sa classification au sein du Bcc et certains phénomènes génétiques liés à son émergence ont été étudiés. Cela a permis d’identifier ce clone comme appartenant à l’espèce Bcen et une forte proximité de celui-ci avec la lignée ET12. L’étude des facteurs transcriptionnels de la famille σ70 au sein du Bcc a mis en évidence une structure génétique similaire entre la lignée ET12 et ce clone, mais différente de celle observée chez les autres espèces du Bcc. L’analyse d’éléments génétiques répétés de la famille des séquences d’insertion (IS) a cependant permis d’observer une organisation génomique distincte de la lignée ET12. Celle-ci a été reliée à des phénomènes d’instabilité génétique notamment à des phénomènes d’acquisition d’éléments génétiques mobiles de type îlot génomique. L’ensemble de ce travail a permis de caractériser un ensemble de phénomènes génétiques pouvant expliquer l’émergence de clones épidémiques tels que le clone B&B

New insights into parasite rhomboid proteases

The rhomboid-like proteins constitute a large family of intramembrane serine proteases that are present in all branches of life. First studied in Drosophila, these enzymes catalyse the release of the active forms of proteins from the membrane and hence trigger signalling events. In protozoan parasites, a limited number of rhomboid-like proteases have been investigated and some of them are associated to pathogenesis. In Apicomplexans, rhomboid-like protease activity is involved in shedding adhesins from the surface of the zoites during motility and host cell entry. Recently, a Toxoplasma gondii rhomboid was also implicated in an intracellular signalling mechanism leading to parasite proliferation. In Entamoeba histolytica, the capacity to adhere to host cells and to phagocytose cells is potentiated by a rhomboid-like protease. Survey of a small number of protozoan parasite genomes has uncovered species-specific rhomboid-like protease genes, many of which are predicted to encode inactive enzymes. Functional investigation of the rhomboid-like proteases in other protozoan parasites will likely uncover novel and unexpected implications for this family of proteases

Iron depletion has different consequences on the growth and survival of Toxoplasma gondii strains

International audienceToxoplasma gondii is an obligate intracellular parasite responsible for a pathology called toxoplasmosis, which primarily affects immunocompromised individuals and developing foetuses. The parasite can scavenge essential nutrients from its host to support its growth and survival. Among them, iron is one of the most important elements needed to sustain basic cellular functions as it is involved in a number of key metabolic processes, including oxygen transport, redox balance, and electron transport. We evaluated the effects of an iron chelator on the development of several parasite strains and found that they differed in their ability to tolerate iron depletion. The growth of parasites usually associated with a model of acute toxoplasmosis was strongly affected by iron depletion, whereas cystogenic strains were less sensitive as they were able to convert into persisting developmental forms that are associated with the chronic form of the disease. Ultrastructural and biochemical characterization of the impact of iron depletion on parasites also highlighted striking changes in both their metabolism and that of the host, with a marked accumulation of lipid droplets and perturbation of lipid homoeostasis. Overall, our study demonstrates that although acute iron depletion has an important effect on the growth of T. gondii, it has a more profound impact on actively dividing parasites, whereas less metabolically active parasite forms may be able to avoid some of the most detrimental consequences

The Conoid Associated Motor MyoH Is Indispensable for Toxoplasma gondii Entry and Exit from Host Cells

Many members of the phylum of Apicomplexa have adopted an obligate intracellular life style and critically depend on active invasion and egress from the infected cells to complete their lytic cycle. Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa, and as such, the invasive tachyzoite contains an organelle termed the conoid at its extreme apex. This motile organelle consists of a unique polymer of tubulin fibres and protrudes in both gliding and invading parasites. The class XIV myosin A, which is conserved across the Apicomplexa phylum, is known to critically contribute to motility, invasion and egress from infected cells. The MyoA-glideosome is anchored to the inner membrane complex (IMC) and is assumed to translocate the components of the circular junction secreted by the micronemes and rhoptries, to the rear of the parasite. Here we comprehensively characterise the class XIV myosin H (MyoH) and its associated light chains. We show that the 3 alpha-tubulin suppressor domains, located in MyoH tail, are necessary to anchor this motor to the conoid. Despite the presence of an intact MyoA-glideosome, conditional disruption of TgMyoH severely compromises parasite motility, invasion and egress from infected cells. We demonstrate that MyoH is necessary for the translocation of the circular junction from the tip of the parasite, where secretory organelles exocytosis occurs, to the apical position where the IMC starts. This study attributes for the first time a direct function of the conoid in motility and invasion, and establishes the indispensable role of MyoH in initiating the first step of motility along this unique organelle, which is subsequently relayed by MyoA to enact effective gliding and invasion