RESUMEN
BACKGROUND: CTX-M-producing Escherichia coli strains are regarded as major global pathogens. METHODOLOGY/PRINCIPAL FINDINGS: The nucleotide sequence of three plasmids (pEC_B24: 73801-bp; pEC_L8: 118525-bp and pEC_L46: 144871-bp) from Escherichia coli isolates obtained from patients with urinary tract infections and one plasmid (pEC_Bactec: 92970-bp) from an Escherichia coli strain isolated from the joint of a horse with arthritis were determined. Plasmid pEC_Bactec belongs to the IncI1 group and carries two resistance genes: bla(TEM-1) and bla(CTX-M-15). It shares more than 90% homology with a previously published bla(CTX-M)-plasmid from E. coli of human origin. Plasmid pEC_B24 belongs to the IncFII group whereas plasmids pEC_L8 and pEC_L46 represent a fusion of two replicons of type FII and FIA. On the pEC_B24 backbone, two resistance genes, bla(TEM-1) and bla(CTX-M-15), were found. Six resistance genes, bla(TEM-1), bla(CTX-M-15), bla(OXA-1), aac6'-lb-cr, tetA and catB4, were detected on the pEC_L8 backbone. The same antimicrobial drug resistance genes, with the exception of tetA, were also identified on the pEC_L46 backbone. Genome analysis of all 4 plasmids studied provides evidence of a seemingly frequent transposition event of the bla(CTX-M-15)-ISEcp1 element. This element seems to have a preferred insertion site at the tnpA gene of a bla(TEM)-carrying Tn3-like transposon, the latter itself being inserted by a transposition event. The IS26-composite transposon, which contains the bla(OXA-1), aac6'-lb-cr and catB4 genes, was inserted into plasmids pEC_L8 and pEC_L46 by homologous recombination rather than a transposition event. Results obtained for pEC_L46 indicated that IS26 also plays an important role in structural rearrangements of the plasmid backbone and seems to facilitate the mobilisation of fragments from other plasmids. CONCLUSIONS: Collectively, these data suggests that IS26 together with ISEcp1 could play a critical role in the evolution of diverse multiresistant plasmids found in clinical Enterobacteriaceae.
Asunto(s)
Elementos Transponibles de ADN , Escherichia coli/genética , Plásmidos , beta-Lactamasas/genéticaRESUMEN
The term peptidomics for a new promising "omics" field was not introduced until the beginning of 2000. The approach has been proven successful in several domains such as neuroendocrine research and biomarker or drug discovery. This review reports on bioinformatics tools and methodologies within the peptidomics field and the application thereof. Obviously, a plethora of proteomics data analysis tools lends themselves to direct use in peptidomics because the latter is a subfield of the former, at least to a certain extent. Nevertheless, peptidomics-specific tool extensions, inventions, and validation procedures have emerged, and certain tools are more suitable for this subfield than others due to small but important differences in peptidomics sample analysis. This paper focuses on these topics. Furthermore, it gives a comprehensive overview of available online tools tailored to the peptidomics field. To conclude, an ideal pipeline for bioactive peptide identification is presented.
Asunto(s)
Biología Computacional/métodos , Péptidos/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Bases de Datos de Proteínas , Humanos , Reproducibilidad de los ResultadosRESUMEN
Peptidomics is the identification and study of the in vivo biologically active peptide profile. A combination of high performance liquid chromatography, mass spectrometry, and bioinformatics tools such as database search engines are commonly used to perform the analysis. We report a methodology based on a database system holding the completed translated genome, whereby de novo sequencing and genome-wide database searching are combined. The methodology was applied to the sea urchin neuropeptidome resulting in a 30% increase in identification rate.
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Bases de Datos de Proteínas , Genoma , Proteínas del Tejido Nervioso/metabolismo , Proteoma , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Erizos de Mar , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
When studying the set of biologically active peptides (the so-called peptidome) of a cell type, organ, or entire organism, the identification of peptides is mostly attempted by MS. However, identification rates are often dismally unsatisfactory. A great deal of failed or missed identifications may be attributable to the wealth of modifications on peptides, some of which may originate from in vivo post-translational processes to activate the molecule, whereas others could be introduced during the tissue preparation procedures. Preliminary knowledge of the modification profile of specific peptidome samples would greatly improve identification rates. To this end we developed an approach that performs clustering of mass spectra in a way that allows us to group spectra having similar peak patterns over significant segments. Comparing members of one spectral group enables us to assess the modifications (expressed as mass shifts in Dalton) present in a peptidome sample. The clustering algorithm in this study is called Bonanza, and it was applied to MALDI-TOF/TOF MS spectra from the mouse. Peptide identification rates went up from 17 to 36% for 278 spectra obtained from the pancreatic islets and from 21 to 43% for 163 pituitary spectra. Spectral clustering with subsequent advanced database search may result in the discovery of new biologically active peptides and modifications thereof, as shown by this report indeed.
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Análisis por Conglomerados , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Algoritmos , Animales , Bases de Datos de Proteínas , Femenino , Islotes Pancreáticos/química , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Péptidos/análisis , Hipófisis/química , Hipófisis/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
Wolbachia is an endosymbiotic bacterium widely present in arthropods and animal-parasitic nematodes. Despite previous efforts, it has never been identified in plant-parasitic nematodes. Random sequencing of genes expressed by the burrowing nematode Radopholus similis resulted in several sequences with similarity to Wolbachia genes. The presence of a Wolbachia-like endosymbiont in this plant-parasitic nematode was investigated using both morphological and molecular approaches. Transmission electronmicroscopy, fluorescent immunolocalisation and staining with DAPI confirmed the presence of the endosymbiont within the reproductive tract of female adults. 16S rDNA, ftsZ and groEL gene sequences showed that the endosymbiont of R. similis is distantly related to the known Wolbachia supergroups. Finally, based on our initial success in finding sequences of this endosymbiont by screening an expressed sequence tag (EST) dataset, all nematode ESTs were mined for Wolbachia-like sequences. Although the retained sequences belonged to six different nematode species, R. similis was the only plant-parasitic nematode with traces of Wolbachia. Based on our phylogenetic study and the current literature we designate the endosymbiont of R. similis to a new supergroup (supergroup I) rather than considering it as a new species. Although its role remains unknown, the endosymbiont was found in all individuals tested, pointing towards an essential function of the bacteria.
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Artrópodos/genética , Genes de Helminto/genética , Nematodos/genética , Simbiosis/genética , Wolbachia/genética , Animales , Artrópodos/parasitología , Perfilación de la Expresión Génica , Interacciones Huésped-Parásitos , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Wolbachia/clasificaciónRESUMEN
We used direct recovery of bacterial 16S rRNA gene sequences to investigate the bacterial diversity under Acacia tortilis subsp. raddiana, a legume tree naturally growing in the dry land part of Senegal (West Africa). Microbial DNA was purified directly from soil samples and subjected to PCR with primers specific for bacterial 16S rRNA gene sequences. 16S rDNA clone libraries were constructed from two soil samples taken at two dates, i.e. June 25th 1999 (dry season) and August 28th 1999 (rainy season) at depths of 0.25-0.50 m and at 3 m distance from the stem. The 16S rDNA of 117 clones was partially sequenced. Phylogenetic analysis of these sequences revealed extensive diversity (100 phylotypes). Comparative sequence analysis of these clones identified members of the Gammaproteobacteria (35% of the phylotypes) as the most important group, followed by the Firmicutes division with 24%. Alphaproteobacteria, Betaproteobacteria, Acidobacteria and Actinobacteria were found to be less represented. Our data suggest that bacterial communities under Acacia tortilis subsp. raddiana might differ according to the season. The relative compositions of the populations is different in both samples: the Acidobacteria are present in a much higher percentage in the dry season than in the rainy season sample while the inverse effect is observed for the members of the other groups. Within the Gammaproteobacteria we found a shift between the dry season and the rainy season from pseudomonads to Acinetobacter and Escherichia related organisms.
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Acacia/microbiología , Actinobacteria/genética , Proteobacteria/genética , ARN Ribosómico 16S/análisis , Microbiología del Suelo , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , Estaciones del Año , SenegalRESUMEN
Fluorescent in situ hybridization with a 16S rRNA probe specific for Verrucomicrobia was used to (i) confirm the division-level identity of and (ii) study the behavior of the obligate intracellular verrucomicrobium "Candidatus Xiphinematobacter" in its nematode hosts. Endosymbionts in the egg move to the pole where the gut primordium arises; hence, they populate the intestinal epithelia of juvenile worms. During the host's molt to adult female, the endosymbionts concentrate around the developing ovaries to occupy the ovarian wall. Some bacteria are enclosed in the ripening oocytes for vertical transmission. Verrucomicrobia in males stay outside the testes because the tiny spermatozoids are not suitable for transmission of cytoplasmic bacteria.