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BACKGROUND DNA uptake is the first step in natural transformation of bacteria, leading to DNA internalization and recombination. It is, therefore, a key determinant in genome evolution. Most bacteria take up DNA indiscriminately, but in two families of Gram-negative bacteria the uptake machinery binds preferentially to short sequences called uptake signal sequences (USS). These sequences are highly enriched in their genomes, which causes preferential uptake of self-DNA over foreign DNA. RESULTS To fully characterize the effects of this preference, and to identify other sequence factors affecting uptake, we carried out a genome-wide analysis of DNA uptake using both measured uptake and the predictions from a sequence-based uptake model. Maps of DNA uptake were developed by recovering and deep sequencing genomic DNA that had been taken up by competent Haemophilus influenzae cells, and comparing sequencing coverage from recovered samples to coverage of the input DNA. Chromosomal DNA that had been sheared into short fragments (50-800bp) produced sharp peaks of uptake centered at USS, separated by valleys with 1000-fold lower uptake. Peaks heights were proportional to the USS scores predicted by the previously measured contribution to uptake of individual bases in each USS, as well as by predicted differences in DNA shape. Uptake of a long-fragment DNA preparation (1.5-17kb) had much less variation, with 90% of positions having uptake within 2-fold of the mean. Although the presence of a second USS within 100bp had no detectable effect on uptake of short fragments, uptake of long fragments increased with the local density of USS. Simulation of the uptake competition between H. influenzae DNA and the abundant human DNA in the respiratory tract DNA showed that the USS-based system allows H. influenzae DNA to prevail even when human DNA is present in 100-fold excess. CONCLUSION All detectable DNA uptake biases arose from sequences that fit the USS uptake motif, and presence of such sequences increased uptake of short DNA fragments by about 1000-fold. Preferred sequences also had rigidly bent AT-tracts and outer cores. Uptake of longer DNA fragments was much less variable, although detection of uptake biases was limited by strong biases intrinsic to the DNA sequencing process.

BackgroundAnalysis of microbiome data involves identifying co-occurring groups of taxa associated with sample features of interest (e.g., disease state). But elucidating key associations is often difficult since microbiome data are compositional, high dimensional, and sparse. Also, the configuration of co-occurring taxa may represent overlapping subcommunities that contribute to, for example, host status. Preserving the configuration of co-occurring microbes rather than detecting specific indicator species is more likely to facilitate biologically meaningful interpretations. In addition, analyses that utilize both taxonomic and predicted functional abundances typically independently characterize the taxonomic and functional profiles before linking them to sample information. This prevents investigators from identifying the specific functional components associate with which subsets of co-occurring taxa.ResultsWe provide an approach to explore co-occurring taxa using “topics” generated via a topic model and then link these topics to specific sample classes (e.g., diseased versus healthy). Rather than inferring predicted functional content independently from taxonomic abundances, we instead focus on inference of functional content within topics, which we parse by estimating pathway-topic interactions through a multilevel, fully Bayesian regression model. We apply our methods to two large publically available 16S amplicon sequencing datasets: an inflammatory bowel disease (IBD) dataset from Gevers et al. and data from the American Gut (AG) project. When applied to the Gevers et al. IBD study, we demonstrate that a topic highly associated with Crohn’s disease (CD) diagnosis is (1) dominated by a cluster of bacteria known to be linked with CD and (2) uniquely enriched for a subset of lipopolysaccharide (LPS) synthesis genes. In the AG data, our approach found that individuals with plant-based diets were enriched with Lachnospiraceae, Roseburia, Blautia, and Ruminococcaceae, as well as fluorobenzoate degradation pathways, whereas pathways involved in LPS biosynthesis were depleted.ConclusionsWe introduce an approach for uncovering latent thematic structure in the context of sample features for 16S rRNA surveys. Using our topic-model approach, investigators can (1) capture groups of co-occurring taxa termed topics, (2) uncover within-topic functional potential, and (3) identify gene sets that may guide future inquiry. These methods have been implemented in a freely available R package https://github.com/EESI/themetagenomics.

Trabajo presentado en el 7th European Conference on Prokaryotic and Fungal Genomics (ProkaGENOMICS), celebrado en Gottingen (Alemania), del 19 al 22 de septiembre de 2017), Introduction. The respiratory pathogen nontypeable Haemophilus influenzae (NTHi) persistently infects a significant proportion of Chronic Obstructive Pulmonary Disease (COPD) patients’ lower airways, and is usually associated with acute exacerbations of this chronic disease. Objectives. Based on the hypothesis that the COPD may be a unique niche favouring NTHi adaptation, this work aimed to unravel NTHi evolutionary dynamics in the COPD lungs over time, with the goal of identifying genomic patho-adaptive features.

Póster presentado en la XI Reunión del Grupo de Microbiología Molecular de la Sociedad Española de Microbiología (SEM), celebrada en Sevilla del 6 al 8 de septiembre de 2016., Haemophilus influenzae (Hi) es un patógeno oportunista causante de infección respiratoria crónica y de exacerbación periódica en pacientes con patologías respiratorias crónicas asociadas al tabaquismo como la Enfermedad Pulmonar Obstructiva Crónica (EPOC). El pulmón EPOC es un nicho ecológico complejo, con niveles elevados de inflamación, mucosidad, enfisema y fibrosis, expuesto a humo de tabaco y a fármacos. Todo ello genera una presión selectiva que modula la evolución, adaptación y persistencia pulmonar de Hi. Para entender la dinámica de la evolución pato-adaptativa de Hi en el pulmón EPOC, se dispuso de una colección prospectiva longitudinal de 86 aislados de Hi procedentes de muestras de esputo de 12 pacientes EPOC en exacerbación, generada entre los años 2005 y 2014. Para identificar rasgos fenotípicos y genotípicos de pato-adaptación, se analizaron crecimiento bacteriano, auto-agregación y resistencia a la muerte por péptidos antimicrobianos, y se realizó secuenciación genómica de la colección de aislados. El análisis bioinformático del material secuenciado estableció la existencia 20 series de aislados clonales. El análisis de polimorfismos en ORFs en las 20 series clonales identificó 231 SNPs no sinónimos en 180 genes, y reveló la existencia de 15 genes que presentan rasgos de evolución paralela (SNPs en varias series clonales aisladas de distintos pacientes). Entre ellos, 6 series clonales mostraron SNPs en el gen fadL (transiciones e inserciones), generando 12 variantes alélicas con cambios en loops extracelulares y 9 variantes alélicas truncadas. FadL es una proteína con función dual, (i) transportador de ácidos grasos exógenos de cadena larga (LCFA), y (ii) ligando del receptor eucariota Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 (hCEACAM1) que facilita la invasión epitelial por Hi. El significado biológico de la variabilidad alélica de FadL en ambas categorías funcionales se analizó mediante el diseño y utilización de (i) un medio mínimo con ácido oleico como fuente de carbono, y (ii) un ensayo de interacción con CEACAM1 mediante línea celular HeLa transfectada con hCEACAM1. En conjunto, este trabajo constituye el primer estudio evolutivo de un patógeno bacteriano adaptado al pulmón EPOC, e identifica rasgos de pato-adaptación y potenciales dianas que serán explotadas en el desarrollo de nuevos antimicrobianos.

The PLOS Pathogens Staff. Notice of Republication.This article was republished on May 13, 2016, to correct the title which was incorrectly published as “Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza”. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference., This work was supported by National Institutes of Health Ruth Kirschstein postdoctoral fellowship F32AI084427 (to JCM); a Canadian Institute of Health Research operating grant (to RJR); Genome British Columbia grant SOF122 (to RJR and JCM); the Faculty of Pharmaceutical Sciences, Canadian Foundation for Innovation (to CN); National Institutes of Heath R01 grant DC002873 (to JWSG); and (to JG) grants from Ministerio Economía y Competitividad-MINECO SAF2012-31166 and SAF2015-66520-R, Dpto. Salud Gobierno Navarra 359/2012 and Ministerio de Educación PRX12/00191.

Póster presentado en la International Pasteurellaceae Conference, celebrada en Prato (Italia) del 13 al 16 de mayo de 2014, Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

Trabajo presentado en el XXIV Congreso de la Sociedad Española de Microbiología, celebrado en L’Hospitalet de Llobregat (Barcelona) del 10 al 13 de julio de 2013., Se ha empleado la transformación natural como estrategia de cribado de genes/agrupamientos genéticos implicados en la invasión del epitelio respiratorio humano por el patógeno respiratorio H. Influenzae. Este trabajo constituye la primera búsqueda sistemática de invasinas de H. Influenzae mediante un cribado positivo de ganancia de función., MINECO SAF2012-31168; Departamento de Salud Gobierno Foral de Navarra; CIBERES, (España); Universidad British Columbia-UBC, (Canadá).