Natural transformation of Vibrio cholerae as a tool - Optimizing the procedure

<p>Abstract</p> <p>Background</p> <p><it>Vibrio cholerae </it>gains natural competence upon growth on chitin. This allows the organism to take up free DNA from the environment and to incorporate it into its genome by homologous recombination.</p> <p>Results</p> <p>Making use of this developmental program in order to use it as a tool to genetically manipulate <it>V. cholerae </it>and potentially also others <it>Vibrio </it>species was envisaged. Therefore, we re-investigated the experimental design for natural transformation of <it>V. cholerae </it>and tested different donor DNA fragments with respect to their source (genomic versus PCR-derived), quantity, and homologous flanking regions. Furthermore, we simplified the procedure in terms of the chitin source used as inducer of natural competence and the composition of the growth medium.</p> <p>Conclusions</p> <p>The current study allows us to recommend a standard protocol to genetically manipulate <it>V. cholerae </it>using commercially available sources of chitin and minimal medium, respectively, as well as PCR-derived donor DNA as transforming material.</p

Genome Analysis of a Transmissible Lineage of Pseudomonas aeruginosa Reveals Pathoadaptive Mutations and Distinct Evolutionary Paths of Hypermutators.

Genome sequencing of bacterial pathogens has advanced our understanding of their evolution, epidemiology, and response to antibiotic therapy. However, we still have only a limited knowledge of the molecular changes in in vivo evolving bacterial populations in relation to long-term, chronic infections. For example, it remains unclear what genes are mutated to facilitate the establishment of long-term existence in the human host environment, and in which way acquisition of a hypermutator phenotype with enhanced rates of spontaneous mutations influences the evolutionary trajectory of the pathogen. Here we perform a retrospective study of the DK2 clone type of P. aeruginosa isolated from Danish patients suffering from cystic fibrosis (CF), and analyze the genomes of 55 bacterial isolates collected from 21 infected individuals over 38 years. Our phylogenetic analysis of 8,530 mutations in the DK2 genomes shows that the ancestral DK2 clone type spread among CF patients through several independent transmission events. Subsequent to transmission, sub-lineages evolved independently for years in separate hosts, creating a unique possibility to study parallel evolution and identification of genes targeted by mutations to optimize pathogen fitness (pathoadaptive mutations). These genes were related to antibiotic resistance, the cell envelope, or regulatory functions, and we find that the prevalence of pathoadaptive mutations correlates with evolutionary success of co-evolving sub-lineages. The long-term co-existence of both normal and hypermutator populations enabled comparative investigations of the mutation dynamics in homopolymeric sequences in which hypermutators are particularly prone to mutations. We find a positive exponential correlation between the length of the homopolymer and its likelihood to acquire mutations and identify two homopolymer-containing genes preferentially mutated in hypermutators. This homopolymer facilitated differential mutagenesis provides a novel genome-wide perspective on the different evolutionary trajectories of hypermutators, which may help explain their emergence in CF infections

Draft Genome Sequences of Pseudomonas aeruginosa B3 Strains Isolated from a Cystic Fibrosis Patient Undergoing Antibiotic Chemotherapy

Pseudomonas aeruginosa frequently establishes chronic infections in the airways of patients suffering from cystic fibrosis (CF). Here, we report the draft genome sequences of four P. aeruginosa B3 strains isolated from a chronically infected CF patient undergoing antibiotic chemotherapy

Draft Genome Sequences of Pseudomonas aeruginosa B3 Strains Isolated from a Cystic Fibrosis Patient Undergoing Antibiotic Chemotherapy

At treatment of wastewater of viscose rayon’s spinning produces the sediment – zinc-bearing sludge, a humidity content of 66 to 95 %. To date, it is deposited up to 2.5 million cubic meters of zinc-bearing sludge in the ponds of plants in Russia. In the paper chemical composition and particle size distribution of sludge of viscose rayon manufacturing (OJSC “Balakovskoye khimvolokno”, OJSC “Ryazanskoye khimvolokno”, OJSC “Sibvolokno”) are presented. The percentage of zinc in sludge is of 6 to 23 %. Investigations by methods of X-ray diffraction (XRD) and scanning electron microscopy (SEM) are shown, that zinc is present in form of hydroxides, carbonates and silicates. Thermodynamic analysis of zinc dissolution of compounds, found in product of sludge roasting at a temperature of 850∘C, was conducted

Is genotyping of single isolates sufficient for population structure analysis of <i>Pseudomonas aeruginosa</i> in cystic fibrosis airways?

The primary cause of morbidity and mortality in cystic fibrosis (CF) patients is lung infection by Pseudomonas aeruginosa. Therefore much work has been done to understand the adaptation and evolution of P. aeruginosa in the CF lung. However, many of these studies have focused on longitudinally collected single isolates, and only few have included cross-sectional analyses of entire P. aeruginosa populations in sputum samples. To date only few studies have used the approach of metagenomic analysis for the purpose of investigating P. aeruginosa populations in CF airways. We analysed five metagenomes together with longitudinally collected single isolates from four recently chronically infected CF patients. With this approach we were able to link the clone type and the majority of SNP profiles of the single isolates to that of the metagenome(s) for each individual patient. Based on our analysis we find that when having access to comprehensive collections of longitudinal single isolates it is possible to rediscover the genotypes of the single isolates in the metagenomic samples. This suggests that information gained from genome sequencing of comprehensive collections of single isolates is satisfactory for many investigations of adaptation and evolution of P. aeruginosa to the CF airways