Formation of genes coding for hybrid proteins by recombination between related, cloned genes in E. coli.

We describe a method for the formation of hybrid genes by in vivo recombination between two genes with partial sequence homology. DNA structures consisting of plasmid vector sequences, flanked by the alpha 2 interferon gene on the one side and a portion of the alpha 1 interferon gene (homology about 80%) on the other, were transfected into E. coli SK1592. Appropriate resistance markers allowed the isolation of colonies containing circular plasmids which arose by in vivo recombination between the partly homologous interferon gene sequences. Eleven different recombinant genes were identified, six of which encoded new hybrid interferons not easily accessible by recombinant DNA techniques.     

SVA: software for annotating and visualizing sequenced human genomes

SUMMARY: Here we present Sequence Variant Analyzer (SVA), a software tool that assigns a predicted biological function to variants identified in next-generation sequencing studies and provides a browser to visualize the variants in their genomic contexts. SVA also provides for flexible interaction with software implementing variant association tests allowing users to consider both the bioinformatic annotation of identified variants and the strength of their associations with studied traits. We illustrate the annotation features of SVA using two simple examples of sequenced genomes that harbor Mendelian mutations. Availability and implementation: Freely available on the web at http://www.svaproject.org.     

Mapping quantitative traits and strategies to find quantitative trait genes

In 1999 a meeting took place at the Jackson Laboratory, a large mouse research centre in Bar Harbor, Maine, to consider the value of systematically collecting phenotypes on inbred strains of mice (Paigen and Eppig (2000) [1]). The group concluded that cataloguing the extensive phenotypic diversity present among laboratory mice, and in particular providing the research community with data from cohorts of animals, phenotyped according to standardized protocols, was essential if we were to take advantage of the possibilities of mouse genetics. Beginning with the collection of basic physiological, biochemical and behavioral data on nine commonly used inbred strains, the project has expanded so that by the beginning of 2010 data for 178 strains had been collected, with 105 phenotype projects yielding over 2000 different measurements (Bogue et al. (2007) [2].     

Genome-scale identification of conditionally essential genes in E. coli by DNA microarrays

Identifying the genes required for the growth or viability of an organism under a given condition is an important step toward understanding the roles these genes play in the physiology of the organism. Currently, the combination of global transposon mutagenesis with PCR-based mapping of transposon insertion sites is the most common method for determining conditional gene essentiality. In order to accelerate the detection of essential gene products, here we test the utility and reliability of a DNA microarray technology-based method for the identification of conditionally essential genes of the bacterium, Escherichia coli, grown in rich medium under aerobic or anaerobic growth conditions using two different DNA microarray platforms. Identification and experimental verification of five hypothetical E. coli genes essential for anaerobic growth directly demonstrated the utility of the method. However, the two different DNA microarray platforms yielded largely non-overlapping results after a two standard deviations cutoff and were subjected to high false positive background levels. Thus, further methodological improvements are needed prior to the use of DNA microarrays to reliably identify conditionally essential genes on genome-scale.     

Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome

Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle.     

Targeting virulence traits: potential strategies to combat extraintestinal pathogenic E. coli infections

Extraintestinal pathogenic Escherichia coli (ExPEC) are able to colonize, invade and induce disease in body niches outside the gastrointestinal tract. The emergence of (multi)resistant ExPEC variants calls for alternative antimicrobial strategies with a reduced pressure on selection of antibiotic resistances. We report on recent research efforts to target such virulence traits or to interfere with ExPEC colonization of extraintestinal niches in order to prevent or treat ExPEC infections and reduce the risk of further emergence of antibiotic resistances.     

Microbial production of R-3-hydroxybutyric acid by recombinant E. coli harboring genes of phbA, phbB, and tesB

Production of R-3-hydroxybutyric acid (3HB) was observed when genes of β-ketothiolase (PhbA), acetoacetyl CoA reductase (PhbB), and thioesterase II (TesB) were jointly expressed in Escherichia coli. TesB, generally regarded as a medium chain length acyl CoA thioesterase, was found, for the first time, to play an important role for transforming short chain length 3-hydroxybutyrate-CoA to its free fatty acid, namely, 3HB. E. coli BW25113 (pSPB01) harboring phbA, phbB, and tesB genes produced approximately 4 g/l 3HB in shake flask culture within 24 h with glucose used as a carbon source. Under anaerobic growth conditions, 3HB production was found to be more effective, achieving 0.47 g 3HB/g glucose compared with only 0.32 g 3HB/g glucose obtained from aerobic process. When growth was conducted on sodium gluconate, 6 g/l 3HB was obtained. In a 24-h fed-batch growth process conducted in a 6-l fermentor containing 3 l glucose mineral medium, 12 g/l 3HB was produced from 17 g/l cell dry weight (CDW). This was the highest 3HB productivity achieved by a one-stage fermentation process for 3HB production. Liu and Ouyang contributed equally to the paper.     

Evaluation of trichloroethylene degradation by E. coli transformed with dimethyl sulfide monooxygenase genes and/or cumene dioxygenase genes

Pseudomonas fluorescens IP01 grown on isopropylbenzene (cumene) and Acinetobacter sp. 20B grown on dimethyl sulfide (DMS) degraded up to 90% and 25% of 1.5 mg trichloroethylene (TCE)/l, respectively. Escherichia coli harboring the DMS monooxygenase genes from strain 20B, the cumene dioxygenase genes from strain IP01 and both oxygenase genes, degraded up to 50%, 75% and 88% of 75 mg TCE/l, respectively. The growth rates of the E. coli recombinants remained nearly unaffected by TCE at 15 150 mg/l. Thus, the E. coli recombinants were indicated to degrade high concentrations of TCE efficiently at least up to 150 mg l–1.