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TraG-Like Proteins of DNA Transfer Systems and of the Helicobacter pylori Type IV Secretion System: Inner Membrane Gate for Exported Substrates?. Gunnar Schröder, 2002.TraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation . They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems . TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such as Helicobacter pylori . Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H . pylori) . These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity . Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity . Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4 . Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus . We predict that multimeric inner membrane protein TraG forms a pore . A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented . Use of the Caulobacter crescentus Genome Sequence To Develop a Method for Systematic Genetic Mapping. Lisandra West , 2002.The functional analysis of sequenced genomes will be facilitated by the development of tools for the rapid mapping of mutations . We have developed a systematic approach to genetic mapping in Caulobacter crescentus that is based on bacteriophage-mediated transduction of strategically placed antibiotic resistance markers . The genomic DNA sequence was used to identify sites distributed evenly around the chromosome at which plasmids could be nondisruptively integrated . DNA fragments from these sites were amplified by PCR and cloned into a kanamycin-resistant (Kanr) suicide vector . Delivery of these plasmids into C . crescentus resulted in integration via homologous recombination . A set of 41 strains containing Kanr markers at 100-kb intervals was thereby generated . These strains serve as donors for generalized transduction using bacteriophage  Cr30, which can transduce at least 120 kb of DNA . Transductants are selected with kanamycin and screened for loss of the mutant phenotype to assess linkage between the marker and the site of the mutation . The dependence of cotransduction frequency on sequence distance was evaluated using several markers and mutant strains . With these data as a standard, previously unmapped mutations were readily localized to DNA sequence intervals equivalent to less than 1% of the genome . Candidate genes within the interval were then examined further by subcloning and complementation analysis . Mutations resulting in sensitivity to ampicillin, in nutritional auxotrophies, or temperature-sensitive growth were mapped . This approach to genetic mapping should be applicable to other bacteria with sequenced genomes for which generalized transducing phage are available .
Insecticidal Activity Associated with the Outer Membrane Vesicles of Xenorhabdus nematophilus. Puneet Khandelwal, 2003.Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane . Transmission electron micrographs of X . nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium . The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X . nematophilus and analyzed . Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm . A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present . The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type . Live cells of X . nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera . The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs . The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H . armigera neonatal larvae . The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay . The OMV proteins showed chitinase activity . This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X . nematophilus into the extracellular medium .
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