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Nucleotide and Amino Acid Sequences of oriT-traM-traJ-traY-traA-traL Regions and Mobilization of Virulence Plasmids of Salmonella enterica Serovars Enteritidis, Gallinarum-Pullorum, and Typhimurium. Chishih Chu, 2002.The virulence plasmid of Salmonella enterica serovar Gallinarum-Pullorum (pSPV) but not those of Salmonella enterica serovars Enteritidis (pSEV) and Typhimurium (pSTV) can be readily mobilized by an F or F-like conjugative plasmid . To investigate the reason for the difference, the oriT-traM-traJ-traY-traA-traL regions of the three salmonella virulence plasmids (pSVs) were cloned and their nucleotide and deduced amino acid sequences were examined . The cloned fragments were generally mobilized more readily than the corresponding full-length pSVs, but the recombinant plasmid containing the oriT of pSPV was, as expected, more readily mobilized, with up to 100-fold higher frequency than the recombinant plasmids containing the oriT of the other two pSVs . The nucleotide sequences of the oriT-traM-traJ-traY-traA-traL region of pSEV and pSTV were almost identical (only 4 bp differences), but differed from that of pSPV . Major nucleotide sequence variations were found in traJ, traY, and the Tra protein binding sites sby and sbm . sby of pSPV showed higher similarity than that of pSEV or pSTV to that of the F plasmid . The reverse was true for sbm: similarity was higher with pSEV and pSTV than with pSPV . In the deduced amino acid sequences of the five Tra proteins, major differences were found in TraY: pSEV's TraY was 75 amino acids, pSTV's was 106 amino acids, and pSPV's was 133 amino acids; and there were duplicate consensus ß  fragments in the TraY of pSPV and F plasmid, whereas there was only a single ß fragment in that of pSEV and pSTV .
Mutations at Residues 282, 286, and 293 of Phage  Integrase Exert Pathway-Specific Effects on Synapsis and Catalysis in Recombination.Troy M. Bankhead, 2003.Bacteriophage integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites . The att sites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur . We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q) . We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions . Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination . The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways . The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways . We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway . Finally, we found that not all catalytic events in excision follow synapsis: the attL site probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA with attR .
Metagenomic Profiling: Microarray Analysis of an Environmental Genomic Library. Jonathan L. Sebat, 2003.Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms . However, conventional library-screening techniques permit characterization of relatively few environmental clones . Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities . A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing  1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls . COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species . The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe . Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes . Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture . Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition . Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms .
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