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Characterization of the Class 3 Integron and the Site-Specific Recombination System It Determines. Christina M. Collis, 2002.Integrons capture gene cassettes by using a site-specific recombination mechanism . As only one class of integron and integron-determined site-specific recombination system has been studied in detail, the properties of a second class, the only known class 3 integron, were examined . The configuration of the three potentially definitive features of integrons, the intI3 gene, the adjacent attI3 recombination site, and the Pc promoter that directs transcription of the cassettes, was similar to that found in the corresponding region (5' conserved segment) of class 1 integrons . The integron features are flanked by a copy of the terminal inverted repeat, IRi, from class 1 integrons on one side and a resolvase-encoding tniR gene on the other, suggesting that they are part of a transposable element related to Tn402 but with the integron module in the opposite orientation . The IntI3 integrase was active and able to recognize and recombine both known types of IntI-specific recombination sites, the attI3 site in the integron, and different cassette-associated 59-be (59-base element) sites . Both integration of circularized cassettes into the attI3 site and excision of integrated cassettes were also catalyzed by IntI3 . The attI3 site was localized to a short region adjacent to the intI3 gene . Recombination between a 59-be and secondary sites was also catalyzed by IntI3, but at frequencies significantly lower than observed with IntI1, the class 1 integron integrase . Mobility of a Restriction-Modification System Revealed by Its Genetic Contexts in Three Hosts. Marc Naderer, 2002.The flow of genes among prokaryotes plays a fundamental role in shaping bacterial evolution, and restriction-modification systems can modulate this flow . However, relatively little is known about the distribution and movement of restriction-modification systems themselves . We have isolated and characterized the genes for restriction-modification systems from two species of Salmonella, S . enterica serovar Paratyphi A and S . enterica serovar Bareilly . Both systems are closely related to the PvuII restriction-modification system and share its target specificity . In the case of S . enterica serovar Paratyphi A, the restriction endonuclease is inactive, apparently due to a mutation in the subunit interface region . Unlike the chromosomally located Salmonella systems, the PvuII system is plasmid borne . We have completed the sequence characterization of the PvuII plasmid pPvu1, originally from Proteus vulgaris, making this the first completely sequenced plasmid from the genus Proteus . Despite the pronounced similarity of the three restriction-modification systems, the flanking sequences in Proteus and Salmonella are completely different . The SptAI and SbaI genes lie between an equivalent pair of bacteriophage P4-related open reading frames, one of which is a putative integrase gene, while the PvuII genes are adjacent to a mob operon and a XerCD recombination (cer) site . Genetic Analysis of Pathway Specificity during Posttranslational Protein Translocation across the Escherichia coli Plasma Membrane. Natascha Blaudeck, 2003.In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane . Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein . The TorA signal peptide selectively and specifically directed MalE into the Tat pathway . The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway . Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked . These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity . Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway . Killing of Bacillus Spores by Aqueous Dissolved Oxygen, Ascorbic Acid, and Copper Ions. J. B. Cross, 2003.An approach to decontamination of biological endospores is discussed . Specifically, the performance of an aqueous modified Fenton reagent is examined . A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions . The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions . Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu2+) and then subsequent conversion to hydroxyl radicals . Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii . A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature . This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O2 is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action . Reduction of Experimental Salmonella and Campylobacter Contamination of Chicken Skin by Application of Lytic Bacteriophages. D. Goode, 2003.Lytic bacteriophages, applied to chicken skin that had been experimentally contaminated with Salmonella enterica serovar Enteritidis or Campylobacter jejuni at a multiplicity of infection (MOI) of 1, increased in titer and reduced the pathogen numbers by less than 1 log10 unit . Phages applied at a MOI of 100 to 1,000 rapidly reduced the recoverable bacterial numbers by up to 2 log10 units over 48 h . When the level of Salmonella contamination was low (< log10 2 per unit area of skin) and the MOI was 105, no organisms were recovered . By increasing the number of phage particles applied (i.e., MOI of 107), it was also possible to eliminate other Salmonella strains that showed high levels of resistance because of restriction but to which the phages were able to attach .
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