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Processing of the Tail Lysozyme (gp5) of Bacteriophage T4. Nanzhang Ye, 2004.The processing site of gp5 has been determined to be betweenresidues Val-390 and His-391, instead of Ser-351 and Ala-352as previously reported [H . Kanamaru, N . C . Gassner, N . Ye, S.Takeda, and F . Arisaka, J . Bacteriol . 181:2739-2744] . Moreover,the maturation of gp5 is abolished by null mutations in otherhub genes, indicating that cleavage requires the interactionsof several baseplate proteins. Variation in the Effectors of the Type III Secretion System among Photorhabdus Species as Revealed by Genomic Analysis. Karine Brugirard-Ricaud, 2004.Entomopathogenic bacteria of the genus Photorhabdus harbor a type III secretion system . This system was probably acquired prior to the separation of the species within this genus . Furthermore, the core components of the secretion machinery are highly conserved but the predicted effectors differ between Photorhabdus luminescens and P . asymbiotica, two highly related species with different hosts . AcrAB-TolC Directs Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium DT104. Sylvie Baucheron, 2004.Multidrug-resistant Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) strains harbor a genomic island, called Salmonella genomic island 1 (SGI1), which contains an antibiotic resistance gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracyclines . They may be additionally resistant to quinolones . Among the antibiotic resistance genes there are two, i.e., floR and tet(G), which code for efflux pumps of the major facilitator superfamily with 12 transmembrane segments that confer resistance to chloramphenicol-florfenicol and the tetracyclines, respectively . In the present study we determined, by constructing acrB and tolC mutants, the role of the AcrAB-TolC multidrug efflux system in the multidrug resistance of several DT104 strains displaying additional quinolone resistance or not displaying quinolone resistance . This study shows that the quinolone resistance and the decreased fluoroquinolone susceptibilities of the strains are highly dependent on the AcrAB-TolC efflux system and that single mutations in the quinolone resistance-determining region of gyrA are of little relevance in mediating this resistance . Overproduction of the AcrAB efflux pump, as determined by Western blotting with an anti-AcrA polyclonal antibody, appeared to be the major mechanism of resistance to quinolones . Moreover, chloramphenicol-florfenicol and tetracycline resistance also appeared to be highly dependent on the presence of AcrAB-TolC, since the introduction of mutations in the respective acrB and tolC genes resulted in a susceptible or intermediate resistance phenotype, according to clinical MIC breakpoints, despite the presence of the FloR and Tet(G) efflux pumps . Resistance to other antibiotics, ampicillin, streptomycin, and sulfonamides, was not affected in the acrB and tolC mutants of DT104 strains harboring SGI1 . Therefore, AcrAB-TolC appears to direct efflux-mediated resistance to quinolones, chloramphenicol-florfenicol, and tetracyclines in multidrug-resistant S . enterica serovar Typhimurium DT104 strains . Initial Reaction(s) in Biotransformation of CL-20 Is Catalyzed by Salicylate 1-Monooxygenase from Pseudomonas sp . Strain ATCC 29352. Bharat Bhushan, 2004.CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C6H6N12O12), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp . strain FA1 and Agrobacterium sp . strain JS71; however, the nature of the enzyme(s) involved in the process was not understood . In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp . strain ATCC 29352, biotransformed CL-20 at rates of 0.256 ± 0.011 and 0.043 ± 0.003 nmol min–1 mg of protein–1 under anaerobic and aerobic conditions, respectively . The disappearance of CL-20 was accompanied by the release of nitrite ions . Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M – H]– at 345 Da, corresponding to an empirical formula of C6H6N10O8, produced as a result of two sequential N denitration steps on the CL- 20 molecule . We also detected two isomeric metabolites with [M – H]– at 381 Da corresponding to an empirical formula of C6H10N10O10 . The latter was a hydrated product of the metabolite C6H6N10O8 with addition of two H2O molecules, as confirmed by tests using 18O-labeled water . The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion . Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase . The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical . Protein Splicing of the Deinococcus radiodurans Strain R1 Snf2 Intein. Maurice W. Southworth, 2002.Adjacent intein fragments fused to a Snf2/Rad54 helicase-related protein and Snf2/Rad54 helicase were reported for Deinococcus radiodurans R1, leading to the speculation that a frameshift was required for splicing or that trans splicing occurred . However, a type strain (ATCC 13939, RF18410) yielded a single protein that splices by the Ala1 protein splicing pathway, with splicing dependent on adjacent residues . An Arsenate Reductase from Synechocystis sp . Strain PCC 6803 Exhibits a Novel Combination of Catalytic Characteristics. Renhui Li, 2003.The deduced protein product of open reading frame slr0946 from Synechocystis sp . strain PCC 6803, SynArsC, contains the conserved sequence features of the enzyme superfamily that includes the low-molecular-weight protein-tyrosine phosphatases and the Staphylococcus aureus pI258 ArsC arsenate reductase . The recombinant protein product of slr0946, rSynArsC, exhibited vigorous arsenate reductase activity (Vmax = 3.1 µmol/min · mg), as well as weak phosphatase activity toward p-nitrophenyl phosphate (Vmax = 0.08 µmol/min · mg) indicative of its phosphohydrolytic ancestry . pI258 ArsC from S . aureus is the prototype of one of three distinct families of detoxifying arsenate reductases . The prototypes of the others are Acr2p from Saccharomyces cerevisiae and R773 ArsC from Escherichia coli. All three have converged upon catalytic mechanisms involving an arsenocysteine intermediate . While SynArsC is homologous to pI258 ArsC, its catalytic mechanism exhibited a unique combination of features . rSynArsC employed glutathione and glutaredoxin as the source of reducing equivalents, like Acr2p and R773 ArsC, rather than thioredoxin, as does the S . aureus enzyme . As postulated for Acr2p and R773 ArsC, rSynArsC formed a covalent complex with glutathione in an arsenate-dependent manner . rSynArsC contains three essential cysteine residues like pI258 ArsC, whereas the yeast and E . coli enzymes require only one cysteine for catalysis . As in the S . aureus enzyme, these "extra" cysteines apparently shuttle a disulfide bond to the enzyme's surface to render it accessible for reduction . SynArsC and pI258 ArsC thus appear to represent alternative branches in the evolution of their shared phosphohydrolytic ancestor into an agent of arsenic detoxification . Impact of Land Use Intensity on the Species Diversity of Arbuscular Mycorrhizal Fungi in Agroecosystems of Central Europe. Fritz Oehl, 2003. Toward an International Standard for PCR-Based Detection of Food-Borne Thermotolerant Campylobacters: Assay Development and Analytical Validation. P. S. Lübeck, 2003.As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C . jejuni, C . coli, and C . lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene . A panel of 150 strains including target and nontarget strains was used in an in-house validation . Only one primer pair, OT1559 plus 18-1, was found to be selective . The inclusivity and exclusivity were 100 and 97%, respectively . In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated . The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme . Based on these results, Tth was selected as the most suitable enzyme for the assay . The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified .
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