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Quinolone-Resistant Haemophilus influenzae in a Long-Term-Care Facility: Nucleotide Sequence Characterization of Alterations in the Genes Encoding DNA Gyrase and DNA Topoisomerase IV. Xinying Li, 2004.Fluoroquinolone-resistant isolates of Haemophilus influenzae, obtained from a long-term care facility, were examined for nucleotide sequence differences in the quinolone-resistance-determining regions of gyrA, gyrB, parC, and parE . Similarities among the resistant isolates, plus multiple differences with susceptible isolates, suggest clonal dissemination involving two resistant subclones . Purification, Cloning, and Sequencing of a 3,5-Dichlorophenol Reductive Dehalogenase from Desulfitobacterium frappieri PCP-1. Jacinthe Thibodeau, 2004.A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1 . The highest dehalogenase activity was observed with the biomass cultured at 22°C, compared to 30 and 37°C, where the cell suspensions were 2.2 and 9.6 times less active, respectively . The reductive dehalogenase was purified 12.7-fold to apparent homogeneity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa . Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN . A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor . Several polychlorophenols were dechlorinated at the meta and para positions . The apparent Km for 3,5-dicholorophenol was 49.3 ± 3.1 µM at a methyl viologen concentration of 2 mM . Six internal tryptic peptides were sequenced by mass spectrometry . One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences . This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase . The corresponding ORF (named cprA5) in D . frappieri PCP-1 was cloned and sequenced . The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion . The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases . This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions . Characterization of Urease-Positive Thermophilic Campylobacter Subspecies by Multilocus Enzyme Electrophoresis Typing. Motoo Matsuda, 2003.Thirty-one urease-positive thermophilic Campylobacter (UPTC) isolates, including three reference strains (NCTC12892, NCTC12895 and NCTC12896), and three Campylobacter lari isolates, which were isolated from several countries and sources, were compared genotypically by using multilocus enzyme electrophoresis (MLEE) . We examined allelic variation around seven enzyme loci, including the adenylate kinase, alkaline phosphatase, catalase, fumarase, malic enzyme, malate dehydrogenase, and L-phenylalanyl-L-leucine peptidase loci . MLEE typing revealed the presence of 23 different electrophoretic types (ETs) among the 31 UPTC isolates, and 14 isolates shared six electrophoretic profiles . Three different ETs were identified for the three C . lari isolates examined, and no ETs were shared by UPTC and C . lari isolates . Quantitative analyses were subsequently performed by using allelic variation data, and the results demonstrated that the mean genetic diversity was 0.655 . In conclusion, MLEE demonstrated that the UPTC isolates examined are genetically hypervariable and form a cluster separate from the C . lari cluster .
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