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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 139 (1996), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract The OmpR protein is a positive regulator specific for the Escherichia coli ompF and ompC genes. This protein functions in a phosphorylation-dependent manner through a presumed interaction with RNA polymerase. We previously isolated OmpR mutants which were suggested to be defective in transcription activation, but not in DNA binding (the so-called positive control (PC) mutant). In this study we isolated mutants of the α-subunit of RNA polymerase which can suppress one of the putative PC mutants of OmpR. A crucial amino acid substitution was identified as [V264G] in the α-subunit, which is located in the helix H1 of the C-terminal domain, which has been claimed, based on mutational and structural analyses, to be involved in the interaction with other positive regulators including the well-characterized cAMP receptor protein.
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Escherichia coli sensory kinase, ArcB, possesses a histidine-containing phosphotransfer (HPt) domain, which is implicated in the His-Asp multistep phosphorelay. We searched for a presumed phosphohistidine phosphatase, if present, which affects the function of the HPt domain through its dephosphorylation activity. Using in vivo screening, we first identified a gene that appeared to interfere with the His-Asp phosphorelay between the HPt domain and the receiver domain of OmpR, provided that the gene product was expressed through a multicopy plasmid. The cloned gene (named sixA) was found to encode a protein consisting of 161 amino acids, which has a noticeable sequence motif, an arginine–histidine–glycine (RHG) signature, at its N-terminus. Such an RHG signature, which presumably functions as a nucleophilic phosphoacceptor, was previously found in a set of divergent enzymes, including eukaryotic fructose-2,6-bisphosphatase, E. coli periplasmic phosphatase and E. coli glucose-1-phosphate phosphatase, and ubiquitous phosphoglycerate mutase. Otherwise, the entire amino acid sequences of none of these enzymes resembles that of SixA. It was demonstrated in vitro that the purified SixA protein exhibited the ability to release the phosphoryl group from the HPt domain of ArcB, but the mutant protein lacking the crucial histidine residue in the RHG signature did not. Evidence was also provided that a deletion mutation of sixA on the chromosome affected the in vivo phosphotransfer signalling. These results support the view that SixA is capable of functioning as a phosphohistidine phosphatase that may be implicated in the His-Asp phosphorelay through regulating the phosphorylation state of the HPt domain.
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  • 3
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S–23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA–DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.
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  • 4
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The bft gene encoding Bacteroides fragilis toxin (BFT) has been devided into two subtypes, bft-1 and bft-2. We found a novel subtype by sequencing a segment of the bft gene from 64 enterotoxigenic B. fragilis (ETBF) strains isolated in Japan. The 1548-bp nucleotide sequences of the new bft, the bft-1, and bft-2 genes were determined for five, four, and four ETBF strains, respectively; the nucleotide sequence was identical among each bft subtype and the degree of identity between each subtype was between 89 and 94%. Most of the variations between the three subtypes were detected in the region encoding mature toxin. A multiplex PCR was developed with a four-primer mix to subtype the bft sequences. The subtyping of 143 ETBF isolates from extraintestinal and stool specimens of humans and cows showed that the bft-1 was the most prevalent subtype, followed by bft-2 and a new bft subtype. No other subtype was found among the strains tested.
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  • 5
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The repeating sequences of the toxin A gene from toxin A-negative, toxin B-positive (toxin A−, toxin B+) strains of Clostridium difficile which were isolated in geographically separated facilities in Japan and Indonesia were determined. All six strains tested had identical repeating sequences with two deletions (1548 and 273 nucleotides in size) in the toxin A gene. A PCR method was designed to detect the deletions and the deletions were confirmed in all 50 toxin A−, toxin B+ strains examined by this method. Western immunoblot analysis revealed that polyclonal antiserum against native toxin A did not react with the concentrated culture filtrates of the toxin A−, toxin B+ strains. These results may suggest that toxin A−, toxin B+ strains have deletions of the two thirds of the repeating regions of the toxin A gene, which encodes the epitopes fully responsible for the reaction with the polyclonal antiserum.
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 192 (2000), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: We investigated the mechanism of the oxidative DNA damage induction by exposure to O2 in Prevotella melaninogenica, a strict anaerobe. Flow cytometry with hydroethidine and dichlorofluorescein diacetate showed that O2 exposure generated O2•− and H2O2. Results of electron spin resonance with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and ethanol showed that O2 exposure also induced •OH radical generation in P. melaninogenica loaded with FeCl2 but not in samples without FeCl2 loading. In P. melaninogenica, O2 exposure increased 8-hydroxydeoxyguanosine (8OHdG), typical of oxidative DNA damage. Catalase inhibited the increase, but the •OH radical scavengers did not. Phenanthroline, a membrane-permeable Fe and Cu chelator, increased the 8OHdG induction. In FeCl2-loaded samples, induction of 8OHdG decreased. Addition of H2O2 markedly increased 8OHdG levels. These results indicate that in P. melaninogenica, exposure to O2 generated and accumulated O2•− and H2O2, and that a crypto-OH radical generated through H2O2 was the active species in the 8OHdG induction.
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  • 7
    Electronic Resource
    Electronic Resource
    [S.l.] : American Institute of Physics (AIP)
    Journal of Applied Physics 66 (1989), S. 2719-2723 
    ISSN: 1089-7550
    Source: AIP Digital Archive
    Topics: Physics
    Notes: Phosphorous coimplantation effect on electrical uniformity of Si-implanted GaAs active layer is investigated for undoped as-grown, undoped ingot-annealed, and In-doped substrates. Pairs of field-effect transistors, fabricated with and without P coimplantation, are placed on whole 3-in.-diam substrates at a 200-μm interval. Threshold voltage measurements reveal that a concentration of 1018 /cm3 coimplanted P reduces the standard deviation in threshold voltage for undoped as-grown substrates to 1/2.5. Undoped ingot-annealed substrates achieve the same uniformity of threshold voltage as In-doped substrates, which showed the best data. Moreover, this method reduces the variation in the mean threshold voltage among substrates by one-half. These results indicate that P coimplantation successfully suppresses the change of As to Ga vacancy concentration ratio.
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford, UK; Malden, USA : Munksgaard International Publishers
    Physiologia plantarum 122 (2004), S. 0 
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′,5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3′5′H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.
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  • 9
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract TheEscherichia coli DNA-binding protein, OmpR, is one of the best characterized of the bacterial positive regulators that enhance the transcriptional ability of RNA polymerase. OmpR, consisting of 239 amino acids, binds to specific sequences located upstream of the cognateompC andompF promoters. The C-terminal half of OmpR, consisting of about 120 amino acids, exhibits an inherent DNA-binding ability. To address the issue of DNA binding by OmpR, we selected a set of OmpR mutants, each of which has a single amino acid substitution in the C-terminal half of OmpR. In particular, we characterized a number of OmpR mutants which are defective in DNA binding and thereby result in an OmpF− OmpC phenotype. Among them, a putative positive control OmpR mutant was also obtained, which appears to be defective in phosphorylation-dependent transcriptional activation, but not in DNA binding. These results are discussed with general emphasis on DNA recognition by theE. coli family of OmpR-like regulatory proteins.
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  • 10
    ISSN: 1432-0991
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Immunoblotting and pulsed-field gel electrophoresis of Clostridium difficile isolates were employed to differentiate reinfection by a newly acquired strain from relapse by an original strain in a 10-year-old patient with four episodes of C. difficile-associated colitis. Immunoblot typing demonstrated subserogroup K-1 of serogroup K for the first and second organisms, subserogroup A-1 of serogroup A for the third organism, and subserogroup G-4 of serogroup G for the fourth organism. PFGE analysis revealed consistent results with immunoblot analysis except that the strains from the fourth episode, whose DNA constantly degraded, were nontypable by this method. Five separate isolates of C. difficile from a specimen of each episode showed identical PFGE patterns, indicating that infections of multiple strains probably did not occur in this patient. These typing results suggested that the second episode after a 17-day course of vancomycin therapy represented a relapse by the strain causing the first episode, and that the third and fourth episodes after tapering vancomycin therapy were reinfections by other strains. Both immunoblot and PFGE typing systems are promising tools for analyzing recurrence of C. difficile infection.
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