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Isolation and Purification of Pyranose 2-Oxidase from Phanerochaete chrysosporium and Characterization of Gene Structure and Regulation. Theodorus H. de Koker, 2004.Pyranose 2-oxidase (POX) was recovered from Phanerochaete chrysosporium BKM-F-1767 solid substrate culture using mild extraction conditions and was purified . 13C-nuclear magnetic resonance confirmed production of D-arabino-hexos-2-ulose (glucosone) from D-glucose with the oxidase . Peptide fingerprints generated by liquid chromatography-tandem mass spectrometry of tryptic digests and analysis of the corresponding cDNA revealed a structurally unusual sequence for the P . chrysosporium POX . Relatively high levels of pox transcript were detected under carbon-starved culture conditions but not under nutrient sufficiency . This regulation pattern is similar to that observed for lignin peroxidases, manganese peroxidases, and glyoxal oxidase of P . chrysosporium, supporting evidence that POX has a role in lignocellulose degradation . Determination of the Efficacy of Two Building Decontamination Strategies by Surface Sampling with Culture and Quantitative PCR Analysis. Mark P. Buttner, 2004.The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood . Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings . An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus ("Bacillus subtilis subsp . niger," also referred to as BG), a Bacillus anthracis surrogate . Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas . Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR) . Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis . Culture results indicated that 105 to 106 CFU per sample were present on surfaces before decontamination . After decontamination with the foam, no culturable B . atrophaeus spores were detected . After decontamination with chlorine dioxide gas, no culturable B . atrophaeus was detected in 24 of 27 samples (89%) . However, QPCR analysis showed that B . atrophaeus DNA was still present after decontamination with both methods . Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed . These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies . Integration and Distribution of Lactobacillus johnsonii Prophages. Marco Ventura, 2003.In Lactobacillus johnsonii strain NCC533, two prophages were integrated into tRNA genes and one was disrupted by integration . In a survey, the prophages were restricted to strains sharing an essentially identical restriction pattern . Microarray analysis showed that the prophage DNA represents about 50% of the NCC533 strain-specific DNA . Role for both DNA and RNA in GTP Hydrolysis by the Neisseria gonorrhoeae Signal Recognition Particle Receptor. Cody Frasz, 2003.The prokaryotic signal recognition particle (SRP) targeting system is a complex of two proteins, FtsY and Ffh, and a 4.5S RNA that targets a subset of proteins to the cytoplasmic membrane cotranslationally . We previously showed that Neisseria gonorrhoeae PilA is the gonococcal FtsY homolog . In this work, we isolated the other two components of the gonococcal SRP, Ffh and 4.5S RNA, and characterized the interactions among the three SRP components by using gel retardation and nitrocellulose filter-binding assays and enzymatic analyses of the two proteins . In the current model of prokaryotic SRP function, based on studies of the Escherichia coli and mammalian systems, Ffh binds to 4.5S RNA and the Ffh-4.5S RNA complex binds to the signal sequence of nascent peptides and then docks with FtsY at the membrane . GTP is hydrolyzed by both proteins synergistically, and the nascent peptide is transferred to the translocon . We present evidence that the in vitro properties of the gonococcal SRP differ from those of previously described systems . GTP hydrolysis by PilA, but not that by Ffh, was stimulated by 4.5S RNA, suggesting a direct interaction between PilA and 4.5S RNA that has not been reported in other systems . This interaction was confirmed by gel retardation analyses in which PilA and Ffh, both alone and together, bound to 4.5S RNA . An additional novel finding was that PpilE DNA, previously shown by us to bind PilA in vitro, also stimulates PilA GTP hydrolysis . On the basis of these data, we hypothesize that DNA may play a role in targeting proteins via the SRP . Energy Conservation in Acetogenic Bacteria. Volker Müller, 2003.
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