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Detection of Enteric Viruses in Shellfish from the Norwegian Coast. M. Myrmel, 2004.Common blue mussels (Mytilus edulis), horse mussels (Modiolus modiolus), and flat oysters (Ostrea edulis) obtained from various harvesting and commercial production sites along the Norwegian coast were screened for the presence of norovirus by a real-time reverse transcription (RT)-nested PCR assay and for possible indicators of fecal contamination, i.e., for F-specific RNA bacteriophages (F-RNA phages) by plaque assay and for human adenoviruses and human circoviruses by nested PCR assay . The aims were to obtain relevant information for assessing the risk of transmission of enteric viruses by shellfish and to investigate the potential of various indicator viruses in routine screening . Noroviruses were detected in 6.8% of the samples, and the indicators were detected in 23.8% (F-RNA phages), 18.6% (adenoviruses), and 8.0% (circoviruses) of the samples . A seasonal variation was observed, with the exception of circoviruses, with more positive samples in the winter . A positive correlation was found between F-RNA phages and noroviruses . However, F-RNA phages were present in only 43% of the norovirus-positive samples . The results show that mussels from the Norwegian coast can constitute a risk of infection with enteric viruses and that routine testing of samples may be justified . Advantages and disadvantages of various options for screening are discussed . Implicating the Glutathione-Gated Potassium Efflux System as a Cause of Electrophile-Induced Activated Sludge Deflocculation. Charles B. Bott, 2004.The glutathione-gated K+ efflux (GGKE) system represents a protective microbial stress response that is activated by electrophilic or thiol-reactive stressors . It was hypothesized that efflux of cytoplasmic K+ occurs in activated sludge communities in response to shock loads of industrially relevant electrophilic chemicals and results in significant deflocculation . Novosphingobium capsulatum, a bacterium consistent with others found in activated sludge treatment systems, responded to electrophilic thiol reactants with rapid efflux of up to 80% of its cytoplasmic K+ pool . Furthermore, N . capsulatum and activated sludge cultures exhibited dynamic efflux-uptake-efflux responses very similar to those observed by others in Escherichia coli K-12 exposed to the electrophilic stressors N-ethylmaleimide and 1-chloro-2,4-dinitrobenzene and the reducing agent dithiothreitol . Fluorescent LIVE/DEAD stains were used to show that cell lysis was not the cause of electrophile-induced K+ efflux . Nigericin was used to artificially stimulate K+ efflux from N . capsulatum and activated sludge cultures as a comparison to electrophile-induced K+ efflux and showed that cytoplasmic K+ efflux by both means corresponded with activated sludge deflocculation . These results parallel those of previous studies with pure cultures in which GGKE was shown to cause cytoplasmic K+ efflux and implicate the GGKE system as a probable causal mechanism for electrophile-induced, activated sludge deflocculation . Calculations support the notion that shock loads of electrophilic chemicals result in very high K+ concentrations within the activated sludge floc structure, and these K+ levels are comparable to that which caused deflocculation by external (nonphysiological) KCl addition . Salicylate 5-Hydroxylase from Ralstonia sp . Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase. Ning-Yi Zhou, 2002.The genes from the oxygenase cluster nagAaGHAbAcAd of naphthalene-degrading Ralstonia sp . strain U2 were cloned and overexpressed . Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with overexpressed nagAaGHAb or in a mixture of three cell extracts containing, respectively, NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin) . Each of the three extracts required for S5H activity was rate limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis . S5H catalyzed the 5-hydroxylation of the aromatic rings of 3- and 4-substituted salicylates . However, the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated, producing 5-chloro-2,6-dihydroxybenzoate . In an assay for the nag naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, and NagAb) . NDO and S5H were assayed in the presence of all possible combinations of the nag proteins and the corresponding nah NDO proteins from the "classical" naphthalene degrader P . putida NCIMB9816 . All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain . The S5H from strain U2 is a unique monooxygenase which shares sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2 . Novel Genomic Rearrangement That Affects Expression of the Streptococcus pyogenes Streptolysin O (slo) Gene. Dragutin J. Savic, 2003.A RecA-independent chromosomal rearrangement in the upstream region of the streptolysin O (slo) gene of Streptococcus pyogenes which affects slo expression was identified . PCR analysis was used to demonstrate that this kind of rearrangement was found in several strains of different lineages . Chromosomal loci involved in the recombination were found to be 746 kb apart on the 1.85-Mb-long chromosome . The primary structure of the splicing region, the reproducibility of the rearrangement, and the fact that reconstructed recombinant molecules fused to erm and lacZ reporter genes affected their expression indicate that this event is not accidental but may play a role in the expression of the slo gene . In addition, the product of the recombining DNAs, including the splicing site, does not follow any example of a known recombination mechanism . The implications of this rearrangement for slo expression are discussed .
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