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Isolation of the Gene for the B12-Dependent Ribonucleotide Reductase from Anabaena sp . Strain PCC 7120 and Expression in Escherichia coli. Florence K. Gleason, 2002.The gene for ribonucleotide reductase from Anabaena sp . strain PCC 7120 was identified and expressed in Escherichia coli . This gene codes for a 1,172-amino-acid protein that contains a 407-amino-acid intein . The intein splices itself from the protein when it is expressed in E . coli, yielding an active ribonucleotide reductase of 765 residues . The mature enzyme was purified to homogeneity from E . coli extracts . Anabaena ribonucleotide reductase is a monomer with a molecular weight of approximately 88,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Superose 12 column chromatography . The enzyme reduces ribonucleotides at the triphosphate level and requires a divalent cation and a deoxyribonucleoside triphosphate effector . The enzyme is absolutely dependent on the addition of the cofactor, 5'-adenosylcobalamin . These properties are characteristic of the class II-type reductases . The cyanobacterial enzyme has limited sequence homology to other class II reductases; the greatest similarity (38%) is to the reductase from Lactobacillus leichmannii . In contrast, the Anabaena reductase shows over 90% sequence similarity to putative reductases found in genome sequences of other cyanobacteria, such as Nostoc punctiforme, Synechococcus sp . strain WH8102, and Prochlorococcus marinus MED4, suggesting that the cyanobacterial reductases form a closely related subset of the class II enzymes . The Quorum Sensing Negative Regulators EsaR and ExpREcc, Homologues within the LuxR Family, Retain the Ability To Function as Activators of Transcription. Susanne B. von Bodman, 2003.Most LuxR homologues function as activators of transcription during the process of quorum sensing, but a few, including EsaR and ExpREcc, negatively impact gene expression . The LuxR-activated luxI promoter and LuxR binding site, the lux box, were used in artificial contexts to assess the potential for transcriptional activation and DNA binding by EsaR and ExpREcc . Although the acyl-homoserine lactone responsiveness of both proteins is the opposite of that shown by most LuxR family members, EsaR and ExpREcc have preserved the ability to interact with RNA polymerase and activate transcription despite their low affinity for the lux box DNA . Thermophily in the Geobacteraceae: Geothermobacter ehrlichii gen . nov., sp . nov., a Novel Thermophilic Member of the Geobacteraceae from the "Bag City" Hydrothermal Vent. Kazem Kashefi, 2003.Little is known about the microbiology of the "Bag City" hydrothermal vent, which is part of a new eruption site on the Juan de Fuca Ridge and which is notable for its accumulation of polysaccharide on the sediment surface . A pure culture, designated strain SS015, was recovered from a vent fluid sample from the Bag City site through serial dilution in liquid medium with malate as the electron donor and Fe(III) oxide as the electron acceptor and then isolation of single colonies on solid Fe(III) oxide medium . The cells were gram-negative rods, about 0.5 µm by 1.2 to 1.5 µm, and motile and contained c-type cytochromes . Analysis of the 16S ribosomal DNA (rDNA) sequence of strain SS015 placed it in the family Geobacteraceae in the delta subclass of the Proteobacteria . Unlike previously described members of the Geobacteraceae, which are mesophiles, strain SS015 was a thermophile and grew at temperatures of between 35 and 65°C, with an optimum temperature of 55°C . Like many previously described members of the Geobacteraceae, strain SS015 grew with organic acids as the electron donors and Fe(III) or nitrate as the electron acceptor, with nitrate being reduced to ammonia . Strain SS015 was unique among the Geobacteraceae in its ability to use sugars, starch, or amino acids as electron donors for Fe(III) reduction . Under stress conditions, strain SS015 produced copious quantities of extracellular polysaccharide, providing a model for the microbial production of the polysaccharide accumulation at the Bag City site . The 16S rDNA sequence of strain SS015 was less than 94% similar to the sequences of previously described members of the Geobacteraceae; this fact, coupled with its unique physiological properties, suggests that strain SS015 represents a new genus in the family Geobacteraceae . The name Geothermobacter ehrlichii gen . nov., sp . nov., is proposed (ATCC BAA-635 and DSM 15274) . Although strains of Geobacteraceae are known to be the predominant Fe(III)-reducing microorganisms in a variety of Fe(III)-reducing environments at moderate temperatures, strain SS015 represents the first described thermophilic member of the Geobacteraceae and thus extends the known environmental range of this family to hydrothermal environments . Development of a PCR-Enzyme Immunoassay Oligoprobe Detection Method for Toxoplasma gondii Oocysts, Incorporating PCR Controls. Kellogg J. Schwab, 2003.Infections caused by Toxoplasma gondii are widely prevalent in animals and humans throughout the world . In the United States, an estimated 23% of adolescents and adults have laboratory evidence of T . gondii infection . T . gondii has been identified as a major opportunistic pathogen in immunocompromised individuals, in whom it can cause life-threatening disease . Water contaminated with feces from domestic cats or other felids may be an important source of human exposure to T . gondii oocysts . Because of the lack of information regarding the prevalence of T . gondii in surface waters, there is a clear need for a rapid, sensitive method to detect T . gondii from water . Currently available animal models and cell culture methods are time-consuming, expensive, and labor-intensive, requiring days or weeks for results to be obtained . Detection of T . gondii nucleic acid by PCR has become the preferred method . We have developed a PCR amplification and detection method for T . gondii oocyst nucleic acid that incorporates the use of hot-start amplification to reduce nonspecific primer annealing, uracil-N-glycosylase to prevent false-positive results due to carryover contamination, an internal standard control to identify false-negative results due to inadequate removal of sample inhibition, and PCR product oligoprobe confirmation using a nonradioactive DNA hybridization immunoassay . This method can provide positive, confirmed results in less than 1 day . Fewer than 50 oocysts can be detected following recovery of oocyst DNA . Development of a T . gondii oocyst PCR detection method will provide a useful technique to estimate the levels of T . gondii oocysts present in surface waters .
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