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Recommendations for Treatment of Human Infections Caused by Bartonella Species. J. M. Rolain, 2004. Automated Purification and Suspension Array Detection of 16S rRNA from Soil and Sediment Extracts by Using Tunable Surface Microparticles. Darrell P. Chandler, 2004.Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps . In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector . The tunable surface detection limits in both low- and high-salt buffers were 1 ng of total RNA (  104 cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA (105 cell equivalents) in hybridizations with the automated system (30-s contact time) . RNA fragmentation was essential for achieving tunable surface suspension array specificity . Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences . Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor . Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads . The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background . The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection . The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system .
DNA Topoisomerase III from the Hyperthermophilic Archaeon Sulfolobus solfataricus with Specific DNA Cleavage Activity. Penggao Dai, 2003.We report the production, purification, and characterization of a type IA DNA topoisomerase, previously designated topoisomerase I, from the hyperthermophilic archaeon Sulfolobus solfataricus . The protein was capable of relaxing negatively supercoiled DNA at 75°C in the presence of Mg2+ . Mutation of the putative active site Tyr318 to Phe318 led to the inactivation of the protein . The S . solfataricus enzyme cleaved oligonucleotides in a sequence-specific fashion . The cleavage occurred only in the presence of a divalent cation, preferably Mg2+ . The cofactor requirement of the enzyme was partially satisfied by Cu2+, Co2+, Mn2+, Ca2+, or Ni2+ . It appears that the enzyme is active with a broader spectrum of metal cofactors in DNA cleavage than in DNA relaxation (Mg2+ and Ca2+) . The enzyme-catalyzed oligonucleotide cleavage required at least 7 bases upstream and 2 bases downstream of the cleavage site . Analysis of cleavage by the S . solfataricus enzyme on a set of oligonucleotides revealed a consensus cleavage sequence of the enzyme: 5'-G(A/T)CA(T)AG(T)G(A)X  XX-3' . This sequence bears more resemblance to the preferred cleavage sites of topoisomerases III than to those of topoisomerases I . Based on these data and sequence analysis, we designate the enzyme S . solfataricus topoisomerase III .
Microbial Populations Stimulated for Hexavalent Uranium Reduction in Uranium Mine Sediment. Yohey Suzuki, 2003.Uranium-contaminated sediment and water collected from an inactive uranium mine were incubated anaerobically with organic substrates . Stimulated microbial populations removed U almost entirely from solution within 1 month . X-ray absorption near-edge structure analysis showed that U(VI) was reduced to U(IV) during the incubation . Observations by transmission electron microscopy, selected area diffraction pattern analysis, and energy-dispersive X-ray spectroscopic analysis showed two distinct types of prokaryotic cells that precipitated only a U(IV) mineral uraninite (UO2) or both uraninite and metal sulfides . Prokaryotic cells associated with uraninite and metal sulfides were inferred to be sulfate-reducing bacteria . Phylogenetic analysis of 16S ribosomal DNA obtained from the original and incubated sediments revealed that microbial populations were changed from microaerophilic Proteobacteria to anaerobic low-G+C gram-positive sporeforming bacteria by the incubation . Forty-two out of 94 clones from the incubated sediment were related to sulfate-reducing Desulfosporosinus spp., and 23 were related to fermentative Clostridium spp . The results suggest that, if in situ bioremediation were attempted in the uranium mine ponds, Desulfosporosinus spp . would be a major contributor to U(VI) and sulfate reduction and Clostridium spp . to U(VI) reduction .
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