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Differential Expression of Two Paralogous Genes of Bacillus subtilis Encoding Single-Stranded DNA Binding Protein. Cordula Lindner, 2004.The Bacillus subtilis genome comprises two paralogous single-stranded DNA binding protein [SSB] genes, ssb and ywpH, which show distinctexpression patterns . The main ssb gene is strongly expressedduring exponential growth and is coregulated with genes encodingthe ribosomal proteins S6 and S18 . The gene organization rpsF-ssb-rpsRas observed in B . subtilis is found in many gram-positive aswell as some gram-negative bacteria, but not in Escherichiacoli . The ssb gene is essential for cell viability, and likeother SSBs its expression is elevated during SOS response . Incontrast, the paralogous ywpH gene is transcribed from its ownpromoter at the onset of stationary phase in minimal mediumonly . Its expression is ComK dependent and its gene product is required for optimal natural transformation. Mutant Prevention Concentrations for Single-Step Fluoroquinolone-Resistant Mutants of Wild-Type, Efflux-Positive, or ParC or GyrA Mutation-Containing Streptococcus pneumoniae Isolates. Heather J. Smith, 2004.Three fluoroquinolone-susceptible and five fluoroquinolone-resistant (two with ParC Ser79Phe mutations, one with a GyrA Ser81Phe mutation, and two that were efflux positive) Streptococcus pneumoniae isolates were exposed to one, two, four, eight, and sixteen times the MICs of ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin . Mutational frequencies were calculated at each multiple of the MIC for which growth was observed . Mutant prevention concentrations (MPCs) and the multiple of the MIC at the MPC (MPMIC) were evaluated . All resulting mutants were sequenced for quinolone resistance-determining region changes in GyrA and ParC and were evaluated for reserpine-sensitive efflux . The MPC order was generally ciprofloxacin > levofloxacin > gatifloxacin > moxifloxacin > gemifloxacin . The MPMIC order varied depending on the genetic constitution of the original isolates from which the mutants were generated . For those mutants created from fluoroquinolone-susceptible isolates (those that had wild-type ParC and GyrA and were efflux negative), the MPMIC order was ciprofloxacin = moxifloxacin > gemifloxacin > levofloxacin > gatifloxacin . The MPMICs of each fluoroquinolone for mutants created from isolates with a ParC mutation (with wild-type GyrA and efflux negative) were similar . A similar occurrence was observed with the mutants created from the efflux-positive isolates (with wild-type ParC and GyrA) . The MPMIC order for the mutants created from the isolate with a GyrA mutation (with wild-type ParC and efflux negative) was ciprofloxacin = gemifloxacin > levofloxacin = moxifloxacin > gatifloxacin . Gatifloxacin, levofloxacin, and moxifloxacin may be intrinsically more able to prevent the development of resistance by fluoroquinolone-susceptible isolates, isolates that are efflux positive, or isolates that carry a GyrA mutation . However, once a ParC mutation is present, the MPC increases dramatically for all fluoroquinolones . Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds. H. Wouter Wisselink, 2004.To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity . High-performance liquid chromatography and 13C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol . Moreover, the formed mannitol was not reutilized upon glucose depletion . Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L . lactis seemed to be the most promising strain for mannitol production . The Purine Repressor of Bacillus subtilis: a Novel Combination of Domains Adapted for Transcription Regulation. Sangita C. Sinha, 2003.The purine repressor from Bacillus subtilis, PurR, represses transcription from a number of genes with functions in the synthesis, transport, and metabolism of purines . The 2.2-Å crystal structure of PurR reveals a two-domain protein organized as a dimer . The larger C-terminal domain belongs to the PRT structural family, in accord with a sequence motif for binding the inducer phosphoribosylpyrophosphate (PRPP) . The PRT domain is fused to a smaller N-terminal domain that belongs to the winged-helix family of DNA binding proteins . A positively charged surface on the winged-helix domain likely binds specific DNA sequences in the recognition site . A second positively charged surface surrounds the PRPP site at the opposite end of the PurR dimer . Conserved amino acids in the sequences of PurR homologs in 21 gram-positive bacteria cluster on the proposed recognition surface of the winged-helix domain and around the PRPP binding site at the opposite end of the molecule, supporting a common function of DNA and PRPP binding for all of the proteins . The structure supports a binding mechanism in which extended regions of DNA interact with extensive protein surface . Unlike most PRT proteins, which are phosphoribosyltransferases (PRTases), PurR lacks catalytic activity . This is explained by a tyrosine side chain that blocks the site for a nucleophile cosubstrate in PRTases . Thus, B . subtilis has adapted an enzyme fold to serve as an effector-binding domain and has used it in a novel combination with the DNA-binding winged-helix domain as a repressor of purine genes . Modifications to United States Environmental Protection Agency Methods 1622 and 1623 for Detection of Cryptosporidium Oocysts and Giardia Cysts in Water. Randi M. McCuin, 2003.Collaborative and in-house laboratory trials were conducted to evaluate Cryptosporidium oocyst and Giardia cyst recoveries from source and finished-water samples by utilizing the Filta-Max system and U.S . Environmental Protection Agency (EPA) methods 1622 and 1623 . Collaborative trials with the Filta-Max system were conducted in accordance with manufacturer protocols for sample collection and processing . The mean oocyst recovery from seeded, filtered tap water was 48.4% ± 11.8%, while the mean cyst recovery was 57.1% ± 10.9% . Recovery percentages from raw source water samples ranged from 19.5 to 54.5% for oocysts and from 46.7 to 70.0% for cysts . When modifications were made in the elution and concentration steps to streamline the Filta-Max procedure, the mean percentages of recovery from filtered tap water were 40.2% ± 16.3% for oocysts and 49.4% ± 12.3% for cysts by the modified procedures, while matrix spike oocyst recovery percentages ranged from 2.1 to 36.5% and cyst recovery percentages ranged from 22.7 to 68.3% . Blinded matrix spike samples were analyzed quarterly as part of voluntary participation in the U.S . EPA protozoan performance evaluation program . A total of 15 blind samples were analyzed by using the Filta-Max system . The mean oocyst recovery percentages was 50.2% ± 13.8%, while the mean cyst recovery percentages was 41.2% ± 9.9% . As part of the quality assurance objectives of methods 1622 and 1623, reagent water samples were seeded with a predetermined number of Cryptosporidium oocysts and Giardia cysts . Mean recovery percentages of 45.4% ± 11.1% and 61.3% ± 3.8% were obtained for Cryptosporidium oocysts and Giardia cysts, respectively . These studies demonstrated that the Filta-Max system meets the acceptance criteria described in U.S . EPA methods 1622 and 1623 . Identification of Naegleria fowleri in Domestic Water Sources by Nested PCR. Francine Marciano-Cabral, 2003.The free-living amoeboflagellate Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system . In the United States, the disease is generally acquired while swimming and diving in freshwater lakes and ponds . In addition to swimming, exposure to N . fowleri and the associated disease can occur by total submersion in bathwater or small backyard wading pools . In the present study, swipe samples and residual pipe water from homes in Arizona were examined for N . fowleri by nested PCR due to the death of two previously healthy children from PAM . Since neither child had a history of swimming in a freshwater lake or pond prior to the onset of disease symptoms, the domestic water supply was the suspected source of infection . Of 19 samples collected from bathroom and kitchen pipes and sink traps, 17 samples were positive for N . fowleri by PCR . A sample from a Micro-Wynd II filter was obtained by passing water from bathtubs through the filter . Organisms attached to the filter also tested positive by PCR . The two samples that tested negative for N . fowleri were one that was obtained from a kitchen sink trap and a swipe sample from the garbage disposal of one home .
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