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The trp RNA-Binding Attenuation Protein of Bacillus subtilis Regulates Translation of the Tryptophan Transport Gene trpP (yhaG) by Blocking Ribosome Binding. Helen Yakhnin, 2004.Expression of the Bacillus subtilis tryptophan biosynthetic genes (trpEDCFBA and pabA [trpG]) is regulated in response to tryptophan by TRAP, the trp RNA-binding attenuation protein . TRAP-mediated regulation of the tryptophan biosynthetic genes includes a transcription attenuation and two distinct translation control mechanisms . TRAP also regulates translation of trpP (yhaG), a single-gene operon that encodes a putative tryptophan transporter . Its translation initiation region contains triplet repeats typical of TRAP-regulated mRNAs . We found that regulation of trpP and pabA is unaltered in a rho mutant strain . Results from filter binding and gel mobility shift assays demonstrated that TRAP binds specifically to a segment of the trpP transcript that includes the untranslated leader and translation initiation region . While the affinities of TRAP for the trpP and pabA transcripts are similar, TRAP-mediated translation control of trpP is much more extensive than for pabA . RNA footprinting revealed that the trpP TRAP binding site consists of nine triplet repeats (five GAG, three UAG, and one AAG) that surround and overlap the trpP Shine-Dalgarno (S-D) sequence and translation start codon . Results from toeprint and RNA-directed cell-free translation experiments indicated that tryptophan-activated TRAP inhibits TrpP synthesis by preventing binding of a 30S ribosomal subunit . Taken together, our results establish that TRAP regulates translation of trpP by blocking ribosome binding . Thus, TRAP coordinately regulates tryptophan synthesis and transport by three distinct mechanisms: attenuation transcription of the trpEDCFBA operon, promoting formation of the trpE S-D blocking hairpin, and blocking ribosome binding to the pabA and trpP transcripts . Efficacies of Amphotericin B (AMB) Lipid Complex, AMB Colloidal Dispersion, Liposomal AMB, and Conventional AMB in Treatment of Murine Coccidioidomycosis. Gloria M. González, 2004.The therapeutic efficacy of three lipid formulations of amphotericin B was compared with that of conventional amphotericin B in treatment of murine coccidioidomycosis . All treatments prolonged survival compared with the no-treatment group (P < 0.0001) . Although conventional amphotericin B was more active than lipid formulations on reducing quantitative fungal load on a milligram-per-kilogram basis (P < 0.003 to 0.0002), the lipid preparations could be administered at higher doses, sterilizing liver and spleen tissues . The efficacies of the lipid preparations were similar in this murine model of coccidioidomycosis . Influence of Precipitation and Soil on Transport of Fecal Enterococci in Fractured Limestone Aquifers. Fulvio Celico, 2004.Limestone aquifers provide the main drinking water resources of southern Italy . The groundwater is often contaminated by fecal bacteria because of the interaction between rocks having high permeability and microbial pollutants introduced into the environment by grazing and/or manure spreading . The microbial contamination of springwater in picnic areas located in high mountains can cause gastrointestinal illness . This study was carried out in order to analyze the interaction between Enterococcus faecalis and the soil of a limestone aquifer and to verify the influence of this interaction on the time dependence of groundwater contamination . E . faecalis was chosen because, in the study area involved, it represents a better indicator than Escherichia coli . The research was carried out through field (springwater monitoring) and laboratory experiments (column tests with intact soil blocks) . The transport of bacterial cells through soil samples was analyzed by simulating an infiltration event that was monitored in the study area . Comparison of laboratory results with data acquired in the field showed that discontinuous precipitation caused an intermittent migration of microorganisms through the soil and produced, together with dispersion in the fractured medium (unsaturated and saturated zones), an articulated breakthrough at the spring . The short distances of bacterial transport in the study area produced a significant daily variability of bacterial contamination at the field scale . Purification, Substrate Range, and Metal Center of AtzC: the N-Isopropylammelide Aminohydrolase Involved in Bacterial Atrazine Metabolism. Nir Shapir, 2002.N-Isopropylammelide isopropylaminohydrolase, AtzC, the third enzyme in the atrazine degradation pathway in Pseudomonas sp . strain ADP, catalyzes the stoichiometric hydrolysis of N-isopropylammelide to cyanuric acid and isopropylamine . The atzC gene was cloned downstream of the tac promoter and expressed in Escherichia coli, where the expressed enzyme comprised 36% of the soluble protein . AtzC was purified to homogeneity by ammonium sulfate precipitation and phenyl column chromatography . It has a subunit size of 44,938 kDa and a holoenzyme molecular weight of 174,000 . The Km and kcat values for AtzC with N-isopropylammelide were 406 µM and 13.3 s-1, respectively . AtzC hydrolyzed other N-substituted amino dihydroxy-s-triazines, and those with linear N-alkyl groups had higher kcat values than those with branched alkyl groups . Native AtzC contained 0.50 eq of Zn per subunit . The activity of metal-depleted AtzC was restored with Zn(II), Fe(II), Mn(II), Co(II), and Ni(II) salts . Cobalt-substituted AtzC had a visible absorbance band at 540 nm (   = 84 M-1 cm-1) and exhibited an axial electron paramagnetic resonance (EPR) signal with the following effective values: g(x) = 5.18, g(y) = 3.93, and g(z) = 2.24 . Incubating cobalt-AtzC with the competitive inhibitor 5-azacytosine altered the effective EPR signal values to g(x) = 5.11, g(y) = 4.02, and g(z) = 2.25 and increased the microwave power at half saturation at 10 K from 31 to 103 mW . Under the growth conditions examined, our data suggest that AtzC has a catalytically essential, five-coordinate Zn(II) metal center in the active site and specifically catalyzes the hydrolysis of intermediates generated during the metabolism of s-triazine herbicides .
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