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Regulation of Ribosomal Protein Synthesis in Vibrio cholerae. Todd D. Allen, 2004.We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae . Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively . Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria . Mutations of Pneumocystis jirovecii Dihydrofolate Reductase Associated with Failure of Prophylaxis. Aimable Nahimana, 2004.Most drugs used for prevention and treatment of Pneumocystis jirovecii pneumonia target enzymes involved in the biosynthesis of folic acid, i.e., dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) . Emergence of P . jirovecii drug resistance has been suggested by the association between failure of prophylaxis with sulfa drugs and mutations in DHPS . However, data on the occurrence of mutations in DHFR, the target of trimethoprim and pyrimethamine, are scarce . We examined polymorphisms in P . jirovecii DHFR from 33 patients diagnosed with P . jirovecii pneumonia who were receiving prophylaxis with a DHFR inhibitor (n = 15), prophylaxis without a DHFR inhibitor (n = 11), or no prophylaxis (n = 7) . Compared to the wild-type sequence present in GenBank, 19 DHFR nucleotide substitution sites were found in 18 patients with 3 synonymous and 16 nonsynonymous mutations . Of 16 amino acid changes, 6 were located in positions conserved among distant organisms, and five of these six positions are probably involved in the putative active sites of the enzyme . Patients with failure of prophylaxis, including a DHFR inhibitor, were more likely to harbor nonsynonymous DHFR mutations than those who did not receive such prophylaxis (9 of 15 patients versus 2 of 18; P = 0.008) . Analysis of the rate of nonsynonymous versus synonymous mutations was consistent with selection of amino acid substitutions in patients with failure of prophylaxis including a DHFR inhibitor . The results suggest that P . jirovecii populations may evolve under selective pressure from DHFR inhibitors, in particular pyrimethamine, and that DHFR mutations may contribute to P . jirovecii drug resistance . Roles of the Conserved Aspartate and Arginine in the Catalytic Mechanism of an Archaeal ß-Class Carbonic Anhydrase. Kerry S. Smith, 2002.The roles of an aspartate and an arginine, which are completely conserved in the active sites of ß-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the ß-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the kcat/Km value depends only on the rate constants for the CO2 hydration step, whereas kcat also depends on rate constants from the proton transfer step (K . S . Smith, N . J . Cosper, C . Stalhandske, R . A . Scott, and J . G . Ferry, J . Bacteriol . 182:6605-6613, 2000) . The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P . Strop, K . S . Smith, T . M . Iverson, J . G . Ferry, and D . C . Rees, J . Biol . Chem . 276:10299-10305, 2001) . The kcat/Km values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO2 hydration step . Parallel decreases in kcat and kcat/Km values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the kcat of the D34A variant was chemically rescued by replacement of 2-(N-morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step . The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34 . Arg-36 variants had both kcat and kcat/Km values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue . Imidazole was unable to rescue the kcat of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this residue is important . Phase Variation in the Phage Growth Limitation System of Streptomyces coelicolor A3(2). Paul Sumby, 2003.The phase-variable phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2) is an unusual bacteriophage resistance mechanism that confers protection against the temperate phage  C31 and homoimmune relatives . Pgl is subject to phase variation, and data presented here show that this is at least partially due to expansion and contraction of a polyguanine tract present within the putative adenine-specific DNA methyltransferase gene, pglX . Furthermore, the pglX paralogue SC6G9.02, here renamed pglS, was shown to be able to interfere with the Pgl phenotype, suggesting that PglS could provide an alternative activity to that conferred by PglX .
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