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Dihydropteroate Synthase Mutations in Pneumocystis jiroveci Can Affect Sulfamethoxazole Resistance in a Saccharomyces cerevisiae Model. Peter Iliades, 2004.Dihydropteroate synthase (DHPS) mutations in Pneumocystis jiroveci have been associated epidemiologically with resistance to sulfamethoxazole (SMX) . Since P . jiroveci cannot be cultured, inherent drug resistance cannot be measured . This study explores the effects of these mutations in a tractable model organism, Saccharomyces cerevisiae . Based on the sequence conservation between the DHPS enzymes of P . jiroveci and S . cerevisiae, together with the structural conservation of the three known DHPS structures, DHPS substitutions commonly observed in P . jiroveci were reverse engineered into the S . cerevisiae DHPS . Those mutations, T597A and P599S, can occur singly but are most commonly found together and are associated with SMX treatment failure . Mutations encoding the corresponding changes in the S . cerevisiae dhps were made in a yeast centromere vector, p414FYC, which encodes the native yeast DHPS as part of a trifunctional protein that also includes the two enzymes upstream of DHPS in the folic acid synthesis pathway, dihydroneopterin aldolase and 2-amino-4-hydroxymethyl dihydropteridine pyrophosphokinase . A yeast strain with dhps deleted was employed as the host strain, and transformants having DHPS activity were recovered . Mutants having both T597 and P599 substitutions had a requirement for p-aminobenzoic acid (PABA), consistent with resistance being associated with altered substrate binding . These mutants could be adapted for growth in the absence of PABA, which coincided with increased sulfa drug resistance . Upregulated PABA synthesis was thus implicated as a mechanism for sulfa drug resistance for mutants having two DHPS substitutions . A Novel Class of Heat and Secretion Stress-Responsive Genes Is Controlled by the Autoregulated CssRS Two-Component System of Bacillus subtilis. Elise Darmon, 2002.Bacteria need dedicated systems that allow appropriate adaptation to the perpetual changes in their environments . In Bacillus subtilis, two HtrA-like proteases, HtrA and HtrB, play critical roles in the cellular response to secretion and heat stresses . Transcription of these genes is induced by the high-level production of a secreted protein or by a temperature upshift . The CssR-CssS two-component regulatory system plays an essential role in this transcriptional activation . Transcription of the cssRS operon is autoregulated and can be induced by secretion stress, by the absence of either HtrA or HtrB, and by heat stress in a HtrA null mutant strain . Two start sites are used for cssRS transcription, only one of which is responsive to heat and secretion stress . The divergently transcribed htrB and cssRS genes share a regulatory region through which their secretion and heat stress-induced expression is linked . This study shows that CssRS-regulated genes represent a novel class of heat-inducible genes, which is referred to as class V and currently includes two genes: htrA and htrB . Prediction of Gene Function in Methylthioadenosine Recycling from Regulatory Signals. Brooke A. Murphy, 2002.The S-box transcription termination control system, first identified in Bacillus subtilis, is used for regulation of gene expression in response to methionine availability . The presence of the S-box motif provided the first indication that the ykrTS and ykrWXYZ genes could play a role in recycling of 5'-methylthioadenosine, a by-product of polyamine biosynthesis that can be converted to methionine . In this study we demonstrate a role for the ykrTS and ykrWXYZ gene products in this pathway . Global Mutational Analysis of NtrC-Like Activators in Myxococcus xanthus: Identifying Activator Mutants Defective for Motility and Fruiting Body Development. Nora B. Caberoy, 2003.The multicellular developmental cycle of Myxococcus xanthus requires large-scale changes in gene transcription, and recent findings indicate that NtrC-like activators play a prominent role in regulating these changes . In this study, we made insertions in 28 uncharacterized ntrC-like activator (nla) genes and found that eight of these insertions cause developmental defects . Hence, these results are consistent with the idea that M . xanthus uses a series of different NtrC-like activators during fruiting body development . Four of the eight developmental mutants we identified have motility defects . The nla1, nla19, and nla23 mutants show S-motility defects, while the nla24 mutant shows defects in both S-motility and A-motility . During development, aggregation of the nla1, nla19, and nla23 mutants is delayed slightly and the nla24 mutant shows no signs of aggregation or sporulation . The nla4, nla6, nla18, and nla28 mutants have no appreciable loss in motility, but they fail to aggregate and to sporulate normally . The nla18 mutant belongs to a special class of developmental mutants whose defects can be rescued when they are codeveloped with wild-type cells, suggesting that nla18 fails to produce a cell-cell signal required for development . The three remaining activator mutants, nla4, nla6, and nla28, appear to have complex developmental phenotypes that include deficiencies in cell-cell developmental signals . Thirteen Years of Building Constraint-Based In Silico Models of Escherichia coli. Jennifer L. Reed, 2003. Infectious Cryptosporidium parvum Oocysts in Final Reclaimed Effluent. Angela L. Gennaccaro, 2003.Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique . Results revealed the presence of infectious C . parvum oocysts in 40% of the final disinfected effluent samples . Sampled effluent contained on average seven infectious oocysts per 100 liters . Thus, reclaimed water is not pathogen free but contains infectious C . parvum .
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