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Pneumocystis jiroveci Dihydropteroate Synthase Polymorphisms Confer Resistance to Sulfadoxine and Sulfanilamide in Saccharomyces cerevisiae. I. Meneau, 2004.Failure of anti-Pneumocystis jiroveci prophylaxis with sulfa drugs is associated with mutations within the putative active site of the fungal dihydropteroate synthase (DHPS), an enzyme encoded by the multidomain FAS gene . This enzyme is involved in the essential biosynthesis of folic acid . The most frequent polymorphisms are two mutations leading to two amino acid changes (55Trp-Arg-57Pro to 55Ala-Arg-57Ser), observed as a single or double mutation in the same P . jiroveci isolate . In the absence of a culture method for P . jiroveci, we studied potential resistance to sulfa drugs conferred by these polymorphisms by using Saccharomyces cerevisiae as a model . Single or double mutations identical to those observed in the DHPS domain of the P . jiroveci FAS gene were introduced by in vitro site-directed mutagenesis into alleles of the S . cerevisiae FOL1 gene, which is the orthologue of the P . jiroveci FAS gene . The mutated alleles were integrated at the genomic locus in S . cerevisiae and expressed by functional complementation in a strain with a disrupted FOL1 allele . The single mutation 55Trp to 55Ala conferred resistance to sulfanilamide, whereas the single mutation 57Pro to 57Ser conferred resistance to both sulfanilamide and sulfadoxine . Both single mutations also separately conferred hypersensitivity to sulfamethoxazole and dapsone . The resistance to sulfadoxine is consistent with epidemiological data on P . jiroveci . The double mutation 55Trp-Arg-57Pro to 55Ala-Arg-57Ser conferred on S . cerevisiae a requirement for p-aminobenzoate, suggesting reduced affinity of DHPS for this substrate . This characteristic is commonly observed in mutated DHPS enzymes conferring sulfa drug resistance from other organisms . However, the double mutation conferred hypersensitivity to sulfamethoxazole, which is not in agreement with epidemiological data on P . jiroveci . Taken together, our results suggest that the DHPS polymorphisms observed in P . jiroveci confer sulfa drug resistance on this pathogen . Regulation of Expression of Cellulosomal Cellulase and Hemicellulase Genes in Clostridium cellulovorans. Sung Ok Han, 2003.The regulation of expression of the genes encoding the cellulases and hemicellulases of Clostridium cellulovorans was studied at the mRNA level with cells grown under various culture conditions . A basic pattern of gene expression and of relative expression levels was obtained from cells grown in media containing poly-, di- or monomeric sugars . The cellulase (cbpA and engE) and hemicellulase (xynA) genes were coordinately expressed in medium containing cellobiose or cellulose . Growth in the presence of cellulose, xylan, and pectin gave rise to abundant expression of most genes (cbpA-exgS, engH, hbpA, manA, engM, engE, xynA, and/or pelA) studied . Moderate expression of cbpA, engH, manA, engE, and xynA was observed when cellobiose or fructose was used as the carbon source . Low levels of mRNA from cbpA, manA, engE, and xynA were observed with cells grown in lactose, mannose, and locust bean gum, and very little or no expression of cbpA, engH, manA, engE, and xynA was detected in glucose-, galactose-, maltose-, and sucrose-grown cells . The cbpA-exgS and engE genes were most frequently expressed under all conditions studied, whereas expression of xynA and pelA was more specifically induced at higher levels in xylan- or pectin-containing medium, respectively . Expression of the genes (cbpA, hbpA, manA, engM, and engE) was not observed in the presence of most soluble di- or monosaccharides such as glucose . These results support the hypotheses that there is coordinate expression of some cellulases and hemicellulases, that a catabolite repression type of mechanism regulates cellulase expression in rapidly growing cells, and that the presence of hemicelluloses has an effect on cellulose utilization by the cell . Lessons from the Genome of a Lithoautotroph: Making Biomass from Almost Nothing. Juan-Luis Ramos, 2003. Analysis of the argK-tox Gene Cluster in Nontoxigenic Strains of Pseudomonas syringae pv . phaseolicola. Ana Isabel González, 2003.The analysis of 46 isolates obtained directly from different and distant common bean fields from the northwestern part of Spain revealed that they do not produce phaseolotoxin . The isolates were classified as race 5, and their analysis revealed that they do not carry the argK-tox gene cluster involved in the biosynthesis of the phaseolotoxin .
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