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Regulation of the Cellulosomal celS (cel48A) Gene of Clostridium thermocellum Is Growth Rate Dependent. Tali W. Dror, 2003.Clostridium thermocellum produces an extracellular multienzyme complex, termed cellulosome, that allows efficient solubilization of crystalline cellulose . One of the major enzymes in this complex is the CelS (Cel48A) exoglucanase . The regulation of CelS at the protein and transcriptional levels was studied using batch and continuous cultures . The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses indicated that the amount of CelS in the supernatant fluids of cellobiose-grown cultures is lower than that of cellulose-grown cultures . The transcriptional level of celS mRNA was determined quantitatively by RNase protection assays with batch and continuous cultures under carbon and nitrogen limitation . The amount of celS mRNA transcripts per cell was about 180 for cells grown under carbon limitation at growth rates of 0.04 to 0.21 h-1 and 80 and 30 transcripts per cell for batch cultures at growth rates of 0.23 and 0.35 h-1, respectively . Under nitrogen limitation, the corresponding levels were 110, 40, and 30 transcripts/cell for growth rates of 0.07, 0.11, and 0.14 h-1, respectively . Two major transcriptional start sites were detected at positions -140 and -145 bp, upstream of the translational start site of the celS gene . The potential promoters exhibited homology to known sigma factors (i.e.,  A
and
B)
of Bacillus subtilis . The relative activity of the two
promoters remained constant under the conditions studied and was in
agreement with the results of the RNase protection assay, in which
the observed transcriptional activity was inversely proportional to
the growth rate .
Functional Genomic Analysis of Fluconazole Susceptibility in the Pathogenic Yeast Candida glabrata: Roles of Calcium Signaling and Mitochondria. Rupinder Kaur, 2004.The pathogenic yeast Candida glabrata exhibits innate resistance to fluconazole, the most commonly used antifungal agent . By screening a library of 9,216 random insertion mutants, we identified a set of 27 genes which upon mutation, confer altered fluconazole susceptibility in C . glabrata . Homologues of three of these genes have been implicated in azole and/or drug resistance in Saccharomyces cerevisiae: two of these belong to the family of ABC transporters (PDR5 and PDR16), and one is involved in retrograde signaling from mitochondria to nucleus (RTG2) . The remaining 24 genes are involved in diverse cellular functions, including ribosomal biogenesis and mitochondrial function, activation of RNA polymerase II transcription, nuclear ubiquitin ligase function, cell wall biosynthesis, and calcium homeostasis . We characterized two sets of mutants in more detail . Strains defective in a putative plasma membrane calcium channel (Cch1-Mid1) were modestly more susceptible to fluconazole but showed a significant loss of viability upon prolonged fluconazole exposure, suggesting that calcium signaling is required for survival of azole stress in C . glabrata . These mutants were defective in calcium uptake in response to fluconazole exposure . The combined results suggest that, in the absence of Ca2+ signaling, fluconazole has a fungicidal rather than a fungistatic effect on C . glabrata . The second set of mutants characterized in detail were defective in mitochondrial assembly and organization, and these exhibited very high levels of fluconazole resistance . Further analysis of these mutants indicated that in C . glabrata a mechanism exists for reversible loss of mitochondrial function that does not involve loss of mitochondrial genome and that C . glabrata can switch between states of mitochondrial competence and incompetence in response to fluconazole exposure .
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