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Genetic Evidence Identifying the True Gluconeogenic Fructose-1,6-Bisphosphatase in Thermococcus kodakaraensis and Other Hyperthermophiles. Takaaki Sato, 2004.Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis . Although FBPase activity has been detected in several hyperthermophiles, no orthologs corresponding to the classical FBPases from bacteria and eukaryotes have been identified in their genomes . An inositol monophosphatase (IMPase) from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel FBPase (FbpTk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed as the "missing" FBPase . For this study, using T . kodakaraensis, we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo . The IMPase/FBPase ortholog in T . kodakaraensis, ImpTk, was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs . We therefore constructed  fbp
and
imp
strains by applying a gene disruption system recently developed for
T . kodakaraensis and investigated their phenotypes . The
fbp
strain could not grow under gluconeogenic conditions while glycolytic
growth was unimpaired, and the disruption resulted in the complete
abolishment of intracellular FBPase activity . Evidently, fbpTk
is an indispensable gene for gluconeogenesis and is responsible for
almost all intracellular FBPase activity . In contrast, the endogenous
impTk gene could not complement the defect of the
fbp deletion, and its disruption did not lead to any
detectable phenotypic changes under the conditions examined . These
facts indicated that impTk is irrelevant to gluconeogenesis,
despite the high FBPase activity of its protein product, probably
due to insufficient transcription . Our results provide strong
evidence that the true FBPase for gluconeogenesis in T . kodakaraensis
is the FbpTk ortholog, not the IMPase/FBPase ortholog .
Molecular Mechanisms of Primary Resistance to Flucytosine in Candida albicans. William W. Hope, 2004.Primary resistance in Candida albicans to flucytosine (5-FC) was investigated in 25 strains by identifying and sequencing the genes FCA1, FUR1, FCY21, and FCY22, which code for cytosine deaminase, uracil phosphoribosyltransferase (UPRT), and two purine-cytosine permeases, respectively . These proteins are involved in pyrimidine salvage and 5-FC metabolism . An association between a polymorphic nucleotide and resistance to 5-FC was found within FUR1 where the substitution of cytidylate for thymidylate at nucleotide position 301 results in the replacement of arginine with cysteine at amino acid position 101 in UPRT . Isolates that are homozygous for this mutation display increased levels of resistance to 5-FC, whereas heterozygous isolates have reduced susceptibility . Three-dimensional protein modeling of UPRT suggests that the Arg101Cys mutation disturbs the quaternary structure of the enzyme, which is postulated to compromise optimal enzyme activity . A single resistant isolate, lacking the above polymorphism in FUR1, has a homozygous polymorphism in FCA1 that results in a glycine-to-aspartate substitution at position 28 in cytosine deaminase . CcpA-Independent Regulation of Expression of the Mg2+-Citrate Transporter Gene citM by Arginine Metabolism in Bacillus subtilis. Jessica B. Warner, 2003.Transcriptional regulation of the Mg2+-citrate transporter, CitM, the main citrate uptake system of Bacillus subtilis, was studied during growth in rich medium . Citrate in the growth medium was required for induction under all growth conditions . In Luria-Bertani medium containing citrate, citM expression was completely repressed during the exponential growth phase, marginally expressed in the transition phase, and highly expressed in the stationary growth phase . The repression was relieved when the cells were grown in spent Luria-Bertani medium . The addition of a mixture of 18 amino acids restored repression . L-Arginine in the mixture appeared to be solely responsible for the repression, and ornithine appeared to be an equally potent repressor of citM expression . Studies of mutant strains deficient in RocR and SigL, proteins required for the expression of the enzymes of the arginase pathway, confirmed that uptake into the cell and, most likely, conversion of arginine to ornithine were required for repression . Arginine-mediated repression was independent of a functional CcpA, the global regulator protein in carbon catabolite repression (CCR) . Nevertheless, CCR-mediated repression was the major mechanism controlling the expression during exponential growth, while the newly described, CcpA-independent arginine-mediated repression was specifically apparent during the transition phase of growth .
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