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Occurrence and Characterization of Mercury Resistance in the Hyperthermophilic Archaeon Sulfolobus solfataricus by Use of Gene Disruption. James Schelert, 2004.Mercury resistance mediated by mercuric reductase (MerA) is widespread among bacteria and operates under the control of MerR . MerR represents a unique class of transcription factors that exert both positive and negative regulation on gene expression . Archaea and bacteria are prokaryotes, yet little is known about the biological role of mercury in archaea or whether a resistance mechanism occurs in these organisms . The archaeon Sulfolobus solfataricus was sensitive to mercuric chloride, and low-level adaptive resistance could be induced by metal preconditioning . Protein phylogenetic analysis of open reading frames SSO2689 and SSO2688 clarified their identity as orthologs of MerA and MerR . Northern analysis established that merA transcription responded to mercury challenge, since mRNA levels were transiently induced and, when normalized to 7S RNA, approximated values for other highly expressed transcripts . Primer extension analysis of merA mRNA predicted a noncanonical TATA box with nonstandard transcription start site spacing . The functional roles of merA and merR were clarified further by gene disruption . The merA mutant exhibited mercury sensitivity relative to wild type and was defective in elemental mercury volatilization, while the merR mutant was mercury resistant . Northern analysis of the merR mutant revealed merA transcription was constitutive and that transcript abundance was at maximum levels . These findings constitute the first report of an archaeal heavy metal resistance system; however, unlike bacteria the level of resistance is much lower . The archaeal system employs a divergent MerR protein that acts only as a negative transcriptional regulator of merA expression . Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli. Marcel Emmerling, 2002.The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model . The general response to disruption of both pyruvate kinase isoenzymes in E . coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme . Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions . In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E . coli, with a turnover that is comparable to the specific glucose uptake rate . Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E . coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate . Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis .
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