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A Gene from the Mesophilic Bacterium Dehalococcoides ethenogenes Encodes a Novel Mannosylglycerate Synthase. Nuno Empadinhas, 2004.Mannosylglycerate (MG) is a common compatible solute found in thermophilic and hyperthermophilic prokaryotes . In this study we characterized a mesophilic and bifunctional mannosylglycerate synthase (MGSD) encoded in the genome of the bacterium Dehalococcoides ethenogenes . mgsD encodes two domains with extensive homology to mannosyl-3-phosphoglycerate synthase (MPGS, EC 2.4.1.217) and to mannosyl-3-phosphoglycerate phosphatase (MPGP, EC 3.1.3.70), which catalyze the consecutive synthesis and dephosphorylation of mannosyl-3-phosphoglycerate to yield MG in Pyrococcus horikoshii, Thermus thermophilus, and Rhodothermus marinus . The bifunctional MGSD was overproduced in Escherichia coli, and we confirmed the combined MPGS and MPGP activities of the recombinant enzyme . The optimum activity of the enzyme was at 50°C . To examine the properties of each catalytic domain of MGSD, we expressed them separately in E . coli . The monofunctional MPGS was unstable, while the MPGP was stable and was characterized . Dehalococcoides ethenogenes cannot be grown sufficiently to identify intracellular compatible solutes, and E . coli harboring MGSD did not accumulate MG . However, Saccharomyces cerevisiae expressing mgsD accumulated MG, confirming that this gene product can synthesize this compatible solute and arguing for a role in osmotic adjustment in the natural host . We did not detect MGSD activity in cell extracts of S . cerevisiae . Here we describe the first gene and enzyme for the synthesis of MG from a mesophilic microorganism and discuss the possible evolution of this bifunctional MGSD by lateral gene transfer from thermophilic and hyperthermophilic organisms . Restoration of a Defective Lactococcus lactis Xylose Isomerase. Joo-Heon Park, 2004.The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp . lactis strains, 210 (Xyl–) and IO-1 (Xyl+), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated . The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs . The purified IO-1 XI was soluble with Km and kcat being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive . Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble . The Km and kcat of this mutant were 4.15 mM and 141/s, respectively . One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI . The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility . The dissolution of XI activity in L . lactis subsp . lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme . Characterization of Genes Involved in Modulation of Conjugal Transfer of the Bacteroides Conjugative Transposon CTnDOT. Gabrielle Whittle, 2002.In previous studies we identified an 18-kb region of the Bacteroides conjugative transposon CTnDOT that was sufficient for mobilization of coresident plasmids and unlinked integrated elements, as well as self-transfer from Bacteroides to Escherichia coli . When this 18-kb region was cloned on a plasmid (pLYL72), the plasmid transferred itself constitutively in the absence of a coresident conjugative transposon . However, when this plasmid was present in a Bacteroides strain containing a coresident conjugative transposon, conjugal transfer was repressed in the absence of tetracycline and enhanced in the presence of tetracycline . These results suggested that a negative and a positive regulator of conjugal transfer were encoded outside the transfer region of the CTnDOT element . In this work, a minimal and inducible transfer system was constructed and used in transfer and Western blot analyses to identify the differentially regulated genes from CTnDOT responsible for the enhancement and repression of pLYL72 conjugal transfer . Both of these regulatory functions have been localized to a region of the CTnDOT element that is essential for CTn excision . In the presence of tetracycline, the regulatory protein RteC activates the expression of a putative topoisomerase gene, exc, which in turn results in an increase in transfer protein expression and a concomitant 100- to 1,000-fold increase in the frequency of pLYL72 transfer . Our results also suggest that since exc alone cannot result in enhancement of transfer, other factors encoded upstream of exc are also required . Conversely, in the absence of tetracycline, a gene located near the 3' end of exc is responsible for the repression of transfer protein expression and also results in a 100- to 1,000-fold decrease in the frequency of pLYL72 transfer . X-Ray Crystal Structure of the Multidomain Endoglucanase Cel9G from Clostridium cellulolyticum Complexed with Natural and Synthetic Cello-Oligosaccharides. David Mandelman, 2003.Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy . To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the ß-1,4-glycosidic bonds in cellulose, is a topic of great interest . The high-resolution X-ray crystal structure of a multidomain endoglucanase from Clostridium cellulolyticum has been determined at a 1.6-Å resolution . The endoglucanase, Cel9G, is comprised of a family 9 catalytic domain attached to a family IIIc cellulose-binding domain . The two domains together form a flat platform onto which crystalline cellulose is suggested to bind and be fed into the active-site cleft for endolytic hydrolysis . To further dissect the structural basis of cellulose binding and hydrolysis, the structures of Cel9G in the presence of cellobiose, cellotriose, and a DP-10 thio-oligosaccharide inhibitor were resolved at resolutions of 1.7, 1.8, and 1.9 Å, respectively . Identification and Characterization of Coenzyme B12-Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures. Anja Knietsch, 2003.To isolate genes encoding coenzyme B12-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions . The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain . In this way, two positive E . coli clones out of 560,000 tested clones were obtained . In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes . The screening of 158,000 E . coli clones by this method yielded five positive clones . Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further . The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria . Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases . We were able to perform high-level production and purification of three of these dehydratases . The glycerol dehydratases purified from E . coli Bl21/pAK2.1 and E . coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E . coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C . freundii . The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C . freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol . Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E . coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol . Stimulating the In Situ Activity of Geobacter Species To Remove Uranium from the Groundwater of a Uranium-Contaminated Aquifer. Robert T. Anderson, 2003.The potential for removing uranium from contaminated groundwater by stimulating the in situ activity of dissimilatory metal-reducing microorganisms was evaluated in a uranium-contaminated aquifer located in Rifle, Colo . Acetate (1 to 3 mM) was injected into the subsurface over a 3-month period via an injection gallery composed of 20 injection wells, which was installed upgradient from a series of 15 monitoring wells . U(VI) concentrations decreased in as little as 9 days after acetate injection was initiated, and within 50 days uranium had declined below the prescribed treatment level of 0.18 µM in some of the monitoring wells . Analysis of 16S ribosomal DNA (rDNA) sequences and phospholipid fatty acid profiles demonstrated that the initial loss of uranium from the groundwater was associated with an enrichment of Geobacter species in the treatment zone . Fe(II) in the groundwater also increased during this period, suggesting that U(VI) reduction was coincident with Fe(III) reduction . As the acetate injection continued over 50 days there was a loss of sulfate from the groundwater and an accumulation of sulfide and the composition of the microbial community changed . Organisms with 16S rDNA sequences most closely related to those of sulfate reducers became predominant, and Geobacter species became a minor component of the community . This apparent switch from Fe(III) reduction to sulfate reduction as the terminal electron accepting process for the oxidation of the injected acetate was associated with an increase in uranium concentration in the groundwater . These results demonstrate that in situ bioremediation of uranium-contaminated groundwater is feasible but suggest that the strategy should be optimized to better maintain long-term activity of Geobacter species .
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