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Regulation of Expression of Cellulosomes and Noncellulosomal (Hemi)Cellulolytic Enzymes in Clostridium cellulovorans during Growth on Different Carbon Sources. Sung Ok Han, 2004.Cellulosomes and noncellulosomal (hemi)cellulolytic enzymes are produced by Clostridium cellulovorans to degrade plant cell walls . To understand their synergistic relationship, changes in mRNA and protein expression in cellulosomes and noncellulosomal (hemi)cellulolytic enzymes (hereafter called noncellulosomal enzymes) of cultures grown on cellobiose, cellulose, pectin, xylan, and corn fiber or mixtures thereof were examined . Cellulase expression, favored particularly by the presence of Avicel, was found with all substrates . Comparison of cellulosome and noncellulosomal enzymes showed that expression profiles were strongly affected by the carbon source . High xylanase or pectate lyase expression was observed when C . cellulovorans was grown on xylan or pectin, respectively . Mixed carbon substrates (cellulose-pectin-xylan mixture or corn fiber) induced a wider variety of enzymes than a single carbon source, such as cellobiose, pectin, or xylan . Cellulosomal proteome profiles were more affected by the carbon source than the noncellulosomal enzymes . Transcription and protein analyses revealed that cellulosomes and noncellulosomal enzymes were expressed simultaneously on mixed carbon sources, but their degree of inducibility varied when the substrate was either cellulose or cellobiose . Cellulosomes and noncellulosomal enzymes had synergistic activity on various carbon substrates . These results indicated that expression of plant cell wall-degrading enzymes is highly influenced by the available carbon source and that synergy between cellulosomes and noncellulosomal enzymes contribute to plant cell wall degradation . Evidence from Terminal Recombination Gradients that FtsK Uses Replichore Polarity To Control Chromosome Terminus Positioning at Division in Escherichia coli. Jacqueline Corre, 2002.Chromosome dimers in Escherichia coli are resolved at the dif locus by two recombinases, XerC and XerD, and the septum-anchored FtsK protein . Chromosome dimer resolution (CDR) is subject to strong spatiotemporal control: it takes place at the time of cell division, and it requires the dif resolution site to be located at the junction between the two polarized chromosome arms or replichores . Failure of CDR results in trapping of DNA by the septum and RecABCD recombination (terminal recombination) . We had proposed that dif sites of a dimer are first moved to the septum by mechanisms based on local polarity and that normally CDR then occurs as the septum closes . To determine whether FtsK plays a role in the mobilization process, as well as in the recombination reaction, we characterized terminal recombination in an ftsK mutant . The frequency of recombination at various points in the terminus region of the chromosome was measured and compared with the recombination frequency on a xerC mutant chromosome with respect to intensity, the region affected, and response to polarity distortion . The use of a prophage excision assay, which allows variation of the site of recombination and interference with local polarity, allowed us to find that cooperating FtsK-dependent and -independent processes localize dif at the septum and that DNA mobilization by FtsK is oriented by the polarity probably due to skewed sequence motifs of the mobilized material . Crystal Structure of a Putative Methyltransferase from Mycobacterium tuberculosis: Misannotation of a Genome Clarified by Protein Structural Analysis. Jodie M. Johnston, 2003.Bioinformatic analyses of whole genome sequences highlight the problem of identifying the biochemical and cellular functions of many gene products that are at present uncharacterized . The open reading frame Rv3853 from Mycobacterium tuberculosis has been annotated as menG and assumed to encode an S-adenosylmethionine (SAM)-dependent methyltransferase that catalyzes the final step in menaquinone biosynthesis . The Rv3853 gene product has been expressed, refolded, purified, and crystallized in the context of a structural genomics program . Its crystal structure has been determined by isomorphous replacement and refined at 1.9 Å resolution to an R factor of 19.0% and Rfree of 22.0% . The structure strongly suggests that this protein is not a SAM-dependent methyltransferase and that the gene has been misannotated in this and other genomes that contain homologs . The protein forms a tightly associated, disk-like trimer . The monomer fold is unlike that of any known SAM-dependent methyltransferase, most closely resembling the phosphohistidine domains of several phosphotransfer systems . Attempts to bind cofactor and substrate molecules have been unsuccessful, but two adventitiously bound small-molecule ligands, modeled as tartrate and glyoxalate, are present on each monomer . These may point to biologically relevant binding sites but do not suggest a function . In silico screening indicates a range of ligands that could occupy these and other sites . The nature of these ligands, coupled with the location of binding sites on the trimer, suggests that proteins of the Rv3853 family, which are distributed throughout microbial and plant species, may be part of a larger assembly binding to nucleic acids or proteins . Large-Scale Cultivation of Acidophilic Hyperthermophiles for Recovery of Secreted Proteins. Penny Worthington, 2003.An electric water heater was modified for large-scale cultivation of aerobic acidophilic hyperthermophiles to enable recovery of secreted proteins . Critical changes included thermostat replacement, redesign of the temperature control circuit, and removal of the cathodic anticorrosion system . These alterations provided accurate temperature and pH control . The bioreactor was used to cultivate selected strains of the archaeon Sulfolobus solfataricus and other species within this genus . Reformulation of a basal salts medium facilitated preparation of large culture volumes and eliminated sterilization-induced precipitation of medium components . Substrate induction of synthesis of the S . solfataricus-secreted alpha-amylase during growth in a defined medium supported the utility of the bioreactor for studies of physiologically regulated processes . An improved purification strategy was developed by using strong cation-exchange chromatography for recovery of the alpha-amylase and the processing of large sample volumes of acidic culture supernatant . These findings should simplify efforts to study acidophilic hyperthermophilic microbes and their secreted proteins .
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