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Characterization of a Cellulase Containing a Family 30 Carbohydrate-Binding Module (CBM) Derived from Clostridium thermocellum CelJ: Importance of the CBM to Cellulose Hydrolysis. Takamitsu Arai, 2003.Clostridium thermocellum CelJ is a modular enzyme containing a family 30 carbohydrate-binding module (CBM) and a family 9 catalytic module at its N-terminal moiety . To investigate the functions of the CBM and the catalytic module, truncated derivatives of CelJ were constructed and characterized . Isothermal titration calorimetric studies showed that the association constants (Ka) of the CBM polypeptide (CBM30) for the binding of cellopentaose and cellohexaose were 1.2 x 104 and 6.4 x 104 M-1, respectively, and that the binding of CBM30 to these ligands is enthalpically driven . Qualitative analyses showed that CBM30 had strong affinity for cellulose and ß-1,3-1,4-mixed glucan such as barley ß-glucan and lichenan . Analyses of the hydrolytic action of the enzyme comprising the CBM and the catalytic module showed that the enzyme is a processive endoglucanse with strong activity towards carboxymethylcellulose, barley ß-glucan and lichenan . By contrast, the catalytic module polypeptide devoid of the CBM showed negligible activity toward these substrates . These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity . Evidence for Horizontal Transfer of the EcoT38I Restriction- Modification Gene to Chromosomal DNA by the P2 Phage and Diversity of Defective P2 Prophages in Escherichia coli TH38 Strains. Keiko Kita, 2003.A DNA fragment carrying the genes coding for a novel EcoT38I restriction endonuclease (R.EcoT38I) and EcoT38I methyltransferase (M.EcoT38I), which recognize G(A/G)GC(C/T)C, was cloned from the chromosomal DNA of Escherichia coli TH38 . The endonuclease and methyltransferase genes were in a head-to-head orientation and were separated by a 330-nucleotide intergenic region . A third gene, the C.EcoT38I gene, was found in the intergenic region, partially overlapping the R.EcoT38I gene . The gene product, C.EcoT38I, acted as both a positive regulator of R.EcoT38I gene expression and a negative regulator of M.EcoT38I gene expression . M.EcoT38I purified from recombinant E . coli cells was shown to be a monomeric protein and to methylate the inner cytosines in the recognition sequence . R.EcoT38I was purified from E . coli HB101 expressing M.EcoT38I and formed a homodimer . The EcoT38I restriction (R)-modification (M) system (R-M system) was found to be inserted between the A and Q genes of defective bacteriophage P2, which was lysogenized in the chromosome at locI, one of the P2 phage attachment sites observed in both E . coli K-12 MG1655 and TH38 chromosomal DNAs . Ten strains of E . coli TH38 were examined for the presence of the EcoT38I R-M gene on the P2 prophage . Conventional PCR analysis and assaying of R activity demonstrated that all strains carried a single copy of the EcoT38I R-M gene and expressed R activity but that diversity of excision in the ogr, D, H, I, and J genes in the defective P2 prophage had arisen . Evidence for the Existence of Psychrophilic Methanogenic Communities in Anoxic Sediments of Deep Lakes. Alla N. Nozhevnikova, 2003.In order to obtain evidence for the existence of psychrophilic methanogenic communities in sediments of deep lakes that are low-temperature environments (4 to 5°C), slurries were first incubated at temperatures between 4 and 60°C for several weeks, at which time they were amended, or not, with an additional substrate, such as cellulose, butyrate, propionate, acetate, or hydrogen, and further incubated at 6°C . Initial methane production rates were highest in slurries preincubated at temperatures between 4 and 15°C, with maximal rates in slurries kept at 6°C . Hydrogen-amended cultures were the only exceptions, with the highest methane production rates at 6°C after preincubation at 30°C . Characterization of the Corrinoid Iron-Sulfur Protein Tetrachloroethene Reductive Dehalogenase of Dehalobacter restrictus. Julien Maillard, 2003.The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 ± 1 kDa, whereas the native molecular mass was 71 ± 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-ß-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 ± 0.1 mol of cobalamin, 0.6 ± 0.02 mol of cobalt, 7.1 ± 0.6 mol of iron, and 5.8 ± 0.5 mol of acid-labile sulfur . Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 ± 12 nkat/mg of protein . In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA . The Km values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 ± 3.2, 23.7 ± 5.2, and 47 ± 10 µM, respectively . The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air . Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced . Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences . Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy .
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