The conjugative streptococcal transposon Tn916 was found to transfer naturally between a variety of gram-positive and gram-negative eubacteria. Enterococcus faecalis hosting the transposon could serve as a donor for Alcaligenes eutrophus, Citrobacter freundii, and Escherichia coli at frequencies of 10(-6) to 10(-8). No transfer was observed with several phototrophic species. Mating of an E. coli strain carrying Tn916 yielded transconjugants with Bacillus subtilis, Clostridium acetobutylicum, Enterococcus faecalis, and Streptococcus lactis subsp. diacetylactis at frequencies of 10(-4) to 10(-6). Acetobacterium woodii was the only gram-positive organism tested that did not accept the transposon from a gram-negative donor. The results prove the ability of conjugative transposable elements such as Tn916 for natural cross-species gene transfer, thus potentially contributing to bacterial evolution.
The pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum was purified to homogeneity and partially characterized. A 9.2-fold purification was achieved in a three step purification procedure: ammonium sulfate fractionation, chromatography on Phenyl Sepharose and on Procion Blue H-EGN12. The pure enzyme exhibited a specific activity of 25 U/mg of protein. Homogeneity of the pyruvate-ferredoxin oxidoreductase was confirmed by native polyacrylamide gel electrophoresis and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight was determined to be 123,000/monomer. The subunit composition of the native enzyme could not be determined because of the instability of the pure enzyme. The pyruvate-ferredoxin oxidoreductase is sensitive to oxygen and dilution during purification. The dilution inactivation could be partially overcome by the addition of 300 microM coenzyme A or 50% ethyleneglycol. A thiamine pyrophosphate content of 0.39 mol per mol of enzyme monomer was found, the iron and sulfur content was 4.23 and 0.91, respectively. The pH-optimum was at pH 7.5 and the temperature optimum was at 60 degrees C. Kinetic constants were measured in the forward reaction. The apparent Km for pyruvate and coenzyme A were 322 microM and 3.7 microM, respectively. With 2-ketobutyrate the pyruvate-ferredoxin oxidoreductase showed 12.5% of the activity compared to pyruvate. No activity was found with 2-ketoglutarate. Ferredoxin from Clostridium pasteurianum could be used as physiological electron acceptor.
Various bacterial isolates from enrichments with isopropylbenzene (cumene), toluene or phenol as carbon and energy sources were tested as to their potential to oxidize trichloroethene (TCE). In contrast to toluene and phenol, all isolates enriched on isopropylbenzene were able to oxidize TCE. Two isolates, strain JR1 and strain BD1, were identified as Pseudomonas spec. and as Rhodococcus erythropolis, respectively. TCE oxidation was accompanied by the liberation of stoichiometric amounts of chloride. Initial TCE oxidation rate increased proportional to the substrate concentration from 25 to 200 microM TCE. Maximal initial TCE-degradation rates found here were 4 to 5 nmol.min-1.mg protein-1. The TCE degradation rate decreased with time. The two isolates showed a temperature optimum for TCE degradation between 10 and 20 degrees C. In addition to TCE, R. erythropolis BD1 degraded only cis- and trans-dichloroethene whereas Pseudomonas spec. JR1 was able to oxidize also 1,1-dichloroethene, vinyl chloride, trichloroethane, and 1,2-dichloroethane.
The development of rapid polymerase chain reaction (PCR)region (CDR3).2,6-10 Clonal expansions of B cells carry ident-
accuracy of PCR results is significantly improved by appli-The specificity was 100% as determined by analysis of 50 concation of the thermostable UITma DNA polymerase with an trols, all of which gave polyclonal PCR results. We found a high inherent 3′ to 5′ exonuclease activity. 24 In the present report escence detection of IgH-CDR3-PCR products is a powerful
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