ABSTRACT
A new method of testing restriction nucleases is proposed. This method is based on high-temperature treatment of crude cell extracts. Disrupted cells were heated at 50-60 degrees C, centrifuged, and assayed for restrictases. This method provides the opportunity for screening new enzymes in microbial strains enriched with nonspecific restrictases. High-temperature treatment of cell extracts of certain producers reduces the number of steps of the procedure used for isolating high-purity restrictases; the resulting preparations are capable of maintaining high enzymatic activity during long-term storage. It was shown that high-temperature treatment can be applied not only to thermophilic but also to mesophilic strains of microorganisms of different taxa.
Subject(s)
Bacteria/enzymology , DNA Restriction Enzymes/isolation & purification , Biotechnology/methods , Hot Temperature , Quality ControlABSTRACT
The proposed method for assessing microorganism sensitivity in solid and semisolid liquid nutrient media provides more accurate results and permits isolation of resistant forms of bacteria and studies of antibiotic interactions. Bacteriological study of latent growth zones of bacteria, although rather long, makes the results more reliable. The method can be used for assessing the microflora sensitivity in patients without resorting to isolation of pure cultures and in biotechnology for isolation of new antibiotics.
Subject(s)
Microbial Sensitivity Tests/methods , Agar , Anti-Bacterial Agents/pharmacology , Culture Media , Escherichia coli/drug effects , Escherichia coli/isolation & purification , Staphylococcus aureus/drug effects , Staphylococcus aureus/isolation & purificationABSTRACT
Using the most effective rapid method for the detection of restriction endonucleases (ENases) in microorganisms, 32 thermophilic producers have been isolated. All strains belong to the genus Bacillus. Thermostable isoschizomers of ENases, such as AvaI, BbvI, BbvII, BclI, BsaBI, BsiYI, BsrI, BstEII, BstNI, Cfr10I, ClaI, FspI, HaeIII, HpaII, Ksp632I and SfeI, were isolated.
Subject(s)
Bacillus/enzymology , DNA Restriction Enzymes/metabolism , Soil Microbiology , Base Sequence , DNA Restriction Enzymes/isolation & purification , Enzyme Stability , Hot Temperature , Molecular Sequence Data , Species SpecificityABSTRACT
Restriction endonucleases have been isolated from 26 strains of thermophilic strains of the Bacillus genus, their recognition sequences were determined, and for 15 of them cleavage sites identified. The enzymes proved to be isoschizomers of known endonucleases BstNI, EarI, HaeIII, HpaII, Cfr10I, BsiYI, BclI, BbvII, BbvI, BstEII, BsaBI, BsrI, FspI, ClaI, SfeI.
Subject(s)
Bacillus/enzymology , DNA Restriction Enzymes/isolation & purification , DNA Restriction Enzymes/metabolism , Substrate SpecificityABSTRACT
Bme 361 I, a new site-specific type II deoxyribonuclease, was purified from Bacillus megaterium 361 by chromatography on phosphocellulose P 11 and hydroxylapatite. The enzyme recognizes and cleaves the nucleotide sequence 5'-GG decreases CC-3' in double-strand DNA. Thus it is a true isoschizomer of deoxyribonucleases Hae III and BspR I.
Subject(s)
Bacillus megaterium/enzymology , Deoxyribonucleases/metabolism , Autoradiography , Bacteriophage lambda/metabolism , Chromatography, Liquid , DNA, Bacterial/metabolism , DNA, Viral/metabolism , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Plasmids , Substrate SpecificityABSTRACT
The method for analysis of microorganisms for the presence of the modification-restriction systems has been developed. The method has permitted to detect more than 10 new producing strains of restrictases including microorganisms of Rhizobium genus. Some of them are promising for practical use. It has been shown that using selection of clones the strain productivity can be increased. The purification process for the majority of restrictases has been proposed. Some physical and catalytic properties of new enzymes have been studied.
Subject(s)
Bacteria/enzymology , DNA Restriction-Modification Enzymes/biosynthesis , DNA Restriction-Modification Enzymes/isolation & purification , Bacteriological Techniques , Bacteriophage lambda , Chemical Phenomena , Chemistry, Physical , DNA, Viral/metabolism , Hydrolysis , Restriction Mapping , Substrate SpecificityABSTRACT
The recognition sequence and cleavage site for restriction endonuclease SsrI have been determined, the latter being 5'-GTT decreases AAC-3'. The enzyme was isolated from Staphylococcus saprophyticus strain and may be used in DNA investigation instead of its isoshizomer HpaI.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/isolation & purification , Staphylococcus/enzymology , Base Sequence , Deoxyribonucleases, Type II Site-Specific/geneticsABSTRACT
On the basis of the data obtained in the study of the properties of different Haemophilus species, their classification as a separate family is proposed. In solving the problem of the systematization of Haemophilus, their ecology and, aside from the known properties of these organisms, their sensitivity to sulfonol and antibiotics have been considered.
Subject(s)
Haemophilus/classification , Anti-Bacterial Agents/pharmacology , Benzenesulfonates/pharmacology , Dose-Response Relationship, Drug , Drug Resistance, Microbial , Gram-Negative Bacteria/classification , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/classification , Gram-Positive Bacteria/drug effects , Haemophilus/drug effects , Microbial Sensitivity TestsABSTRACT
The effect of sodium dodecylbenzene sulfonate (sulfonol) and certain froth breakers on the activity of endonuclease was studied in the cultural broth of Serratia marcescens in order to find out whether sulfonol could be used for limiting the infection. Sulfonol was found to have no effect on the cultural growth; it increased the activity of endonuclease in the cultural broth, and the peak of the activity appeared earlier than in the control medium. Propanol B-400 was shown to be the best froth breaker.
Subject(s)
Antifoaming Agents/pharmacology , Benzenesulfonates/pharmacology , Endonucleases/metabolism , Serratia marcescens/enzymology , Surface-Active Agents/pharmacology , Serratia marcescens/drug effects , Serratia marcescens/growth & developmentABSTRACT
The susceptibility to sodium dodecylbenzene sulfonate, an anion-active detergent, was studied with 10 Gram-positive and 18 Gram-negative bacterial cultures. According to this susceptibility, the cultures were subdivided into two groups identical in their tinctorial properties. The bacteria growing at a 0.05% concentration of sodium dodecylbenzene sulfonate or at its higher concentrations were Gram-negative. The threshold concentration of this compound in the medium at which Gram-positive cultures could grow was 0.008%; some of the bacteria ceased growing even at a 0.002% concentration. The bacteria varied in their susceptibility to the detergent also within one and the same group, and even within one and the same species. The subdivision of bacteria on the basis of their susceptibility to sodium dodecylbenzene sulfonate may be considered as a taxonomic feature.
