ABSTRACT
Streptomyces aureofaciens B96 produces several intra- and extracellular enzymes with deoxyribonuclease activity. According to the sequence of the previously published gene exoSc from S. coelicolor A3(2), the DNA sequence from S. aureofaciens B96 was amplified, cloned and expressed in E. coli. The protein product of exoSa gene, recExoSa, was also an exonuclease with DNAase and 5'-phosphomonoesterase activities at optimum temperature 37 degrees C and pH 8.0. It degraded only linear DNA (chromosomal, double-stranded and single-stranded) and linear plasmid DNA from both ends, with a preference to blunt ends in comparison with overhang ends. The purified enzyme exhibited no RNAase activity. Both exoSc and exoSa genes were interrupted by the apramycin resistance gene; constructed fragments were transformed into particular streptomyces protoplasts. Mutation caused by exoSa disruption in S. aureofaciens chromosome and mutation by interrupted exoSc in S. coelicolor were lethal.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Exonucleases/chemistry , Exonucleases/genetics , Gene Silencing , Streptomyces aureofaciens/enzymology , Streptomyces coelicolor/genetics , Amino Acid Sequence , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Cloning, Molecular , Deoxyribonucleases/chemistry , Deoxyribonucleases/genetics , Deoxyribonucleases/isolation & purification , Deoxyribonucleases/metabolism , Enzyme Stability , Exonucleases/isolation & purification , Exonucleases/metabolism , Molecular Sequence Data , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/isolation & purification , Phosphoric Monoester Hydrolases/metabolism , Sequence Alignment , Streptomyces aureofaciens/chemistry , Streptomyces aureofaciens/genetics , Streptomyces coelicolor/chemistry , Streptomyces coelicolor/enzymology , Substrate SpecificityABSTRACT
Temperate bacteriophages were induced in and released from isolates of Staphylococcus aureus and Streptococcus agalactiae using mitomycin C. Various specific indicator cultures were tested for providing clear plaques after phage infection. Specific lytic mixture of bacteriophages was prepared using the induced, modified and laboratory variants of phages. Under laboratory conditions, the mixture eliminated all isolates from the tested collection of microorganisms. The restriction barrier of some bacterial isolates to bacteriophage infection was overcome either by UV irradiation or in vitro modification of bacteriophage DNA with specific methyltransferases. Conjugative R plasmids, capable of replication in G+ and G- bacteria, were detected and isolated from S. aureus and S. agalactiae antibiotic-resistant strains.
Subject(s)
Staphylococcus Phages/physiology , Staphylococcus aureus/drug effects , Staphylococcus aureus/virology , Staphylococcus/drug effects , Staphylococcus/virology , Animals , DNA, Viral/genetics , DNA, Viral/isolation & purification , Drug Resistance, Multiple, Bacterial/genetics , Humans , In Vitro Techniques , R Factors/genetics , Staphylococcus/genetics , Staphylococcus/isolation & purification , Staphylococcus aureus/genetics , Staphylococcus aureus/isolation & purification , Virus ActivationABSTRACT
Characterization of classic type II restriction-modification systems (RMS) (restriction endonucleases and modification methyltransferases) was carried out in isolates of Staphylococcus aureus and Streptococcus agalactiae obtained from clinical material. Among the 100 isolates of S. aureus two different RMS type II were detected. The first was expressed in isolates 32 and 33 (Sau32 I and Sau33 I); the targeting sequence was determined as 5'-GGN CC-3' (Sau96 I isoschizomer). The second was found in isolates no. 90, 93, 96*, and 98 (Sau90 I, Sau93 I, Sau96* I, Sau98 I) and enzymes recognized sequence 5'-CTY RAG-3' (SmlI isoschizomer). Analysis of 40 isolates of S. agalactiae revealed only one RMS; it was detected in two isolates (no. 16 and 23; Sag16 I and Sag23 I). Restriction endonuclease expressed by these isolates cleaved DNA in sequence 5'-CTG CA/G-3' (PstI isoschizomer). In RMS-positive S. aureus and S. agalactiae isolates plasmid DNA capable of replication in Escherichia coli and Bacillus subtilis was also detected and isolated.
