| A novel DNA modification discovered in Streptomyces lividans 1326 is different from DNA methylation. This unusual modification causes wild type S. lividans 1326 DNA sensitive to site-specific oxidative double-strand cleavage (Dnd phenotype). Preliminary study revealed that such DNA modification involves incorporation of sulphur. The entire functional dnd cluster, 8.3kb in size, including 3 ORFs-dndA, dndB and dndC was involved in this DNA modification. Four plasmids derived from an integrative vector pSET152 were constructed which contain the whole dnd cluster but each ORF has been interrupted individually, and introduced into 5. lividans ZX1, a mutant strain with a chromosome deletion including dnd cluster for complementation test. All of three ORFs were demonstrated to be necessary for the Dnd phenotype.For the survey of the biological relevence, different 5". lividans strains with Dnd+ and Dnd" phenotype were used for testing phage infections, growth under different pH, temperature, concentration of NaCl and an extremely low concentration of O2. No obvious differences could be tested from these strains. The detection of the Dnd phenotype of the total DNA isolated from the mycelie growing at an extremely low concentration of O2 showed no difference from that at normal condition.The Dnd phenotype was also found in DNA of Mycobacterium smegmatis mc2155 by PFGE. Query of the amino acid sequence of dnd cluster from S. lividance to the uncompleted genome sequence of me2155, a homologous gene with a 38% identity to dndA was found and was tentatively named mddl. Two pairs of primers were designed based on the sequence of mddl to get a 0.9kb and an 1.1kb PCR products which were confirmed to be expected sequence from mc2155 by DNA sequencing. The me2155 genomic library was constructed using pHZ132 as the cosmid vector, and 10 positive clones were fished out using 0.9kb PCR product as probe. A construct derived frompHZ2157 is now ready for the constrction of mddl mutant by gene disruption experiment. |
