| Summary: | <p>Abstract</p> <p>Background</p> <p>A novel DNA phosphorothioate modification (DNA sulfur modification), in which one of the non-bridging oxygen atoms in the phosphodiester bond linking DNA nucleotides is exchanged by sulphur, was found to be genetically determined by <it>dnd </it>or <it>dnd</it>-counterpart loci in a wide spectrum of bacteria from diverse habitats. A detailed mutational analysis of the individual genes within the <it>dnd </it>locus in <it>Streptomyces lividans </it>responsible for DNA phosphorothioation was performed and is described here. It should be of great help for the mechanistic study of this intriguing system.</p> <p>Results</p> <p>A 6,665-bp DNA region carrying just five ORFs (<it>dndA-E</it>) was defined as the sole determinant for modification of the DNA backbone in <it>S. lividans </it>to form phosphorothioate. This provides a diagnostically reliable and easily assayable Dnd (DNA degradation) phenotype. While <it>dndA </it>is clearly transcribed independently, <it>dndB</it>-<it>E </it>constitute an operon, as revealed by RT-PCR analysis. An efficient mutation-integration-complementation system was developed to allow for detailed functional analysis of these <it>dnd </it>genes. The Dnd<sup>- </sup>phenotype caused by specific in-frame deletion of the <it>dndA</it>, <it>C</it>, <it>D</it>, and <it>E </it>genes or the enhanced Dnd phenotype resulting from in-frame deletion of <it>dndB </it>could be restored by expression vectors carrying the corresponding <it>dnd </it>genes. Interestingly, overdosage of DndC or DndD, but not other Dnd proteins, <it>in vivo </it>was found to be detrimental to cell viability.</p> <p>Conclusion</p> <p>DNA phosphorothioation is a multi-enzymatic and highly coordinated process controlled by five <it>dnd </it>genes. Overexpression of some proteins <it>in vivo </it>prevented growth of host strain, suggesting that expression of the gene cluster is strictly regulated in the native host.</p>
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