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DNA phosphorothioation in Streptomyces lividans: mutational analysis of the dnd locus.

ABSTRACT: BACKGROUND: 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 dnd or dnd-counterpart loci in a wide spectrum of bacteria from diverse habitats. A detailed mutational analysis of the individual genes within the dnd locus in Streptomyces lividans responsible for DNA phosphorothioation was performed and is described here. It should be of great help for the mechanistic study of this intriguing system. RESULTS: A 6,665-bp DNA region carrying just five ORFs (dndA-E) was defined as the sole determinant for modification of the DNA backbone in S. lividans to form phosphorothioate. This provides a diagnostically reliable and easily assayable Dnd (DNA degradation) phenotype. While dndA is clearly transcribed independently, dndB-E 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 dnd genes. The Dnd- phenotype caused by specific in-frame deletion of the dndA, C, D, and E genes or the enhanced Dnd phenotype resulting from in-frame deletion of dndB could be restored by expression vectors carrying the corresponding dnd genes. Interestingly, overdosage of DndC or DndD, but not other Dnd proteins, in vivo was found to be detrimental to cell viability. CONCLUSION: DNA phosphorothioation is a multi-enzymatic and highly coordinated process controlled by five dnd genes. Overexpression of some proteins in vivo prevented growth of host strain, suggesting that expression of the gene cluster is strictly regulated in the native host.

SUBMITTER: Xu T

PROVIDER: S-EPMC2653506 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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DNA phosphorothioation in Streptomyces lividans: mutational analysis of the dnd locus.

Xu Tiegang T Liang Jingdan J Chen Shi S Wang Lianrong L He Xinyi X You Delin D Wang Zhijun Z Li Aiying A Xu Zhongli Z Zhou Xiufen X Deng Zixin Z

BMC microbiology 20090220

<h4>Background</h4>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 dnd or dnd-counterpart loci in a wide spectrum of bacteria from diverse habitats. A detailed mutational analysis of the individual genes within the dnd locus in Streptomyces lividans responsible for DNA phosphorothioation was performed and is descr ...[more]

PMID: 19232083

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Project description:Strains of Escherichia coli that are non-typeable by pulsed-field gel electrophoresis (PFGE) due to in-gel degradation can influence their molecular epidemiological data. The DNA degradation phenotype (Dnd(+)) is mediated by the dnd operon that encode enzymes catalyzing the phosphorothioation of DNA, rendering the modified DNA susceptible to oxidative cleavage during a PFGE run. In this study, a PCR assay was developed to detect the presence of the dnd operon in Dnd(+) E. coli strains and to improve their typeability. Investigations into the genetic environments of the dnd operon in various E. coli strains led to the discovery that the dnd operon is harboured in various diverse genomic islands.The dndBCDE genes (dnd operon) were detected in all Dnd(+) E. coli strains by PCR. The addition of thiourea improved the typeability of Dnd(+) E. coli strains to 100% using PFGE and the Dnd(+) phenotype can be observed in both clonal and genetically diverse E. coli strains. Genomic analysis of 101 dnd operons from genome sequences of Enterobacteriaceae revealed that the dnd operons of the same bacterial species were generally clustered together in the phylogenetic tree. Further analysis of dnd operons of 52 E. coli genomes together with their respective immediate genetic environments revealed a total of 7 types of genetic organizations, all of which were found to be associated with genomic islands designated dnd-encoding GIs. The dnd-encoding GIs displayed mosaic structure and the genomic context of the 7 islands (with 1 representative genome from each type of genetic organization) were also highly variable, suggesting multiple recombination events. This is also the first report where two dnd operons were found within a strain although the biological implication is unknown. Surprisingly, dnd operons were frequently found in pathogenic E. coli although their link with virulence has not been explored.Genomic islands likely play an important role in facilitating the horizontal gene transfer of the dnd operons in E. coli with 7 different types of islands discovered so far.

Dataset Information

DNA phosphorothioation in Streptomyces lividans: mutational analysis of the dnd locus.

Publications

DNA phosphorothioation in Streptomyces lividans: mutational analysis of the dnd locus.

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