Dataset Information

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.

Project description:DndABCDE-catalyzed DNA phosphorothioation (PT), in which the nonbridging oxygen is swapped with a sulfur atom, was first identified in the bacterial genome. Usually, this modification gene cluster is paired with a restriction module consisting of DndF, DndG, and DndH. Although the mechanisms for the antiphage activity conferred by this Dnd-related restriction and modification (R-M) system have been well characterized, several features remain unclear, including the antiphage spectrum and potential interference with DNA methylation. Recently, a novel PT-related R-M system, composed of the modification module SspABCD paired with a single restriction enzyme, SspE, was revealed to be widespread in the bacterial kingdom, which aroused our interest in the interaction between Dnd- and Ssp-based R-M systems. In this study, we discussed the action of Dnd-related R-M systems against phages and demonstrated that the host could benefit from the protection provided by Dnd-related R-M systems against infection by various lytic phages as well as temperate phages. However, this defense barrier would fail against lysogenic phages. Interestingly, DNA methylation, even in the consensus sequence recognized by the Dnd system, could not weaken the restriction efficiency. Finally, we explored the interaction between Dnd- and Ssp-based R-M systems and found that these two systems were compatible. This study not only expands our knowledge of Dnd-associated R-M systems but also reveals a complex interaction between different defense barriers that coexist in the cell. IMPORTANCE Recently, we decoded the mechanism of Dnd-related R-M systems against genetic parasites. In the presence of exogenous DNA that lacks PT, the macromolecular machine consisting of DndF, DndG, and DndH undergoes conformational changes to perform DNA binding, translocation, and DNA nicking activities and scavenge the foreign DNA. However, several questions remain unanswered, including questions regarding the antiphage spectrum, potential interference by DNA methylation, and interplay with other PT-dependent R-M systems. Here, we revealed that the host could benefit from Dnd-related R-M systems for a broad range of antiphage activities, regardless of the presence of DNA methylation. Furthermore, we demonstrated that the convergence of Dnd- and Ssp-related R-M systems could confer to the host a stronger antiphage ability through the additive suppression of phage replication. This study not only deepens our understanding of PT-related defense barriers but also expands our knowledge of the arms race between bacteria and their predators.

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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