A new type of DNA phosphorothioation-based antiviral system in archaea.
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ABSTRACT:
SUBMITTER: Xiong L
PROVIDER: S-EPMC6459918 | biostudies-literature | 2019 Apr
REPOSITORIES: biostudies-literature
Publications
A new type of DNA phosphorothioation-based antiviral system in archaea.
Xiong Lei L Liu Siyi S Chen Si S Xiao Yao Y Zhu Bochen B Gao Yali Y Zhang Yujing Y Chen Beibei B Luo Jie J Deng Zixin Z Chen Xiangdong X Wang Lianrong L Chen Shi S
Nature communications 20190411 1
Archaea and Bacteria have evolved different defence strategies that target virtually all steps of the viral life cycle. The diversified virion morphotypes and genome contents of archaeal viruses result in a highly complex array of archaea-virus interactions. However, our understanding of archaeal antiviral activities lags far behind our knowledges of those in bacteria. Here we report a new archaeal defence system that involves DndCDEA-specific DNA phosphorothioate (PT) modification and the PbeAB ...[more]
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Project description:Phosphorothioate (PT) modification, a sequence-specific modification that replaces the nonbridging oxygen atom with sulfur in a DNA phosphodiester through the gene products of dndABCDE or sspABCD, is widely distributed in prokaryotes. DNA PT modification functions together with gene products encoded by dndFGH, pbeABCD, or sspE to form defense systems that can protect against invasion by exogenous DNA particles. While the functions of the multiple enzymes in the PT system have been elucidated, the exact role of DndE in the PT process is still obscure. Here, we solved the crystal structure of DndE from the haloalkaliphilic archaeal strain Natronorubrum bangense JCM10635 at a resolution of 2.31 Å. Unlike the tetrameric conformation of DndE in Escherichia coli B7A, DndE from N. bangense JCM10635 exists in a monomeric conformation and can catalyze the conversion of supercoiled DNA to nicked or linearized products. Moreover, DndE exhibits preferential binding affinity to nicked DNA by virtue of the R19- and K23-containing positively charged surface. This work provides insight into how DndE functions in PT modification and the potential sulfur incorporation mechanism of DNA PT modification. IMPORTANCE DndABCDE proteins have been demonstrated to catalyze DNA PT modification with the nonbridging oxygen in the DNA sugar-phosphate backbone replaced by sulfur. In the PT modification pathway, DndA exerts cysteine desulfurase activity capable of catalyzing the mobilization of sulfur from l-cysteine, which involves the ion-sulfur cluster assembly of DndC. This is regarded as the initial step of the DNA PT modification. Moreover, DndD has ATPase activity in vitro, which is believed to provide energy for the oxygen-sulfur swap, while the function of DndE is unknown. However, the exact function of the key enzyme DndE remains to be elucidated. By determining the structure of DndE from the haloalkaliphilic strain Natronorubrum bangense JCM10635, we showed that the archaeal DndE adopts a monomer conformation. Notably, DndE can introduce nicks to supercoiled DNA and exhibits a binding preference for nicked DNA; the nicking is believed to be the initial step for DNA to facilitate the sulfur incorporation.
| S-EPMC9239217 | biostudies-literature
Project description:Phosphorothioate (PT) modification by the dnd gene cluster is the first identified DNA backbone modification and constitute an epigenetic system with multiple functions, including antioxidant ability, restriction modification, and virus resistance. Despite these advantages for hosting dnd systems, they are surprisingly distributed sporadically among contemporary prokaryotic genomes. To address this ecological paradox, we systematically investigate the occurrence and phylogeny of dnd systems, and they are suggested to have originated in ancient Cyanobacteria after the Great Oxygenation Event. Interestingly, the occurrence of dnd systems and prophages is significantly negatively correlated. Further, we experimentally confirm that PT modification activates the filamentous phage SW1 by altering the binding affinity of repressor and the transcription level of its encoding gene. Competition assays, concurrent epigenomic and transcriptomic sequencing subsequently show that PT modification affects the expression of a variety of metabolic genes, which reduces the competitive fitness of the marine bacterium Shewanella piezotolerans WP3. Our findings strongly suggest that a series of negative effects on microorganisms caused by dnd systems limit horizontal gene transfer, thus leading to their sporadic distribution. Overall, our study reveals putative evolutionary scenario of the dnd system and provides novel insights into the physiological and ecological influences of PT modification.
| S-EPMC8569181 | biostudies-literature
Project description:DNA phosphorothioate (PT) modification, in which the nonbridging oxygen in the sugar-phosphate backbone is substituted by sulfur, is catalyzed by DndABCDE or SspABCD in a double-stranded or single-stranded manner, respectively. In Dnd and Ssp systems, mobilization of sulfur in PT formation starts with the activation of the sulfur atom of cysteine catalyzed by the DndA and SspA cysteine desulfurases, respectively. Despite playing the same biochemical role, SspA cannot be functionally replaced by DndA, indicating its unique physiological properties. In this study, we solved the crystal structure of Vibrio cyclitrophicus SspA in complex with its natural substrate, cysteine, and cofactor, pyridoxal phosphate (PLP), at a resolution of 1.80?Å. Our solved structure revealed the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor, suggesting a common binding mode shared by cysteine desulfurases. In addition, although the distance between the catalytic Cys314 and the substrate cysteine is 8.9?Å, which is too far for direct interaction, our structural modeling and biochemical analysis revealed a conformational change in the active site region toward the cysteine substrate to move them close to each other to facilitate the nucleophilic attack. Finally, the pulldown analysis showed that SspA could form a complex with SspD, an ATP pyrophosphatase, suggesting that SspD might potentially accept the activated sulfur atom directly from SspA, providing further insights into the biochemical pathway of Ssp-mediated PT modification.IMPORTANCE Apart from its roles in Fe-S cluster assembly, tRNA thiolation, and sulfur-containing cofactor biosynthesis, cysteine desulfurase serves as a sulfur donor in the DNA PT modification, in which a sulfur atom substitutes a nonbridging oxygen in the DNA phosphodiester backbone. The initial sulfur mobilization from l-cysteine is catalyzed by the SspA cysteine desulfurase in the SspABCD-mediated DNA PT modification system. By determining the crystal structure of SspA, the study presents the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor. To overcome the long distance (8.9?Å) between the catalytic Cys314 and the cysteine substrate, a conformational change occurs to bring Cys314 to the vicinity of the substrate, allowing for nucleophilic attack.
| S-EPMC7188994 | biostudies-literature
Project description:To provide protection against viral infection and limit the uptake of mobile genetic elements, bacteria and archaea have evolved many diverse defence systems. The discovery and application of CRISPR-Cas adaptive immune systems has spurred recent interest in the identification and classification of new types of defence systems. Many new defence systems have recently been reported but there is a lack of accessible tools available to identify homologs of these systems in different genomes. Here, we report the Prokaryotic Antiviral Defence LOCator (PADLOC), a flexible and scalable open-source tool for defence system identification. With PADLOC, defence system genes are identified using HMM-based homologue searches, followed by validation of system completeness using gene presence/absence and synteny criteria specified by customisable system classifications. We show that PADLOC identifies defence systems with high accuracy and sensitivity. Our modular approach to organising the HMMs and system classifications allows additional defence systems to be easily integrated into the PADLOC database. To demonstrate application of PADLOC to biological questions, we used PADLOC to identify six new subtypes of known defence systems and a putative novel defence system comprised of a helicase, methylase and ATPase. PADLOC is available as a standalone package (https://github.com/padlocbio/padloc) and as a webserver (https://padloc.otago.ac.nz).
| S-EPMC8565338 | biostudies-literature
Project description:Unlike nucleobase modifications in canonical restriction-modification systems, DNA phosphorothioate (PT) epigenetic modification occurs in the DNA sugar-phosphate backbone when the nonbridging oxygen is replaced by sulfur in a double-stranded (ds) or single-stranded (ss) manner governed by DndABCDE or SspABCD, respectively. SspABCD coupled with SspE constitutes a defense barrier in which SspE depends on sequence-specific PT modifications to exert its antiphage activity. Here, we identified a new type of ssDNA PT-based SspABCD-SspFGH defense system capable of providing protection against phages through a mode of action different from that of SspABCD-SspE. We provide further evidence that SspFGH damages non-PT-modified DNA and exerts antiphage activity by suppressing phage DNA replication. Despite their different defense mechanisms, SspFGH and SspE are compatible and pair simultaneously with one SspABCD module, greatly enhancing the protection against phages. Together with the observation that the sspBCD-sspFGH cassette is widely distributed in bacterial genomes, this study highlights the diversity of PT-based defense barriers and expands our knowledge of the arsenal of phage defense mechanisms.IMPORTANCE We recently found that SspABCD, catalyzing single-stranded (ss) DNA phosphorothioate (PT) modification, coupled with SspE provides protection against phage infection. SspE performs both PT-simulated NTPase and DNA-nicking nuclease activities to damage phage DNA, rendering SspA-E a PT-sensing defense system. To our surprise, ssDNA PT modification can also pair with a newly identified 3-gene sspFGH cassette to fend off phage infection with a different mode of action from that of SspE. Interestingly, both SspFGH and SspE can pair with the same SspABCD module for antiphage defense, and their combination provides Escherichia coli JM109 with additive phage resistance up to 105-fold compared to that for either barrier alone. This agrees with our observation that SspFGH and SspE coexist in 36 bacterial genomes, highlighting the diversity of the gene contents and molecular mechanisms of PT-based defense systems.
| S-EPMC8092258 | biostudies-literature
Project description:Although the phosphorothioate (PT) modification, in which the nonbridging oxygen in the DNA sugar-phosphate backbone is replaced by sulfur, has been reported to play versatile roles in multiple cellular processes, very little data have been obtained to define the role of PT in epigenetic regulation. In this study, we report that the PT system in Salmonella enterica serovar Cerro 87 is involved in the transcriptional regulation of the torCAD operon encoding the trimethylamine N-oxide (TMAO) respiration machinery that enables the use of TMAO as a terminal electron acceptor for respiration when oxygen is not available. In vitro, PT enhanced the binding of the transcriptional activator of the torCAD operon, namely, TorR, to its DNA substrate (tor boxes). However, in vivo, the PT modification protein complex DndCDE downregulated torCAD transcription through competing with the binding of TorR to the tor boxes. The altered expression of torCAD caused by PT modification proteins affected cell growth that relied on TMAO respiration. To our knowledge, this is the first report supporting that PT proteins participate in transcriptional regulation, showing a new function of PT systems. IMPORTANCE Since the initial discovery of DNA phosphorothioate (PT) modification systems in Streptomyces lividans in the 1980s, explorations of the biological functions of DNA PT systems have advanced and yielded a number of important findings. However, the functions of PT systems, especially in genetic regulation, remain largely unknown. In this study, we report a case in which the PT system participates in the transcriptional regulation of the torCAD operon in Salmonella enterica serovar Cerro 87. While the PT modification enhanced the binding of TorR, the torCAD operon transcriptional activator, to its DNA substrate in vitro, we found that the PT modification protein complex DndCDE directly competed with TorR binding in vivo and subsequently repressed the expression of torCAD and attenuated cell growth that relied on TMAO respiration. These findings provide a deeper understanding of the characteristics of the PT chemical structure and broaden our understanding of the mechanisms by which PT regulates gene expression.
| S-EPMC9239176 | biostudies-literature
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.
| S-EPMC4373003 | biostudies-literature
Project description:BACKGROUND: The Dnd DNA degradation phenotype was first observed during electrophoresis of genomic DNA from Streptomyces lividans more than 20 years ago. It was subsequently shown to be governed by the five-gene dnd cluster. Similar gene clusters have now been found to be widespread among many other distantly related bacteria. Recently the dnd cluster was shown to mediate the incorporation of sulphur into the DNA backbone via a sequence-selective, stereo-specific phosphorothioate modification in Escherichia coli B7A. Intriguingly, to date all identified dnd clusters lie within mobile genetic elements, the vast majority in laterally transferred genomic islands. METHODOLOGY: We organized available data from experimental and bioinformatics analyses about the DNA phosphorothioation phenomenon and associated documentation as a dndDB database. It contains the following detailed information: (i) Dnd phenotype; (ii) dnd gene clusters; (iii) genomic islands harbouring dnd genes; (iv) Dnd proteins and conserved domains. As of 25 December 2008, dndDB contained data corresponding to 24 bacterial species exhibiting the Dnd phenotype reported in the scientific literature. In addition, via in silico analysis, dndDB identified 26 syntenic dnd clusters from 25 species of Eubacteria and Archaea, 25 dnd-bearing genomic islands and one dnd plasmid containing 114 dnd genes. A further 397 other genes coding for proteins with varying levels of similarity to Dnd proteins were also included in dndDB. A broad range of similarity search, sequence alignment and phylogenetic tools are readily accessible to allow for to individualized directions of research focused on dnd genes. CONCLUSION: dndDB can facilitate efficient investigation of a wide range of aspects relating to dnd DNA modification and other island-encoded functions in host organisms. dndDB version 1.0 is freely available at http://mml.sjtu.edu.cn/dndDB/.
| S-EPMC2663466 | biostudies-literature
Project description:Explosive growth in the study of microbial epigenetics has revealed a diversity of chemical structures and biological functions of DNA modifications in restriction-modification (R-M) and basic genetic processes. Here, we describe the discovery of shared consensus sequences for two seemingly unrelated DNA modification systems, 6mA methylation and phosphorothioation (PT), in which sulfur replaces a nonbridging oxygen in the DNA backbone. Mass spectrometric analysis of DNA from Escherichia coli B7A and Salmonella enterica serovar Cerro 87, strains possessing PT-based R-M genes, revealed d(GPS6mA) dinucleotides in the GPS6mAAC consensus representing ∼5% of the 1,100 to 1,300 PT-modified d(GPSA) motifs per genome, with 6mA arising from a yet-to-be-identified methyltransferase. To further explore PT and 6mA in another consensus sequence, GPS6mATC, we engineered a strain of E. coli HST04 to express Dnd genes from Hahella chejuensis KCTC2396 (PT in GPSATC) and Dam methyltransferase from E. coli DH10B (6mA in G6mATC). Based on this model, in vitro studies revealed reduced Dam activity in GPSATC-containing oligonucleotides whereas single-molecule real-time sequencing of HST04 DNA revealed 6mA in all 2,058 GPSATC sites (5% of 37,698 total GATC sites). This model system also revealed temperature-sensitive restriction by DndFGH in KCTC2396 and B7A, which was exploited to discover that 6mA can substitute for PT to confer resistance to restriction by the DndFGH system. These results point to complex but unappreciated interactions between DNA modification systems and raise the possibility of coevolution of interacting systems to facilitate the function of each.
| S-EPMC5410841 | biostudies-literature
Project description:Phosphorothioate (PT) modification of DNA, with sulfur replacing a nonbridging phosphate oxygen, was recently discovered as a product of the dnd genes found in bacteria and archaea. Given our limited understanding of the biological function of PT modifications, including sequence context, genomic frequencies, and relationships to the diversity of dnd gene clusters, we undertook a quantitative study of PT modifications in prokaryotic genomes using a liquid chromatography-coupled tandem quadrupole mass spectrometry approach. The results revealed a diversity of unique PT sequence contexts and three discrete genomic frequencies in a wide range of bacteria. Metagenomic analyses of PT modifications revealed unique ecological distributions, and a phylogenetic comparison of dnd genes and PT sequence contexts strongly supports the horizontal transfer of dnd genes. These results are consistent with the involvement of PT modifications in a type of restriction-modification system with wide distribution in prokaryotes.
| S-EPMC3041111 | biostudies-other