SspABCD–SspE is a phosphorothioation-sensing bacterial defence system with broad anti-phage activities

Xiong, Xiaolin; Wu, Geng; Yue, W.W.; Liu, L.; Zhang, Y.; Su, Ruochen; Jiang, Xingyu; Li, M.; Gao, Haiyan; Tian, Xianxiang; Hu, Lirong; Shi, Chen; Tang, Yulong; Jiang, Shu‐Heng; Huang, Ruhua; Li, Z.; Wang, Yishuang; Deng, Zixin; Wang, J.; Dedon, Peter C.; Wang, L.

doi:10.1038/s41564-020-0700-6

Cited by 106 publications

(151 citation statements)

References 57 publications

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“…As a versatile sulfur donor, cysteine desulfurase activity is also essential to DNA phosphorothioate (PT) modification, in which the nonbridging oxygen atom in the DNA sugar-phosphate backbone is replaced by sulfur (6,7). The "writing" of a PT modification into DNA occurs in a sequence-selective and R P configuration-specific manner governed by Dnd or Ssp machineries in bacteria and archaea (6,8,9). Dnd modification systems consist of five proteins (DndA, B, C, D, and E) and confer PTs in 4-bp complementary motifs, e.g., 5=-G PS AAC-3=/5=-G PS TTC-3= (PS, phosphate-sulfur linkage) in Escherichia coli B7A and 5=-G PS ATC-3=/5=-G PS ATC-3= in Hahella chejuensis KCTC2396 (10,11).…”

mentioning

confidence: 99%

“…Interestingly, we recently characterized a new type of Ssp system that exhibits different genetic organization, biochemical functions, and phenotypic behavior from Dnd systems. In contrast to the typical dsDNA PT modification mediated by Dnd systems, Ssp systems confer single-stranded (ss), high-frequency DNA PT modification (9). For instance, SspABCD confers ssDNA PT modification to 3-bp consensus sequences, e.g., 5=-C PS CA-3= in Vibrio cyclitrophicus FF75, and the PT levels are 3-to 10-fold higher than those in DndBCDE-expressing E. coli B7A and H. chejuensis KCTC2396 (10,20).…”

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confidence: 99%

“…For instance, SspABCD confers ssDNA PT modification to 3-bp consensus sequences, e.g., 5=-C PS CA-3= in Vibrio cyclitrophicus FF75, and the PT levels are 3-to 10-fold higher than those in DndBCDE-expressing E. coli B7A and H. chejuensis KCTC2396 (10,20). In contrast to the simple self-nonself discrimination mechanism in the Dnd system, SspABCD functions together with SspE to provide protection against phage invasion in an unusual PT-dependent manner (9). In parallel with the companion ssDNA PT modification, SspE exerts its toxicity by introducing nicking damage on phage DNA and consequently impairs phage replication and disturbs phage propagation (9).…”

mentioning

confidence: 99%

“…In contrast to the simple self-nonself discrimination mechanism in the Dnd system, SspABCD functions together with SspE to provide protection against phage invasion in an unusual PT-dependent manner (9). In parallel with the companion ssDNA PT modification, SspE exerts its toxicity by introducing nicking damage on phage DNA and consequently impairs phage replication and disturbs phage propagation (9). Additionally, the redox and nucleophilic properties of PT sulfur render the PT modification a versatile player in maintenance of cellular redox homeostasis, epigenetic regulation, and environmental fitness (21,22).…”

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confidence: 99%

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Structural Analysis of an l -Cysteine Desulfurase from an Ssp DNA Phosphorothioation System

Liu

Jiang

Mai

et al. 2020

mBio

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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.

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Structural Analysis of an l -Cysteine Desulfurase from an Ssp DNA Phosphorothioation System

Liu

Jiang

Mai

et al. 2020

mBio

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“…The recently discovered SspE nicking restriction system (nickase/NTPase), coupled with the SspABCD dnd modification enzymes, attenuated unmodified phages. SspE-mediated restriction was alleviated if the phages had been grown in dnd + strain and PT-modified (CpsCA) (Xiong et al, 2020). Thus, the SspE nicking restriction system differs in specificity and type of DNA cleavage from the PTDR systems described in this work although both recognize PT backbone modifications.…”

Section: Biological Function Of the Dnd Modification Enzymes And Pt-dmentioning

confidence: 76%

Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases

et al. 2020

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Modification dependent restriction endonucleases (MDREs) restrict modified DNA, typically with limited sequence specificity (∼2-4 bp). Here, we focus on MDREs that have an SRA and/or SBD (sulfur binding domain) fused to an HNH endonuclease domain, cleaving cytosine modified or phosphorothioated (PT) DNA. We independently characterized the SBD-SRA-HNH endonuclease ScoMcrA, which preferentially cleaves 5hmC modified DNA. We report five SBD-HNH endonucleases, all recognizing GpsAAC/GpsTTC sequence and cleaving outside with a single nucleotide 3 stagger: EcoWI (N7/N6), Ksp11411I (N5/N4), Bsp305I (N6/N4-5), Mae9806I [N(8-10)/N(8-9)], and Sau43800I [N(8-9)/N(7-8)]. EcoWI and Bsp305I are more specific for PT modified DNA in Mg 2+ buffer, and promiscuous with Mn 2+. Ksp11411I is more PT specific with Ni 2+. EcoWI and Ksp11411I cleave fully-and hemi-PT modified oligos, while Bsp305I cleaves only fully modified ones. EcoWI forms a dimer in solution and cleaves more efficiently in the presence of two modified sites. In addition, we demonstrate that EcoWI PT-dependent activity has biological function: EcoWI expressing cells restrict dnd + GpsAAC modified plasmid strongly, and GpsGCC DNA weakly. This work establishes a framework for biotechnology applications of PT-dependent restriction endonucleases (PTDRs).

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Insertion sequence transposition activates antimycobacteriophage immunity through an lsr2‐silenced lipid metabolism gene island

Li,

Wei,

Guo

et al. 2024

mLife

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Insertion sequences (ISs) exist widely in bacterial genomes, but their roles in the evolution of bacterial antiphage defense remain to be clarified. Here, we report that, under the pressure of phage infection, the IS1096 transposition of Mycobacterium smegmatis into the lsr2 gene can occur at high frequencies, which endows the mutant mycobacterium with a broad‐spectrum antiphage ability. Lsr2 functions as a negative regulator and directly silences expression of a gene island composed of 11 lipid metabolism‐related genes. The complete or partial loss of the gene island leads to a significant decrease of bacteriophage adsorption to the mycobacterium, thus defending against phage infection. Strikingly, a phage that has evolved mutations in two tail‐filament genes can re‐escape from the lsr2 inactivation‐triggered host defense. This study uncovered a new signaling pathway for activating antimycobacteriophage immunity by IS transposition and provided insight into the natural evolution of bacterial antiphage defense.

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SspABCD–SspE is a phosphorothioation-sensing bacterial defence system with broad anti-phage activities

Cited by 106 publications

References 57 publications

Structural Analysis of an l -Cysteine Desulfurase from an Ssp DNA Phosphorothioation System

Structural Analysis of an l -Cysteine Desulfurase from an Ssp DNA Phosphorothioation System

Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases

Insertion sequence transposition activates antimycobacteriophage immunity through an lsr2‐silenced lipid metabolism gene island

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