Sspabcd–Sspe Is a Phosphorothioation-Sensing Bacterial Defence System with Broad Anti-Phage Activities
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ARTICLES https://doi.org/10.1038/s41564-020-0700-6 SspABCD–SspE is a phosphorothioation-sensing bacterial defence system with broad anti-phage activities Xiaolin Xiong1,2,5,7, Geng Wu3,7, Yue Wei1,7, Liqiong Liu1,3, Yubing Zhang1,3, Rui Su4, Xianyue Jiang1, Mengxue Li1, Haiyan Gao1,3, Xihao Tian1, Yizhou Zhang1,5, Li Hu1,5, Si Chen1, You Tang1,5, Susu Jiang1,5, Ruolin Huang1,5, Zhiqiang Li5, Yunfu Wang2, Zixin Deng1,3, Jiawei Wang4, Peter C. Dedon 6, Shi Chen 1,2,5 and Lianrong Wang 1,2,5 ✉ Bacteria have evolved diverse mechanisms to fend off predation by bacteriophages. We previously identified the Dnd system, which uses DndABCDE to insert sulfur into the DNA backbone as a double-stranded phosphorothioate (PT) modification, and DndFGH, a restriction component. Here, we describe an unusual SspABCD–SspE PT system in Vibrio cyclitrophicus, Escherichia coli and Streptomyces yokosukanensis, which has distinct genetic organization, biochemical functions and phenotypic behav- iour. SspABCD confers single-stranded and high-frequency PTs with SspB acting as a nickase and possibly introducing nicks to facilitate sulfur incorporation. Strikingly, SspABCD coupled with SspE provides protection against phages in unusual ways: (1) SspE senses sequence-specific PTs by virtue of its PT-stimulated NTPase activity to exert its anti-phage activity, and (2) SspE inhibits phage propagation by introducing nicking damage to impair phage DNA replication. These results not only expand our knowledge about the diversity and functions of DNA PT modification but also enhance our understanding of the known arsenal of defence systems. s a result of intense competition for environmental resources This function is attributable to a unique feature of PT modification and coevolution of bacteriophages and their hosts, pro- in which the replacement of an oxygen atom by sulfur endows the Akaryotes have developed a spectrum of diverse defensive PT-modified DNA backbone with resistance to nucleases15. With a systems. These include restriction-modification (R-M), toxin–anti- loss of the dnd modification genes, which leads to PT deficiency, toxin, abortive infection, prokaryotic Argonaute and the clustered the unrestrained activity of DndFGH causes toxic double-stranded regularly interspaced short palindromic repeats (CRISPR) and breaks of the DNA16,17. The dndABCDE–dndFGH system is thus CRISPR-associated gene systems1–5. The relatively recent discovery regarded as a prokaryotic innate defence system with functional of defence systems such as BREX6, DISARM7 and Zorya8 highlights similarity to methylation-based R-M systems—that is, it restricts the fact that our knowledge of the arsenal of anti-phage measures invading DNA lacking PT modifications18,19. residing in prokaryotic genomes is incomplete and that many cur- Methylation-based R-M systems have consensus sequences that rently unknown defence mechanisms probably await discovery. are typically fully modified to avoid the attack of resident DNA by As a form of prokaryotic epigenetics, R-M systems involve sequence- cognate restriction endonucleases. However, only 12% of the 40,701 specific DNA modification, traditionally a nucleobase methylation, 5′-GAAC-3′/5′-GTTC-3′ consensus sequences in the Escherichia and a restriction endonuclease to discriminate and destroy unla- coli B7A genome are protected with PTs despite the presence of active belled invading DNA9,10. Despite the diversity of the four major restriction by DndFGH. In contrast to the double-stranded DNA types of R-M systems, all associated DNA modifications—such as (dsDNA) PT in B7A, PT modification in Vibrio cyclitrophicus FF75 6 4 5 N -methyl-adenine, N -methyl-cytosine, C -methyl-cytosine and occur on only one strand at 5′-CPSCA-3′ (PS, phosphate–sulfur link- 7-deazaguanine derivatives—were believed to occur exclusively on age) motifs and only 14% of the 160,541 genome-wide 5′-CCA-3′ nucleobase moieties until our discovery of phosphorothioate (PT) sites are PT modified20. Moreover, PTs do not occur consistently at modifications of the DNA sugar-phosphate backbone, in which the a given site in a population of bacterial genomes of both B7A and non-bridging oxygen is replaced by sulfur11–13. FF75, reflecting PT heterogeneity and implying that unusual DNA- DndABCDE proteins catalyse the sulfur substitution to form target-selection mechanisms are used by the Dnd modifying and 20,21 PTs in a sequence-selective and RP stereospecific manner in diverse restricting components . In addition to being a single-stranded bacteria, whereas DndFGH proteins, located in the vicinity of dnd- DNA (ssDNA) PT modification, the PT levels in FF75 are three- to ABCDE, utilize PT to discriminate and restrict foreign DNA11,12,14. tenfold higher than those in B7A, Salmonella enterica serovar Cerro 1Ministry of Education Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Sciences, Wuhan University, Wuhan, China. 2Taihe Hospital, Hubei University of Medicine, Shiyan, China. 3State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, The Joint International Research Laboratory of Metabolic and Developmental Sciences, Shanghai Jiao Tong University, Shanghai, China. 4State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China. 5Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Wuhan, China. 6Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. 7These authors contributed equally: Xiaolin Xiong, Geng Wu, Yue Wei. ✉e-mail: [email protected] NatURE MICROBIOLOgy | www.nature.com/naturemicrobiology ARTICLES NATURE MICROBIOLOGY 87, Hahella chejuensis KCTC2396 and Pseudomonas fluorescens sulfur mobilization pathway. Moreover, SspC possessed an ATPase pf0-1 (refs. 20,22,23). activity of 8.58 ± 3.12 U per mg of protein and feasibly drove PT Based on an exploration of the unusual PT behaviour in FF75, modification by providing energy in a manner similar to that pre- we started with a comparative genomic analysis and characterized dicted for DndD28 (Supplementary Fig. 3). a ssDNA PT modification system—which we termed ssp—with The gene sspB poses a different problem. It is predicted to encode genetic organization, biochemical functions and phenotypic behav- a 321-amino-acid protein of unknown function (DUF4007), which iour distinct from the dnd system. SspABCD and SspE constitute exhibits no sequence similarity to any Dnd proteins, suggesting its a defensive barrier against a diverse array of phages via anti-phage critical role in the ssDNA PT formation. However, the insolubil- activity, which depends on the sensing of sequence-specific PT ity of SspB from FF75 impeded the exploration of its function. motifs and the introduction of nicking damage to phage DNA, Instead, we determined the crystal structure of full-length SspB from highlighting an unusual mechanism of defence. Streptomyces clavuligerus ATCC 27064 to a resolution of 1.75 Å using the single-wavelength anomalous dispersion (SAD) method and a Results selenomethionine (SeMet) derivative (Supplementary Table 1). The Discovery of the Ssp PT modification system. Despite mass- sspABCD homologues in ATCC 27064 are closely clustered and con- spectrometric evidence for PTs in V. cyclitrophicus FF75 (PT-linked fer PT-modified d(CPSC) in a Streptomyces lividans HXY6 strain lack- 3 d(CPSC) dinucleotides, 2.6 per 1 × 10 nucleotides), a BLAST search ing endogenous ssp genes (Fig. 1b and Supplementary Fig. 4). The of the FF75 genome for dnd gene homologues proved negative. We SspB structure contained an unprecedented fold, with the main body extended this analysis to several related Vibrio species for which we largely consisting of an α-helical structure and three β-sheets on the had PT-level data22. Two other Vibrio species with PT levels similar periphery (Supplementary Results and Extended Data Fig. 1). to those of FF75 were also found to lack dnd genes: Vibrio breoganii A DALI search indicated that SspB exhibited similarities to 1C10, with 3.1 PT per 1 × 103 nucleotides, and V. breoganii ZF-29, the endonucleases FokI (Z-score = 6.8; matched residues, 8%) and with 2.2 PT per 1 × 103 nucleotides. In contrast, Vibrio tasmaniensis R.BSPD6I (Z-score = 5.5; matched residues, 9%), inspiring the per- 1F-267 possessed a dndBCDE gene cluster and a previous analysis formance of a nuclease assay. A time-course experiment established revealed fivefold fewer PTs in d(GPSA)/d(GPST) motifs in this organ- that SspB rapidly converted covalently closed circular DNA to an ism than in FF75 (0.6 PT per 1 × 103 nucleotides)22. These differ- open circular form, which was followed by a slower conversion to ences between 1F-267 and the other three strains led us to perform linear DNA in the presence of Mg2+ or Mn2+ (Fig. 2a). Mg2+ ions a comparative analysis of their genomes on the RAST server24. This were indeed found bound directly to E18, and the E18R single-point analysis yielded homologues of three clustered genes—M565_ mutation remarkably impaired the nicking activity of SspB (Fig. 2b ctg1P1907 to M565_ctg1P1909—in FF75, 1C10 and ZF-29 but not and Extended Data Fig. 2). These findings implied that SspB acted in 1F-267 (Fig. 1a,b). The three genes, which bore no sequence as a nickase capable of introducing two or more nicks into pUC19 similarity to DndB, DndD or DndE, were co-transcribed as a single because dsDNA breaks only when two nicking sites on opposite operon in FF75, implying functional linkage (Supplementary Fig. 1). strands are close to each other. However, regular agarose gels cannot The M565_ctg1P1907 gene encoded a protein with unknown func- differentiate open circular plasmid DNA with one nick from that tion, whereas remote homology analysis with HHpred showed that with multiple nicks. Alkaline denaturing agarose gel electrophore- M565_ctg1P1908 had similarity to a domain denoted DUF499, sis was thus exploited to determine the nicking pattern on single a distant version of the AAA + ATPase domain25,26.