Extended Figure 7. SAM lyse

<strong>CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements (MGE) 1,2. Type III CRISPR systems, with the signature gene </strong><em><strong>cas10</strong></em><strong>, use CRISPR RNA (crRNA) to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against MGE either directly, via the integral HD nuclease domain 3-5 or indirectly, via synthesis of cyclic oligonucleotide (cOA) second messengers to activate diverse ancillary effectors 6-9. A subset of type III CRISPR systems encode an uncharacterised CorA-family membrane protein and an associated NrN family phosphodiesterase predicted to function in antiviral defence. Here, we demonstrate that the CorA associated type III-B CRISPR system from </strong><em><strong>Bacteroides fragilis</strong></em><strong> (BfaCmr) provides immunity against MGE when expressed in </strong><em><strong>E. coli</strong></em><strong>. However, BfaCmr does not synthesise cOA species on activation, instead generating a previously undescribed signalling molecule, SAM-AMP (3’-adenylyl-AdoMet) by conjugating ATP to S-adenosyl methionine via a phosphodiester bond. Once synthesised, SAM-AMP binds to the CorA effector, presumably leading to cell death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR associated phosphodiesterases or a SAM lyase, providing an “off switch” analogous to cOA specific ring nucleases 10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts. </strong>

CRISPR系统（CRISPR systems）广泛分布于原核生物界，能够为宿主提供针对可移动遗传元件（mobile genetic elements，MGE）的适应性免疫1,2。第三类CRISPR系统以特征基因cas10为标志性组成，借助CRISPR RNA（crRNA）识别非自身RNA，激活具有酶活性的Cas10亚基，进而通过两种途径为细胞提供针对MGE的防御：一是通过其固有的HD核酸酶结构域直接发挥抗病毒作用3-5；二是合成环状寡核苷酸（cyclic oligonucleotide，cOA）第二信使，以此激活多种辅助效应因子6-9。部分第三类CRISPR系统会编码功能尚未明确的CorA家族膜蛋白，以及与之相关的、被预测可参与抗病毒防御的NrN家族磷酸二酯酶。本研究证实，来自脆弱拟杆菌（Bacteroides fragilis）的CorA关联型第三类-B CRISPR系统（BfaCmr）在大肠杆菌（E. coli）中异源表达时，可赋予宿主针对MGE的免疫防御能力。然而，BfaCmr在激活后并不会合成cOA类分子，而是通过将ATP与S-腺苷甲硫氨酸（S-adenosyl methionine）以磷酸二酯键结合，生成一种此前从未被报道过的信号分子SAM-AMP（3’-腺苷酰基-AdoMet）。该分子合成后会结合CorA效应因子，推测通过破坏细胞膜完整性引发细胞死亡。SAM-AMP可被CRISPR关联的磷酸二酯酶或SAM裂解酶降解，这一过程构成了类似cOA特异性环形核酸酶的“关闭开关”10。综上，SAM-AMP代表了一类全新的抗病毒信号第二信使，有望在不同的细胞环境中发挥多样化的生理功能。