Deinococcus radiodurans is a phylogenetically deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet (UV) radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of D. radiodurans has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in D. radiodurans and related bacteria. Here, we report a electron cryomicroscopy (cryo-EM) structure of the abundant D. radiodurans OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded β-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Furthermore, using protein structure prediction and sequence-based bioinformatic analysis, we show that SlpA-like putative OM–PG connector proteins are widespread in phylogenetically deep-branching Gram-negative bacteria. Finally, combining our atomic structures with fluorescence and electron microscopy of cell envelopes of wild-type and mutant bacterial strains, we report a model for the cell surface of D. radiodurans . Our results will have important implications for understanding the cell surface organization and hyperstability of D. radiodurans and related bacteria and the evolutionary transition between Gram-negative and Gram-positive bacteria.

中文翻译：

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多域连接器连接系统发育深分支细菌的外膜和细胞壁

耐辐射奇异球菌是一种系统发育深分支的极端嗜热细菌，它对许多环境压力具有显着的耐受性，包括大剂量的紫外线 (UV) 辐射和极端温度。它甚至可以在外太空生存数年。这种耐力D. radiodurans部分归因于其非典型细胞包膜，包括内膜、具有厚肽聚糖 (PG) 层的大周质空间和被表面层（S 层）覆盖的外膜 (OM)。尽管进行了大量研究，但控制包膜组织和 OM 稳定的分子原理尚不清楚D. radiodurans和相关细菌。在这里，我们报告了丰富的电子冷冻显微镜 (cryo-EM) 结构D. radioduransOM 蛋白 SlpA，显示其 C 端片段如何在 OM 中形成 30 链 β 桶的同源三聚体，而其 N 端片段如何形成长的同源三聚体卷曲螺旋，通过 S 层同源性 (SLH) 将 OM 连接到 PG 层) 域。此外，使用蛋白质结构预测和基于序列的生物信息学分析，我们表明 SlpA 样推定的 OM-PG 连接蛋白广泛存在于系统发育深分支革兰氏阴性细菌中。最后，将我们的原子结构与野生型和突变型细菌菌株细胞包膜的荧光和电子显微镜相结合，我们报告了一个细胞表面模型D. radiodurans. 我们的结果将对理解细胞表面组织和超稳定性具有重要意义D. radiodurans和相关细菌以及革兰氏阴性菌和革兰氏阳性菌之间的进化转变。