Lactobacillus reuteri is a gut symbiont inhabiting the gastrointestinal tract of numerous vertebrates. The surface-exposed serine-rich repeat protein (SRRP) is a major adhesin in Gram-positive bacteria. Using lectin and sugar nucleotide profiling of wild-type or L. reuteri isogenic mutants, MALDI-ToF-MS, LC–MS and GC–MS analyses of SRRPs, we showed that L. reuteri strains 100-23C (from rodent) and ATCC 53608 (from pig) can perform protein O-glycosylation and modify SRRP_100-23 and SRRP₅₃₆₀₈ with Hex-Glc-GlcNAc and di-GlcNAc moieties, respectively. Furthermore, in vivo glycoengineering in E. coli led to glycosylation of SRRP₅₃₆₀₈ variants with α-GlcNAc and GlcNAcβ(1→6)GlcNAcα moieties. The glycosyltransferases involved in the modification of these adhesins were identified within the SecA2/Y2 accessory secretion system and their sugar nucleotide preference determined by saturation transfer difference NMR spectroscopy and differential scanning fluorimetry. Together, these findings provide novel insights into the cellular O-protein glycosylation pathways of gut commensal bacteria and potential routes for glycoengineering applications.

中文翻译：

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来自罗伊氏乳杆菌的富含丝氨酸的重复蛋白粘附素显示菌株特异性糖基化谱


罗伊氏乳杆菌是一种肠道共生菌，栖息在许多脊椎动物的胃肠道中。表面暴露的富含丝氨酸的重复蛋白（SRRP）是革兰氏阳性菌中的主要粘附素。使用野生型或罗伊氏乳杆菌同基因突变体的凝集素和糖核苷酸分析，以及 SRRP 的 MALDI-ToF-MS、LC-MS 和 GC-MS 分析，我们表明罗伊氏乳杆菌菌株 100-23C（来自啮齿动物）和 ATCC 53608（来自猪）可以进行蛋白O-糖基化并分别用Hex-Glc-GlcNAc和二-GlcNAc部分修饰SRRP _100-23和SRRP _{53608 。}此外，大肠杆菌中的体内糖工程导致 SRRP ₅₃₆₀₈变体与 α-GlcNAc 和 GlcNAcβ(1→6)GlcNAcα 部分发生糖基化。在 SecA2/Y2 辅助分泌系统中鉴定了参与这些粘附素修饰的糖基转移酶，并通过饱和转移差核磁共振波谱和差示扫描荧光测定法确定了它们的糖核苷酸偏好。总之，这些发现为肠道共生细菌的细胞O蛋白糖基化途径和糖工程应用的潜在途径提供了新的见解。