Abstract：
Prokaryotes are known to produce and secrete a broad range of biopolymers with a high functional and structural heterogeneity, often with critical duties in the bacterial physiology and ecology. Among these, exopolysaccharides (EPS) play relevant roles in the interaction of bacteria with eukaryotic hosts. EPS can help to colonize the host and assist in bacterial survival, making this interaction more robust by facilitating the formation of structured biofilms. In addition, they are often key molecules in the specific recognition mechanisms involved in both beneficial and pathogenic bacteria-host interactions. A novel EPS known as MLG (Mixed-Linkage β-Glucan) was recently discovered in rhizobia, where it participates in bacterial aggregation and biofilm formation and is required for efficient attachment to the roots of their legume host plants. MLG is the first and, so far, the only reported linear Mixed-Linkage β-glucan in bacteria, containing a perfect alternation of β (1 → 3) and β (1 → 4) bonds. A phylogenetic study of MLG biosynthetic genes suggests that far from being exclusive of rhizobia, different soil and plant-associated bacteria likely produce MLG, adding this novel polymer to the plethora of surface polysaccharides that help bacteria thrive in the changing environment and to establish successful interactions with their hosts.In this work, a quantification method for MLG is proposed. It relays on the hydrolysis of MLG by a specific enzyme (lichenase), and the subsequent quantification of the released disaccharide (laminaribiose) by the phenol-sulfuric acid method. The protocol has been set up and optimized for its use in 96-well plates, which makes it suitable for high-throughput screening (HTS) approaches. This method stands out by its fast processing, technical simplicity, and capability to handle multiple samples and biological replicates at a time.
摘要：
已知原核生物产生和分泌范围广泛的具有高功能和结构异质性的生物聚合物。通常在细菌生理学和生态学中负有重要职责。其中,胞外多糖（EPS）在细菌与真核宿主的相互作用中起着相关作用。EPS可以帮助定植宿主并协助细菌生存，通过促进结构化生物膜的形成使这种相互作用更加稳健。此外,它们通常是涉及有益和致病细菌-宿主相互作用的特定识别机制中的关键分子。最近在根瘤菌中发现了一种称为MLG（混合连锁β-葡聚糖）的新型EPS，它参与细菌聚集和生物膜形成，并且是有效附着于豆科植物宿主植物根部所必需的。MLG是第一个，到目前为止,唯一报道的线性混合链β-葡聚糖在细菌中，包含β（1→3）和β（1→4）键的完美交替。对MLG生物合成基因的系统发育研究表明，根瘤菌远非唯一，不同的土壤和植物相关细菌可能产生MLG，将这种新型聚合物添加到大量的表面多糖中，帮助细菌在不断变化的环境中茁壮成长，并与宿主建立成功的相互作用。在这项工作中,提出了一种MLG的量化方法。它通过一种特定的酶（地链酶）对MLG的水解进行中继，以及随后通过苯酚-硫酸方法对释放的二糖(拉米马二糖)进行定量。该协议已经建立并优化了其在96孔板中的使用，这使得它适用于高通量筛选（HTS）方法。这种方法因其快速处理而脱颖而出，技术简单，以及一次处理多个样品和生物重复的能力。
