Myxococcus xanthus arranges into two morphologically distinct biofilms depending on its nutritional status, i.e., coordinately spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. A secreted polysaccharide, referred to as exopolysaccharide (EPS), is a structural component of both biofilms and is also important for type IV pilus-dependent motility and fruiting body formation. Here, we characterize the biosynthetic machinery responsible for EPS biosynthesis using bioinformatics, genetics, heterologous expression, and biochemical experiments. We show that this machinery constitutes a Wzx/Wzy-dependent pathway dedicated to EPS biosynthesis. Our data support that EpsZ (MXAN_7415) is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for the initiation of the repeat unit synthesis. Heterologous expression experiments support that EpsZ has galactose-1-P transferase activity. Moreover, MXAN_7416, renamed Wzx_EPS, and MXAN_7442, renamed Wzy_EPS, are the Wzx flippase and Wzy polymerase responsible for translocation and polymerization of the EPS repeat unit, respectively. In this pathway, EpsV (MXAN_7421) also is the polysaccharide copolymerase and EpsY (MXAN_7417) the outer membrane polysaccharide export (OPX) protein. Mutants with single in-frame deletions in the five corresponding genes had defects in type IV pilus-dependent motility and a conditional defect in fruiting body formation. Furthermore, all five mutants were deficient in type IV pilus formation, and genetic analyses suggest that EPS and/or the EPS biosynthetic machinery stimulates type IV pilus extension. Additionally, we identify a polysaccharide biosynthesis gene cluster, which together with an orphan gene encoding an OPX protein make up a complete Wzx/Wzy-dependent pathway for synthesis of an unknown polysaccharide.

中文翻译：

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黄曲霉中外多糖生物合成途径的表征。

黄花粘球菌根据其营养状况分为两个形态不同的生物膜，即在有营养的情况下协调地散布菌落，在没有营养的情况下将孢子充满子实体。分泌的多糖，称为胞外多糖（EPS），是两种生物膜的结构成分，对于IV型菌毛依赖性运动和子实体形成也很重要。在这里，我们使用生物信息学，遗传学，异源表达和生化实验来表征负责EPS生物合成的生物合成机制。我们表明，该机制构成致力于EPS生物合成的Wzx / Wzy依赖途径。我们的数据支持EpsZ（MXAN_7415）是负责重复单元合成起始的聚异戊二烯基磷酸己糖-1-磷酸转移酶。异源表达实验支持EpsZ具有半乳糖-1-P转移酶活性。此外，MXAN_7416更名为Wzx_EPS和MXAN_7442，重命名为Wzy _EPS分别是负责EPS重复单元移位和聚合的Wzx翻转酶和Wzy聚合酶。在此途径中，EpsV（MXAN_7421）也是多糖共聚酶，而EpsY（MXAN_7417）也是外膜多糖输出（OPX）蛋白。在五个相应基因中具有单个框内缺失的突变体具有IV型菌毛依赖性运动的缺陷和子实体形成的条件缺陷。此外，所有五个突变体都缺乏IV型菌毛的形成，遗传分析表明EPS和/或EPS生物合成机制刺激IV型菌毛的延伸。此外，我们确定了多糖生物合成基因簇，它与编码OPX蛋白的孤儿基因一起构成了合成未知多糖的完整Wzx / Wzy依赖性途径。