Clostridium difficile is the leading cause of hospital-acquired antibiotic-associated diarrhea and is the only widespread human pathogen that contains a complete set of genes encoding the Wood-Ljungdahl pathway (WLP). In acetogenic bacteria, synthesis of acetate from 2 CO₂ molecules by the WLP functions as a terminal electron accepting pathway; however, C. difficile contains various other reductive pathways, including a heavy reliance on Stickland reactions, which questions the role of the WLP in this bacterium. In rich medium containing high levels of electron acceptor substrates, only trace levels of key WLP enzymes were found; therefore, conditions were developed to adapt C. difficile to grow in the absence of amino acid Stickland acceptors. Growth conditions were identified that produce the highest levels of WLP activity, determined by Western blot analyses of the central component acetyl coenzyme A synthase (AcsB) and assays of other WLP enzymes. Fermentation substrate and product analyses, enzyme assays of cell extracts, and characterization of a ΔacsB mutant demonstrated that the WLP functions to dispose of metabolically generated reducing equivalents. While WLP activity in C. difficile does not reach the levels seen in classical acetogens, coupling of the WLP to butyrate formation provides a highly efficient system for regeneration of NAD⁺ “acetobutyrogenesis,” requiring only low flux through the pathways to support efficient ATP production from glucose oxidation. Additional insights redefine the amino acid requirements in C. difficile, explore the relationship of the WLP to toxin production, and provide a rationale for colocalization of genes involved in glycine synthesis and cleavage within the WLP operon.

中文翻译：

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艰难梭菌中的多种节能途径：缺乏氨基酸粘着地受体的生长和Wood-Ljungdahl通路的作用。

艰难梭状芽胞杆菌是医院获得性抗生素相关性腹泻的主要原因，并且是唯一广泛传播的人类病原体，其包含编码Wood-Ljungdahl途径（WLP）的完整基因。在产乙酸细菌中，WLP由2个CO ₂分子合成乙酸盐起着末端电子接受途径的作用。然而，艰难梭菌还包含其他多种还原途径，包括严重依赖Stickland反应，这质疑WLP在这种细菌中的作用。在含有高水平电子受体底物的丰富培养基中，仅发现了痕量的关键WLP酶。因此，开发了适应艰难梭菌的条件在没有氨基酸Stickland受体的情况下生长。鉴定出产生最高水平WLP活性的生长条件，这是通过对中心成分乙酰辅酶A合酶（AcsB）的Western印迹分析和其他WLP酶的测定确定的。发酵底物和产物分析，细胞提取物的酶分析以及ΔacsB突变体的表征证明，WLP可以处理代谢产生的还原当量。尽管艰难梭菌中的WLP活性未达到经典产乙酸菌所见的水平，但WLP与丁酸酯形成的耦合为NAD ^+的再生提供了高效系统“乙酰丁酸生成”，仅需通过途径的低通量即可支持葡萄糖氧化产生有效的ATP。其他见解重新定义了艰难梭菌的氨基酸需求，探索了WLP与毒素产生之间的关系，并为WLP操纵子内甘氨酸合成和裂解相关基因的共定位提供了理论依据。