The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. While high butyrate levels induce toxic stress, effects of non-toxic levels on cell growth, health, metabolism, and sporulation remain unclear. Here, we show that butyrate stimulates cellular processes of Clostridium acetobutylicum, a model butyrate forming Firmicute. First, we deleted the 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from the C. acetobutylicum chromosome to eliminate the butyrate synthetic pathway and thus butyrate formation. A xylose inducible Cas9 cassette was chromosomally integrated and utilized for the one-step markerless gene deletions. Non-toxic butyrate levels significantly affected growth, health, and sporulation of C. acetobutylicum. After deleting spo0A, the gene encoding the master regulator of sporulation, Spo0A, and conducting butyrate addition experiments, we conclude that butyrate affects cellular metabolism through both Spo0A-dependent and independent mechanisms. We also deleted the hbd gene from the chromosome of the asporogenous C. acetobutylicum M5 strain lacking the pSOL1 plasmid to examine the potential involvement of pSOL1 genes on the observed butyrate effects. Addition of crotonate, the precursor of butyrate biosynthesis, to the hbd deficient M5 strain was used to probe the role of butyrate biosynthesis pathway in electron and metabolic fluxes. Finally, we found that butyrate addition can enhance the growth of the non-butyrate forming Clostridium saccharolyticum. Our data suggest that butyrate functions as a stimulator of cellular processes, like a growth factor, in C. acetobutylicum and potentially evolutionarily related Clostridium organisms.

中文翻译：

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丁酸盐作为乙酰丁酸梭菌的生长因子


丁酸盐生物合成途径不仅有助于形成丁酸盐的细菌中的电子管理和能量产生，而且还通过抑制周围丁酸盐敏感微生物的生长为宿主赋予进化优势。虽然高丁酸盐水平会诱发毒性应激，但无毒水平对细胞生长、健康、代谢和孢子形成的影响仍不清楚。在这里，我们表明丁酸盐刺激乙酰丁酸梭菌的细胞过程，乙酰丁酸梭菌是一种形成厚壁菌门的模型丁酸盐。首先，我们从乙酰丁酸梭菌染色体中删除了 3-羟基丁酰辅酶 A 脱氢酶基因 （hbd），以消除丁酸盐合成途径，从而消除丁酸盐的形成。木糖诱导型 Cas9 盒被染色体整合并用于一步无标记基因缺失。无毒丁酸盐水平显着影响乙酰丁酸梭菌的生长、健康和孢子形成。在删除编码孢子形成主调节因子 Spo0A 的基因 Spo0A 并进行丁酸盐加成实验后，我们得出结论，丁酸盐通过 Spo0A 依赖性和独立机制影响细胞代谢。我们还从缺乏 pSOL1 质粒的产孢菌乙酰丁酸 C. 5 菌株的染色体中删除了 hbd 基因，以检查 pSOL1 基因对观察到的丁酸盐效应的潜在参与。将丁酸盐生物合成的前体巴豆酸盐添加到 hbd 缺陷的 M5 菌株中用于探测丁酸盐生物合成途径在电子和代谢通量中的作用。最后，我们发现丁酸盐的添加可以促进非丁酸盐形成溶糖梭菌的生长。我们的数据表明，丁酸盐在 C 中起细胞过程的刺激剂，如生长因子。 乙酰丁菌和可能进化相关的梭菌生物。