Layton Alexandria M, McCauley Christopher, Redding Kevin E
School of Molecular Sciences, Arizona State University, Tempe, Arizona, USA.
Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, Arizona, USA.
Appl Environ Microbiol. 2025 Apr 23;91(4):e0177224. doi: 10.1128/aem.01772-24. Epub 2025 Mar 6.
a phototrophic member of the phylum Firmicutes and family Clostridiales, possesses most of the enzymes specific to the reductive tricarboxylic acid (rTCA) cycle, except for the key enzyme, ATP-citrate lyase. It is thought to utilize a split TCA cycle when growing on pyruvate as a carbon source, in which the oxidative TCA (oTCA) direction generates most of the 2-ketoglutarate, but some can be produced in the reductive direction. Although a typical -citrate synthase gene is not found in the genome, it was suggested that gene HM1_2993, annotated as homocitrate synthase, actually encodes -citrate synthase, which would function as the initial enzyme of the oTCA cycle. We deleted this gene to test this hypothesis and, if true, see what effect severing access to the oTCA cycle would have on this organism. The endogenous CRISPR-Cas system was used to replace the open reading frame with a selectable marker. The deletion mutants could grow on pyruvate but were unable to grow phototrophically on acetate + CO as carbon source. Growth on acetate could be rescued by the addition of different electron sources (formate or ascorbate), suggesting that the oTCA cycle is used to oxidize acetate to generate electrons required to drive the carboxylation of acetyl-CoA. The deletion mutants were capable of growing in acetate minimal media without additional organic supplements beyond formate, demonstrating that the rTCA cycle can be employed to support sufficient 2-ketoglutarate production in this organism, unlike citrate synthase mutants in several chemoheterotrophic organisms utilizing the oTCA cycle.
Heliobacteria are a unique group of phototrophic bacteria that are obligate anaerobes and possess a rudimentary system to use light as a source of energy. They do not make oxygen or fix carbon dioxide. Here, we explore their fundamental carbon metabolism to understand the role and operation of the central TCA cycle. This work shows both the role and operation of this cycle under different growth modes and explains how these organisms can obtain electrons to drive their biosynthetic metabolism. This foundational knowledge will be crucial in the future when attempts are made to use this organism as a platform for oxygen-sensitive synthesis of compounds in an anaerobe that can use light as its energy source.
作为厚壁菌门和梭菌目的光合成员，除关键酶ATP - 柠檬酸裂解酶外，拥有还原三羧酸（rTCA）循环特有的大多数酶。据认为，当以丙酮酸作为碳源生长时，它利用分裂的三羧酸循环，其中氧化三羧酸（oTCA）方向产生大部分2 - 酮戊二酸，但也有一些可以在还原方向产生。虽然在基因组中未发现典型的柠檬酸合酶基因，但有人认为注释为高柠檬酸合酶的基因HM1_2993实际上编码柠檬酸合酶，其将作为oTCA循环的起始酶发挥作用。我们删除了该基因以验证这一假设，如果假设成立，则观察切断oTCA循环通路对该生物体有何影响。使用内源性CRISPR - Cas系统用选择标记替换开放阅读框。缺失突变体能够在丙酮酸上生长，但不能以乙酸盐 + CO作为碳源进行光合生长。添加不同的电子源（甲酸盐或抗坏血酸盐）可以挽救在乙酸盐上的生长，这表明oTCA循环用于氧化乙酸盐以产生驱动乙酰辅酶A羧化所需的电子。缺失突变体能够在除甲酸盐外无其他有机补充物的乙酸盐基本培养基中生长，这表明rTCA循环可用于支持该生物体中足够的2 - 酮戊二酸产生，这与几种利用oTCA循环的化学异养生物体中的柠檬酸合酶突变体不同。
嗜盐菌是一类独特的光合细菌，它们是专性厌氧菌，拥有一个将光作为能量来源的基本系统。它们不产生氧气或固定二氧化碳。在这里，我们探索它们的基本碳代谢，以了解中心三羧酸循环的作用和运作。这项工作展示了该循环在不同生长模式下的作用和运作，并解释了这些生物体如何获得电子以驱动其生物合成代谢。当未来试图将这种生物体用作在可利用光作为能源的厌氧菌中进行对氧敏感的化合物合成的平台时，这一基础知识将至关重要。
