Baran Richard, Lau Rebecca, Bowen Benjamin P, Diamond Spencer, Jose Nick, Garcia-Pichel Ferran, Northen Trent R
Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
Center for Circadian Biology and Division of Biological Sciences, University of California, San Diego , La Jolla, California, United States.
ACS Chem Biol. 2017 Mar 17;12(3):674-681. doi: 10.1021/acschembio.6b00890. Epub 2017 Jan 18.
Cyanobacteria are important primary producers of organic matter in diverse environments on a global scale. While mechanisms of CO fixation are well understood, the distribution of the flow of fixed organic carbon within individual cells and complex microbial communities is less well characterized. To obtain a general overview of metabolism, we describe the use of deuterium oxide (DO) to measure deuterium incorporation into the intracellular metabolites of two physiologically diverse cyanobacteria: a terrestrial filamentous strain (Microcoleus vaginatus PCC 9802) and a euryhaline unicellular strain (Synechococcus sp. PCC 7002). DO was added to the growth medium during different phases of the diel cycle. Incorporation of deuterium into metabolites at nonlabile positions, an indicator of metabolite turnover, was assessed using liquid chromatography mass spectrometry. Expectedly, large differences in turnover among metabolites were observed. Some metabolites, such as fatty acids, did not show significant turnover over 12-24 h time periods but did turn over during longer time periods. Unexpectedly, metabolites commonly regarded to act as compatible solutes, including glutamate, glucosylglycerol, and a dihexose, showed extensive turnover compared to most other metabolites already after 12 h, but only during the light phase in the cycle. The observed extensive turnover is surprising considering the conventional view on compatible solutes as biosynthetic end points given the relatively slow growth and constant osmotic conditions. This suggests the possibility of a metabolic sink for some compatible solutes (e.g., into glycogen) that allows for rapid modulation of intracellular osmolarity. To investigate this, uniformly C-labeled Synechococcus sp. PCC 7002 were exposed to C glucosylglycerol. Following metabolite extraction, amylase treatment of methanol-insoluble polymers revealed C labeling of glycogen. Overall, our work shows that DO probing is a powerful method for analysis of cyanobacterial metabolism including discovery of novel metabolic processes.
蓝细菌是全球范围内不同环境中重要的有机物初级生产者。虽然二氧化碳固定机制已得到充分理解，但固定有机碳在单个细胞和复杂微生物群落内的流动分布特征尚不明确。为了全面了解新陈代谢，我们描述了使用氧化氘（D₂O）来测量氘掺入两种生理特性不同的蓝细菌的细胞内代谢物中的情况：一种是陆生丝状菌株（鞘丝藻Microcoleus vaginatus PCC 9802）和一种广盐性单细胞菌株（聚球藻Synechococcus sp. PCC 7002）。在昼夜循环的不同阶段将D₂O添加到生长培养基中。使用液相色谱质谱法评估氘掺入非不稳定位置代谢物的情况，这是代谢物周转的一个指标。不出所料，观察到代谢物之间的周转存在很大差异。一些代谢物，如脂肪酸，在12 - 24小时时间段内没有显示出明显的周转，但在更长时间段内会周转。出乎意料的是，通常被认为作为相容性溶质起作用的代谢物，包括谷氨酸、葡糖基甘油和一种二糖，与大多数其他代谢物相比，在12小时后就显示出广泛的周转，但仅在循环的光照阶段。考虑到相容性溶质作为生物合成终点的传统观点，鉴于相对缓慢的生长和恒定的渗透条件，观察到的广泛周转令人惊讶。这表明某些相容性溶质（例如，进入糖原）可能存在代谢汇，从而允许快速调节细胞内渗透压。为了研究这一点，将均匀¹³C标记的聚球藻Synechococcus sp. PCC 7002暴露于¹³C葡糖基甘油。在代谢物提取后，对甲醇不溶性聚合物进行淀粉酶处理，结果显示糖原被¹³C标记。总体而言，我们的工作表明D₂O探测是分析蓝细菌代谢包括发现新代谢过程的有力方法。
