Table_12_Position-Specific Metabolic Probing and Metagenomics of Microbial Communities Reveal Conserved Central Carbon Metabolic Network Activities at High Temperatures.XLSX

Temperature is a primary driver of microbial community composition and taxonomic diversity; however, it is unclear to what extent temperature affects characteristics of central carbon metabolic pathways (CCMPs) at the community level. In this study, 16S rRNA gene amplicon and metagenome sequencing were combined with 13C-labeled metabolite probing of the CCMPs to assess community carbon metabolism along a temperature gradient (60–95°C) in Great Boiling Spring, NV. 16S rRNA gene amplicon diversity was inversely proportional to temperature, and Archaea were dominant at higher temperatures. KO richness and diversity were also inversely proportional to temperature, yet CCMP genes were similarly represented across the temperature gradient and many individual metagenome-assembled genomes had complete pathways. In contrast, genes encoding cellulosomes and many genes involved in plant matter degradation and photosynthesis were absent at higher temperatures. In situ13C-CO2 production from labeled isotopomer pairs of glucose, pyruvate, and acetate suggested lower relative oxidative pentose phosphate pathway activity and/or fermentation at 60°C, and a stable or decreased maintenance energy demand at higher temperatures. Catabolism of 13C-labeled citrate, succinate, L-alanine, L-serine, and L-cysteine was observed at 85°C, demonstrating broad heterotrophic activity and confirming functioning of the TCA cycle. Together, these results suggest that temperature-driven losses in biodiversity and gene content in geothermal systems may not alter CCMP function or maintenance energy demands at a community level.

温度是调控微生物群落组成与分类学多样性的核心驱动因素之一，但目前尚不清楚温度在群落层面会在多大程度上影响中心碳代谢途径（central carbon metabolic pathways, CCMPs）的特征。本研究结合16S rRNA基因扩增子测序（16S rRNA gene amplicon sequencing）、宏基因组测序（metagenome sequencing），以及针对中心碳代谢途径的¹³C标记代谢物探针技术，对美国内华达州大沸泉（Great Boiling Spring, NV）沿60~95℃温度梯度的群落碳代谢情况进行评估。16S rRNA基因扩增子多样性与温度呈负相关，且古菌（Archaea）在较高温度下占据群落主导地位。KEGG同源基因（KEGG Orthology, KO）的丰度与多样性同样与温度呈负相关，但中心碳代谢途径的基因在各温度梯度下的分布占比均较为一致，且多数单份宏基因组组装基因组（metagenome-assembled genomes, MAGs）均携带完整的代谢途径。与之相对的是，编码纤维小体（cellulosomes）的基因以及诸多参与植物物质降解与光合作用的基因，在较高温度环境下均未检出。对葡萄糖、丙酮酸与乙酸盐的标记同位素异构体对进行原位¹³C-CO₂生成检测的结果显示，60℃环境下的相对氧化戊糖磷酸途径（oxidative pentose phosphate pathway）活性与/或发酵水平较低，而较高温度下的维持能量需求则保持稳定或有所降低。在85℃环境下可检测到¹³C标记的柠檬酸、琥珀酸、L-丙氨酸、L-丝氨酸与L-半胱氨酸的分解代谢过程，这表明群落存在广泛的异养活性，并证实了三羧酸循环（tricarboxylic acid cycle, TCA cycle）的功能运作。综合上述结果可知，地热系统中由温度驱动的生物多样性与基因丰度损失，并不会在群落层面改变中心碳代谢途径的功能或维持能量需求。