Table_4_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多样性的核心驱动因素；然而，目前尚不明确温度在群落层面可在多大程度上影响中央碳代谢途径（central carbon metabolic pathways, CCMPs）的特征。本研究将16S rRNA基因扩增子测序与宏基因组测序相结合，辅以13C标记代谢物探针技术，针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展开评估。结果显示，16S rRNA基因扩增子多样性与温度呈负相关，古菌在较高温度下占据群落主导地位。KEGG同源基因（KEGG Orthology, KO）丰度与多样性同样随温度升高而降低，但中央碳代谢途径相关基因在各温度梯度下的占比均较为稳定，且多数单个宏基因组组装基因组均携带完整的代谢途径。相较而言，编码纤维小体的基因以及诸多参与植物物质降解、光合作用的基因在较高温度下均未检出。通过对葡萄糖、丙酮酸与乙酸的标记同位素异构体对进行原位13C-CO2产生量检测，发现60℃条件下氧化磷酸戊糖途径的相对活性及/或发酵水平更低，且较高温度下的维持能量需求趋于稳定或有所下降。在85℃条件下可检测到13C标记的柠檬酸、琥珀酸、L-丙氨酸、L-丝氨酸及L-半胱氨酸的分解代谢，这表明群落存在广泛的异养活性，并证实了三羧酸循环（TCA cycle）的功能。综合以上结果可知，地热系统中温度驱动的生物多样性与基因含量损失，并不会在群落层面改变中央碳代谢途径的功能或维持能量需求。