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

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 situ ¹³C-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.