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

Abstract

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 13 C-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 13 C-CO 2 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 13 C-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.

Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan

“…The 13 C metabolic probing experiments demonstrated broad heterotrophic activity of T. hugenholtzii JAD2 T , despite the challenge of growing it in pure culture. This result is generally consistent with genomic predictions, and broad heterotrophic activity demonstrated in GBS sediments where Thermoflexus is abundant ( Murphy et al, 2013 ; Thomas et al, 2019 ). One surprising result is the apparent uncoupling of glycolysis and the TCA cycle, as evidenced by the decarboxylation of 13 C 1 of pyruvate but not 13 C 2,3 ( Figure 4 ).…”

“…These thermal environments range from 60 to 85°C and pH from 6.7 to 7.3, which is generally consistent with the very narrow range for laboratory growth of T. hugenholtzii [67.5–75°C; pH 6.5–7.75 ( Dodsworth et al, 2014 )]. The reason for the high relative abundance of T. hugenholtzii in GBS sediments above maximum growth temperature in the laboratory is unknown ( Cole et al, 2013 ; Thomas et al, 2019 ). Additional information about these springs is provided elsewhere ( Hedlund et al, 2012 ; Hou et al, 2013 ; Peacock et al, 2013 ; Kato et al, 2018 ; Thomas et al, 2019 ).…”

“…This result might not be surprising given the presence of nucleotides and nucleosides in yeast extract coupled with predicted nucleoside transporters, the demonstrated uptake of adenine and adenosine (Figure 3), and the presence of a ribose transporter in all Thermoflexus genomes (Figure 1). A similarly high ratio of 13 CO 2 production from universally labeled glucose compared with 13 C 1 -glucose was seen in 60 • C GBS sediments (Thomas et al, 2019). The production of thymine and nicotinic acid are not understood based on incomplete biosynthetic pathways in all Thermoflexus genomes and warrants future work.…”

“…The IMG/M system (Chen et al, 2020), in combination with MAPLE, BlastKOALA, and selected searches and manual curation, was utilized to evaluate the T. hugenholtzii genome. Protein sequences were obtained from IMG (IMG Taxon ID: 2140918011) or from NCBI for MAGs and were submitted to MAPLE (Metabolic and Physiological potentiaL Evaluator, v2.3.1) (Takami et al, 2012(Takami et al, , 2016Arai et al, 2018) to determine Hedlund et al (2012Hedlund et al ( , 2015, Hou et al (2013), and Thomas et al (2019) for site locations and details for MAGs from GBS and China].…”

“…Thermoflexus hugenholtzii JAD2 T was isolated from high-temperature (∼80°C) sediments in Great Boiling Spring (GBS), Nevada, United States, where it can be one of the most abundant organisms (estimated 3.2–60% relative abundance) ( Costa et al, 2009 ; Cole et al, 2013 ; Dodsworth et al, 2014 ; Thomas et al, 2019 ). Similar 16S rRNA gene sequences have been recovered from terrestrial geothermal environments around the world ( Engel et al, 2013 ; Hou et al, 2013 ; Kato et al, 2018 ), ranging from 63 to 85°C at circumneutral pH, where they can be abundant [e.g., >8% of 16S rRNA gene sequences ( Hou et al, 2013 )].…”