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

Abstract

Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing “Candidatus Thermoflexus japonica,” “Candidatus Thermoflexus tengchongensis,” and “Candidatus Thermoflexus sinensis.” Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past

“…Overall, the large number of compounds used by the Tepidiforma strains, but lack of growth on defined sole carbon sources is consistent with the complex nutritional profiles of other Chloroflexota 33,42,59 . This general pattern suggests broad heterotrophic activity and metabolic interdependency with cohabiting microorganisms in nature.…”

“…Chloroflexota 33,34,59,64 . Additionally, both novel strains were positive for urease activity, although only YIM 72310 T encoded a urease cluster of three urease-encoding subunits (ureABC), two urease accessory proteins (ureFG), and five urea ABC transporter genes (urtABCDE), similar to those in T. bonchosmolovskayae.…”

“…Quantitative exometabolomics was performed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) as described previously 59 . YIM 72310 T and G233 T were cultivated in 20 ml volumes of R2A in 165 ml serum bottles with an air headspace until the late-exponential phase of growth (4 days).…”

Incomplete denitrification phenotypes in diverse Thermus species from diverse geothermal spring sediments and adjacent soils in southwest China

“…However, single-cell genomes and metagenome-assembled genomes from members of the Caldarchaeales (synonym: 'Aigarchaeota’) show several members of this often-abundant, but not isolated group to contain putative N 2 O reductase genes (Rinke et al 2013 ; Hedlund et al 2015b ) and no other denitrification genes and are, therefore, possible metabolic partners of Thermus . Similarly, multiple species of Thermoflexus that often cohabit with Thermus have putative N 2 O reductase genes, yet this function was not demonstrated in cultures of Thermoflexus hugenholtzii (Thomas et al 2021 ). A multispecies coupled denitrification pathway is consistent with high rates of complete denitrification in sediments in Great Boiling Spring, NV, inferred from much higher N 2 O fluxes in acetylene block experiments compared to no-acetylene controls (Dodsworth et al 2011b ; Hedlund et al 2011 ).…”

Lipid Profiles From Fresh Biofilms Along a Temperature Gradient on a Hydrothermal Stream at El Tatio (Chilean Andes), as a Proxy for the Interpretation of Past and Present Biomarkers Beyond Earth

“…The relative abundance of the orangish Thermus at the hottest site in Cacao (i.e., the hot spring vent at 72°C) agreed with the temperature distribution reported for the thermophilic genus in other hydrothermal systems from Island, the Yellowstone National Park, or the northern Thailand, where the temperature ranged from 75 to 89°C ( Purcell et al, 2007 ). Other thermophilic components of CE6 were Acetothermia , a recently proposed phylum of thermophilic and chemolithotrophic bacteria likely related to Thermotogae ( Rinke et al, 2013 ), and Thermoflexus , a heterotrophic bacterium from the Chloroflexi phylum ( Thomas et al, 2021 ).…”