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.
Highlights
The bacterial phylum Chloroflexota continues to be expanded, revealing a global distribution containing broad phylogenetic and physiological diversity
The genome project for strain JAD2T was created in the Genomes OnLine Database (Mukherjee et al, 2021) (Go0015989) and genome sequencing, assembly, and annotation performed by the Department of Energy Joint Genome Institute (Berkley, CA, United States) (Huntemann et al, 2015)
Key metabolic features of T. hugenholtzii were predicted from the genome (Figure 1) based on IMG/M annotations, BlastKOALA, and selected manual annotations, and pathways were evaluated for feasibility based on MAPLE module completion ratios (MCRs), where Q-values below 0.5 were considered feasible (Takami et al, 2012, 2016; Arai et al, 2018)
Summary
The bacterial phylum Chloroflexota (synonym Chloroflexi) continues to be expanded, revealing a global distribution containing broad phylogenetic and physiological diversity. Chloroflexota are found in freshwater, marine, and hypersaline environments, contaminated groundwater, and terrestrial geothermal springs, among other habitats (Moe et al, 2009; Krzmarzick et al, 2012; Cole et al, 2013; Dodsworth et al, 2014; Hanada, 2014; Denef et al, 2016; Gomez-Saez et al, 2017; Bayer et al, 2018; Kato et al, 2018; Mehrshad et al, 2018; Ward et al, 2018; Thiel et al, 2019; Kochetkova et al, 2020). Many members of the Chloroflexota are difficult to isolate and grow in the laboratory, making detailed physiological investigations challenging, even when an isolate is obtained (Yamada et al, 2006, 2007; Bowman et al, 2013; Dodsworth et al, 2014).
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