Trace metals have been an important ingredient for life throughout Earth’s history. Here, we describe the genome-guided cultivation of a member of the elusive archaeal lineage Caldarchaeales (syn. Aigarchaeota), Wolframiiraptor gerlachensis, and its growth dependence on tungsten. A metagenome-assembled genome (MAG) of W. gerlachensis encodes putative tungsten membrane transport systems, as well as pathways for anaerobic oxidation of sugars probably mediated by tungsten-dependent ferredoxin oxidoreductases that are expressed during growth. Catalyzed reporter deposition-fluorescence in-situ hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) show that W. gerlachensis preferentially assimilates xylose. Phylogenetic analyses of 78 high-quality Wolframiiraptoraceae MAGs from terrestrial and marine hydrothermal systems suggest that tungsten-associated enzymes were present in the last common ancestor of extant Wolframiiraptoraceae. Our observations imply a crucial role for tungsten-dependent metabolism in the origin and evolution of this lineage, and hint at a relic metabolic dependence on this trace metal in early anaerobic thermophiles.

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.

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea. The emergence of hypervirulent C. difficile strains has led to increases in both hospital- and community-acquired CDI. Furthermore, CDI relapse from hypervirulent strains can reach up to 25%. Thus, standard treatments are rendered less effective, making new methods of prevention and treatment more critical. Previously, the bile salt analog CamSA was shown to inhibit spore germination in vitro and protect mice and hamsters from C. difficile strain 630. Here, we show that CamSA was less active at preventing spore germination of other C. difficile ribotypes, including the hypervirulent strain R20291. Strain-specific in vitro germination activity of CamSA correlated with its ability to prevent CDI in mice. Additional bile salt analogs were screened for in vitro germination inhibition activity against strain R20291, and the most active compounds were tested against other strains. An aniline-substituted bile salt analog, (CaPA), was found to be a better anti-germinant than CamSA against eight different C. difficile strains. In addition, CaPA was capable of reducing, delaying, or preventing murine CDI signs in all strains tested. CaPA-treated mice showed no obvious toxicity and showed minor effects on their gut microbiome. CaPA’s efficacy was further confirmed by its ability to prevent CDI in hamsters infected with strain 630. These data suggest that C. difficile spores respond to germination inhibitors in a strain-dependent manner. However, careful screening can identify anti-germinants with broad CDI prophylaxis activity.

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.

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