Abstract
Many thermophiles that are abundant in high-temperature geothermal systems have never been cultivated and are poorly understood, including deeply branching members of the archaeal phylum Thermoproteota. Here, we describe the genome-guided cultivation of one such organism, Calditenuis ramacidaminiphagus, and show that it has evolved a heterotrophic metabolism focused on branched-chain amino acids (BCAAs). Initially, fluorescence in situ hybridization and nanoscale secondary ion mass spectrometry (FISH-nanoSIMS) showed that Cal. ramacidaminiphagus assimilated amino acids rapidly in casamino acid-amended enrichment cultures. Metagenome and metaproteome analyses showed a high abundance and expression of BCAA transporter genes, suggesting a BCAA-focused metabolism. This inference was supported by the subsequent enrichment of Cal. ramacidaminiphagus in BCAA-fed cultures, reaching 2.66×106 cells/mL and 48.7% of the community, whereas it was outcompeted when polar amino acids were included. Metabolic reconstruction and metaproteomics suggest that BCAAs are channeled into the mevalonate pathway for lipid biosynthesis and fuel ATP production through the TCA cycle coupled with aerobic respiration and through production of branched-chain organic acids by overflow metabolism. Ancestral state reconstructions and phylogenetic analyses of 62 Caldarchaeales genomes revealed multiple horizontal transfers of BCAA transporters to the ancestor of the genus Calditenuis. Our study highlights the crucial role of BCAAs in the early evolution and niche of this genus, and suggests a high degree of resource partitioning even within low-diversity thermophilic communities.
Data availability
Genomes were submitted to eLMSG and/or IMG, with nomenclatural type genomes submitted to GenBank, under accession numbers listed in Supplementary Data 20. Raw sequence data for metagenomes from which type genomes were derived are available in the SRA under the run accessions SRX27892530, SRX27857028, SRX25997117, SRX29285230, SRX29285228, and SRX25997118. Raw files for 16S rRNA gene sequencing are available in the SRA under BioProject accession number PRJNA1219692. Metaproteomics (MSV000093641 [https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=c4115bc81d584c77a217ef174c8fa6ce]) data are available from the Center for Computational Mass Spectrometry’s MassIVE repository via the ProteomeXchange Consortium. Source data are provided with this paper.
Code availability
Custom code is available on GitHub (https://github.com/alexximalayaunlv/Calditenuis_paper.git) and Zenodo with the access code https://doi.org/10.5281/zenodo.18097983. The Python scripts in this repository were used to: (1) process ALE results and (2) select genomes with specific marker proteins for genealogical concordance analysis using Densitree.
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Acknowledgements
We thank Dave and Sandy Jamieson for access to GBS and acknowledge that GBS is on the historical lands of the Cui Ui Ticutta (Cui-ui eaters) band of Northern Paiutes. We also thank the operators of Gongxiaoshe resort and hotel and the operators of Yunnan Tengchong Volcano and Spa Tourist Attraction Development Corporation. Part of this work was carried out at Lawrence Livermore National Lab (LLNL) under Contract DE-AC52-07NA2734 (X.M., J.J., P.W., J.P.-R). Funding was also provided by the U.S. National Science Foundation (DEB 1557042 and 2038420, D.L., D.M., M.P., X.M., J.J., J.K.C, C.O.S., J.A.D., W.S., B.P.H.), NASA (80NNSC17KO548 and 80NSSC25M0046, D.L., D.M., M.P., X.M., J.J., J.K.C, C.O.S., J.A.D., W.S., B.P.H.), the National Natural Science Foundation of China (Nos. 92251302, 32200007, and 32370011), and the Novo Nordisk Foundation (NNF24OC0089849). Data from Yellowstone National Park were generated with funding from a NASA grant to D.R.C. and E.S.B. (80NSSC19M0150), and with sample collection under the National Park Service permit YELL-SCI-5544.
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B.P.H., J.A.D., and D.L. conceived the study. D.M., J.A.D., W.S., and B.P.H. conducted field sampling. D.M., J.A.D., D.L., and B.P.H. conducted lab experiments. D.M., J.A.D., X.M., J.J., J. P.-R., P.K.W., and D.L. analyzed samples with nanoSIMS and performed nanoSIMS data analysis and interpretation. M.P., D.L., T.N., and B.P.H. conducted nomenclature and taxonomic work. J.-Y.J., L.L., D.R.C., M.P., D.L., E.S.B., Z.-S.H., and W.-J.L. were involved in metagenomic sampling, sequencing, assembly, and binning. J.K.C. conducted computed structure modeling. R.M. conducted nitric oxide reductase screening. D.L. and C.O.S. conducted bioinformatics analyses. D.L. wrote the first drafts of the manuscript. B.P.H. contributed to the writing and revision of manuscript. All authors contributed to the final version of the paper.
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Lai, D., Mosier, D., Palmer, M. et al. Branched-chain amino acid specialization drove diversification within Calditenuaceae (Caldarchaeia) and enables their cultivation. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68859-6
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DOI: https://doi.org/10.1038/s41467-026-68859-6
