Monocercomonoides exilis is a model species of the amitochondrial eukaryotic group Oxymonadida, which makes it a suitable organism for studying the consequences of mitochondrial loss. Although M. exilis has an endobiotic lifestyle, it can be cultured in vitro in polyxenic conditions alongside an uncharacterized prokaryotic community, while attempts to create axenic cultures have not been successful. In this study, we used metagenomic sequencing, transcriptomics, and metabolomics to characterize the microbial consortium that supports the growth of M. exilis. We assembled genomes for 24 bacterial species and identified at least 30 species in total. M. exilis accounted for less than 1.5% of the DNA reads, while bacterial species dominated the sequence data and shifted in abundance over time. Our metabolic reconstruction and differential gene expression analyses show that the bacterial community relies on organic carbon oxidation, fermentation, and hydrogen production, but does not engage in methanogenesis. We observed rapid depletion of amino acids, nucleotides, glyceraldehyde, lactate, fatty acids, and alcohols in the medium, indicating a reliance on external nutrient recycling. The nitrogen cycle in this community is incomplete, with limited nitrogen fixation and no ammonia oxidation. Despite detailed metabolic profiling, we did not find any direct biochemical connections between M. exilis and the prokaryotes. Several bacterial species produce siderophores to assist themselves and others in the community in acquiring iron. However, M. exilis does not appear to benefit directly from siderophore-mediated iron transport and lacks known iron uptake pathways. This indicates that M. exilis may rely indirectly on the iron metabolism of other bacteria through phagocytosis. Additionally, some bacteria synthesize polyamines like spermidine and phosphatidylcholine, which M. exilis may need but cannot produce on its own. As the culture ages, M. exilis shows changes in gene expression consistent with starvation responses, including the upregulation of carbohydrate storage pathways and processes related to exocytosis. These findings provide new insights into microbial interactions within xenic cultures and emphasize the complex nature of maintaining amitochondriate eukaryotes in vitro.

Department of Cell and Molecular Biology Molecular Evolution Program Uppsala Biomedicine Centre Uppsala University Uppsala Sweden

Department of Parasitology BIOCEV Faculty of Science Charles University 252 50 Vestec Czech Republic

European Molecular Biology Laboratory Genome Biology Unit Heidelberg Germany

Faculty of Science BIOCEV Charles University 252 50 Vestec Czech Republic

Research Group Insect Gut Microbiology and Symbiosis Max Planck Institute for Terrestrial Microbiology Karl von Frisch Str 10 35043 Marburg Germany

Zobrazit více v PubMed

Jandhyala SM, Talukdar R, Subramanyam C, Vuyyuru H, Sasikala M, Reddy DN. Role of the normal gut microbiota. World J Gastroenterol. 2015;21:8836–47. PubMed PMC

Thursby E, Juge N. Introduction to the human gut microbiota. Biochem J. 2017;474:1823–36. PubMed PMC

Fan Y, Pedersen O. Gut microbiota in human metabolic health and disease. Nat Rev Microbiol. 2021;19:55–71. PubMed

Partida-Rodriguez O, Nieves-Ramirez M, Laforest-Lapointe I, Brown EM, Parfrey L, Valadez-Salazar A, et al. Exposure to parasitic protists and helminths changes the intestinal community structure of bacterial communities in a cohort of mother-child binomials from a semirural setting in Mexico. mSphere. 2021. 10.1128/mSphere.00083-21. PubMed PMC

von Huth S, Thingholm LB, Kofoed P-E, Bang C, Rühlemann MC, Franke A, et al. Intestinal protozoan infections shape fecal bacterial microbiota in children from Guinea-Bissau. PLoS Negl Trop Dis. 2021;15: e0009232. PubMed PMC

Diamond LS. A new liquid medium for xenic cultivation of PubMed

Hamann E, Gruber-Vodicka H, Kleiner M, Tegetmeyer HE, Riedel D, Littmann S, et al. Environmental breviatea harbour mutualistic PubMed PMC

Hamann E, Tegetmeyer HE, Di R, Littmann S, Ahmerkamp S, Chen J, et al. Syntrophic linkage between predatory PubMed PMC

Treitli SC, Peña-Diaz P, Hałakuc P, Karnkowska A, Hampl V. High quality genome assembly of the amitochondriate eukaryote PubMed PMC

Karnkowska A, Treitli SC, Brzoň O, Novák L, Vacek V, Soukal P, et al. The oxymonad genome displays canonical eukaryotic complexity in the absence of a mitochondrion. Mol Biol Evol. 2019;36:2292–312. PubMed PMC

Karnkowska A, Vacek V, Zubáčová Z, Treitli SC, Petrželková R, Eme L, et al. A eukaryote without a mitochondrial organelle. Curr Biol. 2016;26:1274–84. PubMed

Treitli SC, Kotyk M, Yubuki N, Jirounková E, Vlasáková J, Smejkalová P, et al. Molecular and morphological diversity of the oxymonad genera PubMed

Preaxostyla HV. In: Archibald JM, Simpson AGB, Slamovits CH, editors. Handbook of the Protists. Cham: Springer International Publishing; 2017. p. 1139–74.

Novák LVF, Treitli SC, Pyrih J, Hałakuc P, Pipaliya SV, Vacek V, et al. Genomics of preaxostyla flagellates illuminates the path towards the loss of mitochondria. PLoS Genet. 2023;19:1–30. PubMed PMC

Treitli SC, Kolisko M, Husník F, Keeling PJ, Hampl V. Revealing the metabolic capacity of PubMed PMC

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20. PubMed PMC

Nurk S, Meleshko D, Korobeynikov A, Pevzner PA. Metaspades: a new versatile metagenomic assembler. Genome Res. 2017;27:824–34. PubMed PMC

Wu Y-W, Simmons BA, Singer SW. MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets. Bioinformatics. 2016;32:605–7. PubMed

Dick GJ, Andersson AF, Baker BJ, Simmons SL, Thomas BC, Yelton AP, et al. Community-wide analysis of microbial genome sequence signatures. Genome Biol. 2009. 10.1186/gb-2009-10-8-r85. PubMed PMC

Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068–9. PubMed

Chaumeil PA, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk v2: memory friendly classification with the genome taxonomy database. Bioinformatics. 2022;38:5315–6. PubMed PMC

Parks DH, Chuvochina M, Chaumeil PA, Rinke C, Mussig AJ, Hugenholtz P. A complete domain-to-species taxonomy for bacteria and archaea. Nat Biotechnol. 2020;38:1079–86. PubMed

Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010;11. PubMed PMC

Eddy SR. Accelerated profile HMM searches. PLoS Comput Biol. 2011;7 PubMed PMC

Matsen FA, Kodner RB, Armbrust EV. pplacer: linear time maximum-likelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree. BMC Bioinformatics. 2010;11: 538. PubMed PMC

Shaw J, Yu YW. Fast and robust metagenomic sequence comparison through sparse chaining with skani. Nat Methods. 2023;20:1661–5. PubMed PMC

Price MN, Dehal PS, Arkin AP. FastTree 2 – approximately maximum-likelihood trees for large alignments. PLoS ONE. 2010. 10.1371/journal.pone.0009490. PubMed PMC

Ondov BD, Treangen TJ, Melsted P, Mallonee AB, Bergman NH, Koren S, et al. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 2016;17:1–14. PubMed PMC

Sukumaran J, Holder MT. DendroPy: a Python library for phylogenetic computing. Bioinformatics. 2010;26:1569–71. PubMed

Harris CR, Millman KJ, van der Walt SJ, Gommers R, Virtanen P, Cournapeau D, et al. Array programming with NumPy. Nature. 2020;585:357–62. PubMed PMC

Huerta-Cepas J, Szklarczyk D, Heller D, Hernández-Plaza A, Forslund SK, Cook H, et al. eggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses. Nucleic Acids Res. 2019;47:D309–14. PubMed PMC

Cantalapiedra CP, Hernández-Plaza A, Letunic I, Bork P, Huerta-Cepas J. EggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol Biol Evol. 2021;38:5825–9. PubMed PMC

Belcour A, Frioux C, Aite M, Bretaudeau A, Hildebrand F, Siegel A. Metage2metabo, microbiota-scale metabolic complementarity for the identification of key species. Elife. 2020;9:1–38. PubMed PMC

Karp PD, Paley SM, Midford PE, Krummenacker M, Billington R, Kothari A, et al. Pathway Tools version 24.0: Integrated Software for Pathway/Genome Informatics and Systems Biology. arXiv e-prints. 2020.

Karp PD, Latendresse M, Caspi R. The pathway tools pathway prediction algorithm. Stand Genomic Sci. 2011;5:424–9. PubMed PMC

Antipov D, Raiko M, Lapidus A, Pevzner PA. Metaviral SPAdes: assembly of viruses from metagenomic data. Bioinformatics. 2020;36:4126–9. PubMed

Antipov D, Raiko M, Lapidus A, Pevzner PA. Plasmid detection and assembly in genomic and metagenomic data sets. Genome Res. 2019;29:961–8. PubMed PMC

Bushmanova E, Antipov D, Lapidus A, Prjibelski AD. rnaSPAdes: a PubMed PMC

Guo J, Bolduc B, Zayed AA, Varsani A, Dominguez-Huerta G, Delmont TO, et al. VirSorter2: a multi-classifier, expert-guided approach to detect diverse DNA and RNA viruses. Microbiome. 2021;9:1–13. PubMed PMC

Zhou Z, Tran PQ, Breister AM, Liu Y, Kieft K, Cowley ES, et al. METABOLIC: high-throughput profiling of microbial genomes for functional traits, metabolism, biogeochemistry, and community-scale functional networks. Microbiome. 2022. 10.1186/s40168-021-01213-8. PubMed PMC

Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017;14:417–9. PubMed PMC

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9. PubMed PMC

Liao Y, Smyth GK, Shi W. Featurecounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–30. PubMed

Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. PubMed PMC

R Core Team. R: A language and environment for statistical computing. 2021.

Klingenberg H, Meinicke P. How to normalize metatranscriptomic count data for differential expression analysis. PeerJ. 2017;2017. PubMed PMC

Christel S, Herold M, Bellenberg S, Buetti-Dinh A, El Hajjami M, Pivkin IV, et al. Weak iron oxidation by Sulfobacillus thermosulfidooxidans maintains a favorable redox potential for chalcopyrite bioleaching. Front Microbiol. 2018;9:1–12. PubMed PMC

Kaviraj M, Kumar U, Snigdha A, Chatterjee S. Nitrate reduction to ammonium: a phylogenetic, physiological, and genetic aspects in prokaryotes and eukaryotes. Arch Microbiol. 2024;206: 297. PubMed

Sugai Y, Katsuyama Y, Ohnishi Y. A nitrous acid biosynthetic pathway for diazo group formation in bacteria. Nat Chem Biol. 2016;12:73–5. PubMed

Arieli B, Shahak Y, Taglicht D, Hauska G, Padan E. Purification and characterization of sulfide-quinone reductase, a novel enzyme driving anoxygenic photosynthesis in PubMed

Caspi R, Altman T, Billington R, Dreher K, Foerster H, Fulcher CA, et al. The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of Pathway/Genome Databases. Nucleic Acids Res. 2014;42:459–71. PubMed PMC

Iturriaga G, Suárez R, Nova-Franco B. Trehalose metabolism: from osmoprotection to signaling. Int J Mol Sci. 2009;10:3793–810. PubMed PMC

Law KC, Chung KK, Zhuang X. An update on coat protein complexes for vesicle formation in plant post-Golgi trafficking. Front Plant Sci. 2022;13:1–9. PubMed PMC

Shimizu Y, Uemura T. The sorting of cargo proteins in the plant trans-Golgi network. Front Plant Sci. 2022;13 August:1–10. PubMed PMC

Tanaka T, Goto K, Iino M. Diverse functions and signal transduction of the exocyst complex in tumor cells. J Cell Physiol. 2017;232:939–57. PubMed

Bucci C, Thomsen P, Nicoziani P, McCarthy J, Van Deurs B. Rab7: a key to lysosome biogenesis. Mol Biol Cell. 2000;11:467–80. PubMed PMC

Teulière J, Bernard G, Bapteste E. The distribution of genes associated with regulated cell death is decoupled from the mitochondrial phenotypes within unicellular eukaryotic hosts. Front Cell Dev Biol. 2020;8 September:1–8. PubMed PMC

Zhukov A, Popov V. Eukaryotic Cell Membranes: Structure, Composition, Research Methods and Computational Modelling. Int J Mol Sci. 2023;24. PubMed PMC

Michael AJ. Polyamines in eukaryotes, bacteria, and archaea. J Biol Chem. 2016;291:14896–903. PubMed PMC

Braun V, Hantke K. The tricky ways bacteria cope with iron limitation. In: Chakraborty R, Braun V, Hantke K, Cornelis P, editors. Iron Uptake in Bacteria with Emphasis on E. coli and Pseudomonas. Dordrecht: Springer Netherlands; 2013. p. 31–66.

Kramer J, Özkaya Ö, Kümmerli R. Bacterial siderophores in community and host interactions. Nat Rev Microbiol. 2020;18:152–63. PubMed PMC

Leventhal GE, Ackermann M, Schiessl KT. Why microbes secrete molecules to modify their environment: The case of iron-chelating siderophores. J R Soc Interface. 2019;16. PubMed PMC

Butaite E, Baumgartner M, Wyder S, Kümmerli R. Siderophore cheating and cheating resistance shape competition for iron in soil and freshwater Pseudomonas communities. Nat Commun. 2017;8. PubMed PMC

Dailey HA, Dailey TA, Gerdes S, Jahn D, Jahn M, O’Brian MR, et al. Prokaryotic heme biosynthesis: multiple pathways to a common essential product. Microbiol Mol Biol Rev. 2017;81:1–62. PubMed PMC

Bryant DA, Hunter CN, Warren MJ. Biosynthesis of the modified tetrapyrroles—the pigments of life. J Biol Chem. 2020;295:6888–925. PubMed PMC

Layer G. Heme biosynthesis in prokaryotes. Biochimica et Biophysica Acta (BBA)—Mol Cell Res. 2021;1868: 118861. PubMed

O’ Donnell MM, Harris HMB, Ross RP, O’Toole PW. Core fecal microbiota of domesticated herbivorous ruminant, hindgut fermenters, and monogastric animals. Microbiologyopen. 2017;6. PubMed PMC

Crowley EJ, King JM, Wilkinson T, Worgan HJ, Huson KM, Rose MT, et al. Comparison of the microbial population in rabbits and guinea pigs by next generation sequencing. PLoS ONE. 2017. 10.1371/journal.pone.0165779. PubMed PMC

Cabello P, Luque-Almagro VM, Roldán MD, Moreno-Vivián C. Nitrogen cycle. Encyclopedia of Microbiology. 2019;:301–10.

Yarlett N, Martinez MP, Moharrami MA, Tachezy J. The contribution of the arginine dihydrolase pathway to energy metabolism by PubMed

Schofield PJ, Edwards MR, Matthews J, Wilson JR. The pathway of arginine catabolism in PubMed