Comprehensive analysis of the microbial consortium in the culture of flagellate Monocercomonoides exilis

Abstract

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.

Fig. 1

Methods workflow of the cell culturing experiment and growth curve of Monocercomonoides exilis with sampling points. A Summary of the experiment workflow. One day before the start of the experiment, 700 mL of growth medium divided into two flasks was inoculated with Citrobacter portucalensis. This initial step is referred to as the bacterization step. On day 0, the contents of the two flasks were combined, and a 45 mL aliquot was taken for the isolation of DNA, RNA, and metabolomic analyses. The remaining medium was inoculated with M. exilis along with the entire prokaryotic community. The mixture was then divided into 50-mL tubes and allowed to incubate at 37 °C without shaking. Each day, three tubes were removed to isolate DNA and RNA, and to freeze the sample for metabolomic analysis. Data for this study were collected from days 0, 2, 3, and 5. B Growth curve of M. exilis (cells/mL) grown in TYSGM-9 medium for seven days. Samples for thorough analysis were taken on days 0, 2, 3, and 5, as highlighted by red squares. Error bars provide standard deviations of the values from three measurements

Fig. 2

Relative read abundance of the members of the community. Each member of the community is represented as relative read abundance (%) corresponding to its genome on each day and replicate

Fig. 3

Potential contribution of the bacterial community to the biochemical cycling processes of carbon, nitrogen, iron, and sulfur. Labels represent the main steps of the process. Steps supported by the genomic data are indicated in green. Steps, for which the enzymes were not identified are indicated in light grey. MAGs: number of MAGs responsible for a step

Fig. 4

Schematic summary of changes in the culture medium. The flasks display numbers and broad chemical classifications of significant compounds identified in the cell-free medium for each day (refer to Supplementary Table 4 for details). Arrows indicate the number of compounds that show an increase in concentration the following day, referred to as “targets.” It also includes the number of targets that M. exilis can synthesize independently and those that can be synthesized through the interaction between M. exilis and the bacterial community. Additionally, the bacterial species identified by the m2m pipeline as “key species” are shown for each consecutive day of comparison

Fig. 5

Differential expression of genes related to carbohydrate metabolism and transport in Monocercomonoides exilis between day 5 and day 2. The identified sugar transporters, glycolytic pathway, pentose phosphate pathway, and related reactions (adapted from Karnkowska et al., 2019) are illustrated with the enzyme abbreviations enclosed in gray ovals. Up-regulated genes and processes are indicated by a green arrow pointing upwards, while down-regulated genes and processes are marked with a red arrow pointing downwards. Processes that show no significant change in expression are represented by an orange equal sign. adhE: Bifunctional acetaldehyde-CoA/alcohol dehydrogenase; ALDH: Aldehyde dehydrogenase; ACS: Acetyl-CoA synthetase (ADP-forming); ACCT/PycB: putative malonyl-CoA:pyruvate transcarboxylase; ACAT: Acetyl-CoA acetyltransferase; PFOR: Pyruvate-ferredoxin oxidoreductase; HYD: [FeFe]-hydrogenase; NADP-ME: NADP-dependent malic enzyme; PK: Pyruvate kinase; PPDK: Pyruvate phosphate dikinase; PEPCK: Phosphoenolpyruvate carboxykinase; AAT: Aspartate aminotransferase; ASNA: Asparagine synthetase; ENO: Enolase; iPGM: 2,3-bisphosphoglycerate independent phosphoglycerate mutase; PGK: Phosphoglycerate kinase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GAPN: NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase; TPI: Triose phosphate isomerase; DERA: Deoxyribose-phosphate aldolase; RBKS: Ribokinase; FBA: Fructose-bisphosphate aldolase; PFP: Phosphofructokinase (pyrophosphate-based); GPI: Glucose-6-phosphate isomerase; PGM: Phosphoglucomutase; BMY: β-amylase; AMY: α-amylase; treS: Maltose alpha-D-glucosyltransferase/ alpha-amylase; HXK: Hexokinase; RPDK; ribose-phosphate diphosphokinase; RPI: Ribose-5-phosphate isomerase; RPE: Ribulose-phosphate 3-epimerase; TKT: Transketolase; TAL: Transaldolase

Fig. 6

Differential expression of genes involved in amino acid metabolism and transport in Monocercomonoides exilis between day 5 and day 2. Identified amino acid importers, amino acid biosynthesis pathways, and the arginine catabolism pathway are illustrated with the enzyme abbreviations enclosed in gray ovals. Up-regulated genes and processes are indicated by a green arrow pointing upwards, while down-regulated genes and processes are marked with a red arrow pointing downwards. Processes that show no significant change in expression are represented by an orange equal sign. Spontaneous reactions are shown in blue. PGDH: Phosphoglycerate dehydrogenase; PSAT: Phosphoserine transaminase; PSPH: putative phosphoserine phosphatase; SAT: Serine O-acetyltransferase; SEPHS: Selenophosphate synthetase; SEPSECS: O-phospho-L-seryl-tRNA[Sec]:L-selenocysteinyl-tRNA synthase; THRC: Threonine synthase: ASNA: Asparagine synthetase; ALAT: Alanine aminotransferase; ACCT/PycB: putative malonyl-CoA:pyruvate transcarboxylase; AAT: Aspartate aminotransferase; ASNA: Asparagine synthetase; DHE: Glutamate dehydrogenase; GLNA: Glutamine synthetase: ADI: Arginine deiminase: OTC: Ornithine transcarbamylase: CK: Carbamate kinase; OAT: Lysine/Ornithine aminotransferase