Revealing the metabolic capacity of Streblomastix strix and its bacterial symbionts using single-cell metagenomics

Abstract

Lower termites harbor in their hindgut complex microbial communities that are involved in the digestion of cellulose. Among these are protists, which are usually associated with specific bacterial symbionts found on their surface or inside their cells. While these form the foundations of a classic system in symbiosis research, we still know little about the functional basis for most of these relationships. Here, we describe the complex functional relationship between one protist, the oxymonad Streblomastix strix, and its ectosymbiotic bacterial community using single-cell genomics. We generated partial assemblies of the host S. strix genome and Candidatus Ordinivivax streblomastigis, as well as a complex metagenome assembly of at least 8 other Bacteroidetes bacteria confirmed by ribosomal (r)RNA fluorescence in situ hybridization (FISH) to be associated with S. strix. Our data suggest that S. strix is probably not involved in the cellulose digestion, but the bacterial community on its surface secretes a complex array of glycosyl hydrolases, providing them with the ability to degrade cellulose to monomers and fueling the metabolism of S. strix In addition, some of the bacteria can fix nitrogen and can theoretically provide S. strix with essential amino acids and cofactors, which the protist cannot synthesize. On the contrary, most of the bacterial symbionts lack the essential glycolytic enzyme enolase, which may be overcome by the exchange of intermediates with S. strix This study demonstrates the value of the combined single-cell (meta)genomic and FISH approach for studies of complicated symbiotic systems.

Keywords: Bacteroidetes; Streblomastix; ectosymbionts; oxymonads; termite.

Fig. 1.

Transmission electron micrograph of S. strix from the gut of Z. angusticollis showing the stellar-shaped eukaryotic cell with bacteria (b) attached to the surface. The cell nucleus (n) surrounded by the axostyle (ax) is clearly visible. The arrowhead indicates the putative process of phagocytosis of a bacterium by S. strix. (Scale bar, 1 µm.)

Fig. 2.

Maximum likelihood (ML) tree showing the relationship of our isolated bacterial 16S rRNA phylotypes within Bacteroidetes. The tree was constructed using 16S rRNA gene sequences. Values at the nodes represent ML bootstrap support values (>70%) and bayesian inference posterior probabilities (>0.9) (BI). Dots represent full node support (ML: 100%; BI: 1). The tree was rooted with representatives of the genus Flavobacterium and Capnocytophaga (not shown). The symbionts of S. strix are shown in bold. The lineages within the miscellaneous Bacteroidales metagenome are highlighted and labeled from I to IV. The clusters of Bacteroidales bacteria symbionts from the termite gut are defined as Cluster I, II, and V according to Ohkuma et al. (26). Sequences originating from bacterial symbionts of other flagellates from the gut of the termites have their branches colored in red. (Scale bar, 0.2 expected substitutions per site.)

Fig. 3.

FISH identification of the ectosymbionts of Streblomastix strix. (Top Left) Localization of the clades II, III, and IV of Bacteroidetes symbionts of S. strix using ST-BACT-850 (A), ST-BACT-137 (B), and ST-BACT-1261 (C). (D) Merged image of A–C. (E) Enlarged view of the region marked by a rectangle in D. (Top Right) Localization of St1 (Ca. O. streblomastigis) and St2 (Clade I) bacterial ribotypes on S. strix using ST-BACT-1020 (F) and ST-BACT-81 (G). Merged image of F and G is shown in H. (I) Enlarged view of the region marked by a rectangle in H. (Bottom Left) Specific localization of different ribotypes within clade III. (J) General localization of the bacteria from clade III using the general probe ST-BACT-137. (K) Specific localization of ST8 and ST9 ribotypes using the ST-BACT-734 probe. (L) Specific localization of ST8 ribotype using the ST-BACT-196 probe. (M) Merged picture of J–L. (N) Enlarged view of the region marked by a rectangle in M. (Bottom Right) Specific localization of different ribotypes within clade II. (O) General localization of the bacteria from clade II using the general probe ST-BACT-1261. (P) Specific localization of the ST6 ribotype using the ST-BACT-477 probe. (Q) Specific localization of ST3 and ST4 ribotypes using the ST-BACT-845 probe. (R) Merged picture of O–Q. (S) Enlarged view of the region marked by a rectangle in R. (Scale bars, 5 µm.)

Fig. 4.

Metabolic map of Streblomastix strix reconstructed from the draft genome. Links to the pathways of amino acid biosynthesis are shown in blue, and links to vitamin and cofactor biosynthesis are shown in green. AA, amino acid; AA⁰, neutral amino acid; AcAdh, acetaldehyde; AcCoA, acetyl-coenzyme A; dNTP, deoxyribonucleotide triphosphate; Fru6P, fructose-6-phosphate; G3P, glycerate 3-phosphate; Ga3P, glyceraldehyde 3-phosphate; Glc, glucose; GDP-Man, guanosine diphosphate mannose; Man, mannose; NMP, nucleoside monophosphate; NTP, nucleoside triphosphate; Pyr, pyruvate.

Fig. 5.

Metabolic map of Ca. O. streblomastigis reconstructed from the draft genome. Links to the pathways of amino acid biosynthesis are shown in blue, and links to the vitamin and cofactor biosynthesis are shown in green. Dashed arrows indicate pathways that are lacking one or more essential genes. Menaquinone is shown in red because the biosynthetic pathway is incomplete. ABC, ATP-binding cassette transporter; AcCoA, acetyl-coenzyme A; AcP, acetyl phosphate; amt, ammonia transporter; BCAA, branched-chain amino acid; CoA, coenzyme A; DHAP, dihydroxyacetone phosphate; Fdx ox, oxidized ferredoxin; Fdx red, reduced ferredoxin; Frd, fumarate reductase; Fru6P, fructose-6-phosphate; Fum, fumarate; G3P, glycerate 3-phosphate; Ga3P, glyceraldehyde 3-phosphate; Glc, glucose; Mal, malate; MQ, menaquinone; 2OG, 2-oxoglutarate; OxAc, oxaloacetate; PRPP, phosphoribosyl pyrophosphate; Pyr, pyruvate; Rha, rhamnose; Sec, general secretion system; Suc, succinate; Xyl, xylose.

Fig. 6.

Putative metabolic map of a bacterium within the miscellaneous Bacteroidales metagenome reconstructed from the metagenomic data. Links to the pathways of amino acid biosynthesis are shown in blue, and links to vitamin and cofactor biosynthesis are shown in green. Light colors (gray, light blue, or light green) represent steps in the metabolism that we think are not common for all bacteria from the metagenome. Dashed arrows indicate pathways that are lacking one or more essential genes. Menaquinone is shown in red because its biosynthetic pathway is incomplete. ABC, ATP-binding cassette transporter; AcCoA, acetyl-coenzyme A; AcP, acetyl phosphate; amt, ammonia transporter; C1, complex I; CoA, coenzyme A; DHAP, dihydroxyacetone phosphate; FAD, flavin adenine dinucleotide; Fdx ox, oxidized ferredoxin; Fdx red, reduced ferredoxin; Frd, fumarate reductase; Fru6P, fructose-6-phosphate; Fuc, fucose; Fum, fumarate; G2P, glycerate 2-phosphate; G3P, glycerate 3-phosphate; Ga3P, glyceraldehyde 3-phosphate; Glc, glucose; Gly3P, glycerol 3-phosphate; Lac, l-lactate; LacAdh, lactaldehyde; Mal, malate; MG, methylglyoxal; MQ, menaquinone; 2OG, 2-oxoglutarate; OxAc, oxaloacetate; PRPP, phosphoribosyl pyrophosphate; Pyr, pyruvate; Rha, rhamnose; Sec, general secretion system; Suc, succinate; Xyl, xylose.

Fig. 7.

Schematic view of the proposed interaction between the bacterial community and S. strix, in the gut of Z. angusticollis. The bacterial symbionts of S. strix use their complex array of GHs to digest the wood particles generating monosaccharides, which are taken up by the bacteria and protist. The eukaryote ferments the sugars to acetate and ethanol, which are most probably excreted into the termite gut. Ca. O. streblomastigis also ferments sugars to acetate and probably secretes the acetate in the gut. In the case of Bacteroidetes bacteria forming the metagenomic bin, the sugars cannot be fermented to acetate due to the lack of enolase. We hypothesize that these bacteria collaborate with S. strix to convert glycerate 2-phosphate (G2P) to PEP in order to complete the glycolysis or that S. strix provides a source of pyruvate (Pyr) for the bacteria, which would be used to produce ATP. The bacterial community also incorporates nitrogen into ammonia and some amino acids. Parts of the synthesized amino acids and various cofactors are most probably provided to S. strix by the bacteria, using some form of transport or via grazing on bacteria. The acetate that is secreted in the hindgut is used by the termite. Fdx ox, oxidized ferredoxin; Fdx red, reduced ferredoxin.