A Novel Lineage of Endosymbiotic Actinomycetales: Genome Reduction and Acquisition of New Functions in Bifidobacteriaceae Associated With Termite Gut Flagellates

Abstract

Cellulolytic flagellates are essential for the symbiotic digestion of lignocellulose in the gut of lower termites. Most species are associated with host-specific consortia of bacterial symbionts from various phyla. 16S rRNA-based diversity studies and taxon-specific fluorescence in situ hybridization revealed a termite-specific clade of Actinomycetales that colonise the cytoplasm of Trichonympha spp. and other gut flagellates, representing the only known case of intracellular Actinomycetota in protists. Comparative analysis of eleven metagenome-assembled genomes from lower termites allowed us to describe them as new genera of Bifidobacteriaceae. Like the previously investigated Candidatus Ancillula trichonymphae, they ferment sugars via the bifidobacterium shunt but, unlike their free-living relatives, experienced significant genome erosion. Additionally, they acquired new functions by horizontal gene transfer from other gut bacteria, including the capacity to produce hydrogen. Members of the genus Ancillula (average genome size 1.56 ± 0.2 Mbp) retained most pathways for the synthesis of amino acids, including a threonine/serine exporter, providing concrete evidence for the basis of the mutualistic relationship with their host. By contrast, Opitulatrix species (1.23 ± 0.1 Mbp) lost most of their biosynthetic capacities, indicating that an originally mutualistic symbiosis is on the decline.

Keywords: bifidobacteria; flagellates endosymbiosis; genome reduction; gut microbiota; horizontal gene transfer; termites.

FIGURE 1

Rank‐normalized phylogeny of Bifidobacteriaceae, including type species of all genera validly published under the ICNP. Family‐level lineages of Actinomycetales and selected orders of Actinomycetia were included for comparison. The maximum‐likelihood tree is based on a concatenated alignment of 120 single‐copy marker genes generated by GTDB‐Tk and was inferred using IQ‐TREE under the LG + F + I + G4 model of evolution. It was normalized using relative evolutionary divergence (RED) values determined with PhyloRank. Bullets on internal nodes indicate ultrafast bootstrap support (●, ≥ 99%; ○, ≥ 95%; 1000 replicates). The number of genomes in the collapsed clades and the type strains of novel genera (^Ts) are indicated. The family of the host termite is indicated (H, Hodotermitidae; K, Kalotermitidae; R, Rhinotermitidae). For an expanded version of the tree that includes also low‐quality MAGs, see Figure A1.

FIGURE 2

Genome size and genomic GC content of the order Actinomycetales, comparing the termite‐associated genera with other members of Bifidobacteriaceae.

FIGURE 3

Key enzymes of major catabolic pathways encoded by MAGs from the termite‐associated Bifidobacteriaceae and other family members (type species only). Selected representatives of other Actinomycetales were included for comparison. Genome size was estimated based on assembly size and completeness of the genomes. More details are shown in (Figures A2 and A3. ABC, ATP‐binding cassette; OPA, organophosphate transporter, PTS, phosphotransferase system; Xfp, xylose‐5‐phosphate/fructose‐6‐phosphate phosphoketolase; Xpk, xylose‐5‐phosphate phosphoketolase.

FIGURE 4

Comparison of the metabolic pathways of the genera Ancillula and Opitulatrix. Pathways shown in black are present in both genera; the fructose‐6‐phosphate phosphoketolase (F6PPK) pathway (bifidobacteria shunt) is highlighted in red. Elements marked in green are present only in Ancillula; those marked in brown are present only in Opitulatrix. Dashed arrows indicate that a pathway is not present in all MAGs. Fermentation products are highlighted in bold letters, and products of biosynthetic pathways are shown in blue. Detailed biosynthetic pathways for amino acids and cofactors are shown in Figure A12. Non‐standard abbreviations: AAP, amino acid permease; ABC, ATP‐binding cassette transporter; Amt, ammonium transporter; AroP, aromatic amino acid transporter; Fd/Fld, ferredoxin/flavodoxin; GlcN, glucosamine; GlcNAc, N‐acetyl‐glucosamine; GlpT, glycerol‐3‐phosphate transporter; KDPG, 2‐keto‐3‐deoxy‐6‐phosphogluconate; LivGH, branched‐chain amino acid transporter; MetQ, methionine transporter; PLP, pyridoxal 5‐phosphate; PPP, pentose phosphate pathway; PRPP, phosphoribosyl pyrophosphate; PTS, phosphotransferase system; SAM, S‐adenosylmethionine; THF, tetrahydrofolate; ThrE, threonine/serine exporter; Xfp, xylose‐5‐phosphate/fructose‐6‐phosphate phosphoketolase.

FIGURE 5

Capacities for biosynthesis and transport of amino acids and co‐factors, and key genes for the assimilation of ammonia of termite‐associated Bifidobacteriaceae and other family members (type species only). The full pathways and other details are shown in (Figures A7 and A12). Amt, ammonium transporter; AroP, aromatic amino acid transporter; AzlC, branched‐chain amino acid transporter; BioY, biotin transporter; FolT, folate transporter; LysE, lysine exporter; LivGH, branched‐chain amino acid transporter; MetQ, methionine transporter; ProV, glycine/proline transporter; RibU, riboflavin transporter; ThrE, threonine/serine exporter.

FIGURE 6

16S rRNA‐based phylogeny of Bifidobacteriaceae, illustrating the relationship of the phylotypes recovered from whole‐gut libraries of termites and cockroaches and capillary‐picked suspensions of termite gut flagellates to the type species of all other genera. Accession numbers of novel sequences are shown in bold. The number of sequences in the collapsed clades and the type strains of novel genera (Ts) are indicated. The host species are indicated (termite family in parentheses: A, Archotermopsidae, H, Hodotermitidae; Hs, Hodotermopsidae, K, Kalotermitidae; R, Rhinotermitidae). Sequences from MAGs are marked in red. The maximum‐likelihood tree is based on a curated alignment of near full‐length 16S rRNA genes (1462 positions) and was generated using IQ‐TREE under the SYM + R4 model of evolution. Bullets on internal nodes indicate ultrafast bootstrap support (●, ≥ 99%, ○, ≥ 95%, 1000 replicates). Other Actinomycetales were used as outgroups. Symbols created with BioRender.com.

FIGURE 7

Fluorescence in situ hybridization analysis of the hindgut content of Incisitermes tabogae (A) and I. incisus (B). Bacterial cells were hybridised with the general Bacteria probe EUB338 (Fam‐labelled, green) and the probe ACT490 specific for the termite‐associated lineages (Cy3‐labelled, orange). Outlines of Trichonympha cells were drawn based on phase‐contrast images of the same slides. Scale bars: 100 μm (A) and 50 μm (B).

FIGURE A1

Phylogenomic tree of Bifidobacteriaceae, including the names of all genera validly published under the ICNP and low‐quality (LQ) MAGs from termite guts. The maximum‐likelihood tree is based on an alignment of 120 single‐copy marker genes generated by GTDB‐Tk and was inferred with IQ‐TREE under the LG + F + I + G4 model of evolution. Numbers at internal nodes indicate ultrafast bootstrap support (in percent, 1000 replicates). MAGs with 16S rRNA genes are in red typeface, type strains of new species are printed in bold. The host families are indicated in parentheses (A, Archotermopsidae, H, Hodotermitidae; Hs, Hodotermopsidae, K, Kalotermitidae; R, Rhinotermitidae; T, Termitidae).

FIGURE A2

Phylogeny of the bifunctional phosphoketolase (Xfp) of termite‐associated Bifidobacteriaceae (TAB) and other family members, and the homologous monofunctional phosphoketolases (Xpk) of Actinomycetota and other bacterial phyla. Biochemically characterised homologues (SWISS‐PROT) are highlighted. The maximum‐likelihood tree is based on a curated alignment of 490 amino acid positions and was inferred under the LG + G4 model of evolution. Bootstrap values were omitted for clarity.

FIGURE A3

PTS systems for N‐acetyl‐glucosamine (A) and glucose/mannose (B) and their conversion to fructose 6‐phosphate in termite‐associated Bifidobacteriaceae.

FIGURE A4

Phylogeny of the organophosphate antiporter (OPA) of termite‐associated Bifidobacteriaceae (TAB) and their closest relatives from public reference databases. Biochemically characterised homologues (SWISS‐PROT) are highlighted. The maximum‐likelihood tree is based on a curated alignment of 428 amino acid positions and was inferred under the LG + F + R9 model of evolution. Bootstrap values were omitted for clarity.

FIGURE A5

Phylogeny of Group A [FeFe] hydrogenases in the genus Ancillula and Opitulatrix and their homologues in public databases. Homologues from MAGs of termite gut microbiota are marked in bold. The maximum‐likelihood tree is based on a curated alignment of 281 amino acid positions and was inferred under the LG + R8 model of evolution. Numbers at internal nodes indicate ultrafast bootstrap support (1000 replicates).

FIGURE A6

Phylogeny of PFOR in termite‐associated Bifidobacteriaceae and their homologues from public databases. The maximum‐likelihood tree is based on a curated alignment of 323 amino acid positions and was inferred under the LG + R5 model of evolution. Numbers at internal nodes indicate ultrafast bootstrap support (1000 replicates).

FIGURE A7

Enzymes of asparagine biosynthesis encoded by termite‐associated Bifidobacteriaceae and other family members. Abbreviations: AsnA, aspartate–ammonia ligase; AsnB, asparagine synthetase B; AspS, aspartate–tRNA ligase; GatABC, aspartyl/glutamyl‐tRNA(Asn/Gln) amidotransferase.

FIGURE A8

Phylogeny of the threonine/serine exporter (ThrE) of termite‐associated Bifidobacteriaceae and its homologues in public databases. The maximum‐likelihood tree is based on a curated alignment of 259 amino acid positions and was inferred under the LG + F + R6 model of evolution. Numbers at internal nodes indicate ultrafast bootstrap support (1000 replicates).

FIGURE A9

Enzymes of murein biosynthesis encoded by termite‐associated Bifidobacteriaceae and other family members. Abbreviations: MurJ, Lipid II flippase; Ddl, D‐alanine–D‐alanine ligase; MurF, UDP‐N‐acetylmuramoyl‐tripeptide–D‐alanyl‐D‐alanine ligase; Mpl, UDP‐N‐acetylmuramate–L‐alanyl‐gamma‐D‐glutamyl‐meso‐2,6‐diaminoheptandioate ligase; MurA, UDP‐N‐acetylglucosamine 1‐carboxyvinyltransferase; Alr, Alanine racemase; MraY, phospho‐N‐acetylmuramoyl pentapeptide transferase; MurB, UDP‐N‐acetylenolpyruvoylglucosamine reductase; FTSW, peptidoglycan glycosyltransferase.

FIGURE A10

Elements of different DNA repair mechanisms encoded by Bifidobacteriaceae and other Actinomycetales.

FIGURE A11

Phylogeny of the folate transporter (FolT) encoded by members of the genus Ancillula and its homologues from public databases. The maximum‐likelihood tree is based on a curated alignment of 324 amino acid positions and was inferred under the LG + G4 model of evolution. Numbers at internal nodes indicate ultrafast bootstrap support (1000 replicates).

FIGURE A12

Expanded biosynthetic pathways for amino acid (A) and cofactor (B) biosynthesis in the Ancillula and Opitulatrix MAGs. Genes in black are encoded by both genera. Genes in purple are present only in Ancillula, and genes in grey are absent in both genera.