The first part was about Trichoplax, and about one of its symbionts, Grellia incantans. Now it's about the second...
Symbiont #2: Ruthmannia eludens
When searching the metagenomes obtained from single Trichoplax H2 individuals for 16S and 18S rRNA-specific sequences, Gruber-Vodicka et al. found, next to the prevalent Trichoplax and Grellia sequences (see part I), a third sequence at high frequency that matched well with known 16S rRNA sequences from the Marinamargulisbacteria (ZB3), a branch within the phylum Margulisbacteria that comprises in addition the WOR-1 and GWF2-35-9 branches (you can spot WOR-1 in the Hug et al. (2016) tree as branching-off from the Cyanobacteria/Melainibacteria group).
Using the FISH technique, they localized this second endosymbiont of Trichoplax H2 exclusively in cytoplasmic vacuoles of the ventral epithelial cells (see part I., Figure 3). These vacuoles contain, in addition to ~20–50 bacteria per cell, numerous membrane-bound vesicles, probably precursors of outer membrane vesicles (OMVs). Thin, tubular structures resembling fimbriae appear to connect the bacterial cells (wide and stout rod-shaped morphology, size ≤1.2 × 0.47 μm) to the host vacuole membrane (Figure 2).
The authors assembled a nearly complete genome of this bacterium (>25 contigs, ~1.5 Mb genome size, average GC-content 37.2%, 1,356 ORFs). As it has so far only been cultivated by its Trichoplax H2 host and not in the lab, its name has to carry the 'Candidatus' prefix. So its official ID is 'Ca. Ruthmannia eludens'. A similar situation exists for the Saccharibacterium 'Ca. Nanosynbacter lyticus' TM7x that can only be grown in coculture with A. odontolyticus XH001 (see the recent post by Mechas Zambrano and Roberto). For the record: with the detection of R. eludens as intracellular symbiont of Trichoplax, Margulisbacteria is the seventh bacterial phylum with intracellular symbionts of animals (the other six being Alphaproteobacteria, Epsilonbacteraeota (formerly Epsilonproteobacteria), Gammaproteobacteria, Tenericutes, Planctomycetes-Chlamydiae-Verrucomicrobia (PVC), and Cyanobacteria).
An aside: I could easily spot the replication origin, oriC, of R. eludens in the 228 bp-long dnaA-dnaN intergenic region, including a twin-R1 type DnaA box" and the DnaA-trio motif (a twin-R1 type DnaA box is a hallmark of predicted cyanobacterial oriCs). This indicates that R. eludens replicates its chromosome in a DnaA-dependent fashion, like most bacteria.
Gruber‑Vodicka et al. addressed the physiology of R. eludens by an all-three-omics approach, that is, they extracted all DNA, mRNA, and proteins from single (DNA, RNA) or a small number of pooled animals (protein), sequenced everything, and assigned peptide and mRNA sequences quantitatively to the reference genomes of Trichoplax and both symbionts. Knowing which genes were actively transcribed and which proteins were actually made allowed them to model the physiology of the complete symbiosis at the time of sampling (see a diagram of the metabolic pathways present in R. eludens and G. incantans here).
According to their modeling, R. eludens is an aerobic chemoorganoheterotroph, with a complete tricarboxylic acid (TCA) cycle that generates energy and biomass from glycerol and the β-oxidation of fatty acids. The R. eludens genome encodes lipases (enzymes that hydrolyze lipids to glycerol and fatty acids) that would allow it to digest lipids but they found neither transcripts nor peptides for such symbiont lipases. In contrast, Trichoplax H2 expressed several lipases, most probably for the digestion of the algae it feeds on, suggesting that R. eludens relies on the lipases expressed by its host.
R. eludens encodes genes for the synthesis of all amino acids, including the nine considered essential for animals. However, its genome does not encode amino acid exporters. Gruber-Vodicka et al. did not see intracellular lysosomal digestion of the symbiont in the epithelial cells. They assume, therefore, that the host takes up OMVs produced by R. eludens via phagocytosis and thus supplements its diet (Elio mentioned this type of "feeding of the host via OMVs" in the Paracatenula–Riegeria symbiosis). The benefits of such putative amino acid provisioning by Ruthmannia are not clear as the animal's algal diet should contain sufficient amounts of essential amino acids. Perhaps the symbiont is useful as a stockpile when Trichoplax is on the move in search of food?
A not-so-stable Company?
Grellia incantans, the endosymbiont introduced in part I, and Ruthmannia eludens inhabit different cell types in their Trichoplax H2 host and appear perfectly well adapted (integration with host metabolism, moderate proliferation). Thus, it is not surprising that Gruber‑Vodicka et al. found both symbionts faithfully transmitted in the Trichoplax H2 lineage; all animals tested at different time intervals always contained both symbionts in comparable numbers. Yet, surprisingly, neither symbiont was found in the Trichoplax H1 lineage, which separated only a few decades ago from the H2 lineage (both haplotypes differ detectably in their mitochondrial DNA, and also in their genomes).
Ruthmannia eludens was not detected in the H1 lineage and no other relative from the Marinamargulisbacteria either (R. eludens sequences were also not found by screening the SRA database for metagenomic samples from various aquatic habitats). So, was it a one hit wonder to find R. eludens in the Trichoplax spp. H2 "Hawaii" lineage? Did *anyone* check the Trichoplax spp. H2 "Panama" lineage that was studied by Kamm et al. (2019) for the presence of R. eludens or of another second symbiont? (the type of question you get on twitter.)
The situation is no less tricky for the Grellia incantans endosymbiont. Stable in the Trichoplax H2 "Hawaii" lineage but not found in the H1 lineage, which harbors (as replacement?) another Midichloriaceae bacterium, similar to the RETA1 phylotype present in Trichoplax adhaerens. Also, the Trichoplax H2 "Panama" lineage is devoid of Grellia but harbors a Midichloriaceae bacterium more closely related to the RETA1 phylotype.
How and where to draw a line in this game of musical chairs? First, Grellia incantans and the Midichloriaceae bacteria are closely related, at approximately the same level as E. coli and Salmonella. Kamm et al. (2019) assume that the small differences in their respective gene repertoires ultimately determine which of the two symbionts is preferred by their Trichoplax host in certain environments. Second, all partners of this company are likely adapted to this musical chairs game through previous encounters. So, unlike an emergent symbiosis or an evolutionary stable symbiosis, this comes close to a meta-symbiosis, in which the eukaryotic and bacterial partners can be constantly replaced by close relatives, because they're already acquainted with the family (I hesitate to introduce a new term like meta-symbiosis, but this might be temporarily permitted in a blog).
In a Nature short communication, Harald Gruber-Vodicka revealed a fun fact that I definitely have to pass on:"We had to travel halfway around the globe to find Trichoplax [in Hawaii]. However, once we learned how to spot Trichoplax, we recovered the same species with the same two symbionts in the marine coral aquarium in the entrance hall of our institute in Bremen." If there ever was a clear case of everything is everywhere... . And it didn't stop here! They found Grellia sp. sequences in metagenomic samples of virtually all aquatic habitats all over the globe in the SRA database. Quoting from Gruber-Vodicka et al. again:"Where do these Grellia live – in other hosts or are they free-living? Is their intra-ER lifestyle more common than we thought?" Nicole Dubilier, co-author of the Gruber-Vodicka et al. study, added not as a warning but in joyful anticipation:"...at least 20 more [Trichoplax] species have been described, and our first results indicate that each host species has its own, very specific set of symbionts." Maybe she had the proverbial German expression in mind: «...einen Sack voller Flöhe hüten» (guarding a bag of fleas) ?
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Kamm K, Osigus HJ, Stadler PF, DeSalle R, Schierwater B. (2019). Genome analyses of a placozoan rickettsial endosymbiont show a combination of mutualistic and parasitic traits. Sci Rep, 9 (1), 17561. PMID 31772223 (Open Access PDF)
Smith CL, Varoqueaux F, Kittelmann M, Azzam RN, Cooper B, Winters CA, Eitel M, Fasshauer D, Reese TS. (2014). Novel cell types, neurosecretory cells, and body plan of the early-diverging metazoan Trichoplax adhaerens. Curr Biol, 24 (14), 1565–1572. PMID 24954051 (Open Access PDF)