A miniature piñata

by Christoph
 

Can you imagine a children's party in Mexico without a piñata as the main attraction? One of the kids, while blindfolded, hits the suspended piñata with a stick until it bursts and the entire contents, usually sweets, splatter onto those standing around waiting expectantly. Microbiologists sometimes throw a party, too, though rarely with a piñata. But here you have it! Cryptomonas gyropyrenoidosa, the egg-shaped miniature piñata that Emma George and her colleagues from Pat Keeling's lab (University of British Columbia Canada) cracked, is only about 15 µm in size (Figure 1A), but seven genomes popped out. Genomic candy. Go figure!
 

(click to enlarge)
Figure 1. Microscopy of C. gyropyrenoidosa SAG 25.80 with bacterial endosymbionts. (A) DIC; (B) DAPI; (C) FISH-M. polyxenophila probe; (D) FISH-G. numerosa probe; (E) overlay of (C) and (D); (F) endosymbionts clustered in the host cytoplasm, including endosymbionts with virus-like particles (Sv); (G) endosymbiont with virus-like particles within the bacterial cytoplasm and attached to the bacterial cell's surface (arrowhead); and (H) bac­terial endosymbionts and a membrane-like structure (putative autolysosome vacuole) that potentially con­tains virus-like particles (arrowhead). Source

Genome numbers in protists   Please bear with me, I will not step into the jungle of protist taxonomy and phylogeny here. Just this: being eukaryotes, these tiny single-celled organisms have a nucleus containing their genome #1. For simplicity, I'll leave out that some have a micronucleus as "permanent storage" and one or multiple macronuclei as "working copies." Then, like almost all eukaryotes, they have mitochondria, containing genome #2. If the protist is from the "phytoplankton branch," you can continue counting. For photosynthesis, these protists have chloroplasts (often called 'plastids') which, like mitochondria, have their own genome. That's now #3. And this is exactly the "basic equipment of genomes" of Ostreococcus tauri, the smallest known green alga with a cell diameter ~0.8 µm, bacteria size.
 

Now it gets a little convoluted. An ancestor of the miniature piñata Cryptomonas gyro­pyrenoi­dosa of the phylum Cryptophyta once phagocytosed (colloquial: ate) a red alga (Rhodophyta) but forgot to digest it completely. Thus, what remained in the former food vacuole is now the remnant of the red alga, whose nucleus has shrunk to a nucleopmorph (genome #3), and within it the chloroplast of the red alga (genome #4). What is special about C. gyropyrenoidosa is that it has in addition to the "basic equipment" of genomes #1−4 three additional ones: those of its two cytoplasmic bacterial endosymbionts Grellia numerosa (#5) and Megaira polyxenophila (#6) and for good measure that of a phage infecting M. polyxenophila (#7). You see G. numerosa (Figure 1E) and M. poly­xenophila (Figure 1D) identified by FISH, phage particles in Figure 1F−H, and a "group portrait" of the seven genomes in Figure 2. Both endosymbionts contain large plasmids, G. numerosa one (136 kb) and M. poly­xenophila two (173 kb, 100 kb), each ~3x the size of the phage genome (38.5 kb)! However, they are by convention not counted as extra genomes, but assigned to the host genome (see the gene numbers in Figure 2).
 

(click to enlarge)
Figure 2. Putative interactions between the MAnky­pha­ge, two bacterial endosymbionts (M. polyxenophila and G. numerosa), and the cryptomonad host. C. gyro­py­renoidosa harbors 4 genomes (in green): nuclear, mi­tochondrial (MT), nucleomorph (NM), and plastid. Mo­bile elements are found in the endosymbionts' chromo­somes and plasmids, particularly in M. polyxenophila. Only G. numerosa encodes a flagellum, but both endosymbionts have type IV secretion systems (T4SS) and ATP/ADP translocases. The endosymbionts and phage also encode eukaryotic-like protein domains like ankyrin repeats (ANKs) and leucine-rich repeats (LRRs). Other putative eukaryotic interactions include the MAnkyphage-encoded protein (DUF3685) with homo­logy to response regulators in plastids and cyanobac­teria. MAnkyphage harbors a type II toxin‑antitoxin system present in M. polyxenophila. The tally of gen­omes in this single-celled cryptomonad comes to 7 plus 3 bacterial plasmids. Source

Genome sequencing allowed George et al. (2023) to reconstruct six of the seven genomes with state-of-the-art bioinformatic methods. The mitochondrial genome (39 kb), the plastid genome (129 kb), and the nucleomorph genome (3 chromosomes, 482 kb) correspond well with known Cryptomonas genomes in size, gene content and gene order. The nuclear genome (~29,000 genes) remained too fragmented to fully reconstruct it, and the authors could thus not derive evidence by pathway reconstructions if and to which extent C. gyropyrenoidosa depends on or profits from harboring its two endosymbionts.
 

The genomes of Grellia numerosa (1.45 Mb) and Megaira polyxenophila (1.73 Mb) are well within the size range and gene content of other known Rickettsiales. The obligate‑parasitic Rickettsiales are known for their reduced capacity for carbon metabolism. Accordingly, G. numerosa and M. polyxe­no­phila have genes for the citric acid cycle (TCA) and for pyruvate decarboxylation but not for glycolysis. Grellia but not Megaira encodes enzymes for gluco­neogenesis and the biosynthesis of various co-factors. Both rely on host metabolites and therefore harbor an array of transporters, including ATP/ADP translocases to import ATP or ADP from their C. gyropyrenoidosa host. In addition to their host dependence, George et al. (2023) detected one instance of dependency among the two endosymbionts: unlike G. nume­ro­sa, M. poly­xe­no­phila lacks two genes, queE and queC, for the synthesis of queuosine, an essential non-standard base in some tRNAs, and may thus depend on provisioning of the precursor 7-cyano-7-deazaguanine by G. numerosa via the YhhQ transporter.
 

It is worth mentioning that lineages of both Grellia and Megaira infect a variety of single-celled and multicellular eukaryotes (algae, ciliates, corals, cnidarians, placozoans). Both together were found, for example, as symbionts in species of the metazoan Hydra, and Grellia incant­ans as symbiont in the metazoan Trichoplax adhaerens (see here in STC). So, without going into the intriguing question of how the stable coexistence of the Megaira-specific phage with the endosymbionts occurs, I will hold that it is not so much the type of endosymbionts that causes fascination here, but the sheer number of genomes assembled in one cell.
 

Piñata or holobiont?   George et al. (2023) say in the abstract: "the Cryptomonas cell is an en­do­symbiotic conglomeration with seven distinct evolving genomes that all show evidence of inter-lineage conflict but nevertheless remain stable, even after more than 4,000 generations in culture." They stopped short of calling this conglomeration a "holobiont." I recalled what Elio said in his essay Paradigm Shifts, Paradigm Drifts back in 2009: "Our thinking has shifted from viewing symbiosis and parasitism in terms of pairs of organisms engaged in a complex cho­reography to a more unitary view in which the two constitute a new whole, a "holobiont."" (reprinted here in STC). The ongoing debate among evolutionary biologists about the validity and usefulness of the "holobiont concept" has been summarized by Stencel & Wloch‑Salamon (2018) in an open-access review that is worth reading but too extensive to recapitulate even briefly here.
 

Let me mention just two points as to why I think the micro-piñata is an excellent system for thoroughly testing the holobiont concept − and the related hologenome theory of evolution at the same time − for validity by all accounts.
 

Cryptomonas gyropyrenoidosa SAG 25.80 is a single-celled eukaryote, and not a multicellular organism that harbors bacterial endo­sym­bionts in distinct tissue cells. Think of the bacteriocytes of aphids housing Buchnera, or think of Drosophila flies "infected" with Wolbachia that colonize female ovaries and male testes (among other organs) and brings along "its" own phage, WO. And then there is the plethora of extracellular symbionts that colonize the guts of their multicellular hosts, the Plautia stali─Pantoea sp. symbiosis may suffice here as an example. Looking through our extensive collection of posts on symbioses between eukaryotes and bacteria, there are only two where the host is a unicellular organism, the alga Paulinella chromatophora with its cyanobacterial endosymbiont, and the ciliate Pseudoblepharisma tenue with two endosymbionts, a green alga and a purple bacterium. It would be just wonderful, scientifically speaking, to study in more detail how a symbiosis of a single-celled organism with two intracellular bacteria, one with a phage companion, plays out.
 

Cryptomonas sp. SAG 25.80 was isolated by the eminent protistologist Ernst G. Pringsheim (1881−1970) in the pre‑1970s and continuously grown in isolation via serial transfers ever since (no periods of cryopreservation). Although not verifiable in retrospect, George et al. (2023) assume that the bacterial endosymbionts of the Cryptomonas alga and the phage were already present at the time of isolation; symbionts and phage were discovered in 1990. Approximately 4,000 generations of a stable endosymbiotic conglomeration with signs of ongoing inter-lineage conflicts sounds quite like a precariously balanced and therefore lively and evolving system. How I would like to learn how this system reacts to changing environmental/cultivation conditions! Can the endosymbionts be "cured" (by antibiotic treatment) and, maybe, replenished from freshwater Rickettsiales? This could be the decisive point whether it is really a holobiont with tenured symbionts or rather a conglomerate with bacteria as time-contracted seasonal workers.
 

A unique Piñata?   You may wonder whether the Cryptomonas piñata is something special in the protist universe. Most certainly not, although seven different genomes held stably in one unicellular species over many generations is unprecedented. Many of the larger protists, especially ciliates, are known to hoard smaller protists and bacteria, and it is often difficult to distinguish whether such "roommates" are symbionts, even transient ones, or just food. Perhaps the ciliates decide this on a case-by-case basis.
 

Figure 3. The ciliate Paramecium bursaria has a mutu­alistic endosymbiotic relationship with green algae cal­led Zoo­chlo­rella. The algae are clearly visible in ciliate cytoplasm. Light microscopy, differential interference contrast (DIC). Note the size scale. Source

To give you a well known example: see in Figure 3 the ciliate Paramecium bursaria filled with well over 100 Chlorella algae. Kodama & Fujishima (2010) say in their review: "Each symbiotic Chlorella species of Paramecium bursaria is enclosed in a perialgal vacuole (PV) membrane derived from the host digestive vacuole (DV) membrane. Algae-free paramecia and symbiotic algae are capable of growing independently and paramecia can be reinfected experimentally by mixing them." So, Paramecium ciliates appear as (comparatively) huge piñatas but are cer­tainly not holobionts in the strict sense (scientese: sensu stricto), with respect to the Chlorel­las. However, Merry told us at STC in her 2009 post Life in a Big Mac of obligate intracellular bacterial symbionts of several Paramecium species, Holospora obtusa, that "populate" the ciliate's macronuclei. Intriguingly Holospora belongs to the same clade as the Cryptomonas endosymbionts, that is, the Rickettsiales that also includes Midichloria, and Wolbachia. Whether these Holospora symbionts actually make Paramecium a holobiont is doubtful be­cause, as known from Chlamydia, they also have an extracellular life phase that allows them to reinfect their host once they are killed when a Paramecium destroys its macronuclei before cell division, retaining only the symbiont-free micro­nucleus.
 

In conclusion, we are left with the impressive number of genomes contained in the Cryptomonas gyropyre­noi­dosa piñata.... and a similar number of questions regarding a meaningful definition of "holobiont" for protists.
 

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Note   Pictured on the frontispiece are several Cryptomonas gyropyrenoidosa cells, a reproduction of an artwork by Ben Darby. Those of you who have read Merry's book Thinking Like a Phage will certainly recognize his style, and you find more of his phage portraits on his webpage. Worth a visit!