A lesson from the comb jelly, deep-sea bacteria, and some obliging E. coli on how to thrive under pressure

by Janie
 

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Figure 1. Plas­mal­­o­­gens like plas­menyl­ethan­ol­amine have acyl chains linked to the gly­cer­ol by an O-alk-1'-enyl ether bond, as op­posed to an ester bond. Source

I sometimes google "Small Things Considered <a biological term of choice>" looking for older articles, to see if any paradigms have changed in the span of a decade or so. In doing so, I came across this post from 2010 on plasmalogens, membrane lipids with an ether bond linking the glycerol to the acyl chain instead of a typical ester bond (Fig. 1). Plasmalogens evolved early on, but reactive oxygen species (ROS) became a problem for their ether bonds when oxygen increased in the Earth’s atmosphere, hence the loss of plasmalogens in many modern organisms. Interestingly, some animals do retain these odd lipids.
 

The old post posed the following question: "Why are plasmalogens needed by animals?"
 

I was pleased to find that some recent tangents I had managed to get lost in – mechanisms of buoyancy, jellies that are not related to jellyfish, internet-celebrity fishes, and piezophiles – led to an answer. (No such thing as useless knowledge!)
 

 

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Figure 2. An example of a ctenophore (aka "comb jelly"). Source

Deep-sea fish live in what us humans would consider crushing pressure, thriving despite the weight of the oceanic expanse above. One of their physiological adaptations is their lack of the air-filled swim bladders that other fish use to adjust their depth in a column of water. Under less extreme pressure conditions, inflating or deflating these gas-filled sacs allows fish to decrease or increase their density relative to the water surrounding them, and this change in buoyancy enables them to rise or sink as they wish. (Planktonic cyanobacteria and archaea use proteinaceous gas vesicles for the same purpose!) The key problem here for deep-sea denizens is the compressibility of gases – if deep-sea creatures had such air-filled balloons, the gases inside would be compressed by the immense pressure, and the water surrounding these organs would push straight through. (Were we to teleport to the ocean floor with no protective gear, our own air-filled lungs would meet the same fate.) So instead, deep-sea creatures are filled with water and are therefore not subject to fishy implosion, since the pressure inside their bodies matches the pressure outside.
 

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Figure 3. Some ctenophore morphologies. Top left: Bathocyroe fosteri, a Beroid (source). Top right: Mertensia ovum, a Cydippid (source). Bottom left: Cestum veneris, aka the Venus girdle, the largest of all known ctenophores (source). Bottom right: Several Coeloplana astericol, a Platyctenid, attached to a starfish (source).

This is not the case for ctenophores. These "comb jellies" are translucent, bioluminescent marine invertebrates that constitute one of the earliest branching animal lineages. The classic ctenophore looks somewhat like a big iridescent fennel seed, with eight rows of fused cilia that beat rhythmically in order to propel the animal along (Fig. 2; see this mesmerizing video from the Monterey Bay Aquarium!). They come in a whole hodge-podge of shapes, from the classic double-lobed Beroida to the egg-shaped Cydippids to the ribbon-shaped Cestida to the flat and rather mussel-like Platyctenida (Fig. 3). Their size, too, varies widely, from mere millimeters to over a meter in length. The diversity of morphologies is surprising, given that fewer than two hundred species have been identified to date.
 

How these gelatinous creatures regulate their buoyancy as they move around the water column remains a puzzle. They lack swim bladders, for one, like many other fellow fauna in their deep-sea vicinity. A couple species are known to adjust their densities when shifted from higher salinity water to lower salinity water or vice versa, taking between three to twenty hours to return to their natural floating state. This could be via their ability to alter their levels of calcium and sulfate ions in order to maintain proper osmotic pressure. In any case, the depth distribution of ctenophores varies widely, with some species limited to shallow coastal waters, others inhabiting waters deeper than 10,000 meters, and yet some happy-go-lucky others with less of a preference, presumably moving about the depth range with ion-based buoyancy adjustments.
 

These various ctenophores experience not only different pressures but likely also different salinities (generally, the deeper you go into the sea, the saltier it gets, especially if you've hit a halocline). Might an organism's regulation of osmotic pressure also tie into withstanding pressure from the weight of the ocean? Can increasing osmolyte concentration inside of a cell help counteract that immense pressure experienced deep in the sea?
 

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Figure 4. Top: Effect of 10 μM TMAO or TMA on the growth and survival of strain D25 at different pressures. The cell number at the start of the incubation (start) was ca. 1 × 10⁶ CFU/ml. The cultures were incubated at 4°C for 10 days, and then CFU was counted and compared with that of the starting culture (end/start). The red line refers to a complete death of cells in the culture. Control: Strain D25 cultured without TMAO or TMA. Bottom: Effect of expression of TmaT and MpTmm on the growth and survival of E. coli ΔtorCAD (C) and B. subtilis 168 (D) cultured at different pressures for 48 hours. The cell number at the start of the incubation (start) was ca. 1 × 10⁵ CFU/ml. The strains were cultivated at 25°C for 48 hours with or without 20 μM TMA, and then CFU were counted and compared with those of the starting culture (end/start).

The classic fishy smell is linked to one such dual-purpose agent. Trimethylamine N-oxide (TMAO) – the precursor of the "fish smell" culprit molecule trimethylamine that is produced when TMAO meets metabolizing bacteria – is a common osmolyte. Marine animals rely on such molecules to maintain proper cell volume amid a very salty environment. In fish, there's a linear correlation between how much TMAO is stored in its tissues and how deep in the sea it lives, regardless of phylogenetic lineage or geographical location. It turns out that TMAO strengthens the structure of water molecules under pressure and prevents further compaction, thus serving not only as an osmolyte, but also as a "piezolyte." This is certainly taken advantage of by bacteria. Take, for example, the deep-sea bacterium Myroides profundi D25, whose accumulation of TMAO aids survival at high pressures (Fig. 4). Even regular E. coli and B. subtilis equipped with the M. profundi TMAO transporter and monooxygenase system are better able to tolerate high pressures (Fig. 4).
 

Ctenophores, too, might use TMAO for both osmo-protective and piezo-protective purposes. They do seem to possess enzymes for synthesizing TMAO: A quick BLAST search for fish homologs of FMO3, which is a human TMAO synthase, yields many flavin-containing monooxygenase sequences, and then BLASTing those against only ctenophore sequences yields a handful of hits with e-values <10^-15.
 

The incredible resilience of pressure-adapted cells is context-dependent, however. When deep-dwelling species are brought to atmospheric pressure, their tissues often fall apart. Ctenophores are no exception, as reported in a paper published just a few months ago. A more famous example of this might be the fish Psychrolutes marcidus, dubbed the blobfish for its appearance when it is fished up from the deep sea (Fig. 5. But surely moving any creature adapted to pressures of 10 MPa into atmospheric pressure (0.1 MPa) will turn it into something of a blobfish?).
 

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Figure 5. Psychrolutes marcidus, the "blobfish," in its normal deep-sea habitat (left; source) versus at atmospheric pressure (right; source).

The reason for this (at least for ctenophores)? It's what this piece started off with: plasmalogens.
 

Ctenophores have cell membranes chock-full of the plasmalogen plasmenyl phosphatidylethanolamine, whose acyl chains flare outwards at atmospheric pressure. However, in a ctenophore's native deep-sea habitat, the pressure forces these conical lipids into a cylindrical shape, so that they form what looks like a textbook bilayer membrane comprised of phospholipids all neatly lined up (Fig. 6). This plasmalogen behavior is binder-clip-like: You can squeeze down on a clip's steel loops (~phospholipid tails) so that the side profile of the clip becomes a straight line, but as soon as you release the applied pressure, the clip will jump back into its usual flared shape.
 

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Figure 6. (From Figure 4 of the paper) (F) A homeocurvature adaptation model in which a more negative baseline curvature is required to offset the effects of high pressure on lipid shape. (From Figure 5 of the paper) (C) Growth of PPE-lacking (gray) and PPE-containing (green) E. coli in microaerobic culture under pressure. (D) Post-decompression survival was similarly rendered pressure insensitive by PPE. Source.

This is a fascinating twist on membrane strategies for living under high pressure. Upping the proportions of unsaturated fatty acids in membranes, of course, prevents overly tight packing and keeps membrane fluidity intact. But upping conical unsaturated fatty acids levels induces a dramatic membrane curvature that, when tamped down by high pressures, results in a perfectly functional membrane. Thus, conical phospholipids contribute to pressure tolerance in ctenophores. This principle holds up in E. coli, too: When E. coli is engineered to express a reductase that converts the cylindrical membrane lipid phosphatidylethanolamine into the conical plasmenyl phosphatidylethanolamine, the cells tolerate higher pressures (Fig. 6).
 

The paper also managed to disentangle cold adaptation from pressure adaptation. Of ctenophores that live in similarly cold temperatures, those that live in shallower depths have a higher membrane proportion of phosphatidylcholines with short acyl chains, while those that live in deeper depths have higher proportions of conical plasmalogens. So, pressure adaptation is one answer to the question posed at the start of this post, the question of why animals have plasmalogens!
 

It turns out ctenophores also have an answer for a related question: "How do animal cells that make reactive oxygen species avoid the potential damage they can do to plasmalogens?" This is a moment of redemption for the comb jelly's bioluminescence, which has otherwise taken the backseat in the public eye. It may be flashy light-scattering that is responsible for the creatures' photographer-attracting, stunning lightshows, but it is the more unassuming bioluminescence that helps neutralize reactive oxygen species (a phenomenon paralleled in deep-sea bacteria), perhaps facilitating the ongoing existence of plasmalogens in such pressure-resisting membranes.
 

This story of the ctenophore (with the help of some supporting-character bacteria) is a fascinating example of the unexpected ties that can exist between seemingly tangential biological mechanisms.
 

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