Wednesday, May 01, 2013

Deep-Sea Vents: The Mosquito Connection

Quick: What species of life on earth is the most abundant? (Which species has more living members than any other species?) Hint: If an alien probe lands in a random location on earth, chances are better than 70% that the probe will encounter this organism.

If you're thinking in terms of the ocean, you're on the right track. What may surprise you is the connection between the world's-most-populous-organism (to be revealed shortly) and the mosquitoes that've been dive-bombing your neck all week. Equally amazing is the link between the mosquitoes in your back yard and hydrothermal vents in the ocean floor.

The hundreds of bright little particles at the
narrow end of this wasp egg are Wolbachia cells.
I wasn't thinking about marine biology or deep-sea hydrothermal vents when I went online at the other day to do a little nosing around into the genome of Wolbachia pipientis, the ultra-tiny bacterial parasite carried by nearly every mosquito on earth. (Caution: Don't attempt the following DNA-analysis tricks on your own unless you want to become thoroughly addicted to desktop omics. I'm a microbiologist by training. I can do these stunts safely.) "Parasite" is actually the wrong word. Our tiny friend Wolbachia doesn't just parasitize the mosquito; it's an integral part of the mosquito. Wolbachia can't live outside its insect host—and guess what? The host frequently can't live without Wolbachia. The two provide essential services for each other, an arrangement known as mutualism.

I would argue that Wolbachia is more than a mutualistic symbiont: It's a proto-organelle, something very close to what Lynn Margulis had in mind as the ancestor of today's mitochondrion.

Wolbachia can't live on its own in the outside world (as far as anybody knows): it needs to live inside a host (generally an arthropod, although filarial worms also carry Wolbachia). Inside its host it occupies a very special niche: It lives in the nursery cells of the insect's ovary—the cells that will go on to become egg cells.

This is no ordinary symbiosis. I mentioned in an earlier post that Wolbachia carries with it genes for reverse-transcriptases, resolvases, recombinases, transposases, translocases, DNA polymerases, RNA polymerases, and phage integrases—a complete suite of retroviral machinery, designed for export of foreign DNA into host DNA. And indeed, researchers have found that Wolbachia DNA is quite often embedded in the host's own nuclear DNA. (One group, looking at four insect hosts and four nematode hosts, found anywhere from 500 base-pairs to over a million base pairs of Wolbachia DNA residing in the nucleus. Another group found 45 Wolbachia genes incorporated in a fruit-fly host's nuclear DNA.) The situation with Wolbachia thus parallels the situation with mitochondria, where we know that 97% of the gene products that go to make up a mitochondrion are actually encoded in nuclear DNA, not mitochondrial DNA.

When you encounter an organism as baffling as Wolbachia, oftentimes you want to know what its relatives are—what it's most closely related to. When a new or poorly understood organism has a close relative that's already well-studied, sometimes you learn a lot in a hurry. That's particularly true of pathogens (not that Wolbachia is a pathogen per se). Pathogens have virulence strategies of various kinds. Maybe Wolbachia has symbiosis strategies that it learned from a relative?

The problem with a lot of the super-tiny microbes (which Wolbachia definitely is, with only a quarter as much DNA as E. coli) is that their relatedness is not always well understood. Organisms are assigned a taxonomic slot, then the assignment changes a few years later, after they're better-studied. (So for example, Cowdria ruminantium was eventually renamed Ehrlichia ruminantium, and a bunch of former Ehrlichias are now Neorickettsias, except the ones that attack red blood cells, which are now Anaplasmas.) Taxonomy at this end of the evolutionary tree is definitely a work in progress.
Deep-sea thermal vents like this one
are home to organisms like Thiomicrospira
that can grow on sulfide, CO2, and basic salts.

Fortunately, it's easy nowadays (what with so many organisms' DNA sequences available online) to go on the web and compare genomes directly, using a tool like SynMap, which is what I started doing with Wolbachia. I started going down the list of mini-microorganisms and began running DNA similarity tests of Wolbachia against Ehrlichia, Neorickettsia, Anaplasma, Chlamydia, and "the usual suspects" at the ultra-small-chromosome end of the tree of life.

What I found surprised me. A bizarre little bacterium called Thiomicrospira kept showing up in my BLAST searches as having many genes in common with Wolbachia (based on sequence matches in large numbers of genes). None of the taxonomy charts showed the two to be related. But DNA doesn't lie. I kept coming up with matches across hundreds of genes. (Bear in mind, Wolbachia has only about 1300 genes to begin with, which is very small, even for a bacterium.)

What's bizarre about Thiomicrospira is that it's one of those fairly newly discovered microbes that lives on sulfur, heat, and CO2 at the bottom of the ocean, in total darkness, in the vicinity of thermal vents. Thiomicrospira is the kind of life form NASA takes a great interest in, because it could be a prototype for exactly the type of survive-in-the-dark CO2-using organism that might live under the ice crust of Europa (Saturn's moon). In theory, there could be geothermal vents on the floor of the large ocean of liquid water that NASA is pretty sure exists under Europa's ice. If there's life down there, it could very well look like Thiomicrospira.

But why should Thiomicrospira have so many genes in common with a mosquito symbiont? Thiomicrospira organism lives at the bottom of the ocean; Wolbachia lives inside arthropod eggs. One obtains its carbon in the form of CO2; the other produces CO2 as a waste product. One is adapted to live in warm salt water; the other lives in cold-blooded insects. In theory, these two germs couldn't be further apart. And yet, oddly enough, they not only have hundreds of genes in common, the genes are well-matched from a DNA sequence-similarity standpoint. Thiomicrospira's DNA even incorporates a prophage module, and some of its phage genes show a high percentage base-pair similarity with the phage genes of Wolbachia. (See screen shot below.)
Remarkably, Thiomicrospira and Wolbachia share certain phage genes in common, as shown here. The genes have a DNA sequence identity of about 60%.
After doing a little more detective work, I found an organism that might very well form a "missing link" between the mosquito symbiont and the thermal-vent dweller. This organism kept showing up in my analyses as having a high degree of DNA similarity with both Thiomicrospira and Wolbachia. The organism in question is Pelagibacter ubique (now known as Candidatus pelagibacter, although some might question this taxonomic assignment since all other Candidatus members are obligate intracellular symbionts), and it's an astonishing organism in two ways: First, it's the smallest non-parasitic (free-living) bacterium known to science, with only 1.3 million base-pairs in its DNA (making it slightly smaller than Wolbachia and its tiny cousins). Secondly, it's the most numerous living thing on earth. It's present in large amounts in every one of earth's oceans.

Pelagibacter was placed in the Candidatus clade in 2007 due to its small genome and cell size and certain ribosomal markers. It has a very mitochondria-like genetic profile, and in fact some people think Pelagibacter is the ancestor of today's mitochondrion, a theory that's all the more satisfying when you consider that Pelagibacter is both ancient and tied to the sea.

My analysis using SynMap found that Pelagibacter and its thermal-vent-dwelling cousin Thiomicrospira share about 660 genes (out of 1480 or so for Pelagibacter), whereas Wolbachia and Pelagibacter share around 581, and Thiomicrospira and Wolbachia share around 1000. These are so-called non-syntenous point matches between genes; instances where the same gene occurs in both organisms, with a high percentage of base-pair matching. Synteny is a concept that takes gene-matching one step further and says that clusters of similar genes are what count. Synteny at the level of higher plants and animals is one thing, but at the level of a mini-microbe it tends to lack meaning, because the genes of bugs like Wolbachia are notoriously mobile: They find new positions on the chromosome over time (probably because of the large number of transposases, nucleases, and integrases in the genome). Even so, I decided to carry out a bit of syntenic analysis to see what I could find out.

For purposes of my analysis I defined a "syntenon" as three or more co-proximal genes that match three or more genes on the other organism's genome. But to be part of a syntenon, all three genes in a triplet have to occur within a 30-gene span (and match 3 genes in a 30-gene span on the other organism's DNA) plus the genes have to be in the same order in both organisms.

A planet-spanning waterworld is thought to exist under
Europa's icy outer crust. If thermal vents exist at the
bottom, any life that exists may look a lot like Thiomicrospira.
Using SynMap, I found that whereas Wolbachia and Pelagibacter share around 157 syntenic genes, and Thiomicrospira and Wolbachia share around 132, Thiomicrospira and Pelagibacter share 250 (which makes sense in that both are ocean-dwellers). For comparison-and-control purposes, I did a triplet match of Thiomicrospira against another chemoautotroph (an organism that gets energy from inorganic chemicals, and carbon from CO2), namely Methanothermobacter marburgensis. There were only 53 syntenic triplets in common between the two chemoautotrophs. (Between Wolbachia and Methanothermobacter, on the other hand, there were only 3 triplet-matches.) Doing a match between two Wolbachia species (a mosquito-dwelling variety and a fruit-fly-dwelling cousin) produced 522 gene matches in syntenic triplets.

It seems reasonable to me, based not just on the previous sorts of analysis but also direct inspection of the genomes (in terms of their respective protein products), that Thiomicrospira evolved from PelagibacterPelagibacter is the most abundant life form in the ocean, and perhaps the oldest. Pelagibacter is also very mitochondria-like, and so is Thiomicrospira, which has rhodanese-like proteins, the full cytochrome system, redox enzymes, citric-acid-cycle enzymes, plus certain characteristic membrane and sensor proteins, flippases, etc. (For what it's worth, Thiomicrospira has the highest signal-transduction profile I've ever seen at, again making it very mitochondrial-feeling.)

I'm tempted to say, similarly, that Thiomicrospira and Wolbachia are related. They have phage proteins in common. They both have genes for patatin proteins. They share multiple drug resistance genes. (That's not so strange. Antibiotics occur naturally in the environment.) They share genes for Flp-type pilins. Plus many more coincidences, big and small.

At first blush, a deep-sea thermal vent seems pretty far removed, environmentally, from the egg cell of a mosquito. How to reconcile the difference? Actually, I see similarities. Thiomicrospira thrives at temperatures of 28 to 32 degrees Celsius (which is also true of mosquitoes, although they prefer the 28-degree end of the scale). And blood (the preferred food source for mosquitoes) is comparable in pH and salinity to seawater. Also, mosquitoes have an aquatic lifecycle: they require brackish water in which to lay eggs. Mosquitoes and salt marshes go back millions of years.

It's even possible that Wolbachia might live in deep-sea-vent-dwelling host organisms. In fact, I predict they will be found there. Why? Because in addition to inhabiting flying insects, spiders, mites, and ticks (and filarial worms), Wolbachia have also been found in a very high percentage of crustaceans. We know that crustaceans are often found living near deep-sea thermal vents; and many crustaceans show the characteristic feminization of genetic males that's so often the tipoff to a massive Wolbachia presence in insect populations.

Insects and crustaceans represent two of the oldest, most successful, and most widely distributed life forms of the animal kingdom. Would it really be so surprising if the bacteria that colonize these life forms are closely related to the most common marine bacteria on the planet? I don't think so. Stranger things have happened.