A Korarchaeote yields to genome sequencing

Archaea have always been hard to study. The cultured instances of that group tend be from environments that to us seem atrocious, with high temperatures, and hypersaline and anoxic conditions. Molecular studies of environmental gene sequences have taught us that these organisms are all around us, but they almost never can be cultured. Reading the article by Elkins et al. (1) in a recent issue of PNAS, I was reminded that the Archaea have been causing problems since they were recognized by Carl Woese in 1977 (2). Of course, the problems are of the best kind: those that stimulate thought, cause consternation among the establishment, demand that more data be gathered, and result (when new data are presented) in new ideas (and perhaps a renewal of the “cycle of consternation”). As an outsider to the archaeal world who always suspected that such organisms indeed were somewhat “different” from the familiar bacteria on which I work, this entire process has been both interesting and entertaining, and perhaps made me just a little thankful that I was not dealing with any of these phylogenetic “thugs” we now call Archaea.

But why is it that the Archaea are sometimes problematic? For one, with the exception of the extreme halophiles, some methanogens, and a few extreme thermophiles, there are so few of them in culture. Especially among the thermophiles, when an organism is cultivated, the small archaeal scientific community coalesces on that strain rather than exploring others: accordingly, much of our information about the group has focused on comparison of only a few different genera and species, with comparatively few questions asked about variability within a genus or species. At the onset this did not seem to be an important issue, but with time, it has become clear that an immense amount of diversity exists even among strains of a given species, with only a small percentage (usually ≈35%) of the total gene complement being shared by all strains [e.g., when three genomes of Escherichia coli were compared, <40% of their proteins were shared by all three (3)]. It is becoming clear for the few groups with many strains sequenced that this is the rule rather than the exception. Thus, the question begins to loom about how much variability exists between similar species isolates of the deeply branching Archaea—a question that remains unanswered because of the lack of cultivated strains.

With these opening thoughts in mind, the recent article by Elkins et al. (1) puts a new and exciting slant on the Archaea with the sequencing of an uncultivated member of the Korarchaeota, Candidatus Korarchaeum cryptofilum, or “Ca. K. cryptofilum” as it is referred to in their article. First detected in 1996 in Obsidian Pool, a hot pool in Yellowstone Park, and seen as notable by their unusually deeply branching ribosomal RNA sequences in phylogenetic trees (4), korarchaeotes have since been detected molecularly from hot springs around the world. Organisms specifically related to the hot spring korarchaeota have not been detected at low temperatures, and no korarchaeote has been successfully brought into pure culture. This article is of note because it is the first complete sequence of a korarchaeal genome, from an organism only remotely related to any known microbe, and was obtained by whole-genome shotgun (WGS) sequencing of an enrichment culture—pure culture has remained elusive. Nonetheless, with the genome sequence in hand, one can move from the world of conjecture into one of considerable knowledge, about this bug, its metabolism, its phylogeny, and its evolutionary past, all of which pose further puzzles.

Very little diversity within the sequences of Ca. K. cryptofilum was observed.

But some aspects of the article demand comment beyond the intellectual delicacies that have been served up from the sequence data. For example, it is a splendid example of the coupling of solid microbiology with molecular genetics. The star of this show, Ca. K. cryptofilum, yielded to analysis only after more than a decade of enrichment culture work [the organism was first reported in 1997 (4)]. Although a pure culture has not yet been obtained, Elkins et al. (1) were cleverly able to select for it over other microbes in the enrichment with their discovery that this microbe was extraordinarily resistant to lysis by sodium dodecyl sulfate (SDS). It was then possible to treat enrichments at each passage with SDS, thus increasing the abundance of the korarchaeote. When sequences were assembled, >85% of the sequences appeared to be from this microbe alone—a remarkable achievement.

A second remarkable issue is that after WGS sequencing of the enrichment, very little diversity within the sequences of Ca. K. cryptofilum was observed. This is quite different from recent reports of the natural diversity of bacteria in the oceanic niche (5, 6), all of which suggest that there is an immense amount of diversity within a given species phylotype. After WGS sequencing of this enrichment culture, however, ≈85% of the sequences assembled without problem into a coherent sequence. It was as if the Ca. K. cryptofilum was a pure culture. Perhaps it was: either that, or the starting inoculum was of low diversity, and one strain was strongly selected by the enrichment method. Given the long pathway to these results, none of these speculations can be verified, although it would be of great interest to take a specific FISH (fluorescence in situ hybridization) probe for Ca. K. cryptophilum and return to the original sample to confirm the abundance and homogeneity of the sample. If it turns out that there is a low diversity, it may suggest that the archaeal diversity in these environments is fundamentally different from that reported for Bacteria in the marine and other environments. If the diversity is much higher, then one has to admit that the work reported here must be regarded as a study of the one microbe that yielded to the microbial expertise, and perhaps to only one strain of this microbe. The korarchaeotal rRNA sequences obtained from Obsidian Pool are not very diverse; all are related at about the species level of traditional taxonomic rank. But sequences gathered worldwide are much more diverse, appropriate for a deeply branching group such as Korarchaeota. This suggests that if these bugs are like the rest of the microbes so far analyzed, there will be abundant metabolic diversity in this group as well.

The article by Elkins et al. (1) itself reveals fascinating and, to some extent, enigmatic things about the phylogeny and evolution of Ca. K. cryptophilum—on the one hand (based on small and large subunit rRNA sequences, RNA polymerase structure, etc.) the bug is strongly aligned with the Crenarchaeota, whereas on the other hand (cell division, tRNA maturation, and DNA replication) it is more similar to studied examples of the other large group of archaea, the Euryarchaeota. However, it is not particularly satisfying to place this very interesting microbe into a separate lineage based on some of the very important conserved genes usually used to guide such actions. Indeed, in some properties the organism seems to straddle the line between the two groups. For instance, studied euryarchaeotes wrap their DNA in histones much as do eukaryotes, whereas crenarchaeotes use other basic proteins. Ca. K. cryptophilum has genes for both kinds of DNA-binding proteins. Although the organism appears to be more closely aligned in its genetic machinery with the Crenarchaeota, it was not possible to align it specifically with either group, and the suggestion was made (quite properly) that it may in fact be derived from a deeply branching predecessor to both of the other groups. What one ends up with is perhaps a view of the korarchaeotes that is somewhat similar to that shown in Fig. 1; surely, more data will begin to offer more insights, but this article is a huge step forward in our understanding of the phylogeny of the Archaea.

Fig. 1.

One view of the position of the Korarchaeota in the Archael domain. Although in many ways they align closely with the Crenarchaeota, genomic data (1) seem to suggest that they are a hybrid of the Crenarchaeota and the Euryarchaeota as shown here.

Metabolically, the data are, at first glance, much less ambiguous. Ca. K. cryptophilum is apparently a heterotroph, living primarily via anaerobic metabolism of peptides and amino acids. It has a nice array of enzymatic equipment, including transport systems, peptidase, transaminases, etc., along with several modes for proton pumping substantiating this lifestyle, although it lacks a critical proton translocating ATPase. The fact that this much information can be gleaned from the sequence of a microbe that has yet to be cultivated is quite remarkable and is reason enough for rejoicing, and a tribute to the talent and persistence of the authors. However, from the point of view of evolution of metabolism, it is less satisfying: The microbe would appear to be either an obligate symbiont (incapable of synthesizing a wide variety of cofactors and vitamins, as well as purines, and perhaps needing a partner to remove hydrogen) or an excellent scavenger. That is, although the highly conserved genes and molecular machinery suggest that it is a deeply branching archaeon, perhaps aligned with the crenarchaeota, its metabolism looks perplexingly “modern”: Looking at what it can do, it is not easy to imagine early microbes eking out an existence in hot springs via the metabolism of amino acids and magically finding sources of purines, vitamins, etc.

Thus, we are left with a conundrum: How did this apparently anciently diverging microbe evolve to be a heterotroph, dependent on amino acids, and devoid of biosynthetic abilities for many important things? Was it a recent evolution—adapting to a high-temperature niche that yielded abundant supplies of amino acids and peptides as less robust microbes lysed in Obsidian Pool? Is it a symbiont with an autotroph that supplies it with the “essentials” (amino acids, vitamins, purines, etc.) in exchange for hydrogen? Or is it just the first of many Korarchaeotes we will eventually encounter, leading to an appreciation for the entire group?

As with many exciting new findings, we are left with more questions than answers, and one can look forward to, with new enrichments, cultures, and sequenced genomes, answers to some of these questions, and for new questions to appear. As the first published genome of E. coli did not tell us about the vagaries of that group, it is only appropriate that the first Korarchaeal genome leaves us wanting more.