Chiggers, Scrub Tyhpus, and Pseudogenes

If you've ever been bitten by tiny red bugs in the garden, you're familiar with members of the Trombiculidae, a family of mites known variously as berry bugs, harvest mites, red bugs, scrub-itch mites, aoutas, or (in the southern U.S.) "chiggers."

In the United States., the garden-variety chigger is basically harmless, but in much of the world this tiny arthropod comes with a very nasty endosymbiont known as Orientia tsutsugamushi, which is a bacterium related to the Rickettsia organisms that cause various tick-borne diseases. Throughout much of the Orient, O. tsutsugamushi infections (from chigger bites) cause scrub typhus, which begins with a rash and fever but can progress to a cough, intestinal distress, swelling of the spleen, abnormal liver chemistry, and ultimately pneumonitis, encephalitis, and/or myocarditis and even death. Treatment with doxycycline, azithromycin, or chloramphenicol is usually successful.

The "harvest mite" (chigger) can carry scrub
typhus, although U.S varieties are typically harmless.

The sequenced genome for O. tsutsugamushi is available, and if you go to this link and click on "Click for features" at the bottom of the Dataset Information box you should be able to open up a table that shows the organism as having 1,182 protein-coding genes (quite a small number), plus an additional 1,994 pseudogenes (quite a huge number, by comparison). The "DNA Seqs" links in the table will let you download the DNA sequences of all the organism's genes and pseudogenes.

This is an extremely unusual situation, in that we're dealing with a bacterium that has more pseudogenes (switched-off, defunct, damaged genes) than regular genes, something that can be said of no other bacterium of which I'm aware. The leprosy bacterium (Mycobacterium leprae) is famed for having approximately 1100 pseudogenes and 1604 "normal" genes. Astonishingly, Orientia tsutsugamushi reverses that ratio, and then some.

We don't know for sure how old Orientia tsutsugamushi's pseudogenes are. A standard rule of thumb in biology is that microbial genomes experience one spontaneous mutation per chromosome per 300 generations. But this doesn't really help us decide how old Orientia's pseudogenes are, since the pseudogenes probably didn't arise one by one, indepedently, through accumulation of random mutations. More than likely, a massive pseudogenization event caused the simultaneous deactivation of a large, unknown number of the organism's genes (of which 1100 survive today as pseudogenes), much the same as has been hypothesized for M. leprae. We have good reason to believe M. leprae's pseudogenes are at least 9 million years old. It seems likely that the pseudogenes in Orientia are also quite old, or at least not terribly new.

To get more perspective on this, I analyzed Orientia's pseudogenes from a couple of perspectives. What I found, first of all, is that the pseudogenes are shorter than their non-pseudo counterparts, averaging 700 bases in length (versus 879 for normal genes). This is similar to the case with M. leprae (where pseudogenes are 795 bases long and normal genes average 1,098). The average shorter gene length for Orientia vis-a-vis M. leprae is consistent with the fact that this is a greatly gene-reduced low-GC (30.5%) endosymbiont, whereas the Mycobacterium family is (in theory) free-living, with higher GC content (57.8% for M. leprae; 65% or more for tuberculosis species).

I've written before about the fact that in most genes, in most organisms, codons tend to begin with a purine base. Therefore I decided to look at purine usage in base one of normal-gene codons versus pseudogene codons (pseudocodons?), finding the following distribution in normal genes:

Purine usage in base one of codons in Orientia tsutsugamushi (N=346,326 codons). No pseudogenes were included in this graph. See the next graph (below) for pseudogenes.

This graph leaves little doubt that most codons begin with a purine (A or G). The median AG1 value is 63.8%. Very few proteins lie to the left of x=0.50, and frankly some of those are probably misannotated as to reading frame.

The situation with pseudogenes is quite a bit different:

Purines in codon base one (AG1) of pseudogenes (N=462,933 codons) in Orientia.

Here we see that purine usage in codon base one is not as strong (median 58.4%), although clearly, plenty of codons still show AG1 above 60%, implying that many pseudogenes are still "in frame" (not frameshifted).

Interestingly, AG1 is not only higher in normal-gene codons than in pseudogene codons, it's also higher in codons associated with proteins of known function than for "hypothetical protein" genes. Only 41.3% of pseudogene codons have AG1 greater than 60%, whereas 66.7% of "hypothetical protein" genes have AG1 > 60% and 84.3% of genes with functional assignments have codon AG1 greater than 60%. This implies that some genes annotated as hypothetical proteins may, in reality, be pseudogenes that are incorrectly annotated. I'll return to that topic some other time.

The Pseudogene Hall of Fame

For "budget reasons" (supposedly), the Joint Genome Institute now requires all users to be registered and approved before they can use the (taxpayer-funded) https://img.jgi.doe.gov site. Fortunately, my registration was approved and I can use the excellent online genomics tools there, one of which produced the following table of Top Ten Organisms by Pseudogene Count.

Organism	Genes	Pseudogenes
Mus musculus C57BL/6J	60745	11398
Rattus norvegicus BN/SsNHsdMCW	38115	9178
Homo sapiens	38612	7186
Candidatus Burkholderia kirkii	6325	4011
Arabidopsis thaliana Columbia	31392	3818
Nostoc azollae 0708	5379	1670
Stenotrophomonas maltophilia SKK35	4760	1589
Pseudomonas aeruginosa VRFPA01	5775	1523
Stenotrophomonas maltophilia RA8	5016	1507
Mycobacterium leprae TN	2750	1086

Again: Don't expect the above links to work if you're not a registered JGI user. (I don't know if they will work for you or not.) This list was automagically generated by the Department of Energy's Joint Genomes Institute and I thought you might get the same kick out of it that I got. It's eye-opening to see the ratio of pseudogenes to "normal" genes in these organisms. Isn't it?

Taking the top three spots are mouse, rat, and humans. (Note: These counts should be taken with a bit of caution, as some have estimated the number of pseudogenes in the human genome to be much higher than the 7,186 shown here.) All the other spots in the chart except Arabidopsis (which is a leafy plant) are bacteria. The leprosy bacterium, which I've written about before, comes in tenth place.

If you're not familiar with the concept of pseudogenes, you might want to look at this post. Basically we're talking about genes that are thought to be disabled and no longer functional in the normal sense, although they may well be functional in some as-yet-unappreciated sense. (Otherwise, evolutionary theory says they should have been eliminated from most genomes eons ago.)

Personally, I believe pseudogenes are as much a feature of DNA as regular genes; certainly in higher life forms, they occur in great numbers. The vast majority of bacterial genomes in public databases are shown as having no pseudogenes. I find that (how shall I say?) not at all credible. Some day it will be obvious that almost every genome harbors pseudogenes; we simply lack smart enough software to detect them all right now.

Coming to Grips with Pseudogenes

The term pseudogene was coined in 1977, when Jacq et al. discovered a version of the gene coding for 5S rRNA in the African clawed frog (Xenopus laevis) that was truncated yet retained homology with the active gene. Subsequent work has shown that in higher life forms, pseudogenes (genes that have been inactivated through one event or another) are almost as numerous as coding genes, with (for example) the human genome containing 10,000 or more pseudogenes. (A more recent estimate puts the number at 20,000.) Many of these pseudogenes are highly conserved. Looking at pseudogenes in the mouse and human, Svensson et al. found that of a group of 74 such genes that occur in both species, 30 appear to have been conserved since before the evolutionary divergence of mice and humans.

In higher organisms, pseudogenes are sometimes transcribed into RNA, with the RNA filling a regulatory function. For example, Korneev et al. found that simultaneous transcription of neural nitric oxide synthase (nNOS) and the antisense strand of a homologous pseudogene in the same neurons of Lymnaea stagnalis (a snail) leads to the formation of a duplex between the two strands and a reduction in nNOS translation. Further examples can be found in Pink et al. (2007), "Pseudogenes: Pseudo-functional or key regulators in health and disease?"

In bacteria, pseudogenes are somewhat rarer than in eukaryotes, but exist in significant numbers in many pathogens (including many species of Mycobacterium, Shigella, Brucella, Bordetella, and others). A study by Kuo and Ochman (2010) found that pseudogenes are swiftly eliminated from Salmonella. They describe "evidence of a strong deletional bias in Salmonella, such that genes that are not maintained by selection are rapidly inactivated and eliminated by mutational events." In fact, Kuo and Ochman found that pseudogenes are eliminated more rapidly than could be explained by the so-called neutral theory of evolution, indicating that the continued presence of pseudogenes exacts a high cost to the cell.

And yet, many bacteria with slow-evolving genomes (such as Mycobacterium species) retain their pseudogenes with high fidelity across evolutionary timespans. The most celebrated "pseudogene hoarder" of all time, M. leprae (the leprosy bacterium) appears to have acquired its 1000+ pseudogenes 9 to 20 million years ago. Meanwhile, the half-life of pseudogenes in Buchnera aphidicola was measured at 23.9 million years—a staggering number.

So on the one hand, we have work by Kuo and Ochman showing that pseudogenes in bacteria are rapidly eliminated, and on the other hand we have some bacterial lineages in which it seems pseudogenes are not only conserved but actively repaired over periods of tens of millions of years!

In Chapter 5 of Brucella: Molecular Microbiology and Genomics (2012, Caister Academic Press), Garcia-Lobo et al. describe their work with RNA sequence data from the bacterium Brucella abortus:

Twenty-four of the genes selected from the RNAseq data were annotated as pseudogenes in the B. abortus 2308 genome, which was considered a rather unexpected finding. By comparison with other Brucella genomes we can reduce the list of highly expressed pseudogenes to 16 (often, truncated parts of a gene are annotated as different pseudogenes especially in B. abortus 2308). This seems contradictory since high transcription of these genes, which should be not able to translate into functional proteins, will be contrary to biological economy. The high levels of transcription observed for these genes strongly suggest that they could be active genes and their products may perform functions unreported in metabolic reconstructions. High pseudogene expression may also indicate that these are very recently produced pseudogenes that did not turned down transcription yet by accumulation of mutations in their promoter or control regions. It is also possible that these pseudogenes may contain sequencing errors and they are indeed active genes.

It's almost comically obvious from this passage that the authors are troubled by their own finding that some pseudogenes in Brucella are highly transcribed. They try explaining it away by saying it could all be "sequencing errors."

A more parsimonious view is that pseudogenes that haven't been eliminated from a genome are, in fact permanent, legitimate fixtures of the landscape, in microbes just as in higher life forms. And as in higher life forms, pseudogenes in microbes are probably serving perfectly understandable regulatory functions (when they're not actually translated into protein products).

Kuo and Ochman have convincingly shown that useless pseudogenes are quickly eliminated. It follows that any pseudogenes that aren't swiftly eliminated are, in fact, serving a biological purpose, or else they wouldn't be there. This line of reasoning is already well accepted by researchers who study eukaryotic life forms. Those who study bacteria need to take a hint from their up-the-food-chain colleagues.

What could the hundreds of pseudogenes in Bordetella pertussis (or the 1000+ pseudogenes in M. leprae) be doing? First we need to get used to the idea that in bacteria, virtually all genes are transcribed, in both directions. It's been four years since Dornernberg et al. reported finding ~1000 antisense transcripts in E. coli, but no one seems to have gotten the memo.

A section of Rothia mucilaginosa genome (top) and a corresponding portion of Mycobacterium leprae (bottom); click to enlarge. The yellow gene, in each case, is DnaE (error-prone polymerase). Pink bands indicate areas of 65% or more homology between the two organisms. The small-diameter silver genes in the lower panel are M. leprae pseudogenes. "Normal genes" are shown in green. Notice that R. mucilaginosa has open reading frames on both strands of DNA, with many bidirectionally overlapping genes.

A look at the genome of the bacterium Rothia mucilaginosa DY18 shows that a very large proportion of "normal genes" have open reading frames on the opposite strand (see illustration). Bidirectional overlapping genes run throughout the Rothia genome. A massive annotation error? Maybe. Or maybe both strands are transcribed.

If massive wholesale transcription of antisense strands occurs in E. coli, as we know it does, certainly it's no stretch to imagine it occurring in Rothia mucilaginosa. And if it is occurring in Rothia, which is (incidentally) an opportunistic pathogen, how much harder can it be to imagine it occurring in another well-known pathogenic member of the Actinomycetales family, Mycobacterium leprae? We know already that upwards of 40% of M. leprae pseudogenes are transcribed. Antisense transcripts could well be playing a role in silencing certain gene essential genes when attempts are made to grow the organism in defined media. Forward transcripts could be producing nonsense or partial-nonsense/truncated proteins that are excreted as toxins or find their way to the cell wall as surface antigens. Any number of scenarios might be possible.

Some very low-hanging fruit is available to micobiologists who are willing to accept the obvious. Instead of wishing away pseudogenes or imagining them to be useless baggage, we should be looking at them as potential determinants of pathogenicity. We should consider their possible roles in modulating protein expression patterns. We should attempt to learn why they're conserved; what role(s) they're playing in cell physiology. The last thing in the world we should be doing is calling them "junk DNA."

Pseudogenes Are Not Junk DNA

In 2007,  a PLoS ONE paper by Ahmed et al. proposed a phylogeny for Mycobacteria in which M. leprae (the leprosy organism) is shown as a relatively recent branch off a very long tree, with M. tuberculosis depicted (in a decidedly fanciful schematic) as being of relatively recent provenance (35,000 years), diverging from M. canettii (a recently discovered cousin of tuberculosis) 3 million years ago.

The rather fanciful phylogenetic picture of Mycobacterium evolution presented by Ahmed et al. (2007). Click to enlarge.

The only trouble with this picture is that we know it's wrong. More exacting work has shown that M. tuberculosis is at least 3 million years old, and one paper estimates that the common ancestor of TB and leprosy may go back 66 million years. If the latter figure sounds dubious, consider that until recently, M. leprae wasn't thought to have any sister strains that could aid with dating the organism phylogenetically. But in 2008, the situation changed dramatically when it was realized that in Mexico, a distinct form of leprosy known as "diffuse lepromatous leprosy" (DLL) was actually due to a genetically distinct variant of Mycobacterium known as M. lepromatosis. When the genome for the latter organism was analyzed, it was found to contain the same stupendous assortment of pseudogenes contained in M. leprae, but detailed analysis of polymorphisms in the genomes of the two strains led to a surprising finding: Divergence of the strains appears to have occurred around 10 million years ago.

Another team found that the massive "pseudogenization event" that caused M. leprae (and its cousin, M. lepromatosis) to become saddled with a record number (1,116) of pseudogenes probably occurred on the order of 20 million years ago.

The age and stability of the pseudogenes in M. leprae can only be described as stunning. Conventional evolutionary dogma says that pseudogenes will inevitably be degraded and lost over time. Surely M. leprae can't be conserving and repairing pseudogenes over 10-million-year-long timespans? Pseudogenes are discardable junk.

Or are they?

An analysis of Buchnera aphidicola (the tiny Enterobacterial endosymbiont of the pea aphid) put the half-life of pseudogenes in that organism at 23.9 million years.

Human DNA reportedly contains over 12,000 pseudogenes. Some of these pseudogenes are quite old. Parallel nonsense mutations caused a pseudogenization of the uricase gene in apes during the early Miocene era (17 million years ago). We still carry the pseudogene in question—and it gets transcribed. According to a report by James T. Kratzer and colleagues at the University of Texas, Austin:

Despite being nonfunctional, cDNA sequencing confirmed that uricase mRNA is present in human liver cells and that these transcripts have two premature stop codons.

The inevitable conclusion is that pseudogenes are not, and should not be considered by default, "junk DNA." To the contrary, the default assumption should be that pseudogenes are ancient and conserved—because in most cases, that's exactly what they are.

What causes genes to "go pseudo"? Why are they conserved? What are they really doing? I'll tackle some of those questions in a followup post. Stay tuned.