Are Overapping Genes Real?

Bacteria belonging to the Pseudomonas family are a perennial favorite among bacteriology instructors (and students) because of the curious ability of some of its members to produce pigments that fluoresce under an ultraviolet light. If you're unlucky enough to get an infected cut on the arm while working in the garden, it's possible your cut will fluoresce under a black light. That's enough of a diagnosis to pronounce the infectious agent. 

Fluorescent colonies of Pseudomonas.

Silby and Levy, investigating the adaptation of the bacterium Pseudomonas fluorescens to soil, uncovered the existence of at least ten antisense genes in P. fluorescens. They went on to demonstrate experimentally that one of the genes, cosA, produces not just antisense RNA but an associated protein. Tellingly, Silby and Levy commented:

These findings suggest that current genome annotations provide an incomplete view of the genetic potential of a given organism.

The implication is that additional antitranscriptome genes remain to be found, not only in Pseudomonas but in other organisms.

There's a good reason they haven't been found yet. Overlapping genes are automatically rejected by many of the annotation programs that are commonly used to find, identify, and label genes in genome sequences. (The oft-used freeware Glimmer 2 program allows you to set the overlap-rejection threshold.) Many yet-to-be-discovered antisense genes have been deliberately and systematically obscured in published genomes.

Still, once in a while such genes do surface. For example, in Pseudomonas stutzeri A1501, we find a pair of overlapping genes at an offset of 3035137 on the chromosome (see illustration below).

Overapping genes in Pseudomonas stutzeri.

The top gene is annotated merely as a "hypothetical protein," while the underlying gene on the opposite strand is an aspartyl-tRNA synthetase. One's normal inclination is to dismiss a hypothetical protein as being unimportant, but this may not be wise. Twenty percent or more of bacterial genes are annotated as hypothetical proteins; common sense says they can't all be unimportant. In fact, in "Transcriptome Analysis of Pseudomonas syringae Identifies New Genes, Noncoding RNAs, and Antisense Activity" by Filiatrault et al. (2010), researchers found that 818 out of 1,646 protein genes in P. syringae annotated as "hypothetical proteins" were expressed under iron-limited conditions. Many (probably most) genes annotated as "hypothetical protein" are quite real and should probably be re-annotated as PUF: "protein of unknown function."

In this case, the "hypothetical protein" shown in yellow (above) turns up medium-strength protein-BLAST hits with other "hypothetical proteins" from other organisms, including a hit with an E-value of 3.0×10^-49 in Parasutterella excrementihominis YIT 11859 and a comparable hit on a predicted phosphatase/phosphohexomutase in Rothia mucilaginosa DY-18.

In this particular case, the hypothetical-protein gene lacks a strong upstream Shine Dalgarno sequence (a sequence preceding many genes that helps bind a ribosome to the mRNA). But so too does the gene on the opposite strand. (This is not unusual. The SD sequence is not required for translation and in fact, in about half of bacterial species, a Shine Dalgarno sequence is associated with fewer than 50% of genes.) Hence, the jury's out on whether the antigene is expressed. It could be that no protein is made from the top strand but the gene provides RNA-mediated control of the gene on the bottom strand. We won't know for sure until someone investigates.

In Pseudomonas aeruginosa strain PADK2_CF510, we find another instance of a bidirectional overlapping gene pair (see graphic below). In this case, the gene on the top strand (CF510_06030) encodes the large subunit of an isopropylmalate isomerase. The gene on the bottom strand (CF510_06025, shown in yellow) is annotated as "Flp pilus assembly protein TadG." It could very well be a misannotated non-gene. However, five genes away is FimV (CF510_06060), another pilus-assembly (motility) protein. Moreover, the gene marked TadG has a strong upstream SD sequence containing the canonical GGAGG motif. The gene above it has a weaker GGAAA motif.

P. aeruginosa has an overlap of an isopropylmalate isomerase gene and a gene for a motility protein. The latter is shown in yellow.

In previous posts, I've mentioned (and shown data for) the fact that in the overwhelming majority of protein-encoding genes (across every kind of genome), the first base of a codon tends to be purine-rich. One check of whether a bidi-overlap gene is "real" or not ought to be that the first codon base should be purine rich in both reading directions. This is, in fact, the case for the examples shown above. The aspartyl-tRNA synthetase gene for P. stutzeri has AG1 (1st base, purine) content averaging 59.8%, whereas its bidirectional partner gene ("hypothetical protein") has AG1 = 58.5%. The isopropylmalate isomerase of P. aeruginosa has AG1 = 65.9%, while its antisymmetric partner (TadG) has AG1 = 56.2%.

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The Pathogen's Playbook

When comparing pathogenic bacteria with non-pathogenic species of the same genus or family, we often find a common pattern. In the pathogen:

• The genome is often reduced in size (particularly in endosymbionts, but also in others).
• The genome is often shifted in the direction of higher A+T content (lower G+C content).
• Many pseudogenes are present.
• Often, the pathogen is a slow-grower in pure culture (if it can be cultured at all).
• The pathogen has special nutritional needs.

An extreme case that illustrates all of these points is Mycobacterium leprae, the leprosy bacterium. It has fewer genes than its cousin, M. tuberculosis (which in turn has fewer genes than non-pathogenic Mycobacteria); its genomic G+C content is 8% lower than most other Mycobacteria; it contains over 1100 pseudogenes; it has a doubling time of two weeks; and it cannot be grown in pure culture (presumably because of fastidious nutritional requirements).

M. tuberculosis can be grown in the laboratory, but it, and its M. avium-group cousins, are very slow growers, taking anywhere from four days to two weeks to develop colonies on solid media.

It seems likely that some pathogens (certainly members of the Mycobacteria, but also the tiny Tenericutes, e.g. Mycoplasma, among many others) have evolved slow growth as a survival strategy. Certainly, organisms that have evolved an intracellular parasitic lifestyle need to be careful not to out-grow the host, if the relationship is to be a long one.

All of the factors listed above suggest a certain scenario, a "pathogen's playbook," if you will, which can be summarized as follows:

1. The organism invades a warm-booded host.
2. Phagocytes (white blood cells) ingest the organism.
3. The phagocytes undergo a respiratory burst, flooding the microbe(s) with peroxides, hypochlorites, nitrous oxide, and other noxious oxidants.
4. The flood of reactive oxygenated species triggers an SOS response in the microbe.
5. The microbe's DNA undergoes massive damage. 
6. Any surviving microbial cells are now pathogenic.

The SOS response is known to trigger mutagenicity. In Mycobacterium, for example, peroxides (as well as UV light) can induce up-regulation of dnaE, an error-prone polymerase. Since Mycobacteria are known to lack a MutS mismatch repair system, SOS-induced errors in DNA replication will almost certainly include uncorrected frameshift errors leading to the creation of pseudogenes. But that's a good thing, if you're a Mycobacterium interested in forming a longterm relationship with a host cell. The loss of certain genes (as long as they're not essential!) will likely slow your metabolism and make you dependent on host nutrients. Truly non-essential pseudogenes will simply be jettisoned over time, reducing the footprint of the remaining genome. Any pseudogenes that survive will likely have done so because they're now playing an essential gene-silencing role.

Let's expand on that last part. Take the dnaE gene, for example. M leprae has two copies of this gene, only one of which is functional. Suppose both copies were functional at the time of the massive pseudogenization event that converted so many of M. leprae's genes to pseudogenes 9 to 20 million years ago. After the pseudogenization event (probably a phagocytic respiratory burst), one copy of dnaE became a pseudogene. But continued transcription of the pseudogene in the forward direction means the pseudo-mRNA competes with the "normal" dnaE transcript for ribosomal attention. Transcription of the antisense strand of the disabled gene would, of course, create a messenger RNA product that could silence the normal transcript by doublestranded interaction. Either way, once the pseudogenization event is over, dnaE expression is attenuated—as it should be, once pathogenicity has been established.

Is it realistic to think M. leprae transcribes antisense strands of its pseudogenes? Given that E. coli has been found to contain ~1000 antisense transcripts, and given that we know M. leprae transcribes many of its pseudogenes, I think the answer has to be yes.

So the pattern is: infection, respiratory burst, massive mutation, silencing of many genes, and (oh by the way) creation of many brand-new gene products, some of them no doubt quite toxic to the host, as the result of gene truncation and pseudogene expression.

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."