Antibiotic-resistant bacteria have been in the news lately, with the usual scary talk about drug-resistant bacteria taking over the world, with a return to the Dark Ages if we don't Do Something.
Certain facts about drug resistance tend to get lost in these sorts of news stories. There's a common misconception that somehow the introduction of antibiotics (first in medicine, then in agriculture) caused bacteria to invent new drug-resistance genes out of nowhere. That's nonsense, of course. The Lederbergs showed in 1951 that drug resistance genes were/are preexisting, and did not come into being because of antibiotics. Rather, bacteria already equipped with such genes were able to survive treatment with antibiotics. Around 1968 it also became clear that bacteria can share drug resistance genes by exchange of extrachromosomal DNA (plasmids). Bacteria that don't have the magic genes can get them from their buddies.
But, so. How is it that the genes for antibiotic resistance already existed prior to the introduction of antibiotics into the food chain and into medicine? The answer is simple: Antibiotics are natural products produced by common soil and water microorganisms. Penicillin is produced by a mold; streptomycin is produced by the soil bacterium Streptomyces. Certainly hundreds (maybe thousands) of different kinds of antibiotics exist in the natural environment. They've been there for millions of years.
Mycobacterium tuberculosis ATCC 35801 (Erdman strain) has two "multidrug resistance" genes, and they reside on the main chromosome (not on a plasmid). They're shared by other members of the Mycobacterium genus, including the leprosy bacterium, M. leprae. In the latter, one of the genes in question (MLBr_2224) is a pseudogene. It's interesting to compare this pseudogene with its counterpart, Erdman_0866, in Mycobacterium tuberculosis ATCC 35801. The two genes are nearly the same size: 1543 base pairs versus 1638. This is quite interesting in itself, inasmuch as most pseudogenes in M. leprae are truncated (averaging just 795 base pairs). But the comparison gets even more interesting when one does a side-by-side analysis of single nucleotide polymorphisms (changes to individual bases).
First I did a SNP comparison of the tuberculosis drug-resistance gene to its counterpart in M. indicus pranii, where there were 325 total base-pair differences, distributed amongst the 1st, 2nd, and 3rd base pairs of codons as:
98, 72, 155
This is the expected pattern: The largest number of mutations tends to accumulate in the third codon base (the so-called "wobble base"), because mutations in this base give a large percentage of synonymous codon changes owing to codon degeneracy. The next-highest number of mutations is expected in the first base, because there is some (but not much) degeneracy in that position. All mutations in the second base are non-synonymous and likely to affect enzyme function. Hence, the lowest number of mutations occurs in the second base.
When I ran the same check between the M. tuberculosis gene and its counterpart (the pseudogene) in M. leprae, I found that the mutational differences totaled 418 changes and segregated by base as:
119, 103, 196
Unexpectedly, the same pattern emerges. The reason this is unexpected is that one expects a pseudogene to contain frameshifts that would destroy the reading frame, mooting 1st/2nd/3rd-base comparisons. More generally, one expects massive random mutations all along a pseudogene's length (of the kind that would tend to make all three of the above numbers equal, even without frameshifts). So the fact that we still see the familiar pattern of 1st/2nd/3rd-base mutations is interesting.
The genes in question were unequal in length, so to do these comparisons I had to disregard a 112-base leader portion of the aligned genes. (I aligned the genes via ClustalW using Mega6.) I also ignored the unequal-length trailer portions of each gene, analyzing only the interior (aligned) portions from base 112 to base 1418. Also, to facilitate the analysis, I removed one adenine (representing a one-base insertion) in the M. leprae gene, at position 407, to restore the reading frame. But it's interesting that at that position there's a run of six adenines, indicative of a slippery site, of the kind associated with frameshift signalling.
The M. leprae pseudogene MLBr_2224 behaves as if it is still a normal gene, accumulating mutations in the "right places." An alternative explanation is that the M. leprae and M. tuberculosis genes were already in pretty much this orientation relative to each other before the two species diverged, and M. leprae simply accumulated very few additional mutations in the pseudogene after it became a pseudogene. (M. leprae is assumed to have experienced a massive pseudogenization event between 9 and 20 million years ago.)
For more about M. leprae's strange assortment of pseudogenes, see this post and also this post.
Showing posts with label leprosy. Show all posts
Showing posts with label leprosy. Show all posts
Thursday, May 08, 2014
Monday, April 28, 2014
Are Some Leprosy Pseudogenes Turned On?
Most genomes, whether human or bacterial, contain significant numbers of pseudogenes (that is, genes that are presumed to be inactive due to internal stop codons, frameshift errors, or other serious defects). The usual presumption is that such genes are dormant or dead and thus are not expressed as proteins, since the proteins would be severely truncated or contain nonsense regions, etc.
However, we know that in Mycobacterium leprae (the leprosy bacterium), some 43% of the organism's 1,116 pseudogenes are transcribed. While most of the transcripts are no doubt used in some kind of regulatory capacity, it would be surprising if not a single transcript got translated into protein.
One sign that a protein gene is highly expressed is the presence, upstream of the start codon, of a strong Shine Dalgarno sequence. This is a special sequence of bases that serves as a binding area for 16S ribosomal RNA. The Shine Dalgarno sequence serves to increase the translational efficiency of the genes that have such signatures. (Not all do.) Generally, they are found ahead of high-priority/highly-expressed genes (such as genes for ribosomal proteins). Absence of a SD sequence doesn't mean the gene doesn't get translated. Many genes carry no SD signal.
I created scripts that looked at all 1,116 pseudogenes in M. leprae, to detect the occurrence of Shine Dalgarno sequences in the 20-base-pair region upstream of what would normally be the start codons of said genes. Intriguingly, 31% of pseudogenes carry a length-4 SD sequence (nearly 7 times the number of such sequences expected to occur by chance). By comparison, 50.6% of normal genes in M. leprae carry a length-4 SD sequence.
When I looked for length-5 SD signals, I found that 8.6% of pseudogenes carry such a signal, compared to 26.4% for regular genes. Length-6 signals were found for 33 pseudogenes (3% of the total of 1,116 pseudogenes) versus 176 normal genes (representing 10.9% of 1,604 normal genes). These numbers are about eight times higher than expected to occur by chance.
These numbers are summarized in the table below, where I also show similar findings for genes and pseudogenes of Bordetella pertussis.
B. pertussis preserves a higher proportion of SD signals in pseudogenes than does M. leprae. This is expected, since most B. pertussis pseudogenes are still "in frame," whereas most (but not all) M. leprae pseudogenes harbor frameshifts.
Length-5-or-longer SD signals occur at about one third the rate in psuedogenes of M. leprae that they do in normal genes of M. leprae, but still much higher than expected by chance. If 43% of pseudogenes are transcribed (as we know they are), and a third of those transcripts have strong enough SD sequences to facilitate translation, it means about 159 pseudogenes in M. leprae could be expected to have expressed protein products. Those products would, of course, be truncated and/or contain nonsense regions. Presumably, many would be marked for proteolysis (either by the tmRNA system or through other mechanisms).
However, we know that in Mycobacterium leprae (the leprosy bacterium), some 43% of the organism's 1,116 pseudogenes are transcribed. While most of the transcripts are no doubt used in some kind of regulatory capacity, it would be surprising if not a single transcript got translated into protein.
One sign that a protein gene is highly expressed is the presence, upstream of the start codon, of a strong Shine Dalgarno sequence. This is a special sequence of bases that serves as a binding area for 16S ribosomal RNA. The Shine Dalgarno sequence serves to increase the translational efficiency of the genes that have such signatures. (Not all do.) Generally, they are found ahead of high-priority/highly-expressed genes (such as genes for ribosomal proteins). Absence of a SD sequence doesn't mean the gene doesn't get translated. Many genes carry no SD signal.
I created scripts that looked at all 1,116 pseudogenes in M. leprae, to detect the occurrence of Shine Dalgarno sequences in the 20-base-pair region upstream of what would normally be the start codons of said genes. Intriguingly, 31% of pseudogenes carry a length-4 SD sequence (nearly 7 times the number of such sequences expected to occur by chance). By comparison, 50.6% of normal genes in M. leprae carry a length-4 SD sequence.
When I looked for length-5 SD signals, I found that 8.6% of pseudogenes carry such a signal, compared to 26.4% for regular genes. Length-6 signals were found for 33 pseudogenes (3% of the total of 1,116 pseudogenes) versus 176 normal genes (representing 10.9% of 1,604 normal genes). These numbers are about eight times higher than expected to occur by chance.
These numbers are summarized in the table below, where I also show similar findings for genes and pseudogenes of Bordetella pertussis.
| Organism | Data Set |
Motif
Length
|
Expected
|
Found
|
%
of Genes
|
| M.
leprae |
genes (n = 1604) |
SD6
|
5
|
176
|
10.9%
|
SD5
|
25
|
423
|
26.4%
|
||
SD4
|
119
|
812
|
50.6%
|
||
| pseudogenes (n = 1116) |
SD6
|
4
|
33
|
3.0%
|
|
SD5
|
18
|
96
|
8.6%
|
||
SD4
|
83
|
346
|
31.0%
|
||
| B.
pertussis |
genes (n = 3377) |
SD6
|
9
|
493
|
15.0%
|
SD5
|
49
|
1032
|
30.6%
|
||
SD4
|
259
|
1939
|
57.4%
|
||
| pseudogenes (n = 370) |
SD6
|
0
|
38
|
10.20%
|
|
SD5
|
1
|
89
|
24.10%
|
||
SD4
|
11
|
194
|
52.40%
|
B. pertussis preserves a higher proportion of SD signals in pseudogenes than does M. leprae. This is expected, since most B. pertussis pseudogenes are still "in frame," whereas most (but not all) M. leprae pseudogenes harbor frameshifts.
Length-5-or-longer SD signals occur at about one third the rate in psuedogenes of M. leprae that they do in normal genes of M. leprae, but still much higher than expected by chance. If 43% of pseudogenes are transcribed (as we know they are), and a third of those transcripts have strong enough SD sequences to facilitate translation, it means about 159 pseudogenes in M. leprae could be expected to have expressed protein products. Those products would, of course, be truncated and/or contain nonsense regions. Presumably, many would be marked for proteolysis (either by the tmRNA system or through other mechanisms).
Tuesday, April 15, 2014
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:
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 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."
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."
Saturday, April 12, 2014
The Most Deadly Pathogen of All Time
Few bacterial species have had as great an impact on humankind as the members of the Mycobacterium family, which encompass the causative agents of (among other ailments) leprosy, tuberculosis, and Crohn's Disease in humans, and Johne's Disease in farm animals. Leprosy is known from antiquity and continues to strike 200,000 or more people each year worldwide. Tuberculosis, which affects (subclinically) one in three persons worldwide, continues to kill well over a million people a year and has caused a billion deaths in the last two centuries, more than all the wars and genocides of history combined.
The association of M. avium subspecies paratuberculosis (MAP) with Crohn's Disease is still considered controversial by some, but if in fact Koch's criteria have already been met, MAP adds millions more to the toll of human misery caused by Mycobacterial infection.
What are these bacteria? Where did they come from? How have they managed to be so successful in causing death and disease?
The prefix "myco" means fungal, but these are not fungi we're talking about. Mycobacteria are soil- and water-borne bacteria that produce an extraordinarily complex cell wall containing not only the usual (for bacteria) peptidoglycans but also:
The Mycobacterial cell wall is famously complex, but it also has the curious habit of disappearing entirely, under nutrient-starvation conditions. Like many other bacteria, Mycobacteria can, under certain conditions, shed their cell walls and take on a so-called L-form morphology, in which cells (bounded only by a thin and osmotically vulnerable cell membrane containing just 7% of the usual amount of peptidoglycan) exist as protoplasts which are nonetheless able to reproduce and thrive, producing distinctive colonies on solid media and giving rise, in vivo, to tiny spherules that are often confused with Russell bodies in cancer biopsies. The medical significance of the mysterious L-forms is still debated, after more than 100 years.
One thing most Mycobacterial species have in common is slow growth. Cultures of M. tuberculosis and MAP often require weeks to develop, and M. leprae (which can't be grown in pure culture at all; it can be lab-grown only in the footpads of mice or armadillos) has the longest known generation time of any bacterium, at two weeks.
Ironically, pathogenic strains of Mycobacterium seem to have evolved slow growth as a survival strategy. (This certainly makes them hard to treat with antibiotics. Most antibiotics are effective only in disrupting the growth of actively growing cells.) The lack of DNA mismatch repair enzyme systems (MutS, MutL, and MutH) may be an outcome of the fact that slow DNA replication in these organisms, in and of itself, ensures reasonably high-fidelity replication. On the other hand, lack of a mismatch repair system could be why pseudogenes (genes inactivated due to frameshifts or other errors) abound in Mycobacterial species. M. leprae famously has over 1000 pseudogenes; M. smegmatis strain JS623 harbors over 200 pseudogenes; M. canettii (strain CIPT 140010059) and M. rhodesiae (strain NBB3) both have over 100. (For a good review of Mycobacterial DNA repair systems, see this 2011 paper.)
Unlike Yersinia pestis, the plague organism, which may be less than 20,000 years old (very young in bacterial species time), M. tuberculosis, as a species, appears to be at least 3 million years old, although this number should probably be considered a minimum age, subject to upward revision. (The species was thought to be only 35,000 years old as late as 2002, before a more detailed genetic analysis established the 3-million-year estimate of its age. The numbers should be viewed with caution, however, since they're based on mutation-rate assumptions derived from data for E. coli.)
The question of how M. tuberculosis has managed to achieve its distinctive pathogenic profile is a matter of active ongoing research, and likely will be for a long time. A recent review article reminds us: "The [complete genome] sequence of the pathogen Mycobacterium tuberculosis strain H37Rv has been available for over a decade, but the biology of the pathogen remains poorly understood."
Miscellaneous Links
List of famous T.B. victims—Brontë family, Balzac, Kafka, Thoreau, Kant, Chekhov, Orwell, Schrödinger, Vivien Leigh, Arline Feynman (wife of the famous physicist), the list goes on.
Tuberculosis in Literature and the Arts
The T.B. Blues (Jimmie Rodgers, 1931) This song, famously covered by Leon Redbone (among others), was written by Rodgers after he contracted the disease at age 27. He died eight years later.
World Health Organization TB Stats (landing page)
The Tuberculosis Systems Biology Program
The association of M. avium subspecies paratuberculosis (MAP) with Crohn's Disease is still considered controversial by some, but if in fact Koch's criteria have already been met, MAP adds millions more to the toll of human misery caused by Mycobacterial infection.
 |
| Colonies of Mycobacterium have a characteristically waxy consistency. Shown here: colonies of M. tuberculosis. |
The prefix "myco" means fungal, but these are not fungi we're talking about. Mycobacteria are soil- and water-borne bacteria that produce an extraordinarily complex cell wall containing not only the usual (for bacteria) peptidoglycans but also:
- Arabinogalactan
- Mycolic acids
- Lipoarabinomannan
- Extractable lipids including glycolipids, phenolic glycolipids (PGL), glycopeptidolipids (GPL), waxes, acylated trehaloses, and sulfolipids
The Mycobacterial cell wall is famously complex, but it also has the curious habit of disappearing entirely, under nutrient-starvation conditions. Like many other bacteria, Mycobacteria can, under certain conditions, shed their cell walls and take on a so-called L-form morphology, in which cells (bounded only by a thin and osmotically vulnerable cell membrane containing just 7% of the usual amount of peptidoglycan) exist as protoplasts which are nonetheless able to reproduce and thrive, producing distinctive colonies on solid media and giving rise, in vivo, to tiny spherules that are often confused with Russell bodies in cancer biopsies. The medical significance of the mysterious L-forms is still debated, after more than 100 years.
 |
| The very small red filaments here are cells of Mycobacterium avium living inside lymph-node macrophages in an immunocompromised individual. |
Ironically, pathogenic strains of Mycobacterium seem to have evolved slow growth as a survival strategy. (This certainly makes them hard to treat with antibiotics. Most antibiotics are effective only in disrupting the growth of actively growing cells.) The lack of DNA mismatch repair enzyme systems (MutS, MutL, and MutH) may be an outcome of the fact that slow DNA replication in these organisms, in and of itself, ensures reasonably high-fidelity replication. On the other hand, lack of a mismatch repair system could be why pseudogenes (genes inactivated due to frameshifts or other errors) abound in Mycobacterial species. M. leprae famously has over 1000 pseudogenes; M. smegmatis strain JS623 harbors over 200 pseudogenes; M. canettii (strain CIPT 140010059) and M. rhodesiae (strain NBB3) both have over 100. (For a good review of Mycobacterial DNA repair systems, see this 2011 paper.)
Unlike Yersinia pestis, the plague organism, which may be less than 20,000 years old (very young in bacterial species time), M. tuberculosis, as a species, appears to be at least 3 million years old, although this number should probably be considered a minimum age, subject to upward revision. (The species was thought to be only 35,000 years old as late as 2002, before a more detailed genetic analysis established the 3-million-year estimate of its age. The numbers should be viewed with caution, however, since they're based on mutation-rate assumptions derived from data for E. coli.)
The question of how M. tuberculosis has managed to achieve its distinctive pathogenic profile is a matter of active ongoing research, and likely will be for a long time. A recent review article reminds us: "The [complete genome] sequence of the pathogen Mycobacterium tuberculosis strain H37Rv has been available for over a decade, but the biology of the pathogen remains poorly understood."
Miscellaneous Links
List of famous T.B. victims—Brontë family, Balzac, Kafka, Thoreau, Kant, Chekhov, Orwell, Schrödinger, Vivien Leigh, Arline Feynman (wife of the famous physicist), the list goes on.
Tuberculosis in Literature and the Arts
The T.B. Blues (Jimmie Rodgers, 1931) This song, famously covered by Leon Redbone (among others), was written by Rodgers after he contracted the disease at age 27. He died eight years later.
World Health Organization TB Stats (landing page)
The Tuberculosis Systems Biology Program
Wednesday, April 09, 2014
Are dead genes still alive in the leprosy bacillus?
The genome of the leprosy bacterium (Mycobacterium leprae) stands as a remarkable example of DNA in an apparent state of massive, wholesale breakdown. Of the organism's 2720 genes, only 1604 appear to be functional, while 1116 are pseudogenes, which is to say genes that have been "turned off" and left for dead.
Genes can become pseudogenes in any number of ways, including loss of a start codon, loss of promoter regions (or degraded Shine Dalgarno signals), random insertions and deletions, mutations that cause spurious stop codons, and so on. Once a gene gets "turned off," assuming loss of the gene in question isn't fatal, the gene typically undergoes a period of degradation (leading to its eventual loss from the genome), but that's not exactly what we see in the leprosy bacterium. When leprosy germs from medieval skeletons were sampled and their genomes sequenced, researchers found that pseudogenes in M. leprae haven't changed very much in the past thousand years or so. Not only does M. leprae tend to hold onto its pseudogenes, it actively transcribes upwards of 40% of them. Probably not all of the transcripts result in expressed proteins (many lack a start codon!), but some no doubt do get translated into proteins. Let's put it this way: It would be extremely unusual for an organism to conserve this many pseudogenes if none of them was doing anything useful.
To get a better idea of what's going on here, I downloaded the DNA sequences of M. leprae's 1604 "normal" genes as well as the 1116 pseudogenes. In analyzing the codons for these genes, I looked for signs of genes that were still in the normal reading frame. One way to detect this is by measuring the purine content at the various base positions in a gene's codons. In a typical protein-coding gene, around 60% of codons begin with A or G (adenine or guanine). This positional bias will, of course, be lost in a gene that has undergone frameshift mutations. Among M. leprae's 1116 pseudogenes, I found 269 in which codons showed an average AG1 percentage (A+G content, codon base one) of 55% or more. These are pseudogenes that appear to still be mostly "in frame."
Things get a lot more interesting where putative membrane proteins are concerned. In a previous post, I showed that in some genes, the second codon base is pyrimidine-rich (i.e., predominantly C or T: cytosine or thymine); these genes encode proteins with a high percentage of nonpolar amino acids. Bottom line, if a gene's codons are mostly T or C in the second position, that gene most likely encodes a membrane-associated protein. (See my previous post for some data.) This is true for all organisms (viruses, cells) and organellar genes, too, by the way, not just M. leprae. It's a generic feature of the genetic code.
When I segregated M. leprae pseudogenes according to whether or not the second codon base was (on average) less than, or more than, 40% purines, I stumbled onto something quite interesting. I found 51 pseudogenes with AG2 less than 40% (meaning, these are probably membrane-associated proteins). Of those, 32 (or 62%) are still "in frame," with AG1 > 55%. By contrast, the majority (78%) of non-membrane pseudogenes (AG2 > 40%) appear to be turned off, with an average AG1 of 51%.
Long story short: Most non-membrane-associated pseudogenes are out-of-frame (and likely dead), whereas 62% of putative membrane-associated pseudogenes appear to be in-frame, and therefore could still be functional (or at least, undead).
In looking at stop codons, I found that of the pseudogenes that still had stop codons, the average distance to the first stop codon is only 149 bases (whereas the average pseudogene length is 795 bases). Pseudogenes for putative membrane-associated proteins were shorter overall (as membrane proteins often are; 495 bases instead of 795), but the average distance to the first stop codon was 190 bases, significantly longer than for the other pseudogenes. This suggests some of them are still alive.
By now you're probably wondering how the heck a pseudogene can be of any possible use whatsoever when it contains a premature stop codon. The thing we need to ask, though, is why M. leprae tolerates (indeed conserves) so many pseudogenes in the first place. Could it be that the organism has adapted a frameshift-tolerant translation apparatus? Maybe some of the stop codons aren't really stop codons.
We know that a wide variety of organisms (not just viruses, where this phenomenon was first discovered, but bacteria and eukaryotes) have evolved special signals to tell ribosomes to shift in and out of frame by plus or minus one. (See "A Gripping Tale of Ribosomal Frameshifting: Extragenic Suppressors of Frameshift Mutations Spotlight P-Site Realignment," Atkins and Björk, Microbiol. Mol. Biol. Rev. 2009.) Certain tRNAs participate in "quadruplet codon" decoding, making it possible for special frameshift signals to work. The signals usually involve 7-base-long "slippery heptamer" sequences, such as CCCTGAC, right where a stop codon (TGA) appears. In other words, when a stop codon appears inside a slippery heptamer, it's not really a stop codon. Depending on the kinds (and amounts) of tRNAs "on duty," it can be a frameshift signal.
When I looked for CCCTGAC in M. leprae's pseudogenes, I found 16 in-frame occurrences of the sequence in 1116 pseudogenes. (Only 7 occurrences of the hexamer CCCTGA were found, in frame, in M. leprae's "normal" genes.) While this doesn't prove that M. leprae is up to any unusual translation tricks, it's a tantalizing result. Also bear in mind, if M. leprae is indeed up to some unusual tricks, it may very well be using frameshift signals other than (or in addition to) CCCTGAC. The fact that Mycobacterium species lack a MutS/MutL mismatch repair system means M. leprae may have adapted different ways of coping with "slippery repeats."
Further work will be needed to confirm whether M. leprae indeed translates some of its pseudogenes into proteins. The 32 "high likelihood" pseudogenes that, according to my analysis, might still encode functional (or at least expressed) membrane-associated proteins are shown in the table below. Leave a comment if you have additional thoughts.
M. leprae pseudogenes that have codons with overall AG1 > 55% and AG2 < 40%:
Genes can become pseudogenes in any number of ways, including loss of a start codon, loss of promoter regions (or degraded Shine Dalgarno signals), random insertions and deletions, mutations that cause spurious stop codons, and so on. Once a gene gets "turned off," assuming loss of the gene in question isn't fatal, the gene typically undergoes a period of degradation (leading to its eventual loss from the genome), but that's not exactly what we see in the leprosy bacterium. When leprosy germs from medieval skeletons were sampled and their genomes sequenced, researchers found that pseudogenes in M. leprae haven't changed very much in the past thousand years or so. Not only does M. leprae tend to hold onto its pseudogenes, it actively transcribes upwards of 40% of them. Probably not all of the transcripts result in expressed proteins (many lack a start codon!), but some no doubt do get translated into proteins. Let's put it this way: It would be extremely unusual for an organism to conserve this many pseudogenes if none of them was doing anything useful.
 |
| This view of a segment of the two genomes shows how a region of around 80,000 base pairs in M. tuberculosis maps to a similar 68,000-base-pair region of M. leprae. Notice that in the lowermost panel (representing M. leprae), many genes are shown as shrunken silver segments instead of fat green cylinders. The smaller grey/silver segments are pseudogenes. Click to enlarge. |
To get a better idea of what's going on here, I downloaded the DNA sequences of M. leprae's 1604 "normal" genes as well as the 1116 pseudogenes. In analyzing the codons for these genes, I looked for signs of genes that were still in the normal reading frame. One way to detect this is by measuring the purine content at the various base positions in a gene's codons. In a typical protein-coding gene, around 60% of codons begin with A or G (adenine or guanine). This positional bias will, of course, be lost in a gene that has undergone frameshift mutations. Among M. leprae's 1116 pseudogenes, I found 269 in which codons showed an average AG1 percentage (A+G content, codon base one) of 55% or more. These are pseudogenes that appear to still be mostly "in frame."
Things get a lot more interesting where putative membrane proteins are concerned. In a previous post, I showed that in some genes, the second codon base is pyrimidine-rich (i.e., predominantly C or T: cytosine or thymine); these genes encode proteins with a high percentage of nonpolar amino acids. Bottom line, if a gene's codons are mostly T or C in the second position, that gene most likely encodes a membrane-associated protein. (See my previous post for some data.) This is true for all organisms (viruses, cells) and organellar genes, too, by the way, not just M. leprae. It's a generic feature of the genetic code.
When I segregated M. leprae pseudogenes according to whether or not the second codon base was (on average) less than, or more than, 40% purines, I stumbled onto something quite interesting. I found 51 pseudogenes with AG2 less than 40% (meaning, these are probably membrane-associated proteins). Of those, 32 (or 62%) are still "in frame," with AG1 > 55%. By contrast, the majority (78%) of non-membrane pseudogenes (AG2 > 40%) appear to be turned off, with an average AG1 of 51%.
Long story short: Most non-membrane-associated pseudogenes are out-of-frame (and likely dead), whereas 62% of putative membrane-associated pseudogenes appear to be in-frame, and therefore could still be functional (or at least, undead).
In looking at stop codons, I found that of the pseudogenes that still had stop codons, the average distance to the first stop codon is only 149 bases (whereas the average pseudogene length is 795 bases). Pseudogenes for putative membrane-associated proteins were shorter overall (as membrane proteins often are; 495 bases instead of 795), but the average distance to the first stop codon was 190 bases, significantly longer than for the other pseudogenes. This suggests some of them are still alive.
By now you're probably wondering how the heck a pseudogene can be of any possible use whatsoever when it contains a premature stop codon. The thing we need to ask, though, is why M. leprae tolerates (indeed conserves) so many pseudogenes in the first place. Could it be that the organism has adapted a frameshift-tolerant translation apparatus? Maybe some of the stop codons aren't really stop codons.
We know that a wide variety of organisms (not just viruses, where this phenomenon was first discovered, but bacteria and eukaryotes) have evolved special signals to tell ribosomes to shift in and out of frame by plus or minus one. (See "A Gripping Tale of Ribosomal Frameshifting: Extragenic Suppressors of Frameshift Mutations Spotlight P-Site Realignment," Atkins and Björk, Microbiol. Mol. Biol. Rev. 2009.) Certain tRNAs participate in "quadruplet codon" decoding, making it possible for special frameshift signals to work. The signals usually involve 7-base-long "slippery heptamer" sequences, such as CCCTGAC, right where a stop codon (TGA) appears. In other words, when a stop codon appears inside a slippery heptamer, it's not really a stop codon. Depending on the kinds (and amounts) of tRNAs "on duty," it can be a frameshift signal.
When I looked for CCCTGAC in M. leprae's pseudogenes, I found 16 in-frame occurrences of the sequence in 1116 pseudogenes. (Only 7 occurrences of the hexamer CCCTGA were found, in frame, in M. leprae's "normal" genes.) While this doesn't prove that M. leprae is up to any unusual translation tricks, it's a tantalizing result. Also bear in mind, if M. leprae is indeed up to some unusual tricks, it may very well be using frameshift signals other than (or in addition to) CCCTGAC. The fact that Mycobacterium species lack a MutS/MutL mismatch repair system means M. leprae may have adapted different ways of coping with "slippery repeats."
Further work will be needed to confirm whether M. leprae indeed translates some of its pseudogenes into proteins. The 32 "high likelihood" pseudogenes that, according to my analysis, might still encode functional (or at least expressed) membrane-associated proteins are shown in the table below. Leave a comment if you have additional thoughts.
M. leprae pseudogenes that have codons with overall AG1 > 55% and AG2 < 40%:
| Pseudogene | Possible product |
| MLBr00146 | hypothetical protein |
| MLBr00189 | hypothetical protein |
| MLBr00278 | conserved hypothetical protein |
| MLBr00341 | hypothetical protein |
| MLBr00460 | hypothetical protein |
| MLBr00478 | hypothetical protein |
| MLBr00738 | PstA component of phosphate uptake |
| MLBr00836 | hypothetical protein |
| MLBr00846 | ABC transporter |
| MLBr01054 | possible PPE-family protein |
| MLBr01156 | hypothetical protein |
| MLBr01237 | possible cytochrome P450 |
| MLBr01238 | probable cytochrome P450 |
| MLBr01400 | possible membrane protein |
| MLBr01414 | PGRS-family protein |
| MLBr01474 | hypothetical protein |
| MLBr01527 | dihydrodipicolinate reductase |
| MLBr01673 | conserved hypothetical protein |
| MLBr01792 | probable Na+/H+ exchanger |
| MLBr01968 | PE family protein |
| MLBr02003 | probable ketoacyl reductase |
| MLBr02101 | conserved hypothetical protein |
| MLBr02150 | molybdopterin converting factor subunit 1 |
| MLBr02190 | PstA component of phosphate uptake |
| MLBr02216 | dihydrolipoamide dehydrogenase |
| MLBr02363 | 19 kDa antigenic lipoprotein |
| MLBr02477 | PE protein |
| MLBr02484 | transcriptional regulator (LysR family) |
| MLBr02533 | PE-family protein |
| MLBr02656 | conserved hypothetical protein |
| MLBr02674 | possible membrane protein |
Wednesday, April 02, 2014
Frameshift errors in leprosy bacterium DNA
Shocking as it might sound, leprosy continues to strike over 200,000 persons per year worldwide, making it as much of a health problem as cholera or yellow fever. One of the oldest known infectious diseases, leprosy became the first disease to be causally linked to bacteria when Hansen made his famous discovery of the connection to Mycobacterium leprae in 1873. Ever since then, scientists have been trying to grow M leprae in the lab, to no avail. Like most environmental isolates, M. leprae defies attempts at pure culture. The only way to grow it in the lab is to infect mice or armadillos, where it has a doubling time of 14 days, the longest known generation time of any bacterium.
Traditionally, it has been assumed that the difficulty in growing M. leprae in pure culture is due to the organism's complex nutritional requirements. (In humans, the organism is an obligate intracellular parasite that takes up residency in the Schwann cells of the peripheral nervous system.) There is no doubt considerable truth to this assumption, but the reason for the organism's fastidious nutritional requirements wasn't fully known until Cole et al. (2001) showed that half the bacterium's genome is inoperative and undergoing decay. Genomic sequencing revealed that M. leprae has only three quarters the DNA content of its (quite robust) cousin, M. tuberculosis, and of M. leprae's 3,000-or-so remaining genes, only 1,600 are fully functional. The rest are pseudogenes.
Pseudogenes are genes that have become inactivated through loss of start codons, loss of promoter regions, introduction of spurious stop codons, introduction of frameshift errors, or through other causes. Almost all organisms contain pseudogenes in their DNA. (Human DNA reportedly contains over 12,000 pseudogenes.) The leprosy bacterium, however, is unique in having approximately half its genome tied up in pseudogenes. Once a gene becomes a pseudogene, it is effectively useless baggage ("junk DNA") and continues on a long path of deterioration. Evolutionary theory predicts that such genes will eventually be lost from the genome, since the carrying cost of keeping them puts the organism at a disadvantage, energetically. But the curious thing about M. leprae is that it's a hoarder: It not only holds onto its useless genes, it actually transcribes upwards of 40% of them. In fact, a recent study of 1000-year-old M. leprae DNA (recovered from medieval skeletons), comparing the medieval version of the organism's genome with the genome of today's M. leprae, found that pseudogenes are highly conserved in the bacterium.
The fact that the bacterium actually transcribes many of its pseudogenes (and doesn't lose them over time) is striking, to say the least, and suggests that the transcription of certain genes or pseudogenes is resulting in mRNAs that silence other, more deleterious genes. It could be that M. leprae can't be grown in culture because when certain combinations of nutrients are presented to it, the nutrients up-regulate deleterious nonsense genes in otherwise-normal operons (or down-regulate important silencers), directly or indirectly. (Williams et al. found that many M. leprae pseudogenes are located in the middle of operons and are transcribed via fortuitous read-through.) Various scenarios are possible. Much work remains to be done.
In the meantime, I couldn't help doing a little desktop science to characterize M. leprae's "defective genes" problem further. I went to http://genomevolution.org/CoGe/OrganismView.pl and entered "Mycobacterium leprae Br4923" in the Organism Name field. In the Genome Information box, if you click the "Click for Features" link, you can see that 1604 genes are labeled "CDS" (meaning, these are the operative, non-defective genes) while a separate line item shows an utterly astounding 2233 genes as pseudogenes. (Addendum: The FASTA file at genomevolution.org contains duplicates. The actual pseudogene count, it turns out, is 1116, not 2233. But still, 1116 is a huge number of pseudogenes.) The "DNA Seqs" links on the right side of that page allow you to download the FASTA sequences for the respective gene groupings. These are simple text files containing the base sequences (A, T, G, and C) for the coding strands of the genes.
I wrote a few lines of JavaScript to analyze the base compositions of the genes (and pseudogenes), and what I noticed immediately is that the base composition differs for the two groups:
The G+C content for the "normal" genes averages 60.6%, whereas for the pseudogenes it's 55.4%. A typical G+C value for other members of the genus Mycobacterium is 65%. Thus, it's clear that not only the pseudogenes but the "normal" genes of M. leprae have drifted in the direction of more A+T. This has been noted before (by Cole et al. and others). What's perhaps less obvious is that purine content (A+G) has shifted from 50.5% in the normal genes to 49.8% in the pseudogenes. Bear in mind we're looking at data for one strand of DNA: the so-called coding or "message" strand.
Clearly, there is a tendency for pseudogenes to "regress to the mean." But the shift in purine concentration is particularly interesting, because it indicates that purine usage in normal-gene coding regions is perhaps non-randomly elevated. The shift from 50.5% to 49.8% in A+G content may not seem particularly striking on its own, but the difference, it turns out, is highly significant. You can see why in the following graph.
To make this graph, I looked at the DNA of the coding regions of "normal" genes and determined the average purine content as well as the G+C content for positions one, two, and three of all codons. As you can see, the purine content (relative to the G+C content) segregates non-randomly according to codon base position. The red dots represent base one, the gold (or brown) dots represent base two, and the blue dots represent base three (often called the "wobble" base, for historical reasons). Not unexpectedly, the greatest G+C shift occurs in base three (as is usually the case). What's perhaps more surprising is the clear preference for purines in base one. The red cluster centers at y = 0.6051 plus or minus 0.0467 (standard deviation). This means that on average, position one of a codon is occupied by a purine (A or G) over 60% of the time. This is actually quite typical of codons in most organisms. I've looked at over 1,300 bacterial species so far, and in all of them, purines accumulate at codon base one. (Maybe in a future post, I'll present more data to this effect.)
Base two segregates out as having a G+C content significantly below the organism's total-genome G+C content and centers on y = 0.4434 (median) plus or minus 0.0547 (SD).
Now compare the above graph with a similar graph for M. leprae's pseudogenes:
Here, it's evident that base compositions for all three codon positions overlap significantly. The fact that the codon positions are no longer clearly defined in their spatial representation on this graph is consistent with widespread frameshift mutations in the DNA, causing bases that would normally be in position one (or two or three) to be in some other position, randomly.
Hence we can say, with some confidence, on the basis of these graphs, that many (if not most) of the "junk genes" in M. leprae harbor frameshift mutations. The question of which came first—frameshift mutations, or silencing of genes (followed by frameshifts)—is still open. But we know for certain frameshifts are indeed rampant in the M. leprae pseudogenome.
Exactly how or why M. leprae accumulated so many frameshift mutations (and then kept hoarding the mutated genes) is unknown. As I said earlier, much work remains to be done.
Note: Graphs were produced using the excellent service at ZunZun.com. Hand-editing of SVG graphs (before conversion to PNG) enabled easy modification of the data-point colors in a text editor. Data points were plotted with opacity = 0.30 so that areas of high overlap are more apparent visually (with the piling of data points on top of data points).
Bioinformaticists (and others!), feel free to leave a comment below.
Traditionally, it has been assumed that the difficulty in growing M. leprae in pure culture is due to the organism's complex nutritional requirements. (In humans, the organism is an obligate intracellular parasite that takes up residency in the Schwann cells of the peripheral nervous system.) There is no doubt considerable truth to this assumption, but the reason for the organism's fastidious nutritional requirements wasn't fully known until Cole et al. (2001) showed that half the bacterium's genome is inoperative and undergoing decay. Genomic sequencing revealed that M. leprae has only three quarters the DNA content of its (quite robust) cousin, M. tuberculosis, and of M. leprae's 3,000-or-so remaining genes, only 1,600 are fully functional. The rest are pseudogenes.
Pseudogenes are genes that have become inactivated through loss of start codons, loss of promoter regions, introduction of spurious stop codons, introduction of frameshift errors, or through other causes. Almost all organisms contain pseudogenes in their DNA. (Human DNA reportedly contains over 12,000 pseudogenes.) The leprosy bacterium, however, is unique in having approximately half its genome tied up in pseudogenes. Once a gene becomes a pseudogene, it is effectively useless baggage ("junk DNA") and continues on a long path of deterioration. Evolutionary theory predicts that such genes will eventually be lost from the genome, since the carrying cost of keeping them puts the organism at a disadvantage, energetically. But the curious thing about M. leprae is that it's a hoarder: It not only holds onto its useless genes, it actually transcribes upwards of 40% of them. In fact, a recent study of 1000-year-old M. leprae DNA (recovered from medieval skeletons), comparing the medieval version of the organism's genome with the genome of today's M. leprae, found that pseudogenes are highly conserved in the bacterium.
The fact that the bacterium actually transcribes many of its pseudogenes (and doesn't lose them over time) is striking, to say the least, and suggests that the transcription of certain genes or pseudogenes is resulting in mRNAs that silence other, more deleterious genes. It could be that M. leprae can't be grown in culture because when certain combinations of nutrients are presented to it, the nutrients up-regulate deleterious nonsense genes in otherwise-normal operons (or down-regulate important silencers), directly or indirectly. (Williams et al. found that many M. leprae pseudogenes are located in the middle of operons and are transcribed via fortuitous read-through.) Various scenarios are possible. Much work remains to be done.
In the meantime, I couldn't help doing a little desktop science to characterize M. leprae's "defective genes" problem further. I went to http://genomevolution.org/CoGe/OrganismView.pl and entered "Mycobacterium leprae Br4923" in the Organism Name field. In the Genome Information box, if you click the "Click for Features" link, you can see that 1604 genes are labeled "CDS" (meaning, these are the operative, non-defective genes) while a separate line item shows an utterly astounding 2233 genes as pseudogenes. (Addendum: The FASTA file at genomevolution.org contains duplicates. The actual pseudogene count, it turns out, is 1116, not 2233. But still, 1116 is a huge number of pseudogenes.) The "DNA Seqs" links on the right side of that page allow you to download the FASTA sequences for the respective gene groupings. These are simple text files containing the base sequences (A, T, G, and C) for the coding strands of the genes.
I wrote a few lines of JavaScript to analyze the base compositions of the genes (and pseudogenes), and what I noticed immediately is that the base composition differs for the two groups:
| Base | Content (Genes) | Content (Pseudogenes) |
A
|
0.1938
|
0.2119
|
G
|
0.3116
|
0.2867
|
C
|
0.2890
|
0.2778
|
T
|
0.2046
|
0.2223
|
The G+C content for the "normal" genes averages 60.6%, whereas for the pseudogenes it's 55.4%. A typical G+C value for other members of the genus Mycobacterium is 65%. Thus, it's clear that not only the pseudogenes but the "normal" genes of M. leprae have drifted in the direction of more A+T. This has been noted before (by Cole et al. and others). What's perhaps less obvious is that purine content (A+G) has shifted from 50.5% in the normal genes to 49.8% in the pseudogenes. Bear in mind we're looking at data for one strand of DNA: the so-called coding or "message" strand.
Clearly, there is a tendency for pseudogenes to "regress to the mean." But the shift in purine concentration is particularly interesting, because it indicates that purine usage in normal-gene coding regions is perhaps non-randomly elevated. The shift from 50.5% to 49.8% in A+G content may not seem particularly striking on its own, but the difference, it turns out, is highly significant. You can see why in the following graph.
 |
| Base composition of "normal" genes in M. leprae (total purines vs. G+C) by codon base position. (n=1604) Red dots are for base one, gold dots are for base two, blue dots are for base 3 (the "wobble" base). Click to enlarge. See text for discussion. |
To make this graph, I looked at the DNA of the coding regions of "normal" genes and determined the average purine content as well as the G+C content for positions one, two, and three of all codons. As you can see, the purine content (relative to the G+C content) segregates non-randomly according to codon base position. The red dots represent base one, the gold (or brown) dots represent base two, and the blue dots represent base three (often called the "wobble" base, for historical reasons). Not unexpectedly, the greatest G+C shift occurs in base three (as is usually the case). What's perhaps more surprising is the clear preference for purines in base one. The red cluster centers at y = 0.6051 plus or minus 0.0467 (standard deviation). This means that on average, position one of a codon is occupied by a purine (A or G) over 60% of the time. This is actually quite typical of codons in most organisms. I've looked at over 1,300 bacterial species so far, and in all of them, purines accumulate at codon base one. (Maybe in a future post, I'll present more data to this effect.)
Base two segregates out as having a G+C content significantly below the organism's total-genome G+C content and centers on y = 0.4434 (median) plus or minus 0.0547 (SD).
Now compare the above graph with a similar graph for M. leprae's pseudogenes:
 |
| Base composition of M. leprae pseudogenes by codon position. (n=2233) Again, red dots are for base one, gold are for base two, blue are for base three. Click to enlarge. See text for discussion. |
Here, it's evident that base compositions for all three codon positions overlap significantly. The fact that the codon positions are no longer clearly defined in their spatial representation on this graph is consistent with widespread frameshift mutations in the DNA, causing bases that would normally be in position one (or two or three) to be in some other position, randomly.
Hence we can say, with some confidence, on the basis of these graphs, that many (if not most) of the "junk genes" in M. leprae harbor frameshift mutations. The question of which came first—frameshift mutations, or silencing of genes (followed by frameshifts)—is still open. But we know for certain frameshifts are indeed rampant in the M. leprae pseudogenome.
Exactly how or why M. leprae accumulated so many frameshift mutations (and then kept hoarding the mutated genes) is unknown. As I said earlier, much work remains to be done.
Note: Graphs were produced using the excellent service at ZunZun.com. Hand-editing of SVG graphs (before conversion to PNG) enabled easy modification of the data-point colors in a text editor. Data points were plotted with opacity = 0.30 so that areas of high overlap are more apparent visually (with the piling of data points on top of data points).
Bioinformaticists (and others!), feel free to leave a comment below.
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