by Nick Lane
Over time, such gene transfers make a big difference. Just to give a single example, gene transfer has produced two different strains of the bacterial ‘species’ E. coli that differ more radically in their gene content (a third of their genome, or nearly 2000 different genes) than all the mammals put together, perhaps even all the vertebrates! The importance of vertical inheritance, descent with modification, in which the genes are only passed on to the daughter cells during cell division, is often ambivalent among bacteria. Imagine trying to work out our own provenance by examining the heirlooms passed down in the family, only to discover that our ancestors were compulsive kleptomaniacs, forever pilfering each other’s family silver. As the branching ‘tree of life’ is based strictly on vertical inheritance—the erroneous assumption that the heirlooms only pass from parents to children—its veracity is open to question. Among the bacteria at least a network may be a better analogy. As one despairing expert put it, reflecting on the troubles of constructing a tree of life, ‘only God can make a tree’.
So why are bacteria so open-handed with their genes? It might sound like altruistic behaviour, sharing genetic resources for the good of the population as a whole, but it is not; it is still a form of selfishness, what Maynard Smith described as an ‘evolutionarily stable strategy’. Compare lateral transfer with conventional ‘vertical’ inheritance. In the latter case, if an antibiotic threatens a population of bacteria, and only a few cells have retained the genes needed to save their lives, then the rest of the unprotected population will die, and only the offspring of the tattered survivors can thrive to replenish the population. If conditions then change again, favouring a different gene, this surviving population too may be decimated. In swiftly changing conditions, only the cells that retain an enormous repertoire of genes will survive most exigencies, and they will be so large and unwieldy that they can be out-competed by bacteria able to replicate faster in the interim. Such streamlined bacteria, of course, may be threatened by any exigencies at all—but not if they are able to pick up genes from the environment; then they can combine speedy replication with the genetic resilience to cope with almost anything thrown at them. Bacteria that lose and gain genes in this way will thrive in place of either lumbering genetic giants, or bacteria that refuse to pick up any new genes at all. Presumably, the most effective way of picking up new genes is by conjugation, rather than from the dead bacteria whose genes may be damaged, so ultimately an apparently altruistic, though individually selfish, sharing of genes is favoured. Overall, then, we see the dynamic balance of two different trends in bacteria—the tendency to gene loss, which reduces the bacterial genome to the smallest possible size in the prevailing conditions; and the accumulation of new genes by means of lateral gene transfer, according to need.
I have cited examples of gene loss in bacteria like Rickettsia, and in the lab, but beyond the sparseness of their genome (the small number of genes and the lack of junk DNA), it is difficult to prove that gene loss is important in bacteria in ‘the wild’. But the importance of lateral gene transfer among bacteria also testifies to the strength and pervasiveness of the selection pressure for bacteria to lose any superfluous genes—otherwise they would not be under such an obligation to pick them up again. Despite taking up new genes, bacteria don’t expand their genomes, so presumably they must lose genes at the same rate. And they lose genes at this rate because the competition between cells within a species (and between cells in different species) must continually reduce the genome to the smallest size possible in the prevailing conditions.
The upper limit of any known bacterial genome is about 9 or 10 million letters, encoding some 9000 genes. Presumably, any bacteria that acquire more genes than this tend to lose them again, as the time needed to copy the extra genes slows down replication without providing any countering benefits. This is a stark contrast between bacteria and eukaryotes. The more we learn about bacteria, the harder it becomes to make valid generalizations about them. In recent years, we have discovered bacteria with straight chromosomes, with nuclei, cytoskeletons, and internal membranes, all traits once considered to be unique prerogatives of the eukaryotes. One of the few definitive differences that hasn’t evaporated on closer inspection is gene number. Why is it that there are no bacteria with more than 10 million DNA letters, when, as we noted in Chapter 1, the single-celled eukaryote Amoeba dubia has managed to accumulate 670 billion letters—67 000 times more letters than the largest bacteria, and for that matter 200 times more than humans? How did the eukaryotes manage to evade the reproductive constraints imposed on bacteria? The answer that I think gets to the heart of the matter was put forward by Tibor Vellai and Gábor Vida in 1999, and is disarmingly simple. Bacteria are limited in their physical size, genome content, and complexity, they say, because they are forced to respire across their external cell membrane. Let’s see why that matters.
The stumbling block of geometry
Recall from Part 2 how respiration works. Redox reactions generate a proton gradient across a membrane, which is then used to power the synthesis of ATP. An intact membrane is necessary for energy generation. Eukaryotic cells use the inner mitochondrial membrane to generate ATP, while bacteria, which do not have organelles, must use their external cell membrane.
The limitation for bacteria is geometric. For simplicity, imagine a bacterium shaped like a cube, then double its dimensions. A cube has six sides, so if our cubic bacterium had dimensions of one thousandth of a millimetre each way (1 µm), doubling its size would quadruple the surface area, from 6 μm2(1 × 1 × 6) to 24μm2 (2 × 2 × 6)μm2. The volume of the cube, however, depends on its length multiplied by its breadth by its depth, and this rises eightfold, from 1μm3 (1 × 1 × 1) to 8μm3 (2 × 2 × 2). When the cube has dimensions of 1 μm each way, the surface area to volume ratio is 6/1 = 6; with dimensions of 2 μm each way, the surface area to volume ratio is 24/8 = 3. The cubic bacterium now has half as much surface area in relation to its volume. The same thing happens if we double the dimensions of the cube again. The surface area to volume ratio now falls to 96/64 = 1.5. Because the respiratory efficiency of bacteria depends on the ratio of surface area (the external membrane used for generating energy) to volume (the mass of the cell using up the available energy) this means that as bacteria become larger their respiratory efficiency declines hyperbolically (or more technically, with mass to the power of 2/3, as we’ll see in the next Part).
This decline in respiratory efficiency is coupled to a related problem in absorbing nutrients: the falling surface area to volume ratio restricts the rate at which food can be absorbed relative to the requirement. These problems can be mitigated to some extent by altering the shape of the cell (for example, a rod has a larger surface-area-to-volume ratio than a sphere) or by folding the membrane into sheets or villi (as in our own intestinal wall, which is subject to the same need to maximize absorption). Presumably, however, there comes a point when complex shapes are selected against, simply because they are too fragile, or too difficult to replicate with any accuracy. As any spatially challenged plasticine modeller knows, an imperfect sphere is much the most robust and replicable shape. We aren’t alone: most bacteria are spherical (cocci) or rod-like (bacilli) in shape.
In terms of energy, a bacterial cell with double the ‘normal’ dimensions will produce half as much ATP per unit volume, while being obliged to divert more energy towards replicating the cellular constituents, such as proteins, lipids, and carbohydrates, that make up the extra cell volume. Smaller variants, with smaller genomes, will almost invariably be favoured by selection. It is therefore hardly surprising that only a handful of bacteria have achieved a size comparable with eukaryotes, and these exceptions merely prove the rule. For example, the giant sulfur bacterium Thiomargarita namibiensis (the ‘sulfur pearl of Namibia’), discovered in the late 1990s, is eukaryotic in size: 100 to 300 microns in diameter (0.1 to 0.3 mm). Although this caused some excitement, it is actually composed almost entirely of a large vacuole. This vacuo
le accumulates raw materials for respiration, which are continually washed up and swept away by the upwelling currents off the Namibian coast. Their giant size is a sham—they amount to no more than a thin layer covering the surface of a spherical vacuole, like the rubber skin of a water-filled balloon.
Geometry is not the only stumbling block for bacteria. Think again about proton pumping. To generate energy, bacteria need to pump protons across their external cell membrane, into the space outside the cell. This space is known as the periplasm, because it is itself bounded by the cell wall.1 The cell wall presumably helps to keep protons from dissipating altogether. Peter Mitchell himself observed that bacteria acidify their medium during active respiration, and presumably more protons are free to disperse if the cell wall is lost. Such considerations may help to explain why bacteria that lose their cell wall become fragile: they not only lose their structural support but also lose the outer boundary to their periplasmic space (of course they retain the inner boundary, the cell membrane itself). Without this outer boundary, the proton gradient is more likely to dissipate, at least to some extent—some protons appear to be ‘tethered’ to the membrane by electrostatic forces. Any dispersal of proton gradient is likely to disrupt chemiosmotic energy production: energy is not produced efficiently. As energy production runs down, all other aspects of a cell’s housekeeping are forced to run down too. Fragility is the least of what we would expect; it’s more surprising that the denuded cells can survive at all.
How to lose the cell wall without dying
While many types of bacteria do lose their cell wall during parts of their life cycle only two groups of prokaryotes have succeeded in losing their cell walls permanently, yet lived to tell the tale. It’s interesting to consider the extenuating circumstances that permitted them to do so.
One group, the Mycoplasma, comprises mostly parasites, many of which live inside other cells. Mycoplasma cells are tiny, with very small genomes. M. genitalium, discovered in 1981, has the smallest known genome of any bacterial cell, encoding fewer than 500 genes. Despite its simplicity, it ranks among the most common of sexually transmitted diseases, producing symptoms similar to Chlamydia infection. It is so small (less than a third of a micron in diameter, or an order of magnitude smaller than most bacteria) that it must normally be viewed under the electron microscope; and difficulties culturing it meant its significance was not appreciated until the important advances in gene sequencing in the early 1990s. Like Rickettsia, Mycoplasma have lost virtually all the genes required for making nucleotides, amino acids, and so forth. Unlike Rickettsia, however, Mycoplasma have also lost all the genes for oxygen respiration, or indeed any other form of membrane respiration: they have no cytochromes, and so must rely on fermentation for energy. As we saw in the previous chapter, fermentation does not involve pumping protons across a membrane, and this might explain how Mycoplasma can survive without a cell wall. But fermentation produces up to 19 times fewer ATPs from a molecule of glucose than does oxygen respiration, and this in turn helps to account for the regressive character of Mycoplasma—their tiny size and genome content. They live like hermits, with little to call their own.
The second group of prokaryotes that thrive without a cell wall is the Thermoplasma, which are extremophile archaea that live in hot springs at 60°C and an optimal acidity of pH 2. They would probably fare well in a British fish and chip shop, as their preferred living conditions are equivalent to hot vinegar. Lynn Margulis once argued that Thermoplasma may be the archaeal ancestors of the eukaryotic cell, on the grounds that they can survive ‘in the wild’ without a cell wall; but, as we saw in Part 1, stronger evidence supports the methanogens as the putative original host. When the complete genome sequence of Thermoplasma acidophilum was reported in Nature in 2000, it provided no evidence of a close link to the eukaryotes.
How do Thermoplasma survive without a cell wall? Simple: their acidic surroundings fulfil the role of the periplasm, so they have no need of a periplasm of their own. Normally, bacteria pump protons across the external cell membrane into the periplasm outside the cell, which is bounded by the cell wall. This small periplasmic space is therefore acidic, and its acidity is essential for chemi-osmosis. In other words, bacteria normally carry around with them a portable acid bath. In contrast, Thermoplasma already live in an acid bath, which is effectively a giant communal periplasm, so they can relinquish their own portable acid bath. As long as they can maintain neutral conditions inside the cell, they can take advantage of the natural chemiosmotic gradient across the cell membrane. So how do they stay neutral inside? Again, the answer is simple: they actively pump protons out of the cell in the same way as any other bacteria, by cell respiration. In other words, as in most prokaryotes, the energy released from food is used to pump protons out of the cell against a concentration gradient; and the backflow of protons into the cell is used to power the ATPase, driving ATP synthesis.
In principle, the absence of a cell wall should not undermine the energetic efficiency or genome size of Thermoplasma but in practice the cells are somewhat regressive. Although they can measure up to 5 microns in diameter, their genome, of 1 to 2 million letters, encodes only 1500 genes, and is among the smallest of bacterial genomes; indeed, it is the smallest non-parasitic genome known. Perhaps the extra effort needed to keep out a high concentration of protons saps the energy that Thermoplasma can afford to divert to replicating its genome.2
Let’s round this up. The exceptions of Mycoplasma and Thermoplasma only go to prove the rule: the complexity of both bacteria and archaea is curtailed by their need to generate energy across the outer cell membrane. In general, bacteria can’t grow larger because their energetic efficiency falls off quickly as their cell volume increases. If they lose their cell wall, the outer boundary to the periplasm, the proton gradient is more likely to dissipate away, sapping the energy supply and rendering the bacteria fragile. The only prokaryotes that have survived without the cell wall are tiny regressive hermits, such as Mycoplasma, which live by parasitism and fermentation, or specialists like Thermoplasma, which can only survive in acid. Despite losing their cell walls, and so in principle being able to consume particles, neither group shows any tendency towards the predatory eukaryotic habit of engulfing food by phagocytosis. Neither do they show any tendency to develop a nucleus, or for that matter any other eukaryotic traits. These traits, I shall argue, depend on the possession of mitochondria.
Why insider dealing pays
The advantage of mitochondria is that they reside physically inside their host cell. Recall that mitochondria are bounded by two membranes, an outer and an inner membrane, which enclose two distinct spaces, the inner matrix and the inter-membrane space. The respiratory chains and the ATPase complexes are all embedded in the inner mitochondrial membrane, and pump protons from the inner matrix to the inter-membrane space (see Figure 1, page 12). The acid environment needed for chemiosmosis is therefore contained within the mitochondria and does not affect other aspects of cellular function. (Technically it is not actually acidic, as the protons are buffered, but this doesn’t alter the thrust of the argument.)
Internalization of energy generation within the cell means that an external cell wall is no longer needed, and so can be lost without inducing fragility. Loss of the cell wall frees up the external cell membrane to specialize in other tasks, such as signalling, movement, and phagocytosis. Most importantly of all, internalization releases the eukaryotic cell from the geometric constraints that oppress bacteria. Eukaryotes are on average 10 000 to 100 000 times the volume of bacteria, but as they become larger, their respiratory efficiency doesn’t slope off in the same way. To increase energetic efficiency, all that eukaryotic cells need to do is to increase the surface area of mitochondrial membranes within the cell; and this can be done simply by having a few more mitochondria. Internalization of energy production therefore enables both the loss of the cell wall and a much greater cell volume. In the fossil record, the sheer size of eukaryotic cel
ls often helps to distinguish them from bacteria—and this greater size appeared quite suddenly, in geological terms, with the internalization of energy generation in the cell. Suddenly, some 2 billion years ago, large eukaryotic cells appear in the fossil record; presumably this must date with some accuracy the origin of the mitochondria, although they themselves can’t be made out in the fossils.
So bacteria are under a strong selection pressure for small size whereas eukaryotes are not. As eukaryotic cells grow larger, they can maintain their energy balance simply by keeping more mitochondria inside—herding more pigs, as it were. So long as they can find enough food to oxidize—enough to feed the pigs—they are not constrained by geometry. Whereas large size is penalized in bacteria, it actually pays dividends in eukaryotes. For example, large size enables a change in behaviour or lifestyle. A large energetic cell does not have to spend all its time replicating its DNA, but can instead spend time and energy developing an arsenal of protein weapons. It can behave like a fungal cell, and squirt lethal enzymes onto neighbouring cells to digest them before absorbing their juices. Or it can turn predator and live by engulfing smaller cells whole, digesting them inside itself. Either way, it doesn’t need to replicate quickly to stay ahead of the competition—it can simply eat the competition. Predation, the archetypal eukaryotic lifestyle, is born of large size, and it depends on overcoming the energetic barriers to being larger. A parallel with human society is the larger communities made possible by farming: with more manpower, it was possible to satisfy food production and still have enough people left over to form an army, or invent lethal new weapons. The hunter-gathers couldn’t sustain such a high population and were bound to lose out to the numerous and specialized competition.