Path of least resistance

13 September 2013 by Malcolm Campbell, posted in Biology

To make a deep physical path, we walk again and again. To make a deep mental path, we must think over and over the kind of thoughts we wish to dominate our lives." By Henry David Thoreau (1817-1862)

It’s hard to remember precisely when the path came into existence.

At the beginning of the season, the possible routes from one place to the other seemed vast. Any number of paths could be taken from point A to point B. Yet somehow, over a mere matter of months, this particular path revealed itself.

Certainly the lay of the land came into play. The path is situated in a manner that is the easiest to tread from one place to the other. It is relatively flat to the foot, with an incline in one direction, and a decline in the other, that is neither too taxing, nor difficult to negotiate. When it rains, it remains navigatable, not too slippery underfoot. When it is dry, the soil remains intact, neither crumbling nor developing ruts from repeated use. As one walks or runs over it, the path feels right – pace can be maintained, with a comfortable, balanced gait.

The route is, quite literally, the path of least resistance.

So, over time, the route was used and re-used. Grass was incrementally worn away. Bit by bit, the path insinuated itself on the landscape. It became a new feature, honed and refined by continued selection by travellers. It was a perfected, naturally-selected adaptation to travel from one place to the other.

Notably, the discovery of the path is a process that plays itself out year after year. The workings of fall, winter and spring will give the landscape a chance to recover. Grass will refill the route, and next summer the daily passage of walkers and runners will re-establish the path anew. It’s as though the passage of winter rewinds a recording of the path’s genesis, and this recording is re-run on an annual basis.

Remarkably, residing just beneath the path, embedded in the soil, are organisms that can retrace the route to a useful adaptation, much like the annual rediscovery of the path. Those organisms are soil microbes. One particular species can follow an evolutionary path that enables it to colonise a new environment, tracing the same course of change every time.

This species is Pseudomonas fluorescens.

Pseudomonas fluorescens is a soil-borne bacterial species found the world over. It is a species in the larger Pseudomonas genus. Pseudomonas fluorescens gets its name by virtue of the fact that it fluoresces under ultraviolet light. This fluorescent property is derived from a specific compound produced by Pseudomonas fluorescens. The compound, pyoverdin, is a siderophore – a specific class of organic molecules that enable the bacteria to soak up iron.

The Pseudomonas genus includes species that are pathogens, free-living bacteria, and helpful bacteria alike. Generally speaking, Pseudomonas fluorescens falls in the latter two categories. While it has been linked to rare disease in humans, Pseudomonas fluorescens is generally viewed as a bacterium with beneficial properties when it comes to plants. Specifically, Pseudomonas fluorescens has been touted as a plant growth-promoting bacterium. It is not known precisely how Pseudomonas fluorescens promotes plant growth, but it is thought that a good part of the benefit is derived from the ability of Pseudomonas fluorescens to ward off bacteria that are harmful to plants.

On account of its plant growth-promoting properties, Pseudomonas fluorescens was an obvious subject for scientific investigations. Once it entered the laboratory environment, Pseudomonas fluorescens revealed itself to be an attractive study organism for other reasons – it was relatively easy to culture, it grew well, and it was amenable to addressing questions that spanned microbiology, molecular genetics, botany, ecology and evolution.

Amongst the people who capitalised on the strengths of Pseudomonas fluorescens research was a young microbiologist, Paul Rainey. Originally hailing from New Zealand (to which he has now returned), Rainey established a powerful research team at Oxford University where they dissected the intricacies of Pseudomonas fluorescens biology.

The strength of Rainey’s research lies in its simplicity. Specifically,  Rainey and his team merely seeded a very simple broth medium with a few Pseudomonas fluorescens bacteria and left the bacteria to their own devices – dividing, generation over generation, for days on end.

The manner in which Rainey grew Pseudomonas fluorescens, contrasts markedly with how most bacteria are normally grown in laboratories. Normally, bacteria are introduced to broth medium in a vessel, and then the vessel is swirled vigorously. The swirling of the vessel is very similar to what wine experts do before they inhale the bouquet of the wine – the swirling aerates the wine in the glass, releasing the odour of the wine as it does so. In the case of bacterial culture, swirling the vessel also aerates the broth medium. This is very important for bacteria like Pseudomonas fluorescens, as they require oxygen to grow. The lower the oxygen in the medium, the less they are able to grow. Swirling the broth medium ensures the bacteria grow in a uniformly aerated, homogeneous environment – that every bacterium within the culture has an equivalent chance to grow.

The vessels in which Rainey and his team grew their Pseudomonas fluorescens were not like normal bacterial growth vessels. Instead of being flask-shaped, they were short, straight-sided, flat-bottomed vessels – more shot glass than wine glass.

Between the shape of the growth vessel, and the fact that the vessel was not swirled, the Rainey team presented a very different set of growth circumstance to Pseudomonas fluorescens than they would encounter in most laboratories. In fact, Rainey was growing bacteria in a manner that most laboratories would find unusual, if not outright useless. As one colleague put it, Rainey “actually studies what most of us would throw away”.

But this was the beauty of the system.

By growing the bacteria in un-swirled medium, in simple, straight-sided vessels, Rainey and his colleagues presented different niches for the bacteria to live in. One could envisage minimally three niches. One would be at the air-broth interface – there would be lots of oxygen there. The next would be throughout the vessel – there would be less oxygen there, but conditions would still support growth. The final niche would be at the bottom of the vessel – this would be the most oxygen-deprived niche – virtually an anaerobic zone.

By leaving the bacteria alone to divide, generation after generation, in the multi-niche vessels, Rainey’s team were conducting a small experiment in evolution. They were asking a simple question. Could the bacteria evolve such that new forms of the bacteria could occupy any of the three different niches?

All of the cultures were established with a single isolate of Pseudomonas fluorescens. Normally, this isolate would live in the moderately aerated zone in the middle of the vessel. If these bacteria were removed from the broth, and made to grow on a semi-solid, gelatine-like surface they grew as nice smooth, round colonies. These bacteria were used as the ancestors in the evolution experiment.

What Rainey and his team discovered was amazing. Left on their own, with nothing but periodic mutation and the natural selection of the three niches, the bacterial cultures would evolve new strains that could occupy the three niches. Ancestral-like bacteria were found, as expected, in the middle of the vessel. By contrast, new, very slow growing bacteria were found that preferred to grow in the relatively anaerobic conditions at the bottom of the vessel. Most dramatically, there were entirely new-looking bacteria growing at the air-broth interface.  These new bacteria adhered together forming a mat. You could take a pair of tweezers and remove the mat as one clump at the end of the experiment.

The new bacteria that grew at the air-broth interface were really rather special. If individual cells from the mat were transferred to the semi-solid, gelatine-like medium they grew faster than the ancestral bacteria, so that the colony was larger. The colony also had a wrinkly appearance. Consequently, these newly evolved bacteria were referred to as “wrinkly spreaders”.

Wrinkly spreaders were a stunning innovation. Their success in occupying the air-broth interface lay in two key factors. First, they produced a lot of a sticky polymer on the outside of the cells that enabled them to adhere to each other. The sticky polymer was a modified form of cellulose – a large polymer that not only enabled adhesion, but also formed a raft-like structure suitable for floating atop the broth. The other factor that contributed to the success of wrinkly spreaders was a system that enabled the bacteria to sense the presence of other bacteria, and to “cooperate” with them. That is, based on the perception of like bacteria, the wrinkly spreaders modified their cellular orientation and production of the polymer so as to create a multi-cellular mat that enabled them to occupy their niche.

Wrinkly spreaders are truly a wonder. They illustrate how complex, multi-cellular structures can emerge from simple single-celled organisms. They speak to the evolution of multicellularity, cooperativity, and niche modification. After all, wrinkly spreaders not only occupy a niche, but they actually modify the niche by constructing a new structure. They are not merely passive occupiers of an environment, but active creators of a new environment.

Now, as if the emergence of wrinkly spreaders was not marvellous enough, there are two other astonishing elements to this story.

Rainey and his team found that they could rerun their evolution experiment time and again with the same outcome. That is, if they started with the same ancestral bacterium, following the same protocol, they could reproduce the evolution of the wrinkly spreaders every time. Rainey and his colleagues had experimentally tested a long-standing question originally posed by Stephen Jay Gould. That question was, “If one was to rewind the tape of evolution and start it over again, would one see the same types of organisms emerge again?”. Rainey’s experiments suggested that, at least for simple organisms, under well-defined, highly reproducible environmental conditions, the answer is “yes”.

Crucially, Rainey and his colleagues also found that the evolutionary path to becoming a wrinkly spreader also was highly similar every time. Evolution occurs when random mutations provide a reproductive advantage under a selective condition. Rainey and his colleagues could control the selective condition – the niches in the vessel. They could also monitor the reproductive advantage – bacterial growth. Finally, the also had control over the starting material – the ancestral bacterium was always the same. That left them to monitor the nature of the mutations that selection chose to advantage the wrinkly spreaders.

Rainey’s experiments showed that mutations in only two gene clusters were necessary for wrinkly  spreaders to emerge. These clusters, known as operons, are aggregates of genes that work together to accomplish particular tasks. In the case of the wrinkly spreaders, the operons in question are completely in keeping with the features of the wrinkly spreader bacteria. One operon is involved in making the sticky cellulose-like polymer that enables the cells to adhere to each other. The other operon is involved in detecting other cells, and using this information to control cellular processes like deposition of the cellulose-like polymer.

In some ways, the two operons that are mutated to create wrinkly spreaders are obvious when one knows how wrinkly spreaders work. This said, one can envisage that there might be other ways for cells to occupy the air-broth interface, and different genes involved in other pathways that might enable them to accomplish this. Instead, the centrality of the two operons in the evolution of wrinkly spreaders suggest that they comprise a form of genetic “path of least resistance”. Mutations in these genes are the easiest way to travel from being an ancestral bacterium to an air-broth-interface-adapted descendant.

The amazing thing about evolution is that its ability to find paths of least resistance is not constrained to unicellular organisms in the laboratory under highly defined experimental conditions. Evolution travels on paths of least resistance with multicellular organisms, for complex adaptations, in the real world. This is beautifully illustrated by considering one of the most unlikely of pairings, bats and dolphins.

The most recent common ancestor of bats and dolphins was a land-dweller that lived some 60 million years ago. Since then, bats and dolphins emerged from branches of the mammalian family tree that had taken radically different trajectories. Over time, bats’ ancestors took to the air. About 10-20 million years ago, some bats specialised in catching insects on the wing, using echolocation to target their prey. Other bat lineages didn’t acquire echolocation, and identified their food by other means. Simultaneously, dolphin ancestors, which includes the progenitors to all whales, took to the sea. Numerous evolutionary innovations emerged in this branch of the mammal family tree as well – including migration of nostrils to the top of the head, modified limbs for swimming, and, in the toothed whale lineage, which includes dolphins, echolocation. As was the case for bats, echolocation likely emerged as a novel, completely-independent innovation some 10-20 million years ago.  Also as was the case for bats, some whale lineages, notably the baleen whales, did not acquire echolocation as a prey-capturing innovation.

Echolocation is effectively a highly-specialised form of noise-making and hearing. It requires a mechanism to generate high frequency sound waves, a means to capture the sound waves when they return to the ear after bouncing off an object, and cognitive capacity to process the sound wave information so as to develop a three-dimensional mental picture of the object. The mammalian ear in particular has to be modified to detect sound waves outside of the normal range of hearing.

Echolocating bats and dolphins both have modified cochlear hairs in their ears that enable them to detect high frequency sound waves. Notably, in both types of echolocators the specialisation of the hairs is imparted by a particular protein, known as prestin. In echolocating bats and dolphins, the gene that encodes the prestin protein has mutations that confer crucial biophysical properties on the ear hairs enabling them to amplify incoming sound waves. What is truly astonishing is that the nature of the mutations that confer these properties on the bat prestin are the same as those that have conferred the same properties on the dolphin prestin. It is notable that these same mutations are not found in either bats that are not echolocators or in baleen whales. This suggests that evolution has discovered the same path of least resistance to acquire one part of the echolocation adaptation. This has occurred in lineages that are separated by 60 million years, where echolocation didn’t emerge for 40 million years after the lineages split. This is what is known as convergent evolution – where two distantly-related lineages acquire similar, or, as in this case, identical evolutionary innovations independently.

The convergent evolution of prestin is astonishing in and of itself.

But this is only the tip of the iceberg.

Recent comparison of a large number of genes from echolocating bats and dolphins has suggested that convergent evolution is widespread in the genomes of these animals. Crucially, this convergent evolution isn’t just randomly distributed in the genome. Instead, mutations were centred on specific genes. As one might expect in a comparison of echolocators, many of these genes were related to hearing. Surprisingly, many were also implicated in vision. It may be that genes related to the circuitry that is normally used to interpret three-dimensional visual data are modified to accommodate the interpretation of three-dimensional aural data. In any case, what is clear is that evolution has cleared an equivalent, multistep path to echolocation in two very distinct lineages. There is no need to rewind the evolutionary tape here – the side-by-side convergence shows that paths of least resistance are readily discovered by evolution.

Many thinkers throughout human history have been rather derisive about paths of least resistance. There is a notion that this is the way of the weak, and that nothing is to be gained by following such paths. Evolution counters that. Evolution shows us that, contingent on the forces that shape them, paths of least resistance can lead to stunning innovation – adaptations that confer great advantage. It suggests that shouldn’t eschew paths of least resistance. They can get us where we need to go with the least of bother, and the greatest of progress.

References:

Bantinaki E, Kassen R, Knight CG, Robinson Z, Spiers AJ, & Rainey PB (2007) Adaptive divergence in experimental populations of Pseudomonas fluorescens. III. Mutational origins of wrinkly spreader diversity. Genetics 176: 441-453

Goymer P, Kahn SG, Malone JG, Gehrig SM, Spiers AJ, & Rainey PB (2006) Adaptive divergence in experimental populations of Pseudomonas fluorescens. II. Role of the GGDEF regulator WspR in evolution and development of the wrinkly spreader phenotype. Genetics 173: 515-526

Li G, Wang J, Rossiter SJ, Jones G, Cotton JA, & Zhang S (2008) The hearing gene Prestin reunites echolocating bats. Proceedings of the National Academy of Sciences 105: 13959-13964

Liu Y, Cotton JA, Shen B, Han X, Rossiter SJ, & Zhang S (2010) Convergent sequence evolution between echolocating bats and dolphins. Current Biology 20: 53-54

Parker J, Tsagkogeorga G, Cotton JA, Liu Y, Provero P, Stupka E & Rossiter SJ (2013) Genome-wide signatures of convergent evolution in echolocating mammals. Nature (epub ahead of print)

Rainey PB, & Travisano M (1998) Adaptive radiation in a heterogeneous environment. Nature 394: 69-72

Spiers AJ, Kahn SG, Bohannon J, Travisano M, & Rainey PB (2002) Adaptive divergence in experimental populations of Pseudomonas fluorescens. I. Genetic and phenotypic bases of wrinkly spreader fitness. Genetics 161: 33-46

Spiers AJ, Bohannon J, Gehrig SM, & Rainey PB (2003) Biofilm formation at the air–liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. Molecular Microbiology 50: 15-27

Spiers AJ, & Rainey PB (2005) The Pseudomonas fluorescens SBW25 wrinkly spreader biofilm requires attachment factor, cellulose fibre and LPS interactions to maintain strength and integrity. Microbiology 151: 2829-2839

Images: All photographs by Malcolm M. Campbell.

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