Rise of the Other Kind(s): Part III

Maliha Tanjum Chowdhury

Freshman

School of Life Sciences

Independent University, Bangladesh

July 13th, 2017

This the concluding part of  a three-part series of articles that aim to introduce the study of evolution using microbes as model systems while focusing on a recent study on speciation in bacteriophages.

After looking at infectivity of the bacteria by the differently evolved bacteriophages, the next factor studied was the adsorption rate – how well the phages can bind to either receptor protein. Surely enough, every specialist bacteriophage that had evolved had a binding affinity greater than the original or ancestral bacteriophage to their preferred host. Additionally, the scientists have also inferred that the gains in adsorption rate for the preferred receptor were greater than the losses on the alternative.

Now, to dig even deeper, the researchers meticulously sequenced phage alleles and measured the differences in gene sequences. All the specialists were found to have mutations in the host recognition gene J (the gene that codes for the protein that is responsible for binding to the bacterial host) and all individual mutations were seen to be non-synonymous – that is, the mutations resulted in changes in protein structure (which can be expected to ultimately affect binding). Interestingly, regardless of evolving by allopatry or sympatry, specialists for either type of receptor showed stronger genetic relatedness between themselves than with specialists of the other type.

Finally, the scientists resorted to cross-checking their findings to verify that their inferences were indeed true. They created new bacteriophages by artificially constructing the mutated alleles of either specialist, the ancestor EvoC strain, and a hybrid with all the mutations from the specialists for both receptors. Reality met expectations as the mutations found in either evolved strain did prove to be responsible for their respective host specializations and the genetic configuration of the ancestral EvoC was indeed seen to be expressing the generalist trait. The hybrid-child, sadly, did not prove to be viable. These observations not only confirm the J allele mutations as the cause of diversification, but also show how species may begin to emerge through mutations that result in reproductively incompatibility, proven by the production of non-sustainable hybrid progeny.

While I know all you science-mad kids are getting totally dizzy and starry-eyed marveling this experiment’s successes, it is always sensible to remember that even the coolest experiments are, to some degree, chained down by assumptions and reality. For instance, in this study, even with the substantial dissimilarities between them, the specialists are still much more similar than the cut-off for different species, that is, less than 70% sequence similarity. Not ones to be disheartened, the scientists argued that they did indeed observe the trademark processes that lead to eventual speciation, but did not let it run long enough for actual, classifiable species to emerge. 

Aside from this, there also surfaced some confusion over the incidence of genetic reversion – did the EvoC phage simply “go back” to being its ancestor, the predominant LamB-specialist? In depth analysis, however, put this matter to rest as among the 12 sequenced alleles, only 1 was seen to have reverted at a single site of the 5 mutations that set EvoC apart from its earlier ancestor. The LamB specialist can thus be described as much an independently evolved phage as the OmpF specialist. Another experimental drawback was in that Lambda phages are not completely sexual; also, phages need only a few mutations in a single gene to become reproductively isolated. As a consequence, it’s logical to think that these conclusions may not apply to speciation that requires more genetic change.

Apart from the specific constraints of this particular experiment, there always remain some basic unanswered questions – how likely is this effect to be a noteworthy and widespread one in nature? What key factors are responsible in speeding up or slowing down speciation? And, most importantly, what parameters can be measured to directly test the limits of speciation? These will be important open questions in evolutionary for a long time.

It’s been a riveting journey observing how life-forms function in response to the winds of change over the course of this experiment. It is no doubt that we owe our heartfelt gratitude to these brilliant minds as they have been able to physically show us the baby steps of a transition as complex and mind-boggling as evolution. Sure, saying that, in 50 more years, we might be able to “see” the monstrous Triceratops transform into the ethereal, enchanting peacock (for example) is a stretch. But because of such breakthroughs occurring ever so often in this millennium of miracles, I dare to dream that we understand the process much more clearly.

Maliha is a weirdo who somehow believes she's from a different planet. But she likes Earth just fine, and is fascinated by the science and beauty of life and has made it her purpose to explore it. Besides this, her most burning desires include becoming a synthetic biologist/ genetic engineer and running away with a heavy metal band.

Rise of the Other Kind(s): Part II

Maliha Tanjum Chowdhury

Freshman

School of Life Sciences

Independent University, Bangladesh 

July 6th, 2017 

This is the second in a three-part series that will broadly introduce and describe the study of evolution using microbes as model systems, and specifically focus on a recent study on speciation.

Practically, the fastest shift from one type of organism to another that can be observed in a laboratory is that of asexual microbes, e.g. bacteria and viruses. It is needless to say that for uncomplicated, single-celled chaps like viruses, bacteria and the like, rapid transition through numerous generations in a couple of hours or less is a walk in the park. Recall that any sort of genetic variation is simply the outcome of random, independent mutations in nucleotide or gene sequences. The accumulation of major changes which can possibly be observed between consecutive generations is far more evident in the case of microbes, as the hereditary fate of a given microbe is often entirely wielded by a single strand of DNA or RNA. We can select for some of these changes by providing different selection pressures.

However, speciation, based on our textbook definition, requires the incidence of sexual reproduction for organisms to diverge into distinct species (for them to no longer be reproductively compatible). Therefore, it is harder to define species when it comes to asexual microbes. 

In a recent study exploring sympatric and allopatric speciation, bacteriophage (viruses that infect bacteria) lambda was chosen as the model system to study the processes, as it not only divides rapidly asexually, but also has an exceptional ability to recombine with phages that coinfect the same host, thereby creating progeny and exchanging genes sexually. This allowed the conclusion of the study to be at least partially relevant to sexually reproducing species. Now, even though the rates and mechanisms of speciation may seem to vary for viruses and multicellular organisms, some features are comparable. For instance, reproductive isolation and incompatibility, which are concepts we learnt about earlier, mean much the same for viruses. In this specific case, reproductive incompatibility refers to the inability to recombine with other viruses whose nucleotide composition has evolved to differ considerably. Hence, all things considered, this was a clever model to work with.

We’re now going to delve deep into the experiment itself, so hold on to your seats, because it’s going to get much more science-y from here onwards. The researchers basically tried to observe the two kinds of speciation, allopatric (due to geographical separation) and sympatric (within the same environment), respectively, in the bacteriophage populations. The lab-generated bacteriophage lambda strain EvoC was the focus of the study. Bacteriophages begin their replication cycles by binding to receptor proteins on the host cell, and injecting their genomes into the cell. Individual bacteriophages tend to be very specific to the type and structure of the receptor proteins they can bind to. This virus, however, was a “generalist”- a bacteriophage with the ability to bind to both the OmpF and LamB receptor-proteins on the bacterial surface of Escherichia Coli.  

For this study, two different hosts were utilized: an E. coli strain carrying the OmpF receptor, and an E. coli strain carrying the LamB receptor. A broad summary of the experimental results is as follows: the bacteriophages, when supplied with just one of the two hosts, specialized in binding to the available receptor on that host while steadily losing the ability to bind to the other (allopatry).  More excitingly, when propagated on equal amounts of both hosts (and therefore in the presence of both receptors) together, the bacteriophages still divided into two distinct lineages with different host preferences (sympatry). In the light of these findings, the results shine through as compelling evidence that for the advent of distinct species, both allopatry and sympatry could play significant roles.

I personally feel that this article would remain terribly incomplete without including a walk-through of the methods used. So, here they are as follows:

1. Twelve bacteriophage (EvoC) populations, initially exactly the same, were grown with either one or other type of host, that is, six populations were grown in OmpF-expressing bacteria and the other six were grown in the LamB-expressing ones.
2. The bacteriophage populations were systematically passaged through the host populations for 35 cycles of dilution (the experiment took roughly a month in real world time).
3. In 8-hour intervals, bacteriophages were collected and stored.
4.  A fresh cycle of viral reproduction was kicked off by the transfer of 1% of the phage into a brand new population of host bacteria the next day.
5. Six other bacteriophage (EvoC) populations, initially exactly the same, were exposed to a culture of both types of host populations present in equal amounts, i.e. both OmpF and Lamb-carrying bacteria.
6. Steps 2-4 were conducted for these as well.

Step 1 is the allopatric set-up, as the isolated flasks containing only kind of host receptor represent geographical separation and different conditions from viruses growing only with the other type of receptor. Step 5 describes the sympatric experimental set-up, as viruses are allowed to switch between both available hosts and this may allow recombination between viruses that co-infect a given bacteria at some point. Lastly, to ensure a higher chance of co-infection – and thus, recombination between viruses – a high virus to bacteria ratio was maintained in all experimental units.

 A typical plaque assay. ASM

The results, as I passingly mentioned above, were beyond satisfactory. The scientists made their primary inferences based on observing clear regions, or “holes”, in lawns of bacterial colonies grown on standard agar plates.  This experimental method is known as the plaque assay – where the term “plaque” refers to the “holes” caused by viral growth. The plaques represent the ability of the bacteriophage to bind to the bacterial receptor. If there are no plaques, there has been no binding or infection.

Considerable significant receptor specialization evolved in all 12 bacteriophage populations which were grown on single bacterial hosts, and this conclusion was drawn on observation that bacteriophages that produced plaques on OmpF-expressing bacteria failed to do so on LamB-expressing ones, and vice versa. Again, more surprisingly, this was seen to be true for bacteriophages that were grown with both kinds of hosts together.

Therefore, even when both receptors were available, bacteriophages tended to become specialized for one kind of host. How and why might that be the case? What do these results really say about speciation? Find out the in the concluding part of this series next week. 

To be continued

Maliha is a weirdo who somehow believes she's from a different planet. But she likes Earth just fine, and is fascinated by the science and beauty of life and has made it her purpose to explore it. Besides this, her most burning desires include becoming a synthetic biologist/ genetic engineer and running away with a heavy metal band.

Rise of the Other Kind(s): Part I

Maliha Tanjum Chowdhury

Freshman

School of Life Sciences

Independent University

July 1st, 2017

This is the first in a three-part series that will broadly introduce and describe the study of evolution using microbes as model systems, and specifically focus on a recent study on speciation.

If you’re still one of those people who constantly pick their brains trying to figure out how a small, often seemingly benign creature like the bird could possibly be descended from the titan-like dinosaurs who once ruled the planet, you are not alone. The word evolution is generously and rightly paraded around to explain this phenomenon, but it is difficult for most to visualize. However, speciation, a word – an idea – much less known to the general public, comes much closer to explaining such transitions. Speciation describes the complex and extremely slow-paced chain of events that directly bring about this incredible metamorphosis from one creature to another over the course of millions of years.

Now, the term “species”, from which “speciation” has been derived, can be described as a group of organisms with strongly similar physical and biochemical properties. In more bookish terms, speciation is defined as the divergence of a single species into two (or more) groups of organisms so different from each other (and from the original species) that they can no longer produce viable, fertile young together. Allopatric speciation is when new species emerge due to a geographical rift between factions of the same population thereby exposing them to different selection pressures and thus, different responses to them. On the other hand, sympatric speciation is the emergence of divergent species from a single, original species in the same geographical region. The latter form of speciation is relatively harder to conceive as the incidence of reproductive isolation (wherein members of the same species stop interacting to reproduce) within closely knit communities is a much rarer phenomenon. However, this can be explained by the fact that separation often occurs due to separation into different ecological niches. For instance, individuals of an aquatic species may prefer to live near the surface or at the bottom of a pond, thereby leading to separation into different niches or locales within the same broad geographical location.

It is quite difficult to imagine how small changes in the characteristics of living organisms in response to different selection pressures could lead to the vast amount of biodiversity we see on earth. But a few billion years on the course of speciation, and magic happens – ancient amoeba-like creepy crawlers may transform to graceful sea-creatures, simple algal ancestors may flourish into magnificent flowering plants, and according to some, the ancestors of the apes that you go visit at the zoo may even turn into a person. The odds are as endless as the universe itself, and so, evolution is a beautiful thing – something quite poetic. It has helped and will continue to help scientists trace back to the ancestors of organisms that exist now, thereby creating a bridge between the present and some long-forgotten, illusory time in the past, and just simply help understand the dynamics of the living world better. Just as boundless oceans are formed from the assemblage of billions of droplets, a steady accumulation of mutations, products of recombination and the like generate more and more diversity and the uninterrupted influence of natural selection continually increases the frequency of fitter variants among this generated diversity. At the current moment, we see a snapshot of life on earth that is very far along, according to our sense of time. We see millions of different species that have evolved from a focal common ancestor.

Honestly, who wouldn’t want to play god and observe such enchanting changes under the microscope in their own little petri-dish? Sadly, and quite obviously, speed-racing through billions of years in a lab is NOT feasible, and thus we cannot hope to observe processes like the evolution of humans and birds. It is this powerlessness of humankind that has driven evolutional theorists and biologists to more often try and establish links between larger, multicellular species based on fossils, geological evidence, DNA sequences (when available), and mathematical modeling.

However, there does happen to exist a way of observing evolution in the lab, by using organisms that go through generations much, much faster than us...

To be continued

Maliha is a weirdo who somehow believes she's from a different planet. But she likes Earth just fine, and is fascinated by the science and beauty of life and has made it her purpose to explore it. Besides this, her most burning desires include becoming a synthetic biologist/ genetic engineer and running away with a heavy metal band.