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.

How to Not Get Away with Murder

Iffat Ara Sharmeen

Senior

School of Life Sciences

Independent University, Bangladesh

April 4th, 2017

The Timeline

In 1998, Dr. Richard Schmidt, a physician from Lafayette, Louisiana was proven guilty of second-degree attempted murder. He was sentenced to 50 years in prison. 

In 1984, Dr. Richard Schmidt began an extramarital affair with a married nurse called Janice Trahan that lasted 10 years. In 1994, Trahan divorced her husband, and then ended the relationship with Schmidt when he did not divorce his wife. On the night of August 4th, 1994, the doctor went to her house and gave her a shot while she was almost asleep, saying that it was a “vitamin B12 injection” and immediately fled. The shot was very painful, more so than the previous vitamin shots she had received from him. On August 16th, unusual symptoms started occurring. A series of visits to different specialists and tests finally revealed on January 3rd, 1995, that she was HIV-positive and Hepatitis C-positive.

Rising Suspicions and Mounting Evidence

Trahan concluded that the injection she received six months ago had not been a vitamin shot, and accused Schmidt of injecting her with infected blood during his visit on August 4th. To rule out other possibilities, prosecution had all of Trahan’s former sexual partners (she had had 7 sexual partners including Schmidt and her ex-husband between 1984 and 1995) tested for HIV. All of them were found to be HIV-negative. Trahan had also been a regular blood donor. The last time she donated blood was April 1994, and this was tested to be HIV-negative. This meant that she was infected with HIV after this, possibly via that injection. Prosecution also discovered that the doctor had drawn blood from an HIV patient under his care on August 4th, and this blood draw was recorded differently from the standard procedure in the hospital records.

A Tool from Evolutionary Biology

Genes are instructional codes found inside all organisms that determine the physical and biochemical characteristics of an organism, and the genome of an organism is its complete set of genes. Mutations refer to any changes in a genetic sequence. HIV mutates or changes very rapidly, and the fittest variants are selected for, and constantly replace older variants. This property of HIV lent this investigation to a new approach of establishing transmission in forensic investigations [1]. Since HIV mutates so fast, it was hypothesized that the viral genomic sequences from the doctor’s patient and Trahan would be more closely related to each other than to HIV viral sequences found in the rest of the population (if the virus had been transmitted between them). The hypothesis was explored using phylogenetic analysis, a tool commonly used in evolutionary biology to establish relationships between different species. 

During phylogenetic analyses, physical traits or genetic sequences are compared to infer the evolutionary relationships between organisms. Based on the similarities and differences in these characteristics, an evolutionary tree is produced, with similar sequences assumed to originate from a common ancestral sequence. In this investigation, HIV genomic sequences were used to construct phylogenetic trees. If HIV genomic sequences in all HIV-positive individuals were the same, determining the relatedness between viruses from different individuals would be impossible. However, owing to its high mutation rate and the resulting diversity, it was possible to infer with high levels of confidence the relationships between HIV viruses found in different individuals, using various statistical and tree-building methods.

A simple phylogenetic tree (left) and an example (right). These trees were generated for viruses for these analyses. Open curriculum

The Analysis

Researchers set out to investigate whether the viral genomes isolated from Trahan and the HIV- positive patient were closely related. HIV viral sequences from 32 other HIV positive patients in the same local area were also included for comparison. The comparisons were carried out by the Baylor College of Medicine and University of Michigan simultaneously to reduce risk of misinterpretation from laboratory errors. The HIV genome contains, among other genes, the env gene and the pol gene. The env gene produces proteins expressed on the surface of the virus, while the pol gene produces reverse transcriptase, the enzyme that initiates viral replication in the host cell. The two sequences are known to have different rates of evolution, and were compared between the victim, patient, the 32 individuals, and additional HIV viral sequences from elsewhere found in medical and research databases.

Results

As hypothesized, the gene sequences of the HIV viruses isolated from the patient and Trahan were more similar to each other than to the rest of the sequences included in the investigation. Baylor College of Medicine identified up to 99.87% similarity between the patient’s and victim’s env gene sequences, while University of Michigan identified up to 99.36% similarity. When they did the analysis for the pol gene, it turned out that the victim’s pol sequences formed a subset within the patient’s pol sequences. HIV viruses are tremendously diverse even within the same host; the pol sequences from  Trahan had branched out from a subset of the pol sequences from the patient that had presumably been successfully transferred in the blood sample collected by the doctor.

AZT is a drug used to treat AIDS. Viral pol sequences from Trahan and the patient had similar mutations that provided resistance against AZT. None of the others included in the investigation had the same mutations for resistance, suggesting that these mutations had evolved once and been transmitted between the two individuals.

All of this was consistent with the charge that Dr. Schmidt had deliberately injected HIV-infected blood into Trahan in an attempt to murder her. The phylogenetic analysis was ultimately a major component of the set of evidence used to build the case against the doctor. It remains a striking reminder of how tools and knowledge built from basic science can find more immediate application in the real world.

Bibliography:

[1] M. L. Metzker, D. P. Mindell, X.-M. Liu, R. G. Ptak, R. A. Gibbs, and D. M. Hillis, “Molecular evidence of HIV-1 transmission in a criminal case,” Proc. Natl. Acad. Sci. U. S. A., vol. 99, no. 22, pp. 14292–14297, Oct. 2002.

 Sharmeen is a fourth-year Biochemistry student at IUB. She loves to explore the world through a scientific lens.