Whether by Stealth or Aggression, Viruses Get to Make a Meal of it

The molecular processes leading to such devastating human diseases are an extension of a fundamental paradigm represented by obscure bacteria-eating viruses.

This article is the second of a two-part series on viruses and bacteriophages. The first part was published on November 26.

Bacteria-eaters, genetic engineers, disease causers. Credit: Zappys/Flickr, CC BY 2.0

Bacteria-eaters, genetic engineers, disease causers. Credit: Zappys/Flickr, CC BY 2.0

A paper published by a London-based scientist of limited financial means named Frederick Twort in The Lancet on December 4, 1915, described the discovery of an infectious agent with the power to kill bacteria. Twort was not sure what this agent might be, but proposed that it could be “an ultra-microscopic virus (that) belongs somewhere in (the) vast field of life more lowly organised than the bacterium or amoeba”.

He was right in this conjecture, but in the absence of assuring evidence, wondered if “it (might) be living protoplasm that forms no definite individuals, or an enzyme with power of growth.” Most scientific endeavours are not complete and always leave room for more interrogations. Twort “regret(ed) that financial considerations have prevented my carrying these researches to a definite conclusion, but I have indicated the lines along which others more fortunately situated can proceed.”

Though Twort was employed in a clinical setting, this first known description of a virus capable of infecting and killing bacteria didn’t arise from a vision to discover antibacterial therapies or natural genetic engineers immediately usable for human betterment – but from Twort’s esoteric interest in discovering viruses that could not cause disease and to develop methods to grow viruses in the laboratory in the absence of host cells (“an unfortunate avenue of research”). Today, viruses which infect bacteria are referred to as bacteriophages – phagein being Greek for ‘devour’. They’re the viral elements that are responsible for the disease causing traits of many bacteria, though this was certainly not suspected 100 years ago.

As an aside, it’s believed that a mysterious anti-bacterial agent whose presence in the Ganges was reported by Ernest Hankin in the 19th century might in fact have been a bacteriophage, but the author didn’t report any such suspicions. This study appears to have its origins in the author’s curiosity about the manner in which the Ganges, uniquely worshipped by the locals and treated as a medium of purification but ridiculed by Europeans, might differ from the “equally cloudy” European rivers.

Anyway, the story of the discovery of the bacteriophage is not without its controversies. Another scientist, Felix d’Herelle, of probably greater fame than Twort given his professorship at Yale and medical explorations in different parts of the world, reported his discovery of the bacteriophage two years after Twort’s publication. In fact, d’Herelle was the one who had coined the term “bacteriophage”.

Leading up to the ‘phage group’

D’Herelle was probably unaware of Twort’s discovery though some suspect that he was not being honest in making this claim. Nevertheless, d’Herelle was also more sure of his identification of his infectious antibacterial agent as a virus. He performed significant work suggesting that growth in the numbers of bacteriophages in the body of a patient was correlated with a decline in the severity of the bacterial infection that the patient was suffering from. This suggests the use of bacteriophage in therapies against bacterial infections, a concept which had mixed success.

The French-Canadian also made a big stride forward in showing that a bacteriophage of a certain type was highly choosy in which bacterium it could infect; it could evolve and attain properties that enabled it to infect related bacterial varieties; but could not – within the scope of his experiment – gain the ability to infect a widely different variety of bacteria. This finding in fact resonates with today’s fears that a virus that causes disease in an animal could evolve to become a pathogen of humans (read: swine flu or bird flu).

Just like living with bacterial and viral pathogens is not doom and gloom for the human race, living with numerous predatory bacteriophages is not the end of the world for bacteria as a class of organisms. As everyone ultimately ends up co-existing. Just as we have immune responses to pathogens, bacteria carry their own molecular immune systems to counter predation by bacteriophages.

We’ve come a long way from the early 20th century in many ways, including in our understanding of bacteriophages, but this is the result of the intellect and toil of many. The most famous among these was a group of scientists known as the ‘phage group’. It was nucleated in the 1930s and 1940s in the United States by the physicist Max Delbrück, who was interested in understanding the fundamental aspects of life. Members of the group and their colleagues believed that bacteriophages – given their simplicity as “lowly organised” creatures, their rapid multiplication and ease of handling – would serve as a perfect ‘model system’ for studying the foundational features of life. The term ‘model system’ and its applicability to science and biology in particular deserves special treatment.

We don’t have the space to discuss all the contributions of the phage group and their illustrious contemporaries, not even all their major findings, so we’ll limit ourselves to select discoveries pertaining to the lead-up to our present-day understanding of bacteriophages as nature’s genetic engineers.

First: a crash-course of what was known by the 1920s. A bacteriophage, after entering its target bacterium and before killing it, exists in a noninfectious form. In other words, if a bacterium newly infected by a bacteriophage is broken up and its internal contents applied on other healthy bacterial cells, the bacteriophage from the now-broken bacterial cell wouldn’t be able to infect the healthy cells. This existence of the bacteriophage in a noninfectious form inside the host bacterium was called lysogeny. It was also known that bacteriophages comprised DNA and protein (though the now-accepted wisdom that DNA is the genetic material of most life forms was not known then).

Hit, run and transfer genes

In the 1950s, two scientists from the phage group, Alfred Hershey and Martha Chase, demonstrated that during the infection of a bacterium by a bacteriophage, the viral DNA was injected into the bacterium while the protein coat that surrounded the DNA in an intact virus was left behind. The finding contributed to our understanding of bacteriophage biology as well as the establishment of DNA as the genetic material. Since the protein coat is the equipment that the virus uses to attach to a bacterium, its DNA on its own turns out to be noninfectious and thus defining lysogeny. This, to the best of our knowledge today, is a universal truth behind bacteriophage-bacteria antagonism.

We also know that the DNA injected into the bacterial cell contains information that enables the virus to go through a complete infection cycle: hijack the host bacterium’s resources to replicate the viral DNA, produce viral proteins, assemble many viral protein coats, break the host bacterial cell and its DNA into pieces, and assemble many live viruses by packaging the newly replicated viral DNA into the newly produced protein coats. The account book at the end of the process will tell us that a single virus infecting a single bacterium will end up producing the broken remains of a dead bacterium and about a hundred viruses looking for more bacterial victims.

This process is essentially a hit and run mode of predation with manifold reproduction built in, and incorporates features that enables a virus to engineer bacterial chromosomes. How? Using classical and simple bacterial growth experiments, Norton Zinder and Joshua Lederberg at the University of Wisconsin showed in 1952 that bacteria harbouring a certain special property could transfer this trait to otherwise similar bacteria, not necessarily by direct contact as demonstrated previously by Lederberg and Edward Tatum in the 1940s, but through a small intermediate particle – which was shown to be the bacteriophage.

In other words, bacteriophages can mediate transfer of genetic material from one organism to another. Such experiments in gene transfer using what are called nutritional auxotrophs had earlier led to the establishment of  the ‘one gene, one enzyme’ hypothesis by Tatum and George Beadle, a cornerstone in the development of molecular genetics.

How does the hit-and-run mode of viral predation result in gene transfer? When, during the infection of a bacterium by a bacteriophage, the bacterial cell breaks open and DNA gets packaged into the viral protein coat, the virus makes mistakes. Sometimes, it ends up packaging pieces of the bacterial chromosome instead of its own DNA into its protein coat. In these defective bacteriophages, the protein coat makes sure that it can attach to other bacteria but the DNA that it injects into these bacteria does not encode features that enable the virus to reproduce and kill the bacterium. Instead, this DNA of bacterial origin gets integrated into the host chromosome, and if many consequent events add together nicely, ends up introducing a new trait to this host bacterium.

Clearly, this trait transfer is not part of the normal reproduction of a parent to give progeny, and so represents an instance of horizontal gene transfer. In this mechanism, any piece of DNA can be moved around but only between very closely related bacteria. The constraint emerges from the limitation of the bacteriophage in being able to infect only a small set of closely-related bacterial varieties, a finding that can be traced back to d’Herelle. For example, it will have the ability to transfer a virulence trait from a bacterial pathogen to its benign relative.

Under controlled laboratory conditions, we can use manipulative techniques to observe such gene transfer events, as was done by Tatum and Lederberg. However, its true ecological impact might be tricky to estimate because in nature it rarely leaves a detectable signature on recipient chromosomes.  

Viruses in stealth mode

Yet another mode of bacteriophage-mediated genetic engineering of a bacterium is even more devious.

Experiments reported in 1956 by Lederberg, his wife Esther, and Melvin Morse showed that a type of bacteriophage dubbed lambda could also mediate gene-transfer. However, this lambda bacteriophage was able to transfer only a limited set of traits, and not all and sundry as resulting from the hit-and-run mode of gene transfer. This, further supported by later experiments by June Rothmann, Allan Campbell, Sankar Adhya and others, indicated that the lambda DNA was physically linked to the bacterial chromosome and also established that this linkage deterministically occurred at a specific site on the host chromosome.

In fact, the lambda DNA becomes part of the bacterial chromosome. It replicates along with the host chromosome and does not kill the host cell unless induced to do so. The presence of the DNA of such viruses can be easily identified in the genome sequence of a bacterium hosting it. Therefore, we can retrospectively identify traits conferred on a bacterium by a virus via this mechanism. The part of the source bacterium’s DNA that lambda is able to transfer to a second recipient bacterium is always the segment that is immediately adjacent to the site at which lambda DNA gets integrated.

The induction of lambda to kill its host is fascinating in itself, and its study has played a central role in establishing paradigms for how cells decide which proteins to produce when. Though killing off the host bacterium by a lambda is necessary for it to transfer genetic material from one bacterium to another, we will deal with this fantastic chapter of molecular biology in a separate instalment in this series.

Genetic engineering by bacteriophages such as lambda, which are capable of integrating with the host chromosome, isn’t limited to what they can transfer from one bacterium to another. The DNA of these viruses themselves carry many genes as a result of their long histories of mixing and matching their DNA with other viruses and bacteria. At times these genes might deliver beneficial and novel traits to the source bacterium. Thus, the integration of such viral DNA into the host chromosome can immediately give a novel characteristic to its host organism. The conferment of the ability to produce the disease-inducing toxin to Vibrio cholerae by a bacteriophage illustrates this. The bacteriophage DNA integrated into the host chromosome might never get induced to kill its host, and over time even lose the ability to do so, thus becoming an integral part of the host!

In the human context, the genetic material of HIV gets integrated in the human chromosome, stays there and secretly gets itself replicated alongside the human chromosome during the long latency period between infection and the expression of symptoms. Similarly, the integration of the DNA of human papilloma virus into the human genome plays a role in the series of consequent events that culminate in cervical cancer. The molecular processes leading to such devastating human diseases are an extension of a fundamental paradigm represented by obscure bacteria-eating viruses, first discovered and studied in the early to mid 20th century by a motley group of researchers searching for some basic truths of life.

Aswin Sai Narain Seshasayee runs a laboratory researching bacterial biology at the National Centre for Biological Sciences, Bengaluru. Beyond science, his interests are in classical art music and history.