12 April 2022

Bacteriophages as a microcosm

What contribution have small viruses made to big science

Rostec Blog, Naked Science

Bacteriophages are the most numerous living organisms on Earth, they have an impact on the entire ecosystem of the planet. For people, first of all for experts from the world of medicine, they are interesting for their unique features — phage—based drugs are an important tool in the fight against the growing resistance of dangerous bacteria to antibacterial drugs — antibiotic resistance. In the first article, which will open a series of publications about the features and process of studying bacteriophages, Alexander Zharnikov, an expert of the holding "Natsibio" of Rostec State Corporation, talks about the contribution phages have made to the development of modern fundamental science.

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Today, any high school student has at least a basic idea of what genes are, how DNA works, how it is copied and how information is read from it. We can say that modern molecular biology has been able to penetrate into the very essence of life.

However, few people think about how scientists found out about all this. It is extremely difficult to study such processes on human cells and other highly organized organisms. Simple model systems are needed here. In the last century, they became bacteriophages – viruses that parasitize bacteria.


Invisible assistants of biologists

The phage particle is arranged elementary: DNA and a protein shell. There are few genes in them, it's easy to study them, and if necessary, you can get a huge number of copies. It is thanks to these microscopic "aliens", as well as the flight of scientific thought and a series of elegant experiments, that many details of the work of genes in the human body have become known.

In Russia, thanks to many years of clinical practice, bacteriophages are well known, first of all, as antibacterial drugs. The company NPO Microgen (part of the holding "Natsibio" of Rostec State Corporation) is the only one in the world that produces them on an industrial scale, devotes a lot of resources to their research and even created the first Bioresource Center in Russia. On the basis of this structure, a fund of bacteriophages circulating in Russia has been formed.

Are mutations random?

Can mutations in living cells occur by chance, or do they necessarily need ultraviolet rays, radiation, viruses, toxins or other factors? Today, the answers to these questions are included in the school curriculum, once none of the luminaries of science could say this.

At the same time, it was extremely important to find answers, because understanding the work of genes, heredity and the theory of evolution depended on it. The solution was discovered in 1943 (when no one even knew how DNA works yet!) two talented American scientists — microbiologist Salvador Luria and biophysicist Max Delbruck. They received the Nobel Prize for their discovery, and bacteriophages helped them in this.

By 1943, scientists already knew that bacteria could very quickly become resistant to phages due to mutations. Trying to explain this phenomenon, biologists of that time were divided into two camps. Some believed that phages become a kind of "vaccination": they themselves cause mutations, due to which bacteria become immune to their descendants. Others have argued that mutations occur spontaneously (randomly) even before the microorganisms meet the phages.

When Luria and Delbruck first tried to test these hypotheses, they were disappointed: mutations in bacteria appeared so chaotically that it was absolutely impossible to understand them. Then everything happened according to the plot of a good Hollywood movie. An accident helped: one day Luria watched his colleague win three dollars on a slot machine and received a prize in dimes. An association flashed in the scientist's head, and he realized that he needed to count the number of colonies with different mutations. Mutations that occur more often are most likely to occur in the earliest generations of microorganisms (before meeting with the virus).

Luria shared his guess with Delbruck, and he used all the power of statistics to test it. The hypothesis that mutations occur only under the action of phages has been refuted. They appear constantly, which means that errors in copying genes can occur accidentally. The main engine of evolution never stops.

Back in the middle of the last century, the Czech monk and biologist Gregor Mendel proved with his famous experiments with peas that living organisms do not inherit traits from their parents directly. This happens according to certain laws through certain substrates — genes.

Where is the "code of life"?

But what are genes? Scientists quickly realized that these mysterious keepers of hereditary information are located in chromosomes. However, chromosomes are not so simple either: they have DNA and protein in them. Now it seems surprising, but until the middle of the last century, scientists gave the palm to proteins. Such views were also held by the American biologist Alfred Hershey. Nevertheless, he decided to double-check his guesses with the botanist Mark Chase. They conducted experiments with bacteria and phages, which later became legendary.

Scientists knew that bacteriophages attach to the host cell and inject some substance into it – DNA or protein. This substance became the instruction for the synthesis and assembly of new phage particles. To understand what exactly the phage introduces into the bacterium, the researchers derived two types of phages. Some were produced in the presence of the radioactive isotope sulfur 35S. Sulfur is present in proteins, but it is not present in DNA. Thus, only the protein component of phage particles turned out to be "labeled". The second population of bacteriophages was removed in the presence of the radioactive isotope phosphorus 32P. On the contrary, it is included only in the DNA.

Individual bacterial cultures were infected with different phages, and then loaded into a blender and thoroughly shaken to clear free viruses. Then the samples were centrifuged, and as a result, only bacteria remained on the bottom, in the sediment, and only the remains of phages remained in the broth. When Hershey and Chase conducted an analysis, they saw that there was a lot of 32P in the sediment, where the bacteria were. This means that phages inject DNA into them. It turns out that it was there that the "code of life" was hiding from scientists all this time.

How many "letters" in the "word" of the genetic code?

On December 30, 1961, an article entitled "The general nature of the genetic code of proteins" was published in the journal Nature, which soon turned into a classic of molecular biology and was declared "one of the most remarkable works in biology." Subsequently, other scientists quoted her in their writings more than 900 times. So how did the authors of this publication – molecular biologist Francis Crick, biologist Sidney Brenner and two of their colleagues – manage to impress the scientific world so much?

By the time this article was published, scientists knew that DNA consists of four types of nitrogenous bases ("letters" of the genetic code), and proteins consist of 20 types of amino acids. Simple calculations suggested that only one nitrogenous base could not encode one amino acid – there would not be enough for everyone. The "words" of two nitrogenous bases are not enough: 16 different combinations are obtained, still less than the number of amino acids. But the "words" of three "letters" are quite suitable. 64 possible combinations more than cover all the amino acid diversity. In theory it is logical, but in practice no one could prove it.

The long-awaited evidence was discovered by Francis Crick and colleagues in 1961. The scientists reasoned like this. If you "cut" one "letter" from DNA, then the genetic code will become meaningless, because the entire reading frame will shift. For example, there is a code AAT GCA AAA TCG. Remove the first "letter" from it and get ATG CAA AAT TSG. That is, all the subsequent "words" also shifted, and a completely different gene turned out.

We decided to test the hypothesis on T4 phages. Mutations were caused in them using a compound called proflavin – it just leads to the removal of individual "letters" of the genetic code or the insertion of superfluous ones. It turned out that if you remove only one or two "letters", then the gene stops working, reads completely incorrectly, and because of this, the phage can no longer infect the bacterium. But if you add or remove three "letters" at once, then the structure of the protein almost does not change, and it still works. So it was proved that each amino acid is encoded by a combination of three nitrogenous bases – a triplet. The importance of this discovery for the entire subsequent development of molecular biology and genetics is difficult to overestimate.

DNA carriers

In 1958, American geneticist and biochemist Joshua Lederberg received the Nobel Prize for discovering conjugation – "bacterial sex". The scientist discovered that bacterial cells can directly exchange genetic material among themselves, and this plays an important role in their evolution.

Lederberg used E. coli in his experiments, and later his work was continued by biologist Norton Zinder. He decided to check how conjugation works in salmonella – the causative agents of intestinal infection. Zinder took two strains of microorganisms unable to synthesize some compounds and grew them in an environment poor in nutrients, and even with penicillin.

Only the fittest had to survive, thanks to new mutations. But the trick that previously worked with E. coli, with salmonella did not work. It was possible to obtain only one strain, in which mutants appeared, capable of synthesizing all the necessary substances for themselves.

However, even such a small success at first glance turned into a defeat. The analysis showed that the new mutant strain turned out without "bacterial sex". But the scientists did not give up and continued their research. They suggested that if conjugation has nothing to do with it, then another mechanism should work. And indeed, it was soon discovered: it turned out that bacteriophages were endowed with mutant salmonella genes.

So in 1966, phage transduction was discovered. This superpower of viruses makes them excellent carriers of genetic material, which is very useful in genetic engineering. With the help of phages, a certain gene can be loaded into bacteria, and they will produce the necessary compound.

"Scissors" for genes

Indirectly, bacteriophages have given modern scientists a simple, fast and very effective way to edit genes. Without it, genetic engineering would hardly have been able to achieve such impressive successes. It all started back in 1987, when Japanese scientist Yoshizumi Ishino accidentally discovered strange areas in the DNA of E. coli, where there were repeating sequences interrupted by unique ones.

These sequences do not encode any proteins, and at first the researchers thought that this was nothing more than "genetic garbage". However, mysterious sequences were later discovered in other bacteria. They were called short palindromic repeats, regularly arranged in groups, abbreviated CRISPR. For a long time it was believed that this was some kind of repair system ("repair") of damaged DNA. But in 2000, it was discovered that CRISPR actually contains fragments of bacteriophage genes. So scientists realized that bacteria have their own "immune system".

This protective mechanism works like this. After a particular phage has been in a bacterial cell, it can save a fragment of the pathogen's DNA and include it in CRISPR. An RNA molecule is synthesized on the matrix of this DNA. The latter floats inside the cell and, like a policeman with an orientation, tracks "familiar" phage genes. As soon as they are detected, the Cas nuclease enzyme is activated and literally cuts the viral DNA. This protective system is called CRISPR-Cas. It somewhat resembles antibodies in the human body: they also know how to specifically recognize foreign particles.

Of course, it is interesting to study the arms race between bacteria and phages, but there was also a practical application for the CRISPR-Cas system. In 2012, scientists figured out how to use it to cut the genes of any organisms in any places. The new method turned out to be much faster and more efficient than the previously used ones. The CRISPR-Cas system is now used for the creation of genetically modified organisms, the production of medicines, and genetic diagnostics. It also has the potential to treat genetic diseases such as sickle cell anemia, cystic fibrosis. 

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