Bacteriophage Genetics - Online Tutor, Practice Problems & Exam Prep

Bacteriophages are viruses that infect bacteria and are crucial for studying bacterial genetics. A common laboratory technique is the plaque assay, which visualizes areas of bacterial lysis, known as plaques. There are three types of phages: prophages, which integrate into the bacterial genome; virulent phages, which immediately lyse the host; and temperate phages, which can remain dormant before lysis. Bacteriophages can also be used for genetic mapping through recombination frequencies, allowing researchers to determine the proximity of genes based on recombinant colony ratios.

Plaques and Experiments

Okay. So in this video, we're going to be talking about bacteriophage genetics. So bacteriophages, if you have ever heard this term before, you may know what they mean, but essentially they're viruses that infect bacteria. Bacteriophages are often used to study bacterial genetics and also bacteriophage genetics, and so I want to talk a little bit about how you, you know, how you actually work with these in a lab. One of the main assays that you use to work with viruses in a laboratory is called a plaque assay. And so, how you do this is you infect the bacterial culture, put the bacterial culture onto a petri dish, and grow the bacteria, so now we're growing colonies of bacteria. Then you can count the number of plaques, and so what is a plaque? It's sort of this like dot on the plate that's formed through the lysis, the breaking open, of the infected bacteria. Right? Because the bacteria were infected, so the virus gets in. It makes a bunch of copies of itself, and then it lyses it so that the virus can get out. So when it lyses it, it forms this hole on the plate, and that hole is called a plaque. So what this looks like here, you can see that there is a bacterial plate here, and all throughout here, anywhere where it's, sort of that, like, kind of light gray yellow color that's covering the most part, that's bacteria. And all of these are plaques. This is where a phage has actually infected a bacteria colony, essentially at this point, and lysed this bacteria so it forms these plaques on the plate. And so a viral plaque can be used to study lots of different things including calculating how much virus you have or how infected the virus is or different types of mutations, and so they're super these are super important assays.

Now there are 3 types of phages. There's the prophage, and this is a virus that has integrated its genetic material into the bacterial genome. So this is a virus that has its own genetic material. Right? And when it infects, sometimes that genetic material can actually be integrated into the bacterial genome. So now you have this hybrid of virus and bacterial chromosome, in the bacterial chromosome, and this portion of that that's the viral DNA is called a prophage, and it can lie there for a really long time and then later become active. These are super important. Then you have the virulent phages, and these are ones that immediately lyse and kill the host cell. So the virus gets inside the cell, it reproduces so quickly, it sort of starts swelling the cell with how fast it's been reproducing, how many virus offspring it's made, and eventually that's going to, you know, pop the cell, it's going to lyse the cell and release that virus into the environment. Then you have the temperate phages, and these are actually viruses that remain inside the host for a period of time. It can be a few minutes, it can be a few years, it can be a few decades. It just sort of sits there, and it's just like I'm just going to chill here for a little while. I'm not in any hurry. I'm just going to enjoy the environment before I start having kids. And so it does that for a period of time, and then something triggers it. And it's like, okay, I'm ready to produce a bunch of offspring now. And so it does. And so it will eventually lyse and kill the cell, but for a very long time, it can actually just stay inside the cell, and that's called a temperate phage. So those are the 3 types that you need to know those vocab words definitely. So with that, let's turn the page.

Mapping Bacteriophages

Okay. So now let's talk about bacteriophages and mapping. Bacteriophages can actually be used to map bacteriophage genes or genomes. So, how do you do this? It's essentially the same way as with humans or other types, such as plants. You use recombination frequencies. Here is how you do this; you take a bacterial culture—they infect bacteria. You perform mixed infection, which means that you have two strains of bacteriophages that have different phenotypes, you mix them together, and infect this one bacterial culture. In this case, we have Virus 1, which has the H+ and R+ genes, and Virus 2, which has H− and R−. If it has H+, it's going to create purple colonies, and if it has R+, it's going to create small colonies. So now we can phenotypically look at the bacteria for the parental and the recombined. The parental will be purple small, and the other parental will be not purple large. The recombinants would be purple large or non-purple small.

We are looking for mixtures of positive and negative signs in the genotypes. The recombination frequency, instead of looking at plants, seeds, animals, or whatever the offspring itself, is observed based on the effect on the bacteria. Here we count the number of recombinant colonies versus the total number of colonies. As an example, let's consider these two viruses: H+R+ and H−R−. The h+r+ will show two parental phenotypes. If there are two recombinant phenotypes, such as purple large or black small, these can only form through recombination. You can use the recombination frequencies to map the bacterial genes and determine their proximity. The closer they are, the smaller the recombination frequency. In this case, we count six recombinants out of twelve trials, a 50% frequency, indicating that these genes are very far apart.

Bacteriophages have a unique form of recombination called intragenic recombination, which does not occur in humans or plants; entire genes can recombine, but not just parts of a gene. However, in bacteriophages, portions of a gene can recombine. This ability was studied by a scientist named Fenzer, particularly at the r11 locus of the T4 bacteriophage. Over 20,000 independent r11 mutants were collected, and he crossed them to analyze recombination. Through his study, he determined distances between mutations in terms of recombination frequency. This tedious process allowed him to identify specific mutations within a gene, marking a significant milestone in genetics. Today, this mapping can be done much more easily through sequencing, but at the time, it was a novel and crucial approach to understanding recombination frequencies and mapping bacteriophage genetics.

With that, let's move on.

A plaque assay studies viruses through what measurement?

• A. The number of bacterial colonies that grow on a plate
• B. The number of bacterial colonies that are lysed upon infection of a bacteriophage
• C. Comparison between the number and size of bacterial colonies
• D. Rate of Infection

A mixed infection of two bacteriophage strains is often used for what purpose?

• A. To sequence bacteriophage genomes
• B. To measure bacteria resistance
• C. To study conjugation
• D. To map bacteriophage genes

A mixed infection of two bacteriophage strains was performed. Infection of bacteriophage strain 1 causes the bacteria to be red and large, while infection of bacteriophage strain 2 causes the bacteria colony to be black and small. The following results were obtained. Using this data, determine the distance between the color and size genes. 

• A. 45.3 m.u
• B. 15.7 m.u
• C. 7.9 m.u
• D. 2.2 m.u

Here’s what students ask on this topic:

What are bacteriophages and how do they infect bacteria?

Bacteriophages, or phages, are viruses that specifically infect bacteria. They attach to the bacterial cell surface and inject their genetic material into the host. This genetic material can either integrate into the bacterial genome (as in prophages) or remain separate. In the case of virulent phages, the viral DNA takes over the bacterial machinery to produce new phage particles, leading to cell lysis and release of new viruses. Temperate phages can remain dormant within the host for extended periods before eventually causing lysis.

What is a plaque assay and how is it used in bacteriophage research?

A plaque assay is a laboratory technique used to quantify and study bacteriophages. In this method, a bacterial culture is infected with phages and spread on a petri dish. As phages infect and lyse bacterial cells, they create clear zones called plaques. Each plaque represents a site where a single phage infected a bacterium and produced progeny. By counting the number of plaques, researchers can determine the concentration of phages in a sample and study various aspects of phage biology, such as infectivity and mutation rates.

What are the different types of bacteriophages?

There are three main types of bacteriophages: prophages, virulent phages, and temperate phages. Prophages integrate their genetic material into the bacterial genome and can remain dormant for long periods. Virulent phages immediately take over the host's machinery to produce new phages, leading to rapid cell lysis. Temperate phages can exist in a dormant state within the host for varying lengths of time before eventually causing lysis. Each type plays a unique role in bacterial and phage genetics.

How are bacteriophages used in genetic mapping?

Bacteriophages are used in genetic mapping through recombination frequencies. By infecting bacteria with two different phage strains and analyzing the resulting phenotypes, researchers can determine the frequency of recombination events. This frequency helps map the relative positions of genes on the phage genome. For example, if two genes recombine frequently, they are likely far apart; if they recombine rarely, they are close together. This method allows for detailed genetic mapping, even within individual genes, as demonstrated by studies on the rII locus of the T4 bacteriophage.

What is intragenic recombination in bacteriophages?

Intragenic recombination in bacteriophages refers to recombination events occurring within a single gene. Unlike in higher organisms, where recombination typically involves entire genes, bacteriophages can undergo recombination within specific regions of a gene. This allows for precise mapping of mutations within a gene. A notable example is the study of the rII locus in the T4 bacteriophage, where extensive recombination mapping revealed the positions of numerous mutations, providing insights into the genetic structure and function of the gene.