How is Wormels’ classification as a lytic bacteriophage, due to its treatment and lysis of bacteria at the site of infection, important to your research?
Alyssa, thanks for your question! We look to categorize our phage as either lytic or temperate largely because of the effect that this could have should our phage be of interest for use in phage therapy. The entire purpose of phage therapy is to infect and kill host bacterium – therefore, introducing a temperate phage into a system of treatment would be an ineffective means of addressing the infection since not all of the bacteria would be killed off when infected (in fact, prophages, or phage genomes that have been integrated into their host’s chromosome, have the capacity to imbue their particular bacterial host cell with some level of immunity to infection by other of the same phage or phages of the same cluster). Classification as lytic or temperate can also sometimes be helpful in cluster narrowing since phages of the same cluster tend to have conserved ability to either enter the lysogenic lifecycle or not. Hope that’s helpful!
Great question Rachel – temperate phages, because they can enter the lysogenic lifecycle, do not always kill their host cell upon infection. When they enter the lysogenic lifecycle these phage genomes will be integrated into the host’s chromosome and repress the expression of their own lytic genes by means of a repressor protein. This repressor protein binds to a specific location in the phage’s genome in order to do so…a location that will be found in all units of the particular phage. If another of the same type of phage tries to infect a host cell that already contains a prophage (which is what we call a phage that has been integrated into the host’s genome), it will be bound by the repressor protein that is actively being made by the prophage already present. As a result, this new infection will be repressed and the host cell remains alive. Despite there existing a high concentration of phages in a particular region of the plate, then, bacterial cells in that area may not all be killed off because some are immune to the phage by virtue of being hosts to a prophage. The plaques formed by temperate phages therefore appear ‘cloudy’ as some bacteria are lysed and some are not.
You successfully narrowed down your phage type to several possible classes. How could you more conclusively classify your phage if genome sequencing were unavailable?
Nickles – a question my group has considered extensively given that sequencing is not available for all of the phages we isolate in this class. We performed cluster-specific PCR amplification of Wormels’ genome with six different sets of primers – all results from these amplification attempts were negative. However, several of the controls run alongside our PCR products also showed unexpected results and so it might be worth repeating attempts at amplification with some of these sets of primers. Unfortunately, though, sequencing is the only way that we can conclusively cluster our phage. Clustering is based on genome similarity levels at the base pair level so, without sequencing data, we can’t actually compare Wormels to other clustered phages and determine where it should be grouped. There are a number of characteristics that can be used other than sequencing in order to hint at cluster – such as plaque morphology (as an indicator of either a lytic or temperate phage, of which only one type is usually present in any given subcluster) or EM morphotype. Some clusters display very distinct constructions – for example, O cluster phages have a high incidence of prolate heads (especially in comparison to other clusters). Characterization as either Siphoviridae or Myoviridae can therefore sometimes play a role in preliminary clustering. A partnership with a lab on campus will hopefully allow us to begin sequencing more of our isolated phages by means of nanopore technology and thus let more groups definitively cluster their phages in the future!
Hi Gavin, great question! A phage’s cluster can tell us a lot about its characteristics even if we haven’t tested them ourselves. For instance, a phage’s cluster might give us an indication as to its host range. Host range is defined as the different types of bacteria any given phage can infect; in this class in particular, we’re interested in studying phages that infect M. smegmatis (nonpathogenic). If researchers were to look to treat a human pathogen closely related to M. smeg, they might consider using a phage that can infect this near evolutionary cousin. If they find that phages in a given cluster can successfully infect their given strain of said pathogen they might then be interested in testing other phages of the same cluster before looking to other clusters (since so much is conserved between phages of the same sub/cluster). Additionally, phages of the same cluster/subcluster often share a number of other genes that might characterize their method of infection or means of replication and escape from the host cell. Lysins, used to degrade the bacterial cell membrane and wall to release the newly assembled phages, are often conserved within a cluster but may slightly differ between them. Additionally, different families of phages (eg. Siphoviridae versus Myoviridae) can inject their genetic material into a host differently and most phages of a given cluster will belong to the same family. These are just some examples, but I hope that helps answer your question!
How is Wormels’ classification as a lytic bacteriophage, due to its treatment and lysis of bacteria at the site of infection, important to your research?
Alyssa, thanks for your question! We look to categorize our phage as either lytic or temperate largely because of the effect that this could have should our phage be of interest for use in phage therapy. The entire purpose of phage therapy is to infect and kill host bacterium – therefore, introducing a temperate phage into a system of treatment would be an ineffective means of addressing the infection since not all of the bacteria would be killed off when infected (in fact, prophages, or phage genomes that have been integrated into their host’s chromosome, have the capacity to imbue their particular bacterial host cell with some level of immunity to infection by other of the same phage or phages of the same cluster). Classification as lytic or temperate can also sometimes be helpful in cluster narrowing since phages of the same cluster tend to have conserved ability to either enter the lysogenic lifecycle or not. Hope that’s helpful!
Why would a lysogenic phage make plaques with a cloudy edge?
Great question Rachel – temperate phages, because they can enter the lysogenic lifecycle, do not always kill their host cell upon infection. When they enter the lysogenic lifecycle these phage genomes will be integrated into the host’s chromosome and repress the expression of their own lytic genes by means of a repressor protein. This repressor protein binds to a specific location in the phage’s genome in order to do so…a location that will be found in all units of the particular phage. If another of the same type of phage tries to infect a host cell that already contains a prophage (which is what we call a phage that has been integrated into the host’s genome), it will be bound by the repressor protein that is actively being made by the prophage already present. As a result, this new infection will be repressed and the host cell remains alive. Despite there existing a high concentration of phages in a particular region of the plate, then, bacterial cells in that area may not all be killed off because some are immune to the phage by virtue of being hosts to a prophage. The plaques formed by temperate phages therefore appear ‘cloudy’ as some bacteria are lysed and some are not.
You successfully narrowed down your phage type to several possible classes. How could you more conclusively classify your phage if genome sequencing were unavailable?
Nickles – a question my group has considered extensively given that sequencing is not available for all of the phages we isolate in this class. We performed cluster-specific PCR amplification of Wormels’ genome with six different sets of primers – all results from these amplification attempts were negative. However, several of the controls run alongside our PCR products also showed unexpected results and so it might be worth repeating attempts at amplification with some of these sets of primers. Unfortunately, though, sequencing is the only way that we can conclusively cluster our phage. Clustering is based on genome similarity levels at the base pair level so, without sequencing data, we can’t actually compare Wormels to other clustered phages and determine where it should be grouped. There are a number of characteristics that can be used other than sequencing in order to hint at cluster – such as plaque morphology (as an indicator of either a lytic or temperate phage, of which only one type is usually present in any given subcluster) or EM morphotype. Some clusters display very distinct constructions – for example, O cluster phages have a high incidence of prolate heads (especially in comparison to other clusters). Characterization as either Siphoviridae or Myoviridae can therefore sometimes play a role in preliminary clustering. A partnership with a lab on campus will hopefully allow us to begin sequencing more of our isolated phages by means of nanopore technology and thus let more groups definitively cluster their phages in the future!
Thank you for the response. You did a great job answering this in your talk! Nice delivery!
What is the significance of a phage belonging to a certain class?
Hi Gavin, great question! A phage’s cluster can tell us a lot about its characteristics even if we haven’t tested them ourselves. For instance, a phage’s cluster might give us an indication as to its host range. Host range is defined as the different types of bacteria any given phage can infect; in this class in particular, we’re interested in studying phages that infect M. smegmatis (nonpathogenic). If researchers were to look to treat a human pathogen closely related to M. smeg, they might consider using a phage that can infect this near evolutionary cousin. If they find that phages in a given cluster can successfully infect their given strain of said pathogen they might then be interested in testing other phages of the same cluster before looking to other clusters (since so much is conserved between phages of the same sub/cluster). Additionally, phages of the same cluster/subcluster often share a number of other genes that might characterize their method of infection or means of replication and escape from the host cell. Lysins, used to degrade the bacterial cell membrane and wall to release the newly assembled phages, are often conserved within a cluster but may slightly differ between them. Additionally, different families of phages (eg. Siphoviridae versus Myoviridae) can inject their genetic material into a host differently and most phages of a given cluster will belong to the same family. These are just some examples, but I hope that helps answer your question!