Structural Studies of CI and MOR in Temperate Phages

A bacteriophage (phage) is a type of virus that infects bacteria. The name literally means bacteria eater and comes from the ability of the virus to kill the host bacteria. All phages are composed of a nucleid acid genome enclosed in a capsid of proteins. Phages attach themselves to the surface of a susceptible bacterium and infect the host by injecting their genome into it. Following this infection, the viral genome hijacks the cellular machinery of the host and forces it to copy its genome and produce viral components necessary for assembling new phage particles. Eventually the number of new phage particles will make the host cell burst in a process called lysis, which releases all the newly formed phages to the surroundings, ready to infect other bacteria. This reproduction cycle is called the lytic cycle and is available to all phages.
Temperate phages have the option to choose an additional cycle, which is called the lysogenic cycle. Here, the viral genome is instead integrated into the host chromosome, where it is replicated and passed on to the host daughter cells without killing them. When integrated, the viral genome is called a prophage and it comes with a variety of genes to be expressed in the host, which can lead to change in the phenotype. The prophage can stay dormant in the host as long as the host conditions are favorable. If the host gets exposed to for example antibiotics, the prophage will induce and switch to the lytic cycle and thereby try to escape the host before it is killed.
Both cycles are implicated in the transfer of genes between bacterial populations and sometimes these genes code for virulence or resistance to antibiotics. Antibiotic resistance has been rising steadily for the last decades and could be responsible for as many as 10 million deaths each year by 2050 if we do not find other ways to treat infections.
Entry into the lytic or lysogenic cycle is controlled by a bi-stable genetic switch, which is composed of transcription factors, promoters and operator sites that together makes it possible for the phage to regulate gene expression. The key regulators in the phages investigated here are the CI repressor and the MOR antirepressor.
This thesis presents structural and biochemical studies of CI and MOR in two different phages: TP901-1 which infects Lactococcus lactis bacteria used in the dairy industry, and Φ13 which infects the human pathogen Staphylococcus aureus.
The studies have helped confirm the significance of the identified TP901-1 CI-NTD:MOR complex by carrying out in vitro mutagenesis studies. Furthermore, we have characterized the interdomain linker region in TP901-1 CI and found that the specific length and flexibility of the linker appears delicately tuned to make CI execute its DNA-binding function as efficient as possible, which highlights the importance of linkers in the biological function of transcription factors. Lastly, we have helped establish the existence of a functional genetic switch in the previously uncharacterized Φ13, by expressing wild-type CI and variants and wild-type MOR and confirming that CI binds the putative operator sites with high affinity, that CI and MOR interact in solution and that bacteria infected with
Φ13 are able to switch between the lytic and lysogenic cycles.
The work provides new knowledge into the relationship between CI and MOR, which will help us understand the mechanisms involved in phage induction, expression of virulence genes and transfer of antibiotic resistance genes and thereby find ways to avoid and fight these harmful events.