Lysis protein requirements of an RNA phage
Linnea Peterson-Bunker
Lysis of the bacterial host is the critical final step in infection for most phages. The decision of when and how to lyse are key determinants of phage fitness. The best-studied mechanisms of lysis are those of the double-stranded DNA phages, which encode multiple proteins to subvert the different layers of the host cell wall. On the other hand, small, single-stranded RNA phages, such as the leviviruses, encode single lysis proteins as small as 34 amino acids. These phages provide insight into the minimal requirements for phage lysis. Levivirus phage lysis proteins are small, highly diverse, and have evolved multiple times. The only commonality among them is the presence of a transmembrane helix within the protein.
MS2 is a long-studied levivirus with a genome roughly 3.5 kb in size, encoding only 4 genes. Using MS2 as a model, I am determining minimal sequences that function as levivirus lysis proteins, as well as RNA secondary structures used to regulate expression and timing of lysis. Using a cDNA clone of the full-length MS2 genome, I am testing MS2’s tolerance for alternative genome organizations by moving the lysis coding region. These rearrangements destroy the existing secondary structures that regulate translation. By passaging these mutagenized phages, I can see how the phage evolves new regulatory RNA secondary structures or improves upon existing ones.
MS2 is a long-studied levivirus with a genome roughly 3.5 kb in size, encoding only 4 genes. Using MS2 as a model, I am determining minimal sequences that function as levivirus lysis proteins, as well as RNA secondary structures used to regulate expression and timing of lysis. Using a cDNA clone of the full-length MS2 genome, I am testing MS2’s tolerance for alternative genome organizations by moving the lysis coding region. These rearrangements destroy the existing secondary structures that regulate translation. By passaging these mutagenized phages, I can see how the phage evolves new regulatory RNA secondary structures or improves upon existing ones.
Figure 1. Levivirus lysis proteins have evolved multiple times and exist in a variety of overlapping and non-overlapping genomic locations.
Figure 2. Levivirus lysis proteins vary in size and sequence, with the only commonality being the presence of a transmembrane helix.
Lambda—E. coli co-evolution
Bryan Andrews
The use of phages as medical or biotechnological reagents is likely to be restricted by the rapid coevolution that occurs between phages and their bacterial prey. We seek to understand the processes that drive phage evolution, particularly the strategies by which phages overcome mutations or expression changes of their preferred receptor. We use lambda and E. coli as a model co-evolutionary system, in part because lambda has been demonstrated to switch receptor usage when under extreme selective pressure. By introducing large libraries of variants into lambda, we can study the mutational landscape of the virus-host interface. One specific question I would like to answer is whether beneficial mutations in lambda are highly dependent on the mutational status of the receptor or whether mutations that increase affinity in a ‘wild type context’ are beneficial in all contexts. I am in the process of optimizing recombineering techniques to support integration of large libraries.
