A variable single amino acid position in bacteriophage DNA polymerase I affects in vitro enzyme biochemistry and in vivo infection dynamics

• Victoria Barbone, Applied Molecular Biology & Biotechnology, University of Delaware

• Shawn Polson, Computer Science, University of Delaware
• Eric Wommack, Plant and Soil Sciences, University of Delaware

Viruses are the most genetically diverse and environmentally abundant biological entities, however they are vastly understudied. Viruses impact host communities and ecosystems in many ways that can be differentiated by distinct infection dynamics. Lytic viruses will lyse the host cell during infection, affecting nutrient cycling and community composition. Temperate viruses incorporate their genomes into the hosts during infection, contributing to horizontal gene transfer and affecting host biology. Bacteriophages—viruses that infect bacteria—are being studied to understand their infection dynamics and predict their roles in marine ecosystems. The DNA polymerase I gene (PolA) is used in investigating phage infection phenotypes, as variations in the PolA 526 residue (T7 numbering) correlate with the speed and accuracy of DNA replication, a vital process in phage infection. Three residues have been identified at the 526 position—phenylalanine, tyrosine, and leucine—that correlate with different phage infection phenotypes. Faster replicating PolAs, carrying a phenylalanine or tyrosine, are found in lytic phage, while slower replicating PolAs, with a leucine, are found in predominantly temperate phage. A novel histidine 526 variant has been identified with currently unknown biochemical properties. In this study, we altered the PolA 526 residue from the T7 wild type, tyrosine, to histidine to study the effect on replication. The histidine mutant enzyme had lower primer extension activity and specific activity than the wild type, indicating that this mutation hinders the processivity of the enzyme. When the mutated His526 PolA gene was inserted into the T7 genome, His526 phages replicated at much lower rates than the wild type phage, showing that phage replication is also influenced by this change. Characterizing the function of different genotypes of PolA will help build a connection to DNA replication phenotype in phage. This aids in predicting the infection strategies of viral populations and understanding the role viruses play in their communities.