A pair of DNA glucosyltransferases elevate counter-defense in bacteriophage T4

Bacteriophages encode diverse nucleotide-modification pathways to evade host restriction-modification (RM) and CRISPR-Cas systems. On the other hand, modifications can also serve as a target for host defense systems, illustrating the complexity of the defense and counter defense landscape. Bacteriophage T4 encodes two glucosyltransferases (GTs), α-GT and β-GT, that post-replicatively add a glucose moiety to the hydroxymethylated deoxycytosines (5-hmC) on phage DNA in the α- and β-conformation, respectively. Among all fully sequenced phages, only six closely related phages encode both α-GT and β-GT. Here, through biochemical and genetic analysis, we show that β-GT has higher catalytic activity, whereas α-GT is more strongly expressed. During T4 infection, these factors determine the contributions of both GTs, with 66% of all 5-hmC α-glucosylated and 33% β-glucosylated. Encoding a single GT is sufficient to overcome the Escherichia coli RM systems, unless the glucosylation levels decrease below 80%, which constitute a complete protection threshold. However, when encountering a host encoding DNA glycosylase Brig1, in addition to type I and type IV RM systems, a second GT is necessary to enable Brig1 escapers to resist RM systems. These results demonstrate that encoding multiple GTs serves as a counter-defense mechanism when simultaneously confronted with several antiphage defense systems.