A phage mechanism for selective nicking of dUMP-containing DNA

Abstract

Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.

“This training is bound for glory” : selection by experimental evolution of a bacteriophage with expanded host-range and increased virulence

“…In fact, dUTPase allows to decrease uracil incorporation in new phage genomes and thus prevents cleavage of phage DNA by the Uracil DNA glycosylase (Ung) host enzyme which explains the fact that the less uracil is incorporated during phage replication, the more the phages multiply (Warner et al, 1979). Moreover, dUTPase action is facilitated by the action of early expression of phage T5.015 gene whose protein links to the bacterial Ung which blocks bacterial cell division and leads to bacteria death (Mahata et al, 2021). To test our hypothesis that parallel mutations observed in dUTPase participate to phage adaptation, we predict that, by integrating by reverse-genetics the mutated dUTPase into the ancestral phage genome, we shall observe an increased phage virulence after infection of bacteria of the two sequence types.…”

Time to lysis determines phage sensitivity to a cytidine deaminase toxin/antitoxin bacterial defense system

“…The growth of several well-known phages is inhibited when their DNA contains dUMP and Ung is present in the host cells. For example, to counter this negative effect T5 encodes its own dUTPase for reducing the dUTP level such that dUMP is limited in its genome [43] .…”

“…The growth of several well-known phages is inhibited when their DNA contains dUMP and Ung is present in the host cells. For example, to counter this negative effect T5 encodes its own dUTPase for reducing the dUTP level such that dUMP is limited in its genome [43]. However, the presence of the AvcID system prevents this T5 infection even in the absence of Ung, indicating that dUMP incorporation into the phage genome may not be the cause for the phage viability defect.…”

A Natural Fungal Gene Drive Enacts Killing via DNA Disruption

“…Because new replication forks are initiated before previous replication forks have completed this results in a ratio of oriC / ter greater than one in actively dividing cells ( 29 ). Treatments which block the progression of the replicon e.g., hydroxyurea (which inhibits RNR and damages DNA) or antibiotics such as trimethoprim (which inhibit DNA synthesis) are known to increase the oriC / ter ratio ( 30 , 31 ). qPCR and whole-genome Illumina sequencing confirmed that the RNA-seq expression patterns were a result of DNA perturbations ( Fig.…”