Phage T5 infects Escherichia coli and injects its genome in an original two-step mechanism: in the First Step Transfer (FST), only 8% of the phage DNA enters the host cell. The transfer pauses then for several minutes before DNA entry resumes to completion (Second Step Transfer, SST). The FST is accompanied by a very rapid and massive destruction of bacterial DNA (50 % decrease in labeled DNA within 4 min of infection). The identity of the phage T5 nuclease has remained elusive for sixty years, as none of the phage proteins encoded on the FST-DNA resemble known nucleases. However, A1, a gene carried by FST-DNA, appears to control host DNA degradation as well as the SST. The C-terminal half of A1 carries several motifs that are conserved in a large family of metallophosphatases including the DNA repair and recombination nucleases Mre11/SbcD/gp46. Purified A1 exhibits manganese-dependent DNase activity on linear or plasmid DNA in vitro. In this study we investigated the role of the 62-kDa protein A1 in DNA degradation in the bacterial cell.

Upon ectopic expression of A1 (cloned under the control of an arabinose-inducible promoter), there was a dramatic decrease in genomic DNA recovered from bacterial cultures. Using fluorescence microcopy of E. coli cells, we observed a rapid decrease in bacterial DNA staining with DAPI within 15 min of induction. Moreover, we frequently saw the formation of fluorescence foci, suggesting a major reorganization of the bacterial nucleoid. Mutations in putative catalytic amino-acid residues abolished nuclease activity in vitro as well as in vivo. Taken together, our results indicate that A1 is the long elusive early-encoded DNase of phage T5. Interestingly, T5 phage DNA is not modified and is sensitive to A1 in vitro. How the DNase activity of A1 is regulated to control the SST without digesting the T5 genome remains to be elucidated.