The Phospholipase D (PLD) superfamily of proteins includes a group of enzymes with nuclease activity on various nucleic acid substrates. Here, with the aim of better understanding the substrate specificity determinants in this subfamily, we have characterised the enzymatic activity and the crystal structure of NucT, a nuclease implicated in Helicobacter pylori purine salvage and natural transformation and compared them to those of its bacterial and mammalian homologues. NucT exhibits an endonuclease activity with a strong preference for single stranded nucleic acids substrates. We identified histidine124 as essential for the catalytic activity of the protein. Comparison of the NucT crystal structure at 1.58 Å resolution reported here with those of other members of the sub-family suggests that the specificity of NucT for single-stranded nucleic acids is provided by the width of a positively charged groove giving access to the catalytic site.

The ComFC protein is essential for natural transformation, a process that plays a major role in the spread of antibiotic resistance genes and virulence factors across bacteria. However, its role remains largely unknown. Here, we show that Helicobacter pylori ComFC is involved in DNA transport through the cell membrane, and is required for the handling of the single-stranded DNA once it is delivered into the cytoplasm. The crystal structure of ComFC includes a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for the protein’s in vivo activity. Furthermore, we show that ComFC is a membrane-associated protein with affinity for single-stranded DNA. Our results suggest that ComFC provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.

Natural transformation plays a major role in the spreading of antibiotic resistances and virulence factors. Whilst bacterial species display specificities in the molecular machineries allowing transforming DNA capture and integration into their genome, the ComF(C) protein is essential for natural transformation in all Gram- positive and - negative species studied. Despite this, its role remains largely unknown. Here, we show that Helicobacter pylori ComF is not only involved in DNA transport through the cell membrane, but it also required for the handling of the ssDNA once it is delivered into the cytoplasm. ComF crystal structure revealed the presence of a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for its in vivo activity. ComF is a membrane-associated protein with affinity for single-stranded DNA. Collectively, our results suggest that ComF provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.