The Hna bacteriophage defence system

Abstract

This work details the discovery and characterization of a new bacteriophage (phage) defence system called Hna. The Hna system consists of a single protein with superfamily II helicase motifs near its N terminus and a PD-(D/E)XK nuclease motif near its C terminus, all of which are required for the protein’s antiphage activity. Although Hna was first identified in the nitrogen-fixing bacterium Sinorhizobium meliloti, homologues are found across many bacterial phyla and appear to have undergone extensive horizontal transfer. We find that a homologous protein from Escherichia coli also provides defence against phages. We also demonstrate that the efficacy of Hna-mediated protection is sensitive to modest changes in expression level, and that Hna expression is affected indirectly by the NolR transcriptional regulator. Hna provides phage defence through an abortive infection mechanism, meaning that infected cells with the system die but no new phages are produced. The expression of a phage single-stranded DNA binding protein (SSB) is sufficient to provoke cell death in cells with Hna. This cell death response is recreated when Hna and phage SSB are expressed in a heterologous host (E. coli) and leads to dramatic changes in gene expression. These results suggest that the Hna system is activated by phage SSB, although it is not clear whether the proteins interact directly or whether Hna responds to physiological stress that may occur upon SSB expression. Additionally, we show that purified Hna protein can bind ssDNA and has ssDNA-stimulated ATPase and DNA helicase activities. However, despite its nuclease domain, Hna has displayed no nuclease activity either on genomic DNA in vivo or on various DNA and RNA substrates in vitro.