Gene transfer agents: Another step towards a mechanistic understanding of expression

Gene transfer agents (GTAs) are small viruses that package and transfer random pieces of the producing cell’s genome but are unable to transfer all the genes required for their own production. GTAs are able to spread any DNA in the host cell and so their potential impact upon bacterial evolution and antimicrobial resistance is immense. Our discovery that the product of gene rcc01865 is a specific GTA activation factor (GafA) for the model Rhodobacter capsulatus GTA (RcGTA) and that GafA is essential for RcGTA production, has provided the link between GTA production and host regulatory pathways. However, while GafA has significantly improved our understanding of GTA regulation the complete mechanism is unclear. Our goal was to investigate the GafA mechanism of action in more detail. We demonstrate direct protein-protein interaction between GafA and the RNA polymerase omega subunit (RNAP Ω) using bacterial-two-hybrid and pull down assays. Further evidence for the interaction has come from random and site directed mutagenesis of gafA and targeted truncations. GafA mutants were also tested to assess their impact on RcGTA production. RNAP Ω is thought to recruit alternative sigma factors to the RNAP holoenzyme. Regions of GafA also share sequence homology with known sigma factor proteins, and we propose that GafA acts as an alternative sigma factor to co-ordinate expression of disparate RcGTA genes. Our results advance our understanding of this fascinating mode of horizontal gene transfer, not only in the model species but also in other potential GTA producing species that contain gafA homologues.