Coil’n’Roll – Secret Life of Circular DNA in biofilms

Bacteria and other pathogens are often studied as stand-alone entities, whereas, in nature, they mostly live in multispecies associations such as biofilms: multicellular communities encased in a shared extracellular matrix composed of polysaccharides, proteins, DNA and RNA. The matrix provides a shield to biofilm against antimicrobials and host immune defenses, and recent research has shown a particularly important role of extracellular DNA (eDNA) adapting different secondary structures with potentially novel functions in the extracellular environment. It has been assumed that eDNA in the biofilm is genomic DNA that arises from lysis of bacterial cells. However, another potential source of DNA involves viruses (phages) that attack, replicate in, and lyse bacteria. 

The interplay between bacteria and their viruses in biofilm development is largely overlooked. In fact, phage therapy of biofilms results in thicker biofilms. I hypothesize that bacterial lysis from phage infection will release nucleotides and a phage replication machinery that will be retained and remain active in the extracellular matrix of biofilms, leading to production of DNA concatemers that strengthen biofilm formation by the remaining viable bacteria. 

The aim of this project is to show the presence of extracellular DNA replication via phage-induced rolling circle replication (RCR) in bacterial biofilms, and to understand its impact on biofilm formation. To address this aim, I will establish an in vitro model, in which the RCR will occur in the extracellular environment in close association with bacteria, and I will determine how DNA concatemers produced in this way provide unique properties to the biofilm matrix. I will then investigate whether phage infections lead to the same phenomenon in biofilms.