Functional amyloids as inhibitors of plasmid DNA replication.

The molecular mechanisms of DNA replication in Gram-negative bacteria have been the subject of intense research for four decades. In the case of most plasmids, a plasmid-encoded protein (Rep) triggers replication in a regulated way1. In RepA from the Pseudomonas pPS10 plasmid replicon2, its N-terminal winged-helix dimerization domain (WH1) is structurally remodelled upon binding to DNA, resulting in the transformation of stable transcriptional repressor dimers into metastable replication-competent monomers3. In the plasmid replication origin (oriV) RepA monomers assemble the initiation complex at specific directly repeated sequences (iterons)4,5. Once replicated, two iteron-containing plasmid DNA molecules get coupled in a ‘handcuffed’ complex through interactions mediated by origin-bound Rep molecules, sterically hindering premature replication rounds6,7,8,9,10. In previous work, we characterized that the handcuffed complexes responsible for negative regulation of pPS10 replication were mediated by RepA monomers coupled through their WH1 domains, albeit involving a distinct interface to that found in the repressor RepA dimers11. Tracking the molecular basis of the intrinsic tendency of RepA towards aggregation, we found that monomers of the isolated RepA-WH1 domain assemble into amyloid fibres in vitro12,13. A plasmid-specific dsDNA sequence acts as allosteric effector of amyloidosis12,14. When fused to a fluorescent protein marker, RepA-WH1 behaves as a proteinopathic, vertically transmissible (from mother cell to daughter cells) prionoid in Escherichia coli thus enabling bacteria as a model system for approaching protein amyloidosis15,16. We have recently described a monoclonal antibody (B3h7) specific for an oligomeric conformation of RepA-WH1 on pathway towards building amyloid fibres17. B3h7 thus overcame limitations imposed by the poor reactivity of RepA-WH1 towards commercially available anti-amyloid antibodies (such as A11 and OC)17. Using B3h7, we discovered that pre-amyloidogenic RepA-WH1 oligomers assemble at the bacterial nucleoid17, as expected from the DNA-promoted amyloidogenesis of the protein in vitro12,14. Protein amyloids, in their fibrillar or oligomeric aggregated states, are infamous as the causative agents of human degenerative proteinopathies spanning from Alzheimer’s, Parkinson’s, Huntington’s, prion diseases and amyotrophic lateral sclerosis to dialysis-related amyloidosis and type-II diabetes18,19. However, work performed on yeast prions20 and bacterial biofilms21 have clearly shown that amyloids can also be functional, i.e. provide microorganisms with quickly selectable epigenetic, gain of function phenotypes22. Here we explore the link between regulation of pPS10 replication by RepA-mediated origin handcuffing and DNA-promoted RepA-WH1 amyloidosis. We have found that the regulatory RepA handcuffs actually are, to our notice, the first intracellular functional amyloids found in bacteria or involved in DNA replication.