Functional amyloids as inhibitors of plasmid DNA replication.

DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is 'handcuffing', i.

Pre-amyloid oligomers of the proteotoxic RepA-WH1 prionoid assemble at the bacterial nucleoid.

Upon binding to short specific dsDNA sequences in vitro, the N-terminal WH1 domain of the plasmid DNA replication initiator RepA assembles as amyloid fibres. These are bundles of single or double twisted tubular filaments in which distorted RepA-WH1 monomers are the building blocks. When expressed in Escherichia coli, RepA-WH1 triggers the first synthetic amyloid proteinopathy in bacteria, recapitulating some of the features of mammalian prion diseases: it is vertically transmissible, albeit non-infectious, showing up in at least two phenotypically distinct and interconvertible strains.

Direct assessment in bacteria of prionoid propagation and phenotype selection by Hsp70 chaperone.

Protein amyloid aggregates epigenetically determine either advantageous or proteinopathic phenotypes. Prions are infectious amyloidogenic proteins, whereas prionoids lack infectivity but spread from mother to daughter cells. While prion amyloidosis has been studied in yeast and mammalian cells models, the dynamics of transmission of an amyloid proteinopathy has not been addressed yet in bacteria.

RepA-WH1 prionoid: a synthetic amyloid proteinopathy in a minimalist host.

The intricate complexity, at the molecular and cellular levels, of the processes leading to the development of amyloid proteinopathies is somehow counterbalanced by their common, universal structural basis. The later has fueled the quest for suitable model systems to study protein amyloidosis under quasi-physiological conditions in vitro and in simpler organisms in vivo. Yeast prions have provided several of such model systems, yielding invaluable insights on amyloid structure, dynamics and transmission.

A DNA-promoted amyloid proteinopathy in Escherichia coli.

Protein amyloids arise from the conformational conversion and assembly of a soluble protein into fibrilar aggregates with a crossed β-sheet backbone. Amyloid aggregates are able to replicate by acting as a template for the structural transformation and accretion of further protein molecules. In physicochemical terms, amyloids arguably constitute the simplest self-replicative macromolecular assemblies.

Negative regulation of pPS10 plasmid replication: origin pairing by zipping-up DNA-bound RepA monomers.

In many plasmid replicons of gram-negative bacteria, Rep protein dimers are transcriptional self-repressors of their genes, whereas monomers are initiators of DNA replication. Switching between both functions implies conformational remodelling of Rep, and is promoted by Rep binding to the origin DNA repeats (iterons) or chaperones. Rep proteins play another key role: they bridge together two iteron DNA stretches, found either on the same or on different plasmid molecules.

Twenty years of the pPS10 replicon: insights on the molecular mechanism for the activation of DNA replication in iteron-containing bacterial plasmids.

This review focuses on the contributions of the Pseudomonas replicon pPS10 to understanding the initiation of DNA replication in iteron-containing plasmids from Gram-negative bacteria. Dimers of the pPS10 initiator protein (RepA) repress repA transcription by binding to the two halves of an inverted repeat operator. RepA monomers are the active initiator species that bind to four directly repeated sequences (iterons).

Modulation of pPS10 host range by plasmid-encoded RepA initiator protein.

We report here the isolation and analysis of novel repA host range mutants of pPS10, a plasmid originally found in Pseudomonas savastanoi. Upon hydroxylamine treatment, five plasmid mutants were selected for their establishment in Escherichia coli at 37 degrees C, a temperature at which the wild-type form cannot be established. The mutations were located in different functional regions of the plasmid RepA initiation protein, and the mutants differ in their stable maintenance, copy number, and ability to interact with sequences of the basic replicon.

Modulation of pPS10 host range by DnaA.

Narrow-host-range plasmid pPS10, originally found in Pseudomonas savastanoi, is unable to replicate in other strains such as Escherichia coli. Here, we report that the establishment of pPS10 in E. coli can be achieved by a triple mutation in the dnaA gene of E.