ABSTRACT

Subjecting the mesophilic Escherichia coli bacterium to a pressure of ~100 MPa (1kbar) elicits a bona fide DNA damage (SOS) response, despite the fact that pressure itself cannot compromise DNA covalent integrity. While E. coli is not naturally subjected to high hydrostatic pressure (HP) in its environment, food products are treated by pascalization to inactivate food-borne pathogens. Screens for resistance to pressure shock revealed the constitutive presence in several strains of E. coli of an inactive, endogenous restriction endonuclease type IV, Mrr (Methylated adenine Recognition and Restriction), that cleaves methylated DNA. Since the enzyme is present and non-toxic in the absence of pressure treatment, activation must be pressure dependent. We investigated the mechanism of pressure dependent activation of Mrr in E. coli MG1655 strains expressing a chromosomal GFPmut2-Mrr fusion as well as Mrr catalytic mutants identified by screens. We used highly quantitative fluorescence fluctuation microscopy that provides a spatial map of the absolute values for the concentration (molecules number /PSF) and stoichiometry (molecular brightness, e) of fluorescently labeled complexes. We also examined the behavior of a phage methyltransferase HhaII, whose expression also leads to Mrr activation. Our results suggest that pressure-mediated triggering of Mrr activity and the concomitant generation of DNA damage involve the conversion of inactive Mrr tetramers into active Mrr dimers by pressure. This could occur either via a direct effect of pressure on the Mrr tetramer-dimer equilibrium, a common effect of pressure on oligomers, or via a pressure effect on structural features of the chromosome leading to more efficient recruitment of active dimeric Mrr. Homology modeling of the tetrameric Mrr catalytic domain supports the proposed mechanism of action drawn from the in vivo results of the mutants.