High pressure activation of the Mrr restriction endonuclease in Escherichia coli involves tetramer dissociation.

A sub-lethal hydrostatic pressure (HP) shock of ∼100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M.HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M.HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M.HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M.HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer.

Construction and validation of the Tn5-PLtetO-1-msfGFP transposon as a tool to probe protein expression and localization.

In this study we report the design, construction and validation of a novel transposon aimed to systematically screen for protein localization and expression patterns in prokaryotes using fluorescence microscopy. Upon random insertion in an open reading frame in the proper frame and orientation, the transposon creates an N-terminal fluorescent protein fusion to the msfGFP reporter. Moreover, in order to examine the localization of fusion proteins whose native expression might be too low or absent, the transposon was fitted with a PLtetO-1 promoter that makes the expression of the generated fluorescent protein fusions controllable by anhydrotetracycline. Importantly, upon flipping out the PLtetO-1 promoter and neighboring antibiotic resistance marker, an in-frame "sandwich" msfGFP fusion is created in which the N- and C-terminal portions of the targeted protein are again controlled by its native promoter.