In vivo architecture and action of bacterial structural maintenance of chromosome proteins

Abstract

SMC (structural maintenance of chromosome) proteins act ubiquitously in chromosome processing. In Escherichia coli, the SMC complex MukBEF plays roles in chromosome segregation and organization. We used single-molecule millisecond multicolor fluorescence microscopy of live bacteria to reveal that a dimer of dimeric fluorescent MukBEF molecules acts as the minimal functional unit. On average, 8 to 10 of these complexes accumulated as "spots" in one to three discrete chromosome-associated regions of the cell, where they formed higher-order structures. Functional MukBEF within spots exchanged with freely diffusing complexes at a rate of one complex about every 50 seconds in reactions requiring adenosine triphosphate (ATP) hydrolysis. Thus, by functioning in pairs, MukBEF complexes may undergo multiple cycles of ATP hydrolysis without being released from DNA, analogous to the behavior of well-characterized molecular motors.

Fig. 1

MukBEF imaging. (A) Representative frame-average and slimfield MukB-YPet cell images (yellow), brightfield and cell outline overlaid (white). (B) Photobleaching of MukBEF-YPet spots, high (upper row) and low stoichiometry data (expanded sections, lower row), raw (blue) and filtered (red). (C) Stoichiometry distributions, N=51-84 cells. 4-mer interval grid-lines, power spectra (arbitrary units) inset. (D) False-color plots for mean 2D spatial distributions for slimfield images with a 3 ms integration time, N=197-237 spots. Estimates for FWHM <σ> and σ_x/σ_y for Gaussian fits parallel to x and y axes (s.d. error). (E) Live-cell PALM, diffusing (gray brightfield, tracks colored) and immobile MukB-PAmCherry (different clusters colored), expanded indicating tracks (black) and clusters (red).

Fig. 2

Dual color single molecule millisecond imaging. (A) Brightfield (gray) and 3 ms fluorescence green (left panel) and red (middle panel) channels, overlaid (right panel) for dual label strain. (B) Unbiased kernel density stoichiometry estimation on mCherry (red) and GFP (blue) components for two dual label strains, 4-mer spaced grid-lines. (C) Stoichiometry of mCherry versus GFP component for each spot, dotted line gradients of 1.0 and 0.5; distribution of ratio of stoichiometry for mCherry and GFP components (gray) with Gaussian fit (red), mean ± s.d. indicated.

Fig. 3

Turnover of MukBEF complexes. (A) FRAP of MukB-YPet (upper panel) and ATP hydrolysis mutant MukB_EQ-YPet (lower panel), laser focus (orange circle) and FRAP (red arrow) and FLIP (blue arrow) indicated; steady-state cells. (B and C) Mean FRAP (red) and FLIP (blue) traces for cephalexin-elongated cells, s.d. errorbounds (shaded), pre bleach levels shown (arrows), N=10-14 traces.