Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein

Abstract

We show that translation initiation factor EF-Tu plays a second important role in cell shape maintenance in the bacterium Bacillus subtilis. EF-Tu localizes in a helical pattern underneath the cell membrane and colocalizes with MreB, an actin-like cytoskeletal element setting up rod cell shape. The localization of MreB and of EF-Tu is interdependent, but in contrast to the dynamic MreB filaments, EF-Tu structures are more static and may serve as tracks for MreB filaments. In agreement with this idea, EF-Tu and MreB interact in vivo and in vitro. Lowering of the EF-Tu levels had a minor effect on translation but a strong effect on cell shape and on the localization of MreB, and blocking of the function of EF-Tu in translation did not interfere with the localization of MreB, showing that, directly or indirectly, EF-Tu affects the cytoskeletal MreB structure and thus serves two important functions in a bacterium.

Fig. 1.

Coomassie-stained SDS/PAGE experiments showing the interaction of MreB and EF-Tu. The identities of coeluting bands are indicated by triangles or lines. (A) TAP-tag experiment of cells expressing TAP-MreB (JS82); shown are the elution fractions of the second affinity column. Control, extracts from cells expressing TAP-tag only; TAP-MreB, extracts from cells expressing TAP-MreB. (B) TAP-tag experiments of cells expressing EF-Tu-TAP or TAP-tag only (“control”); shown are second elution fractions. (C) Strep-MreB and EF-Tu-6His coelution from streptavidin columns: overexpression of both proteins (“strep-MreB”) and or just of EF-Tu-6His (“control”) in E. coli cells. Shown are main MreB elution fractions. (D) Coelution of purified EF-Tu-6His from Streptavidin columns preloaded with purified strep-MreB (“strep-MreB”) or with elution fractions from a Streptavidin column from E. coli cell extract lacking any overexpressed protein (“control”).

Fig. 2.

Localization of EF-Tu in B. subtilis cells. (A–C) Cells expressing EF-Tu-CFP in B. Subtilis (JS88) at the original locus early exponential phase (A) (septa between cells are indicated by white lines and can usually be seen due to a lack of fluorescence between the cells), mid exponential phase (B), late exponential phase (C). (D) Three-dimensional deconvolution images. Arrow, direction of the turning angle of the cell; white arrowhead, an apparent helix. (E) Immunofluorescence on B. subtilis wild-type cells (late exponential growth) using anti-EF-Tu antiserum. (F) Two-dimensional deconvolution images showing colocalization of YFP-MreB and EF-Tu-CFP (strain JS89, late exponential growth). Red arrowheads, YFP-MreB foci (red in the overlay) that do not colocalize with EF-Tu-CFP foci (green in the overlay image). (G–J) BiFC interaction studies. Yn, N-terminal part of the split YFP; Yc, C-terminal part [note that coexpression of EF-Tu-Yc and just the Yn fragment (JS92) or of Yn-MreB and just the Yc fragment (JS93) did not show any defined signal]. (G) Expression of Yn-MreB (JS71; Left) or of EF-Tu-Yc (JS90; Right). (H) Cells expressing both EF-Tu-Yc and Yn-MreB (JS94). (I) Cells expressing of both EF-Tu-Yc and Yn-Mbl (JS95). (J) Cells expressing both EF-Tu-Yc and Yn-MreC (JS96). Cells were grown in S7₅₀ minimal media at room temperature supplemented with necessary antibiotics or inducers (0.5% xylose for YFP-MreB or 1 mM IPTG for Yn-fusions). (Scale bars: 2 μm.)

Fig. 3.

Dynamics of EF-Tu-CFP filaments and effect of the loss of MreB. (A) FRAP experiment on EF-Tu-CFP-expressing cells (JS88). pre, before bleaching; dotted circle, area of bleaching; numbers, minutes after bleaching. (B) FRAP profiles of EF-Tu-CFP (JS88) and of GFP-MreB (JS12). (C) Two-dimensional deconvoluted images of the localization of EF-Tu-CFP in mreB null cells (JS97). (D) Two-dimensional deconvoluted image of EF-Tu-CFP in wild-type cells (JS88). (E) Z-stack through mreB mutant cells expressing EF-Tu-CFP (JS97). White lines through circles, position of the focal plane. For FRAP experiments, strains JS12 and JS88 were grown in S7₅₀ minimal media at room temperature supplemented with necessary antibiotics or inducers (0.5% xylose for GFP-MreB). Strains JS88 and JS97 were grown in PAB/SMM medium (high magnesium and sucrose concentration). Late exponentially growing cells (OD 1.5–3) were used for the microscopy. (Scale bars, 2 μm.)

Fig. 4.

Effect of the depletion of EF-Tu in B. subtilis cells. (A) Western blot using EF-Tu antiserum showing levels of EF-Tu in wild-type cells (WT) and in the pxyl-tufA strain (JS91, OD = 0.5, M9 minimal medium plus fructose and xylose). (B) Western blot using MreB antiserum showing levels of MreB in wild-type (WT) cells and in the pxyl-tufA strain (JS91, OD = 0.5). (C) Translation efficiency (incorporation of radioactive methionine into cellular proteins) in wild-type and pxyl-tufA (JS91) cells. Cells were grown in M9 medium with fructose and xylose until early exponential phase (OD₆₀₀ = 0.5), where wild-type and JS91 cells showed the same doubling time (Fig. S3). See SI Materials and Methods for details. (D–G) Fluorescence microscopy of early exponential phase (OD₆₀₀ = 0.5) growing B. subtilis cells in M9 medium with fructose and xylose. (D) Wild-type cells. (E) pxyl-tufA cells (JS91, EF-Tu depletion). (F) Localization of YFP-MreB in wild-type cells (JS36). (G) pxyl-tufA cells (JS98). Arrowhead, irregular nonhelical filament; white lines, ends of cells. (H) Cells of strain JS98 (YFP-MreB, pxyl-tufA) growing in SMM/PAB medium (high magnesium and sucrose). (I–J) Cells 1 h after addition of kirromycin, where growth had ceased, after growth in S7₅₀ minimal medium to late exponential phase. (I) Cells expressing YFP-MreB (JS36). (J) Cells expressing EF-Tu-CFP (JS88). (Scale bars, 2 μm.)