Determination of bacterial rod shape by a novel cytoskeletal membrane protein

Abstract

Cell shape is critical for growth, and some genes are involved in bacterial cell morphogenesis. Here, we report a novel gene, rodZ, required for the determination of rod shape in Escherichia coli. Cells lacking rodZ no longer had rod shape but rather were round or oval. These round cells were smaller than known round mutant cells, including mreB and pbpA mutants; both are known to lose rod shape. Morphogenesis from rod cells to round cells and vice versa, caused by depletion and overproduction of RodZ, respectively, revealed that RodZ could regulate the length of the long axis of the cell. RodZ is a membrane protein with bitopic topology such that the N-terminal region including a helix-turn-helix motif is in the cytoplasm, whereas the C-terminal region is exposed in the periplasm. GFP-RodZ forms spirals along the lateral axis of the cell beneath the cell membrane, similar to the MreB bacterial actin. Thus, RodZ may mediate spatial information from cytoskeletal proteins in the cytoplasm to a peptidoglycan synthesis machinery in the periplasm.

Figure 1

Cell shape and cell growth of the rodZ deletion mutant. (A) Phase-contrast image of wild-type BW25113 cells. (B) Phase-contrast image of ΔrodZ mutant cells of JW2500. (C) Phase-contrast image of JW2500 harbouring the vector plasmid pWM2784. Arrowhead shows two spheres stuck together. (D) Phase contrast image of JW2500 harbouring pDS69. Scale bars indicate 5 μm. (E) Growth of wild-type and ΔrodZ mutant on a plate incubated at 30°C for 18 h. (F) Magnified colonies of wild-type BW25113. (G) Magnified colonies of ΔrodZ mutant JW2500.

Figure 2

Characterization of cell proportions in round mutant cells. (A) Cell proportions of the ΔrodZ mutant of JW2500. The average length and s.d. of the length of the long axis is in blue, and those of the short axis is in magenta. (B) Phase-contrast image of BW25113 cells. (C) Phase-contrast image of JW2500 cells. Arrowhead shows two spheres stuck together. (D) Phase-contrast image of BW25113 cells treated with A22. (E) Phase-contrast image of BW25113 cells treated with mecillinam. (F) Comparison of cell proportions among wild-type, ΔrodZ, and wild-type cells treated with A22 or mecillinam. The average length and s.d. of the major axis is in blue and that of the minor axis is in magenta. Scale bar indicates 5 μm.

Figure 3

Cell morphogenesis under controlling expression of RodZ. (A) Depletion of RodZ in DS151, which harbours pDS111. Cells were grown in the presence of 0.002% arabinose overnight at 30°C. Cells were diluted into fresh L supplemented with (+) or without (−) 0.002% arabinose (time 0). (B) Cell proportions were measured at the indicated time points. The major axis is in blue and the minor axis is in magenta. (C) Overproduction of RodZ in wild-type BW25113 carrying either pDSW208F or pDS63 (FLAG–RodZ). RodZ was induced by 1 mM IPTG. (D) Cell proportions were measured at the indicated time points. The major axis is in blue and the minor axis is in magenta. (E) Restoration of rod-shaped DS151 cells by RodZ. DS151 cells were grown in the presence of 0.1% glucose overnight. Cells were diluted into fresh L supplemented with 0.2% arabinose (time 0), incubated and observed at the indicated time points. Scale bars indicate 5 μm.

Figure 4

Subcellular localization of RodZ. (A) Immunoblotting analysis of RodZ in cell fractionations. T, total lysates; S, soluble fractions; IM, inner-membrane fractions; OM, outer-membrane fractions. (B) PhoA fusion assay. TH1276 (ΔphoA) cells producing RodZ, PhoA, PhoA_Δss or RodZ–PhoA_Δss were streaked onto L supplemented with 50 μg ml⁻¹ BCIP. The plate was incubated at 30°C for 18 h.

Figure 5

Cytoskeletal protein structures in round cells. (A) Immunoblotting analysis of MreB using anti-MreB antibody. BW25113 (WT), JW2500 (BW25113: ΔrodZ), PA340 (WT), PA340–678 (PA340: ΔmreBCD), and DS165 (PA340: ΔrodZ). Arrowhead indicates MreB protein. The upper band of the MreB protein shows a non-specific protein that cross-reacts with anti-MreB antibody. (B) Fluorescent image of GFP–MreB in JW2500. (C) Fluorescent image of GFP–MreB in JW2500 after the addition of A22. (D) Sectional fluorescent images of GFP–RodZ in JW2500. (E) Three-dimensional reconstitution of GFP–RodZ on the basis of sectional fluorescent images. (F) Fluorescent images of RodZ–mCherry and GFP–MreB in a single cell treated with none (left) or A22 (right). (G) Fluorescent image of GFP–RodZ in BW25113 treated with A22. (H) Fluorescent image of GFP–RodZ in BW25113 treated with mecillinam. (I) Fluorescent image of GFP–RodZ in WM2767 in the absence of sodium salicylate, which is needed to express ftsZ. (Inset) Enlarged fluorescent image of GFP–RodZ in WM2767. Scale bars indicate 2 μm.

Figure 6

Functionality of RodZ and its deletion mutants. (A) Schematic illustrations of RodZ and its deletion mutants and summary of their localization and complementations. Black and green rectangles indicate the transmembrane (TM) and the helix-turn-helix domains of RodZ, respectively. Pink and purple rectangles indicate the transmembrane (MalF_TM1) and the periplasmic domains of MalF. Localization of GFP–RodZ and cell shape were observed in JW2500 harbouring pDS62 (GFP–RodZ) or in its derivatives that encoded truncated GFP–RodZ. In the ‘Localization' column: H, helix; Dm, diffuse in membrane; Dc, diffuse in cytoplasm. In the ‘Shape' column: NR, normal rod; FR, fatter rod; SFR, shorter and fatter rod; S, sphere. Growth defect was measured by colony size on L plate incubated at 30°C for 20 h. In the ‘Growth' column: +, large colonies; −, small colonies; +/−, medium colonies. (B) Localization of GFP–RodZ deletion mutants in the indicated strain. (C) Morphology of cells expressing various RodZ mutants in JW2500. Scale bar indicates 5 μm.

Figure 7

Schematic diagram of defects in cell morphogenesis and function of RodZ. (A) RodZ (blue) and MreB (black) form regular helical filaments along the long axis of the cell to maintain the lengths of the long and short axes, respectively. A defect in maintaining the long axis results in smaller round cells (top). On the other hand, a defect in maintaining the short axis or strength of the horizontal cell wall results in a larger round cell (bottom). For simplicity, the irregular MreB and RodZ filaments shown in Figure 5G and H were omitted in rodZ⁻ or mreB⁻ or pbpA⁻ cells. Thus, rodZ mutant becomes a smaller round cell, and both mreB and pbpA mutants become larger round cells. (B) RodZ is a bitopic membrane protein, and the C-terminal domain (orange) in the periplasm can interact with a factor(s) that is involved in peptidoglycan synthesis. The N-terminal domain (blue), including the HTH motif, can interact with another RodZ molecule, MreB, and/or other molecules to form a helical structure.