A dynamically assembled cell wall synthesis machinery buffers cell growth - PubMed (original) (raw)

A dynamically assembled cell wall synthesis machinery buffers cell growth

Timothy K Lee et al. Proc Natl Acad Sci U S A. 2014.

Abstract

Assembly of protein complexes is a key mechanism for achieving spatial and temporal coordination in processes involving many enzymes. Growth of rod-shaped bacteria is a well-studied example requiring such coordination; expansion of the cell wall is thought to involve coordination of the activity of synthetic enzymes with the cytoskeleton via a stable complex. Here, we use single-molecule tracking to demonstrate that the bacterial actin homolog MreB and the essential cell wall enzyme PBP2 move on timescales orders of magnitude apart, with drastically different characteristic motions. Our observations suggest that PBP2 interacts with the rest of the synthesis machinery through a dynamic cycle of transient association. Consistent with this model, growth is robust to large fluctuations in PBP2 abundance. In contrast to stable complex formation, dynamic association of PBP2 is less dependent on the function of other components of the synthesis machinery, and buffers spatially distributed growth against fluctuations in pathway component concentrations and the presence of defective components. Dynamic association could generally represent an efficient strategy for spatiotemporal coordination of protein activities, especially when excess concentrations of system components are inhibitory to the overall process or deleterious to the cell.

Keywords: Pencillin binding proteins; bacterial cell wall; multienzyme complexes; superresolution microscopy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Single-molecule dynamics reveal that the cell wall synthesis enzyme PBP2 undergoes fast, diffusive motion, unlike the directed motion of the MreB cytoskeleton. (A) Schematic of sptPALM. Photoactivatable fluorescent protein fusions are used to monitor the movement of individual proteins in a crowded environment. A small population of molecules is activated by UV laser light, and then imaged over time to track molecular positions. (B) TIRF images of MreBsw-PAmCherry single molecules in live E. coli TKL039 (MG1655 mreB sw -PAmCherry) cells taken every 2.5 s, overlaid on phase-contrast images. The solid lines in the last panel are representative MreB molecular tracks. (Scale bar: 1 μm.) (C) Mean squared displacement (MSD) of MreB molecules in live (n = 382 molecules) and fixed (n = 105) E. coli TKL039 cells. The shaded area represents SEM. (Inset) The distribution of MreB track speeds (28.6 ± 15.4 nm⋅s−1, SD) at 37 °C and angles relative to the cell midline (99.1 ± 29.4°, SD). Most trajectories were approximately perpendicular to the long axis of the cell, with a slight right-handed bias quantitatively consistent with previous studies of left-handed twisting during growth (42). (D) MSD of MreB molecules (n = 1,018) in TKL039 cells and PBP2 molecules (n = 854) in TKL112 (∆mrdA Pmrda-PAmCherry-mrdA) imaged at high frame rates. The shaded area represents SEM. The linear MSD of PBP2 indicates that PBP2 molecules move diffusively, with a diffusion constant of D = 0.06 ± 0.006 µm2/s.

Fig. 2.

Fig. 2.

Rapid PBP2 diffusion is independent of catalytic activity, and buffers cell growth rate from fluctuations in PBP2 concentration. (A) Representative image of MreB and PBP2 colocalization in a TKL233 (mreB sw -sfGFP ∆mrdA PmrdA-PAmCherry-mrdA) cell filamented with cephalexin. (Upper) MreBsw-sfGFP and PAmCherry-PBP2 were coimaged via TIRF. (Lower) The fluorescence profiles over the length of the cell, averaged over the transverse direction. MreB and PBP2 profiles have a small, but significant correlation [two-sample t test: experimental data vs. scrambled data, t(76) = 2.79, P = 0.0068; Materials and Methods;

Fig. S6

]. (B) The apparent PBP2 diffusion constant is unaffected by antibiotic treatment. SEM is shown (n = 5 experiments). Mecillinam binds the active site of PBP2, A22 disrupts MreB polymerization, and an S330A mutation in PBP2 ablates the catalytic active site. Concentrations are given in micrograms per milliliter. The diffusion constant increases roughly 25-fold when the transpeptidase domain is truncated. (C) Model for PBP2 interaction with MreB. PBP2 moves rapidly within the inner membrane (IM), forming transient interactions with sites of peptidoglycan (PG) synthesis through its transpeptidase (TP) domain, either with the peptidoglycan elongation machinery (PGEM), or with the wall itself. PBP2 also contains transmembrane (TM) and nonpenicillin binding (nPB) domains, which may mediate interactions with other morphogenetic proteins.

Fig. 3.

Fig. 3.

Growth rate is unaffected by substantial PBP2 depletion. (A) Time-lapse microscopy of E. coli TKL141 (∆mrdA Para-mrdA) cells growing during PBP2 depletion. Time is specified in minutes. Even though cells lose their rod-shaped morphology, they continue to grow at a rate comparable to wild-type cells until two to three divisions have taken place. (B) Growth rate during PBP2 depletion is quantitatively unaffected over more than two doubling times. Ara+ represents the nondepleted control, and Ara− represents cells undergoing PBP2 depletion. The shaded area represents SE. (Scale bar: 1 µm.)

Fig. 4.

Fig. 4.

PBP2 activity is required throughout MreB-directed peptidoglycan synthesis. (A) Potential models for the role of PBP2 activity during cell wall synthesis. (Left) PBP2 is required for initiating strand synthesis by the rest of the cell wall synthesis machinery, but is not required for further synthesis-dependent MreB motion. (Right) Diffusing PBP2 molecules rapidly associate and disassociate with MreB-associated proteins to drive the progress of strand incorporation and MreB motion. (B) Cell growth rate decreases under mecillinam treatment in a concentration-dependent manner. Exponential phase cells (MG1655) were placed on agarose pads containing the indicated concentration of mecillinam and imaged for 15 min. To calculate growth rate, cell contours were extracted and cell length over time was fit to an exponential function. (C) MreB speed in TKL039 cells also decreases under mecillinam treatment in a concentration-dependent manner, indicating a requirement for active PBP2 molecules during the insertion of glycan strands.

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