A Dynamically Assembled Cell Wall Synthesis Machinery Buffers Cell Growth

A dynamically assembled cell wall synthesis machinery buffers cell growth

Timothy K. Leea, Carolina Tropinia,b, Jen Hsina, Samantha M. Desmaraisa, Tristan S. Ursella, Enhao Gonga, Zemer Gitaic, Russell D. Mondsa,1,2, and Kerwyn Casey Huanga,b,d,1

aDepartment of Bioengineering and bBiophysics Program, Stanford University, Stanford, CA 94305; cDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544; and dDepartment of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305

Edited by Carol A. Gross, University of California, San Francisco, CA, and approved January 15, 2014 (received for review July 30, 2013) Assembly of protein complexes is a key mechanism for achieving support for the hypothesis that coordination between MreB and spatial and temporal coordination in processes involving many the wall synthesis machinery is achieved through colocalization enzymes. Growth of rod-shaped bacteria is a well-studied example into large, moving, multienzyme complexes (4, 5). However, bio- requiring such coordination; expansion of the cell wall is thought chemical isolation of such a complex has remained elusive (6), to involve coordination of the activity of synthetic enzymes raising the possibility that this picture is incomplete, particularly with the cytoskeleton via a stable complex. Here, we use single- in E. coli, in which dynamic imaging of the PBPs has not yet molecule tracking to demonstrate that the bacterial actin homolog been achieved. MreB and the essential cell wall enzyme PBP2 move on timescales PBP2 is thought to be the major enzyme responsible for co- orders of magnitude apart, with drastically different characteristic valently cross-linking new glycan strands into the cell wall during motions. Our observations suggest that PBP2 interacts with growth (7). PBP2 is essential; cells enlarge and eventually lyse the rest of the synthesis machinery through a dynamic cycle of upon depletion of PBP2 (8). In E. coli, PBP2 was previously transient association. Consistent with this model, growth is robust observed to localize as dispersed puncta and at septa during to large fluctuations in PBP2 abundance. In contrast to stable division (9). PBP2 concentration affects cell size (10), and complex formation, dynamic association of PBP2 is less dependent overexpression is lethal (11). Although MreB binds to the cyto- on the function of other components of the synthesis machinery, plasmic face of the inner membrane, PBP2 is a transmembrane and buffers spatially distributed growth against fluctuations in protein with a cytoplasmic N-terminus and a periplasmic domain MICROBIOLOGY pathway component concentrations and the presence of defective required for transpeptidase activity. Given the requirement of components. Dynamic association could generally represent an a functional PBP2 for MreB motion and peptidoglycan synthesis, efficient strategy for spatiotemporal coordination of protein activi- we sought to determine whether it could be a component of a ties, especially when excess concentrations of system components cell wall-synthesizing, multienzyme complex moving with MreB. are inhibitory to the overall process or deleterious to the cell. In this study, we applied single-particle tracking photoactivated localization microscopy (sptPALM) (12), a technique based Pencillin binding proteins | bacterial cell wall | multienzyme complexes | on the limited photoactivation of fluorescent proteins, to quan- superresolution microscopy tify the dynamics of MreB and PBP2 in E. coli. In contrast to MreB, we find that PBP2 exhibits rapid, diffusive motions that or the wide variety of organisms that have large internal os- do not depend on PBP2 catalytic activity. These data suggest Fmotic pressures, the cell wall is essential for maintaining cellular integrity and morphology (1). As a consequence, the Significance bacterial cell wall is an important antibiotic target. Much research has been performed to elucidate the chemical composi- For complex biological processes, the formation of protein tion of the cell wall and identify the enzymes required for its complexes is a strategy for coordinating the activities of many synthesis, yet we still know little about the dynamics of cell wall enzymes in space and time. It has been hypothesized that assembly in vivo. The cell wall is composed of peptidoglycan, growth of the bacterial cell wall involves stable synthetic a network of long glycan strands that are cross-linked by short complexes, but neither the existence of such complexes nor the peptides. During growth, existing bonds in the peptidoglycan consequences of such a mechanism for growth efficiency have network are broken, and new glycans are polymerized, inserted, been demonstrated. Here, we use single-molecule tracking to and cross-linked. This process depends on a host of enzymes, demonstrate that the association between an essential cell most notably the penicillin-binding proteins (PBPs) (2). It is wall synthesis enzyme and the cytoskeleton is highly dynamic, presumed that the spatial and temporal coordination between which allows the cell to buffer growth rate against large fluc- these proteins is responsible for the maintenance of cell shape tuations in enzyme abundance. This indicates that dynamic during growth and division, although the strategies used to ro- association can be an efficient strategy for coordination of bustly achieve such a task that spans both molecular and cellular multiple enzymes, especially those for which excess abundance scales have not yet been elucidated. can be harmful to cells. Recent live-cell imaging studies in the rod-shaped bacteria Escherichia coli (3) and Bacillus subtilis (4, 5) showed that clus- Author contributions: T.K.L., C.T., J.H., S.M.D., Z.G., R.D.M., and K.C.H. designed research; ters of MreB, a bacterial actin homolog that is essential for rod- T.K.L., C.T., S.M.D., and E.G. performed research; T.K.L., C.T., J.H., S.M.D., T.S.U., R.D.M., and K.C.H. contributed new reagents/analytic tools; T.K.L., C.T., J.H., S.M.D., and K.C.H. like shape, move in linear tracks oriented in the circumferential analyzed data; and T.K.L., C.T., J.H., S.M.D., T.S.U., Z.G., R.D.M., and K.C.H. wrote direction. These dynamics are coupled to cell wall synthesis; the paper. exposure to cell wall-targeting antibiotics or depletion of cell wall The authors declare no conflict of interest. – E. coli precursors halts MreB motion (3 5). In , MreB motion was This article is a PNAS Direct Submission. β stopped by exposure to high concentrations of the -lactam an- 1To whom correspondence may be addressed. E-mail: [email protected] or rmonds@ tibiotic mecillinam, which inhibits the transpeptidase PBP2 (3), syntheticgenomics.com. suggesting a model in which PBP2 activity is necessary for MreB 2Present address: Synthetic Genomics Inc., La Jolla, CA 92037. B. subtilis motion, possibly by directing new cell wall insertion. In , This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tracking of MreB-associated proteins (MreC/D) and PBPs provided 1073/pnas.1313826111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1313826111 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 a model in which PBP2 transiently associates with sites of cell proteins (15), particularly those in low abundance, these results wall synthesis, and hence can act in a distributed manner and indicate that the circumferential motion of single molecules is a need not be rate limiting for growth as was previously hypothe- requisite signature for colocalization with MreB. sized (3). In support of this model, growth was unaffected for more than two doublings during depletion of PBP2. Finally, we Rapid, Diffusive Motion of PBP2 Molecules. To determine the dy- show that both growth rate and MreB speed decrease during namics of PBP2 in E. coli, we constructed an N-terminal fusion mecillinam treatment in a dose-dependent manner, indicating of PAmCherry to PBP2 expressed from the endogenous pro- that a catalytically active PBP2 molecule is required during the moter and verified that this fusion rescued cells from depletion D–G incorporation of a glycan strand. of the native, unlabeled PBP2 (Fig. S1 ). As the sole source of PBP2, either expressed from a low-copy plasmid or integrated Results at the native chromosomal locus, PAmCherry-PBP2 complemented Single-Molecule Tracking of MreB Circumferential Motion in E. coli. We wild-type growth rate with slightly higher average cell width F G tagged E. coli MreB with PAmCherry (13) as an internal sandwich (Fig. S1 and ). Bocillin gels and Western blots demonstrated Materials fusion (14) and expressed this fusion (MreBsw-PAmCherry) as that the fusion remained stable and intact inside the cell ( and Methods A the sole copy at the native chromosomal locus. E. coli cells ; Figs. S3 and S4 ). expressing MreBsw-PAmCherry fusions were viable and remained We observed striking qualitative and quantitative differences rod-shaped, although cells were slightly wider (Fig. S1 A–C), as between the motions of MreB and PBP2. PBP2 molecules moved previously observed for mCherry sandwich fusions (14). We much faster and more erratically than MreB. Reconstructing the imaged single cells using total internal reflectance fluorescence trajectory of a PBP2 molecule required imaging on a timescale 100 times faster (∼10 ms) than that required to observe linear (TIRF) microscopy through several hundred iterations of a cycle Materials and Methods in which a single molecule was activated, followed by time-lapse MreB trajectories ( ; Fig. S5). Moreover, imaging of the molecule’s dynamics every 2.5 s until it photo- the mean squared displacement (MSD) of PBP2 motion was linear (Fig. 1D), indicating diffusive behavior with an apparent bleached (Fig. 1A, Fig. S2, and Materials and Methods). Our diffusion constant of 0.06 ± 0.006 μm2/s. The difference in the sptPALM measurements revealed directed, circumferential mobilities of MreB and PBP2 excluded the possibility that MreB motion of single MreB molecules (Fig. 1 B and C), similar to and PBP2 function together in a long-lived synthesis complex. previous measurements of diffraction-limited clusters of MreB To directly quantify the colocalization of MreB and PBP2, in E. coli (3) and B. subtilis (4, 5). For other cell wall synthesis sw we constructed a strain expressing both MreB -sfGFP and PAmCherry-PBP2. Taking into account the likelihood of random association of diffraction-limited foci, individual cells exhibited A a weak, although significant, correlation between the fluores- activate image image track cence signals from these fusion proteins (Fig. S6; Materials and Methods), suggesting that PBP2 spends at most a small fraction A single-molecule of its time interacting with MreB (Fig. 2 ). repeat trajectories B The Transpeptidase Domain but Not Catalytic Activity Influences PBP2 0 2.5 5 7.5 10 12.5 Motion. PBP2 transpeptidase activity is essential for cell wall growth; mutations or drugs that interfere with transpeptidation are lethal (8, 16). One such drug is the antibiotic mecillinam, CD which specifically inhibits PBP2 by acting as a substrate mimic 0.3 0.1 that irreversibly locks the active site of PBP2 (Fig. S3) (16). To 0.2 0.2 0.1 0.1 0.08 determine whether PBP2 dynamics are dependent on its catalytic Fraction 0 0 PBP2 0 40 80 0 90 180 activity, we tracked PBP2 motion over a range of mecillinam 0.2 -1 Speed (nm s ) 0.06 concentrations (Fig. 2B). In each case, we observed diffusive MreB Live 0.04 motion with no observable difference in the diffusion constant. 0.1 Similarly, a mutation that disrupts the PBP2 active site (S330A) 0.02 MreB MreB Fixed (11) did not impact PBP2 diffusivity (Fig. 2B; Materials and 0 0 Methods). These data indicated that the process of trans- 0 5 10 15 20 0 0.1 0.2 0.3 0.4 0.5 Time (s) Time (s) peptidation does not measurably inhibit PBP2 motion. Our observed diffusion constant of PBP2 is substantially lower Fig. 1. Single-molecule dynamics reveal that the cell wall synthesis enzyme than measurements for transmembrane proteins of similar size PBP2 undergoes fast, diffusive motion, unlike the directed motion of the (2–5 μm2/s) (17). Given the small correlation between MreB and MreB cytoskeleton. (A) Schematic of sptPALM. Photoactivatable fluorescent PBP2 localization, it was possible that PBP2 diffusion is affected protein fusions are used to monitor the movement of individual proteins in by its transient interaction with MreB or indirectly through an a crowded environment. A small population of molecules is activated by UV MreB-associated component such as MreC (Fig. S6). However, laser light, and then imaged over time to track molecular positions. (B) TIRF sw disruption of MreB polymerization with the antibiotic A22 (18) images of MreB -PAmCherry single molecules in live E. coli TKL039 B (MG1655 mreBsw-PAmCherry) cells taken every 2.5 s, overlaid on phase- had little effect on PBP2 diffusivity (Fig. 2 ). In addition, dis- contrast images. The solid lines in the last panel are representative MreB rupting the activity of PBP1a, which binds PBP2 in vitro (19), molecular tracks. (Scale bar: 1 μm.) (C) Mean squared displacement (MSD) of either through inhibition with cefsulodin or deletion, had no MreB molecules in live (n = 382 molecules) and fixed (n = 105) E. coli TKL039 effect on diffusivity (Fig. S7). To test whether PBP2 diffusion cells. The shaded area represents SEM. (Inset) The distribution of MreB track could be increased by removing potential sites of interactions −1 speeds (28.6 ± 15.4 nm·s , SD) at 37 °C and angles relative to the cell midline with other periplasmic components (Fig. 2C), we constructed ± (99.1 29.4°, SD). Most trajectories were approximately perpendicular to the a PAmCherry-PBP2 fusion with the entire periplasmic, trans- long axis of the cell, with a slight right-handed bias quantitatively consistent peptidase domain truncated. We observed a large increase in with previous studies of left-handed twisting during growth (42). (D)MSDof B MreB molecules (n = 1,018) in TKL039 cells and PBP2 molecules (n = 854) in diffusivity (Fig. 2 and Fig. S8), approximately to the levels

TKL112 (ΔmrdA Pmrda-PAmCherry-mrdA) imaged at high frame rates. The expected based on other similarly sized transmembrane proteins shaded area represents SEM. The linear MSD of PBP2 indicates that PBP2 (17). Importantly, this increase was substantially more than molecules move diffusively, with a diffusion constant of D = 0.06 ± 0.006 μm2/s. would be predicted from the Stokes–Einstein relation (D ∼ 1/R)

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1313826111 Lee et al. Downloaded by guest on September 29, 2021 A B transient association could passively buffer growth rate against 1.5 fluctuations in PBP2 concentration. To measure growth rate )

-1 1.2 during changes in PBP2 concentration, we imaged cells during s 2 0.09 PBP2 depletion through several divisions. The depletion of PBP2

m 0.06 µ 0.03 was confirmed by Western blot, with levels equivalent to the

1 MreB D ( 0 chromosomal fusion 15 min after the removal of inducer (Fig. S4 1 PBP2 1 10 10 0.1 0.1 A B

Ctrl and ). The rate of depletion qualitatively agreed with the loss 0.5 mecillinam A22 trunc A S330A of rod-shape morphology (Fig. 3 ). During the course of de- PG pletion (Fig. S4 A and B), cells gradually became wider and 0 C PBP2 TP Signal (a.u.) 0 5 10 15 20 rounder over the course of several divisions (Fig. 3A), consistent nPB Cell length (µm) PGEM with previous observations (11). Despite these morphological IM TM changes indicative of substantial PBP2 depletion, we detected no slowing of the growth rate, defined as the areal strain rate (1/A MreB dA/dt), through two rounds of division (Fig. 3B). It was only after this interval that growth rate became drastically compromised, Fig. 2. Rapid PBP2 diffusion is independent of catalytic activity, and buffers cell growth rate from fluctuations in PBP2 concentration. (A) Representative consistent with our theoretical prediction of the robustness of image of MreB and PBP2 colocalization in a TKL233 (mreBsw-sfGFP ΔmrdA growth rate to large reductions in the number of PBP2 molecules sw Materials and Methods PmrdA-PAmCherry-mrdA) cell filamented with cephalexin. (Upper) MreB - ( ); regardless of the initial abundance sfGFP and PAmCherry-PBP2 were coimaged via TIRF. (Lower) The fluorescence before depletion, the rapid drop in growth rate distinguishes our profiles over the length of the cell, averaged over the transverse direction. model from one in which growth is directly proportional to PBP2 MreB and PBP2 profiles have a small, but significant correlation [two-sample abundance. Quantitatively similar behavior was observed in cells = = t test: experimental data vs. scrambled data, t(76) 2.79, P 0.0068; Materials during PAmCherry-PBP2 depletion (Fig. S4C), and during PBP2 and Methods; Fig. S6]. (B) The apparent PBP2 diffusion constant is unaffected depletion in minimal medium (Fig. S4D) and in filamentous cells by antibiotic treatment. SEM is shown (n = 5 experiments). Mecillinam binds (Fig. S4E; Materials and Methods), providing further indication the active site of PBP2, A22 disrupts MreB polymerization, and an S330A mutation in PBP2 ablates the catalytic active site. Concentrations are given in that growth rate is robust to substantial changes in PBP2 levels. micrograms per milliliter. The diffusion constant increases roughly 25-fold when the transpeptidase domain is truncated. (C) Model for PBP2 interaction Mecillinam Treatment Reduces Growth Rate and MreB Speed in a

with MreB. PBP2 moves rapidly within the inner membrane (IM), forming Dose-Dependent Manner. Because overexpression of catalytically MICROBIOLOGY transient interactions with sites of peptidoglycan (PG) synthesis through its inactive PBP2 is lethal (11), we expected that mecillinam treat- transpeptidase (TP) domain, either with the peptidoglycan elongation ma- ment would be functionally distinct from depletion due to the chinery (PGEM), or with the wall itself. PBP2 also contains transmembrane interference of inactive PBP2 with the cell wall synthesis ma- (TM) and nonpenicillin binding (nPB) domains, which may mediate inter- chinery. Although both treatments result in a lower concentra- actions with other morphogenetic proteins. tion of active PBP2 molecules, we expect that mecillinam-bound PBP2 could block the action of active enzymes. Inhibition of based solely on the decrease in mass of PBP2 due to truncation, PBP2 activity with high levels of mecillinam resulted in a con- and is likely an underestimate, as molecules moving at that speed centration-dependent reduction in the average growth rate (Fig. 4B and Fig. S9A), consistent with a requirement for active PBP2 cannot be well localized with single-molecule fluorescence. to either (i) initiate peptidoglycan insertion events (with sub- These data indicate that PBP2 is slowed by interactions through sequent cross-linking along the glycan strands carried out by its transpeptidase domain, possibly with other proteins such as another enzyme), or (ii) cross-link glycan strands throughout the MreC (20) and/or the wall itself through its transpeptidase do- process of new cell wall incorporation (Fig. 4A). To differentiate main. Nonetheless, these interactions must have sufficiently between these possibilities, we measured MreB speeds during rapid kinetics that PBP2 motion appears diffusive on the fast mecillinam treatment. If PBP2 were required only for initiation, timescale of our measurements. Based on these observations, we the speeds of processive MreB molecules should be maintained hypothesized a model in which PBP2 rapidly binds and dis- and the reduction in growth rate would be due to decreased sociates from active sites of wall synthesis through the trans- initiation, i.e., there would be fewer processive MreB molecules peptidase domain (Fig. 2C). Importantly, the independence of PBP2 motion from MreB in addition to PBP2’s lack of directed motion provide further evidence that E. coli cell wall assembly is not mediated by a stable, MreB-associated, multienzyme com- A B )

plex containing PBP2. -1 0.06

0.05 +ara Growth Rate Is Maintained During Depletion of PBP2. Transient 0 10 20 interactions between MreB and PBP2 may explain how E. coli 0.04 maintains robust cell growth despite only expressing ∼100 PBP2 0.03 molecules per cell (21). If PBP2 were part of a stable multien- 30 40 50 -ara zyme complex, then previous calculations have indicated that 0.02 ∼ Growth rate (min 100 PBP2 molecules would be required to maintain a 20-min 0 20 40 60 80 doubling time (3), and reductions in PBP2 levels would lead to 60 70 80 Time (min) a decrease in growth rate. In contrast, based on our measured diffusive motion of PBP2 and previous experimentally de- Fig. 3. Growth rate is unaffected by substantial PBP2 depletion. (A) Time- Δ termined estimates for the density of peptide cross-links in the lapse microscopy of E. coli TKL141 ( mrdA Para-mrdA) cells growing during cell wall (22), we estimated that as few as ∼30–40 PBP2 mole- PBP2 depletion. Time is specified in minutes. Even though cells lose their rod- cules would be sufficient to move among all active sites of syn- shaped morphology, they continue to grow at a rate comparable to wild- Materials and Methods type cells until two to three divisions have taken place. (B) Growth rate thesis spread across the cell surface ( ), during PBP2 depletion is quantitatively unaffected over more than two indicating that PBP2 abundance should not be limiting for doubling times. Ara+ represents the nondepleted control, and Ara− repre- growth in wild-type cells. Therefore, we hypothesized that the sents cells undergoing PBP2 depletion. The shaded area represents SE. (Scale combination of high diffusivity, rapid catalytic activity, and bar: 1 μm.)

Lee et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 A 1ledoM 2ledoM cross-linked peptidoglycan should be independent of mecillinam concentration, with the rate of cell wall growth slowing to match MreB MreB the rate of glycan cross-linking. We confirmed this prediction TP using ultra performance liquid chromatography (UPLC) (Fig. PBP22 MreB MreB Materials and Methods PBP22 S10; ). Therefore, because cross-linking TP affects cell wall elasticity (23), E. coli cells also appear to buffer MreBB MreB the mechanical strength of the cell wall against mecillinam TP Elongation PBP2PBP2 treatment, despite the resultant changes in cell shape that may be

MreB Initiation due to the spatial pattern of material or subtle changes in glycan

PBP2PBP2 strand length (22). Our study also indicates that the coordination of cell wall synthesis does not necessitate the colocalization of the proteins B C involved. In fact, transient associations are beneficial for buff- 1 µg mL-1 0.1 µg mL-1 10 µg mL-1 ering growth against fluctuations in enzyme abundance. At a 10 µg mL-1 0.45 -1 0.2 untreated 1 µg mL PBP2 abundance of ∼100 enzymes per cell (21), one would ex- 0.1 µg mL-1 pect 1/√N ∼ 10% fluctuations, yet growth rate remains consis- 0.15 0.3 untreated tent through cell division (24). In addition, transient association 0.1 loosens the requirement for an MreB complex to spatially and Fraction 0.15 0.05 temporally order the steps of cell wall synthesis. This line of reasoning is supported by the observation that growth rate is 0 0 unaffected by A22 treatment (25), despite disruption of MreB 0.02 0.03 0.04 0 20 40 60 spatial organization. In vitro interactions between E. coli PBP2 Growth rate (min-1) -1 MreB speed (nm s ) and PBP1a (19) and between Helicobacter pylori PBP2 and MreC Fig. 4. PBP2 activity is required throughout MreB-directed peptidoglycan (20) have been identified, and in the latter case these proteins synthesis. (A) Potential models for the role of PBP2 activity during cell wall appear to form a complex in vivo. Although perturbation of synthesis. (Left) PBP2 is required for initiating strand synthesis by the rest of PBP1a does not affect PBP2 mobility (Fig. S8), it remains pos- the cell wall synthesis machinery, but is not required for further synthesis- sible that some components of the cell wall synthesis machinery dependent MreB motion. (Right) Diffusing PBP2 molecules rapidly associate interact and move together, but not stably with MreB. In- and disassociate with MreB-associated proteins to drive the progress of terestingly, studies in B. subtilis suggest that the converse is also strand incorporation and MreB motion. (B) Cell growth rate decreases under possible: coordination can occur through an MreB-based com- mecillinam treatment in a concentration-dependent manner. Exponential B. subtilis phase cells (MG1655) were placed on agarose pads containing the indicated plex (4, 5). However, in , PBP2A and PBP2H engage in concentration of mecillinam and imaged for 15 min. To calculate growth diffusive motion when MreB is depleted (4). Whether these rate, cell contours were extracted and cell length over time was fit to an differences can be attributed to a specialization for Gram posi- exponential function. (C) MreB speed in TKL039 cells also decreases under tive-specific cell wall growth remains to be resolved. mecillinam treatment in a concentration-dependent manner, indicating a Although PBP2 activity is required throughout MreB-medi- requirement for active PBP2 molecules during the insertion of glycan strands. ated cell wall synthesis, the origin of directed MreB movement is yet unknown. It is presumed that MreB dynamics are related to the known biochemical steps in peptidoglycan synthesis, but but their speed would be unaffected. If PBP2 activity is required a detailed mechanism by which they couple to or drive MreB throughout strand insertion, MreB motion would be contingent motion is unclear. Observing the dynamics of the cell wall syn- on the arrival of an active PBP2, and thus mecillinam would thesis components will clarify their role in the mechanochemical decrease MreB speeds. Supporting the second hypothesis, single- cycle underlying MreB dynamics, and the use of single-molecule molecule measurements showed mecillinam concentration- C techniques will be essential. In our study, sptPALM was critical dependent decreases in MreB speed (Fig. 4 ). The fraction of for gaining insight into PBP2 dynamics rather than relying on MreB molecules moving in a directed fashion also decreased B epifluorescence colocalization to determine whether MreB and (Fig. S9 ), perhaps indicating a concomitant decrease in initia- PBP2 interact in a stable complex. The importance of single- tion or reflecting a decrease in average glycan strand length. molecule techniques has been highlighted in other studies of These quantitative measurements linking perturbations of PBP2 multienzyme processes, such as the observed exchange of poly- activity to reductions in growth rate and MreB speeds confirm merases during DNA replication (26). Our results indicate that that the activity of PBP2 molecules is required throughout the similar dynamic association occurs in peptidoglycan synthesis process of insertion of glycan strands, and provide further evi- and has an important role in maintaining the rate of this spatially dence that MreB motion is directly coupled to cell wall synthesis. distributed process in the presence of concentration fluctuations, Discussion especially important given the low abundances of the enzymes involved. More generally, random links between the processing Our single-molecule measurements demonstrate that PBP2 elements of distributed, multistep processes have been proposed undergoes fast, diffusive dynamics, and indicate that PBP2 ac- to suppress fluctuations in processing times (27); diffusive PBP2 tivity is coupled to MreB motion and cell wall synthesis. These motion between sites of peptidoglycan synthesis may be a bi- data are consistent with a model in which individual glycan ological realization of this principle. Given the numerous cellular strands are synthesized through the interchange of diffusing processes and timescales involved in cell growth, dynamic asso- PBP2 molecules, predicting that growth rate can be maintained ciation between key players may be central to strategies for op- in the face of fluctuations in PBP2 concentration without re- timizing spatiotemporal coordination during growth. quiring large excess. During PBP2 depletion, growth rate was maintained until a sharp decrease after several doubling times. Materials and Methods Our diffusivity measurements suggest that mecillinam-bound Strain Construction. The strains and plasmids used in this study are described PBP2 molecules are still recruited to the site of synthesis, but in Tables S1 and S2, respectively. Plasmids were constructed using enzymatic their inability to catalyze cross-linking decreases the rate of assembly methods (28). Expression plasmids were constructed using a low- insertion due to the requirement for a functional PBP2. Thus, copy plasmid with a pSC101 origin (pRM102) (29), and coding sequences for our model of diffuse PBP2 activity predicts that the fraction of the relevant genes were amplified from E. coli MG1655 with the appropriate

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1313826111 Lee et al. Downloaded by guest on September 29, 2021 homology regions for assembly (Table S3). Gene deletions and fluorescent routines were modified to use multicore processors. The fitted particle protein fusions were introduced into the chromosome using allelic exchange locations were corrected for drift using the cross-correlation between bright methods with suicide plasmids (30). The desired sequences for integration fluorescent beads after smoothing using the “spaps” MATLAB function. were amplified by PCR and cloned into pDS132 (30). MFDpir (31) cells were Tracks were calculated using the “costMatLinearMotion” cost function in transformed with the resulting plasmids and used for conjugative transfer u-track. Only MreB tracks of a certain length (5–15 time points) were into the recipient strain. Resulting merodiploids were selected on lysogeny retained for further analysis. To identify the subset of MreB molecules that broth (LB) (32) plates supplemented with chloramphenicol. Strains that had lost showed directed motion, the MSD of each individual track was fit to the sum the integrated plasmid (and sacB gene) by homologous recombination were of linear and quadratic terms [MSD(t) = c + 4Dt + at2], and particles that fit selected on LB plates containing 5% (wt/vol) sucrose. All chromosomal modifi- well (R2 > 0.9) with a > 0 were considered to undergo directed motion with cations were confirmed by amplification and sequencing of the targeted region. velocity v = a1/2. The MreB molecules that could not be fit well were either moving diffusively or too ambiguous to be classified as directed motion Bocillin Labeling. To verify that PAmCherry-PBP2 fusions were stable and according to the limits set by the temporal and spatial resolution of our remained intact, bocillin gels were carried out (Fig. S3). Overnight cultures measurements. The angle of motion was calculated by fitting the coor- were diluted 1:200 in LB and grown to an optical density at 600 nm dinates of the track to a line and measuring the angle as this linear fit – (OD600) of 0.5, pelleted, and resuspended in PBS. One millimolar phenyl- crossed the cell midline. For PBP2 molecules, tracks with a length of 3 15 methanesulfonylfluoride (Sigma), 10 μM Bocillin-FL (Invitrogen), and 500 μg/mL time points were considered for further analysis. An estimate for the dif- polymixin B (Fisher Scientific) were added to the cells and incubated fusion constant was determined from a linear fit to the first four points of at 37 °C for 1.5 h. Cells were pelleted and washed three times with cold the mean squared displacement. PBS, resuspended in Laemmli Sample Buffer (Bio-Rad), and boiled at 95 °C

for 10 min, and total protein content was quantified using a DC protein assay Colocalization of PBP2 and MreB. E. coli MG1655 ΔmrdA pKC137(PmrdA- (Bio-Rad) according to manufacturer’s instructions. Each sample was normalized PAmCherry-mrdA) cells were transformed with a plasmid expressing MreBsw- to 10 μg of total protein and separated on a 4–15% precast polyacrylamide mVenus (pRMmreBmVenus) or transduced with P1 lysate made from cells gel. The gel was rinsed with water 10 times and then visualized on a Typhoon containing MreBsw-sfGFP::kan. These cells were filamented with 35 μg/mL 9410 Variable Mode Imager (GE Healthcare) with 488-nm excitation. cephalexin and prepared on 1% agarose pads with EZ-RDM plus 0.2% glucose. The population of PAmCherry-PBP2 molecules was photoactivated Immunoblotting. Overnight cultures from strains containing the PAmCherry- with a 3-s burst from the 405-nm laser, and 50-ms exposure images were PBP2 fusion were diluted 1:100 in EZ-RDM plus 0.2% glucose and supple- acquired with 488-nm and 561-nm lasers. Cell outlines were calculated using mented with 0.1% arabinose when appropriate. Cultures were grown to an a custom MATLAB package, and the average fluorescence along the length

approximate OD600 of 0.7, and then 1 mL of culture was pelleted and of the cell was calculated. A moving average of 5 pixels (530 nm) was used to resuspended in 100 μL of 4% (wt/vol) SDS. For Para-PAmCherry-mrdA de- smooth the signal, and an average over 100 pixels was used to correct for

pletion experiments, exponential phase cultures were rinsed three times background, after which the signal was normalized. The Pearson correlation MICROBIOLOGY with media lacking arabinose and samples were drawn at the indicated time coefficient was calculated between these two normalized signals. To esti- points. Whole-cell lysates were prepared by incubating at 95 °C for 10 min, mate the error bars, the order of the signal was randomized once per cell and total protein content was quantified with a DC protein assay (Bio-Rad) and the correlation coefficient was calculated on the randomized signal. according to manufacturer’s instructions. Lithium dodecyl sulfate (LDS) sample buffer (4×) was added to each sample and incubated at 95 °C for 10 Estimate of the Number of PBP2 Molecules Required for Growth. The doubling min, and then 12.5 μg of total protein from each sample were loaded onto of an E. coli cell involves a length increase from ∼2to4μm, with a com- the gel. Proteins were transferred to a PVDF membrane (Immobilon, Millipore), mensurate surface area increase of ∼6 μm2. Each pair of disaccharides and first stained with rabbit anti-mCherry antibodies (ab167453; Abcam), and then associated cross-link corresponds to ∼10 nm2 in surface area (23). Therefore, stained with IRDye 800CW goat anti-rabbit antibodies (Li-Cor) for detection on given the high degree of in-plane cross-linking, ∼6 × 105 cross-links are a Li-Cor Odyssey imaging system according to manufacturer’s instructions. added per doubling. Taken together, for a doubling time of 20 min, ∼500 cross-links per second need to be added during elongation. Microscopy. Single-particle tracking was performed on a TIRF microscope built Previous studies have estimated that there are ∼100 PBP2 molecules per with a Ti-E Eclipse stand (Nikon Instruments). The objectives used were either E. coli cell (21). To estimate whether each PBP2 could participate in five cross- an Apo TIRF 100× (N.A. 1.49) or a Plan Apo Lambda 100× DM (N.A. 1.45) linking events per second, we assume that cell wall material is synthesized in (Nikon), depending on whether phase-contrast images were acquired con- a spatially uniform fashion across the cylindrical portion of the cell, which currently. CUBE diode 405-nm and Sapphire OPSL 561-nm lasers (Coherent) has an average length of 3 μm and an average surface area of ∼9 μm2. Thus, were combined into an optical fiber and into a TIRF illuminator (Nikon) at- each of the 500 new cross-links corresponds to a region with area of ∼0.02 tached to the microscope stand. Shuttering of the laser illumination was μm2. Assuming that the transpeptidation reaction is fast relative to the controlled by an acoustooptic tunable filter (AA Optoelectronics) before the speed of PBP2 motion across this area, a PBP2 molecule has to achieve an fiber coupler. Images were acquired with an iXon3+ 887 EMCCD (Andor MSD of at least 0.1 μm2 within 1 s to visit all five cross-linking sites. Our Technology) camera, and synchronization between components was ach- measured diffusion constant of ∼0.06 μm2/s corresponds to an MSD of 0.24 ieved using μManager (33) with a microcontroller (Arduino). μm2 in 1 s based on the relationship MSD(t) = 4Dt for 2D diffusion, and is therefore sufficient for coverage of cross-linking across the entire cell sur- Single-Particle Imaging. Cells expressing fluorescently labeled proteins were face during cell elongation. grown to saturation overnight in the rich medium EZ-RDM (Teknova) (34) with 0.2% glucose and then diluted 1:100 in fresh medium and incubated Single-Cell and Colony-Growth Imaging. Exponentially growing cells were with shaking at 37 °C for 2 h. Cells were spotted onto 1% agarose pads with spotted onto 1% agarose pads and then imaged every 30 s in a temperature- EZ-RDM plus 0.2% glucose and covered with argon plasma-cleaned cover- controlled chamber (HaisonTech) at 37 °C. For experiments involving slips. For drift correction, Tetra-speck 100-nm fluorescent beads (Invitrogen) mecillinam treatment, cells were transferred from liquid culture with no were added at a dilution of 1:1,000. To measure MreB speeds, we used an antibiotic directly onto agarose pads with the appropriate concentration of imaging sequence consisting of a 50-ms exposure with an activation laser mecillinam and imaged for 15 min. Cell contours were automatically (405 nm at ∼0.05 kW/cm2) followed by capture of 10 images with an imaging extracted from phase-contrast images using a custom MATLAB package. The 2 laser (561 nm at ∼0.5 kW/cm ) every 2.5 s, which was repeated for a total of cell length over time was fit to an exponential [L(t) = L0 exp(kt)] to calculate 500–1,000 cycles. To capture the more rapid PBP2 dynamics, we simulta- the growth rate k. neously exposed the cells to both activation and imaging lasers (405 nm at To analyze the growth rates of single cells in a microcolony during PBP2 ∼0.05 kW/cm2 and 561 nm at ∼1 kW/cm2) for 10-ms exposures every 45.3 ms depletion, cells grown in media supplemented with 0.1% arabinose were (except for the truncation mutant, which required a shorter imaging in- rinsed three times with media lacking arabinose and then spotted onto terval; Fig. S8). In single-particle tracking experiments under antibiotic agarose pads lacking arabinose. Cells were imaged every 30 s for 2 h. These treatments, agarose pads were cast with the appropriate antibiotic, and cells cells were segmented and analyzed using the MATLAB package Microbe- were directly spotted from liquid culture. Tracker (36). Growth rate was defined as the fractional change in cell outline area over time and averaged with a sliding window of eight frames. Growth Single-Particle Analysis. Images were analyzed computationally to generate rates were averaged over all cells for a given treatment at each time point, single-particle tracks using the u-track package (35) in MATLAB (MathWorks). and SEs were calculated based on the number of cells measured at each Gaussian mixture-model fitting was used for particle detection and the time point.

Lee et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 To analyze the growth rates of single filamentous cells during PBP2 de- Peaks were quantified and identified as particular muropeptide species, pletion, cells were grown with 0.1% arabinose and 7 μg/mL cephalexin for 30 from which the cross-linking density and strand length were calculated (40, 41). min, rinsed without arabinose, and then imaged on agarose pads with 7 μg/ mL cephalexin. These cells were segmented and analyzed using a custom ACKNOWLEDGMENTS. We thank Ned Wingreen, Josh Shaevitz, Alex Dunn, MATLAB package, and growth rate was measured as the fractional change David Ehrhardt, and Sven van Teeffelen for their feedback. We also thank in area as described above with an average sliding window of 15 frames. Piet de Boer and Felipe Bendezu for providing strains, and Tom Bernhardt and Monica Markovski for assistance with bocillin labeling. This work was Purification of Sacculi and UPLC Analysis of Peptidoglycan Composition. supported by a Siebel Scholars Graduate Fellowship (to T.K.L.), support from Overnight cultures of E. coli MG1655 were diluted 1:100 in LB and supple- a National Institutes of Health (NIH) Biotechnology Training Grant (to T.K.L.), mented with 0, 0.01, 0.1, and 1 μg/mL mecillinam. The cultures were grown a Stanford Interdisciplinary Graduate Fellowship (to C.T.), a Bio-X Senior

at 37 °C to an OD600 of 0.7. The cultures were then harvested by centrifu- Postdoctoral Fellowship (to R.D.M.), NIH Ruth L. Kirschstein National Re- gation at 5,000 × g for 10 min at room temperature and resuspended in 3 mL search Service Award 1F32GM100677-01A1 (to J.H.), a Stanford School of of LB. Cell suspensions were lysed by boiling in SDS, and SDS-insoluble ma- Medicine Dean’s Postdoctoral Fellowship (to J.H.), support from the Stanford terial was collected by several rounds of ultracentrifugation at 400,000 × g. School of Engineering Chinese Undergraduate Visiting Research Program (to Samples were prepared for UPLC analysis as previously described (37) and E.G.), NIH Director’s New Innovator Awards DP2OD004389 (to Z.G.) and injected onto a Waters H Class UPLC system equipped with a BEH C18 1.7-μm DP2OD006466 (to K.C.H.), and National Science Foundation CAREER Award column (Waters), using elution conditions previously described (38, 39). 1149328 (to K.C.H.).

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