Class A Penicillin-Binding Protein-Mediated Cell Wall Synthesis Promotes Structural Integrity during Peptidoglycan Endopeptidase Insufficiency in Vibrio cholerae

Synthesis and turnover of the bacterial cell wall must be tightly coordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines, the aPBPs and the Rod system.

IMPORTANCE Synthesis and turnover of the bacterial cell wall must be tightly coordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines, the aPBPs and the Rod system. Our results suggest that in Vibrio cholerae, one class of turnover enzymes, the endopeptidases, are necessary for proper cell elongation and division. aPBPs become essential for maintaining structural integrity during EP insufficiency, while the Rod system remains active but contributes little to cell expansion under these conditions. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the coordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes. morphology of an EP-deficient V. cholerae strain. Our data suggest that aPBP activity does not require wild-type levels of cross-link cleavage for PG incorporation (and consequently cell expansion), while the Rod system relies more on EP activity to contribute substantially to proper rod-shaped growth and cell expansion. Our cross-species complementation experiments intriguingly raise the possibility that direct coordination between EPs and cell wall synthases might not be necessary at all, at least under standard laboratory growth conditions.

RESULTS
V. cholerae continues to increase in mass during endopeptidase insufficiency. Endopeptidase depletion was previously shown to preclude insertion of new cell wall material in Escherichia coli, resulting in rapid cell lysis (23). In contrast, we noticed that EPdepleted Vibrio cholerae did not lyse, even in the absence of all six of its major D,D-EPs. This D6 endo strain (DshyA DshyB DshyC Dvc1537 Dvc0843 Dvca1043 P IPTG :shyA) has the remaining, conditionally essential EP ShyA under the control of an isopropyl-b-D-thiogalactopyranoside (IPTG)-inducible promoter and is thus suitable for depletion experiments. Upon growing the D6 endo strain in the absence of inducer for 2 h, ShyA was reduced to less than 10% of initial levels (see Fig. S1A and B in the supplemental material), and the percentage of PG cross-links was significantly increased (35.6% 6 2.1% after depletion compared to 31.6% 6 0.6% when shyA is expressed) (Fig. S1C and D). In this state of EP insufficiency, cell mass measured by either optical density (Fig. 1A) or bacterial dry weight ( Fig. 1B) continued to increase at a rate similar to when shyA was expressed. When we plated these cells on solid medium containing inducer, however, we found that the number of viable cells (CFU per milliliter [CFU/ml]) in the EP-depleted culture remained static or declined slightly (Fig. 1C). This indicates that EP-insufficient cells are not readily dividing but are able to recover and form colonies on a plate if resupplied with inducer (see Movie S1 in the supplemental material). We have previously shown that EP depletion in the D6 endo strain results in a dramatic increase in cell size and ultimately the generation of giant, bulky, and contorted cells (36). Here we have quantified these morphology defects and shown that ShyA-depleted D6 endo cells have significantly greater cell areas, lengths, and widths ( Fig. 1D to G). Overall, these observations show that EP-insufficient V. cholerae cells suffer severe morphology and division defects but still increase in mass and retain structural integrity and viability.
To rule out contributions from other predicted endopeptidases, we additionally deleted the genes encoding PBP4, PBP7, and VC1269 (which have predicted EP activity but are, based on work in E. coli, not required for growth and cell elongation [39,40]). These gene deletions did not appreciably affect mass increase (except for a slight decrease in final yield after 6 h; for an unknown reason, the strain also exhibited a more pronounced drop in CFU/ml than D6 endo) ( Fig. S1E and F), demonstrating that the mass increase phenotype did not simply reflect the ability of these putative EPs to substitute for ShyA.
Cell wall incorporation continues during EP insufficiency. We next probed the impact of endopeptidase insufficiency on PG incorporation and composition. To determine whether these enlarged cells elaborated a wild-type PG cell wall, we cultured D6 endo in the presence of a fluorescent D-amino acid derivative, HADA (3-[[(7-hydroxy-2oxo-2H-1-benzopyran-3-yl)carbonyl]amino]-D-alanine hydrochloride), as a cell wall stain (41). Addition of HADA to ShyA-replete D6 endo cells resulted in an even distribution of staining along the cell wall (Fig. 1H), as expected from wild-type cell wall synthesis. In contrast, depleting ShyA resulted in a strikingly different pattern, where large patches of HADA-reactive material accumulated throughout the cell. In principle, these patches could be a remnant of incompletely degraded cell wall material synthesized before ShyA was completely depleted, or they could reflect the activity of L,D-transpeptidases (which are able to incorporate HADA into the cell wall independent of cell wall synthesis [41,42]). We repeated the staining experiment in a D6 endo strain lacking were washed twice and diluted 100-fold into growth medium with IPTG (ShyA 1) or without IPTG (ShyA -). At the indicated time points, optical density (OD 600 ) (A), dry mass (mg/liter) (B), and viable cell counts (CFU/ml) (C) were measured. Data are means of at least three biological replicates, and error bars represent standard deviations. (D to G) Cells were imaged at 3 h, and representative cells are shown in panel D. ImageJ was used to measure cell area (E), cell length (F), and cell width (G). Raw data points are shown, and error bars represent standard deviations. Asterisks denote statistical difference relative to ShyA 1 via a Mann-Whitney test (****, P , 0.0001). (H) D6 endo strain was grown in the presence of HADA (100 mM) for 3 h, washed twice, and then imaged. All bars, 5 mm. (I) After 2 h of growth, relative PG content of D6 endo strain (normalized to OD 600 ) was measured via UPLC analysis (see Materials and Methods for details). Bar graphs show data normalized to Shy A1. Error bars represent standard deviations of three biological replicates, and asterisks denote statistical difference via unpaired t test (**, P , 0.01). (J) After 3 h of growth, cells were pelleted and resuspended in 20 mM NaCl (osmotic shock treatment) for 5 min. Shock treatment was stopped by adding PBS to 180 mM. Percent survival is CFU/ml after treatment divided by CFU/ml before treatment. Raw data points of three biological replicates are shown. Asterisks denote statistical difference via unpaired t test (****, P , 0.0001). (Continued on next page) Endopeptidase Depletion Effects on Cell Wall Synthesis ® While accumulation of HADA is difficult to interpret quantitatively (due to multiple possible upstream events that could result in increased HADA signal [43]), these data qualitatively show that during endopeptidase insufficiency, PG synthesis and incorporation continue in an aberrant, nondirectional way.
Quantification of PG confirmed and expanded these observations-after 2 h of ShyA depletion, cells accumulated approximately two-to threefold-more PG than ShyA-replete cells (when normalized to an optical density at 600 nm [OD 600 ]) (Fig. 1I). Since these cells are nondividing, we speculate that PG is accumulating within these individual, drastically enlarged ShyA-depleted cells. Consistent with a higher cell wall content of individual cells, ShyA-depleted D6 endo cells were almost 10-fold more resistant to osmotic shock treatment (Fig. 1J). Thus, ShyA-depleted D6 endo cells not only incorporate PG but retain higher levels of PG than the wild type (WT), possibly reflecting the lack of EP-initiated turnover processes. Similar observations have been made in autolysin-inactivated Bacillus subtilis, a Gram-positive bacterium (27,44,45). While we cannot rule out that residual ShyA remains in the cell following depletion (at levels too low to detect above background of the nonspecific band we observed via Western blotting in the same size range as ShyA [ Fig. S1A and B]), we can at a minimum conclude that wild-type levels of V. cholerae's principal EPs are not necessary to facilitate mass increase and incorporation of PG per se. Rather, EPs are essential for maintenance of width homeostasis and cell division, and likely key for the proper, directional integration of PG into the V. cholerae sacculus.
Cell wall incorporation and mass increase in EP-deficient cells rely primarily on aPBPs. We next addressed whether EP insufficiency affected the two cell wall synthases, the Rod system and the aPBPs, differentially. To this end, we grew the D6 endo strain in the presence or absence of inducer for 2 h and then treated with either moenomycin (an aPBP glycosyltransferase inhibitor [46]) or one of two Rod system inhibitors: MP265 (an inhibitor of MreB [47]) or amdinocillin (an inhibitor of PBP2 [48]).
When ShyA was expressed, mass increase (proxied by OD 600 , which correlates strongly with dry weight [ Fig. 1AB]) proceeded at similar rates for all antibiotic treatments and the dimethyl sulfoxide (DMSO) control ( Fig. 2A), while CFU/ml plateaued in the presence of antibiotic (Fig. S2A). The continued OD 600 increase upon antibiotic exposure is consistent with our previous observations that V. cholerae (as well as many clinically significant Gram-negative pathogens) is remarkably tolerant to inhibitors of cell wall synthesis (32,49). Exposure to such agents causes V. cholerae to form cell walldeficient spheroplasts (in the presence of aPBP inhibitors) or spheroid cells containing cell wall material (in the presence of MreB and PBP2 inhibitors) (32,49). Importantly, both sphere cell types continue to increase in mass (32, 49) but fail to divide. Thus, OD 600 continues to increase while CFU/ml remains unchanged.
Upon ShyA depletion, mass increase (OD 600 ) in the presence of MP265 or amdinocillin-treated cells continued at a similar rate compared to untreated and ShyA-replete conditions (Fig. 2B). Measurements of bacterial dry weight at 2 h posttreatment corroborated this finding (Fig. 2C). These data suggest that the Rod system is not required for the observed mass increase in EP-insufficient cells. ShyA depletion still generated enlarged cells in the presence of either Rod system inhibitor; however, the cells were noticeably rounder than untreated cells (cells not treated with ShyA [ShyA-cells]) (Fig. 2D). Cell dimension analysis in ImageJ (50) revealed that neither Rod system inhibitor affected the two-dimensional (2D) area of EP-insufficient cells (Fig. 2E); however, both drugs significantly decreased cell length ( Fig. 2F; additional cell size analyses are presented in Fig. S2C to E). Altogether, these data suggest that the Rod system is not required for mass increase but still contributes to cell shape during EP insufficiency. We next visualized and quantified cell wall material in the presence of Rod system inhibitors. After 2 h of treatment with either MP265 or amdinocillin, we observed sub- h after drug treatment, relative PG content of D6 endo strain relative to OD 600 was measured via UPLC analysis (see Materials and Methods for details). Data are normalized to the ShyA1 DMSO sample. Error bars represent standard deviations of three biological replicates. Asterisks denote statistical difference via unpaired t tests (****, P , 0.0001; *, P , 0.05). stantial HADA incorporation in ShyA-cells (Fig. 2G), consistent with our interpretation that the Rod system is not absolutely required for PG synthesis during EP insufficiency. PG quantifications revealed that when EP-insufficient cells were treated with either Rod system inhibitor, they still accumulated more PG than similarly treated cells treated in the presence of ShyA (ShyA1 cells), albeit to a lower degree (Fig. 2H). Importantly, neither treatment resulted in PG degradation.
In striking contrast to Rod system inhibition, aPBP inhibition via moenomycin exposure completely abrogated growth (measured by OD 600 and dry mass) of ShyA-depleted D6 endo cells ( Fig. 2B and C). This coincided with accumulation of small cells and debris, indicative of lysis (Fig. 2D). In addition, cell viability declined rapidly in early stages (consistent with our previous observations [32]), though ultimately exhibited levels of survival similar to MP265-or amdinocillin-treated, ShyA-depleted D6 endo cells (Fig. S2B). We observed comparable results when the aPBP inhibitor cefsulodin was used at high concentrations ( Fig. S2F to H). Notably, aPBP-inhibited cells lacked strong HADA incorporation (Fig. 2H), though these data are difficult to interpret due to the loss of cell wall integrity. However, our data clearly suggest that during EP insufficiency, the aPBPs are required to maintain structural integrity and are major contributors to both mass increase and sustained PG incorporation, while the Rod system primarily influences cell morphology.
MreB movement continues in EP-insufficient cells. The Rod system, in conjunction with the actin homolog MreB, deposits new cell wall material during cell elongation while performing a rotational movement around the cell, apparently driven by aPBP-independent cell wall synthesis (51)(52)(53). Our data indicate that the Rod system plays a minor role during EP insufficiency, so we asked whether EP depletion altered the mobility of Rod complexes similar to what has been observed during inhibition of cell wall synthesis (52). We constructed a functional (Fig. S3A) MreB msfGFP sandwich fusion in a D6 endo background and measured MreB msfGFP velocity using epifluorescence and total internal reflection fluorescence (TIRF) microscopy (see Materials and Methods for details). We determined MreB velocities through particle analysis and mean square displacements (MSD) calculations in ImageJ (54,55). We confirmed that MP265 stopped MreB movement (Fig. 3B) as a positive control, as expected from what has been reported in E. coli. Mean square displacement values indicated mixed populations of diffusive MreB particles and those exhibiting directed motion under both ShyA-replete and -depleted conditions ( Fig. S3B and C). Interestingly, MreB movement continued even after 3 h of ShyA depletion ( Fig. 3D and Movie S2), albeit with a substantially altered pattern of movement and a reduced velocity (;44 6 34 nm/s) compared to ShyA-replete conditions (;72 6 38 nm/s) (Fig. 3C). This velocity reduction was statistically significant (P , 0.0001, Mann-Whitney test). Our estimates of MreB velocity under ShyA-replete conditions were somewhat higher than what has been reported previously for other bacteria (55 nm/s for B. subtilis [56] and 6.7 6 2.7 nm/s to 30 nm/s for E. coli [52,56]), perhaps reflecting species-specific differences or different properties of our sandwich fusion. We additionally determined the MreB cluster size and number using ImageJ TrackMate (54) and particle analysis (57, 58) (see Materials and Methods for details). Interestingly, the average cluster size and number increased under ShyA depletion conditions (Fig. 3F and G and Fig. S3D), suggesting that EP depletion might affect Rod complex assembly dynamics and possibly explain the altered pattern of MreB movement. While we currently do not have a mechanistic explanation for those pattern differences, the key observation here is simply that MreB movement persists upon prolonged EP depletion. We thus conclude that similar to what has been observed in B. subtilis (24), EP insufficiency does not result in immediate inactivation of the Rod system but changes its velocity and potentially the dynamics of its assembly.
Complementation of EP insufficiency in V. cholerae by expression of heterologous EPs. So far, our results suggested that during EP insufficiency, PG synthesis via aPBPs promotes cell integrity, whereas the Rod system remains functional but is not absolutely required for mass increase or PG incorporation. It has been hypothesized that PG synthases require a physical association with one or more EPs for insertion of nascent PG material, but alternatively, EPs might catalyze PG insertion independently, for Endopeptidase Depletion Effects on Cell Wall Synthesis ® example through recognition of intrinsic PG substrate cues. To gain a better understanding of the necessity for a physical interaction, we conducted cross-species complementation experiments using an EP from Neisseria gonorrhoeae (henceforth "MepM Ngo "). This distantly related EP (a BLAST alignment indicated 29% identity between MepM Ngo and ShyA [ Fig. S4]), when heterologously expressed, is unlikely to interact with any native V. cholerae enzymes, and should thus allow us to isolate its EP activity from the interaction networks it might be embedded in. We expressed arabinose-inducible MepM Ngo in D6 endo cells and measured differential growth in the presence of IPTG (ShyA expression) versus arabinose (MepM Ngo expression). We found that wild-type MepM Ngo was unable to rescue growth of D6 endo cells during ShyA depletion conditions (Fig. 4A). However, we recently demonstrated that EPs from diverse organisms (including E. coli and N. gonorrhoeae) are produced predominantly in an inactive form due to the inhibitory function of their domain 1 and are likely activated in vivo by an unknown mechanism (36). Heterologously expressed enzymes may not be subject to this activation pathway in V. cholerae (especially if the activator is a protein). We thus expressed EP mutant versions with their inhibitory domain 1 deleted (DDom1), rendering them constitutively active, and provided a signal sequence (ss) to ensure export to the periplasm. Surprisingly, ssMepM Ngo DDom1 fully complemented growth of the D6 endo strain to a similar degree as the native ShyA (Fig. 4A). Visual inspection of D6 cells relying on heterologous expression for growth (arabinose-positive [ara1] condition), revealed that complementation with ssMepM Ngo DDom1 (but not its active site mutant derivative H373A) promoted both growth (Fig. 4A) and the generation of rod-shaped cells (Fig. 4B). Interestingly, we found that heterologous expression of a signal sequence fusion or activated MepM from E. coli (ssMepM Eco and ssMepM Eco DDom1 , respectively) were able to restore growth, but not the rod shape, to D6 endo V. cholerae (Fig. S5A and B). Thus, for an unknown reason, intrinsic properties of specific EPs (or rather these mutant derivatives) define their ability to complement the D6 endo cell shape. We sought to confirm that this apparent complementation of the rod shape was still dependent on MepM Ngo DDom1 (rather than a mutation derepressing shyA in D6 endo strain). Thus, we plated all strains on agar Endopeptidase Depletion Effects on Cell Wall Synthesis ® containing IPTG, arabinose, or no inducer at the end of the experiments where we visualized cells relying on MepM Ngo DDom1 for growth. All strains had the same low level of spontaneous suppressors able to grow in the absence of inducer (Fig. S5C), confirming that the majority of the rod-shaped cells observed when only MepM Ngo DDom1 was expressed are not suppressors. In summary, these data demonstrate that heterologous expression of an activated EP can be sufficient to restore both growth and (in the case of the N. gonorrhoeae EP) proper cell shape to D6 endo cells.

DISCUSSION
Bacteria must maintain a careful balance between cell wall cleavage and synthesis to promote cell elongation/division, but the exact relationship between the two cell wall synthases (Rod system versus aPBPs) and cell wall hydrolases (e.g., endopeptidases) is poorly understood, at least in Gram-negative bacteria. Here, we have used EP depletion and chemical inactivation experiments to dissect the interplay between cell wall cleavage and synthesis in the cholera pathogen V. cholerae. Our key observation is that in V. cholerae, cell wall synthesis and cell expansion (but not cell division) continue aberrantly upon EP depletion (Fig. 5). This poses an apparent contradiction to data obtained in E. coli, where cell wall incorporation was drastically reduced after EP depletion and cells started to lyse (23). While ostensibly fundamental aspects of the coordination between cell wall synthesis and cleavage may simply not be as well conserved as one might expect, these observations might also reflect species-specific differences in EP-independent cell wall turnover rates, and not necessarily the consequences of EP depletion per se. It is possible that lysis under EP-insufficient conditions in E. coli reflects generally higher PG degradation rates (E. coli, for example, encodes three amidases [59], while V. cholerae possesses only one [60]). This would mask the underlying continued incorporation of new cell wall material in the absence of EPs. Importantly, EP depletion in E. coli did result in a cell volume increase prior to lysis (23), also supporting at least a transient continuation of PG synthesis during EP insufficiency in this species. Last, it is possible that V. cholerae simply elaborates a more structurally robust cell envelope than E. coli, which seems ecologically appropriate given the wide range of osmolarities (from pond water to seawater) the cholera pathogen resides in.
The amount of cell wall expansion occurring during EP insufficiency was surprising, since presumably any form of cell wall synthesis that promotes the degree of cell expansion we observed in EP-deficient V. cholerae might be expected to require some form of cleavage. This cleavage is likely catalyzed by other autolysins; however, the incisions resulting from such cleavage, and/or the autolysin(s) involved, appear to be of limited utility to the Rod system, while they can be exploited by the aPBPs. This suggests that aPBPs are more versatile in recognizing a variety of cell wall cuts required for structural integrity (independent of an actual physical connection with EPs), while the Rod system primarily relies on either EP-mediated cleavage or a physical association with EPs (see a more detailed discussion below) to promote proper cell elongation and width homeostasis. These interpretations are in line with some old and several recent proposals based on data from E. coli that aPBPs and SEDS have separate (yet perhaps overlapping) functions during cell elongation (15,61,62). Interestingly, upregulated EP activity promotes aPBP function in E. coli, likely indirectly through the creation of PG incisions that allow for an interaction between aPBPs and their OM-localized activators (30). Thus, EP cleavage may not be strictly necessary for, but can promote, aPBP activity. It is possible that under EP-insufficient conditions, the lytic transglycosylases (the other major group of cell wall cleavage enzymes that cut the polysaccharide backbone of PG [63]) create large open areas in PG that can be recognized, and patched, by aPBPs; however, undiscovered EPs might also play a role.
The observation (consistent with what has been shown in B. subtilis [24]) that MreB continues directed movement at least for some time during EP insufficiency suggests that the Rod system does not actually require wild-type EP activity for RodA's glycosyltransferase activity (which likely drives MreB movement). This suggests that EPs may not be a functionally integral part of the Rod system in V. cholerae. Similar to what has been proposed for B. subtilis, it is thus tempting to speculate that V. cholerae may use a "make-before-break" model as proposed by Höltje (38) and Koch (37) for cell elongation via the Rod system. In this model, the Rod system creates a second layer of PG that is incorporated via EPs during or after synthesis. Generation of this second layer could at first proceed independently of wild-type EP activity, but incorporation into the growing sacculus would require cross-link cleavage.
The results of our cross-species complementation experiments with an activated N. gonorrhoeae EP further suggest that a physical association between the Rod system and EPs might not be strictly necessary, unless the heterologously expressed (and truncated) enzyme does somehow directly interact with the V. cholerae Rod system. We thus consider a model plausible where rather than (or in addition to) coordinating with cell wall synthases directly, EPs can somehow specifically recognize and preferentially cleave old PG that is adjacent to nascent PG. Though highly speculative, our observation that the corresponding E. coli homolog does not complement cell shape might reflect different levels of activity-since EPs can promote aPBP activation (at least in E. coli) (30), overexpression of a more active EP might divert PG precursor flux away from the Rod system toward aPBPs to a higher degree than the N. gonorrhoeae enzyme, incapacitating the cells' ability to elaborate a rod shape.
An important caveat to the complementation experiments is that the D6 endo strain still maintains a copy of shyA under IPTG control. While the lac promoter is tightly repressed in the absence of inducer, a small number of molecules under its control might still be produced (64). ShyA is produced predominantly as an inactive precursor, and the signal for activation is unknown (36). It is conceivable that complementation with a heterologously expressed EP might somehow enhance activation of this leaky background of ShyA molecules, if there is, for example, a positive feedback loop between cell wall cleavage and native EP activation.
In summary, our data suggest that two main cell wall synthases, the aPBPs and the Rod system have differential relationships with autolysins, and especially endopeptidases. As such, our data provide additional support for the emerging theme of at least partially differential roles of the aPBPs and the Rod system during cell elongation.
Endopeptidase Depletion Effects on Cell Wall Synthesis ® Plasmid and strain construction. All bacterial strains and oligonucleotides used in this study are summarized in Table S1 in the supplemental material. All Vibrio cholerae strains are derivatives of El Tor strain N16961 (65) or E7946 (66); the latter strain was used for chitin-induced transformation.
Construction of D6 endo strain is reported elsewhere (32). Other strains were constructed by chitininduced transformation of linear PCR products as described in reference 67. A chloramphenicol (chl) resistance cassette insertion into the gene vc1807 (a well-established neutral locus) was used as the primary selector. The transforming fragment for vc1807::chl was constructed by amplifying upstream and downstream homology regions using primers PD079/PD097 and PD098/PD082, respectively. The chl gene coding for chloramphenicol acetyltransferase was amplified from pBAD33 (68) with primers PD095/PD096 and fused with the flanking homologies of vc1807 via isothermal assembly. For antibiotic resistance gene swapping, a vc1807::trim allele was also produced by amplifying upstream (using primers TDP597/598) and downstream (primers TDP601/602) homologies of vc1807 and fusing them with a trimR cassette amplified from V. cholerae Haiti (69) (primers TDP599/600) using splicing by overlap extension PCR (SOE PCR) with primers TDP603/604.
To construct a functional MreB-msfGFP-MreB sandwich fusion, upstream (primers PD056/PD074) and downstream (primers PD071/PD057) homologies were amplified from the V. cholerae genome and fused via isothermal assembly with monomeric superfolder green fluorescent protein (msfGFP) (amplified with primers PD054/PD055). Analogous to a published E. coli MreB-msfGFP sandwich fusion (52), we replaced glycine 228 of MreB with this msfGFP. To enhance the probability of success of finding a functional fusion, we used semidegenerate primers to generate a library of possible linker sequences. Flanking homologies, MreB and msfGFP were first fused using isothermal assembly (70) and then amplified using nesting primers PD104/PD105. The resulting upstream-MreB-linker-msfGFP-linker-MreB-downstream PCR fragments were transformed into E7946 cells using chitin transformation with vc1807::chl as the primary selector. Ninety-six colonies were tested for growth rate, and clone M2C was chosen for further experiments due to its wild-type growth behavior. The linkers of this fusion construct were sequenced (coding for DGVGG upstream of msfGFP and GTPIP downstream).
Bacterial dry weight. One hundred twenty milliliters of culture was harvested (4,830 Â g, 10 min), resuspended in ;1 ml, and transferred to a preweighed 2-ml centrifuge tube. Samples were pelleted at maximum speed (16,100 Â g, 2 min) again to remove any residual supernatant. Tubes were incubated on a heat block at 65°C with the lids open until the pellets were completely dry and mass measurements stabilized (;24 h). Values were transformed to milligram per liter (mg/liter) units.
Single particle tracking by TIRF imaging. The D8 endo mreB::mreBmsfGFP sw strain with chromosomally expressed MreB-msfGFP was grown shaking at 37°C in LB medium supplemented with 100 mM IPTG overnight. The saturated cells were diluted (1:100) into fresh LB in two groups (with 100 mM IPTG for ShyA expression or without IPTG for ShyA depletion). After 2 h of shaking (220 rpm) incubation at 37°C, cells were harvested and spotted on a 0.8% agarose pad containing M9 medium. Time-lapse TIRF (total internal reflection fluorescence) imaging was performed on a Zeiss Elyra equipped with an inverted Axio Observer.Z1 microscope and a 100Â oil objective (numerical aperture, 1.46). The objective was heated at 37°C during image acquisition. The exposure time was 100 ms, and interframe intervals were 2 s over a 2-min recording. The measurement of cluster size and number was performed using ImageJ TrackMate (54,57,58). The background of fluorescent images was subtracted using Image Calculator to reduce background noise. A threshold range was set to distinguish the objects of interest from the background and convert the image to binary (via Adjust Threshold). Automatic particle analysis was performed using Analyze Particles. The mean square displacements (MSD) of particle trajectories were calculated using the msdanalyzer package, and the motion types were analyzed through log-log fitting (55). By setting the R 2 coefficient . 0.8, individual MSD curves were fitted, and the values of anomalous diffusion coefficient (a) indicate that MreB particles exhibit a mix of dynamic behaviors (confined diffusion, 0.1 # a , 0.9; simple diffusion, 0.9 # a , 1.1; directed motion, a $ 1.1) (72).
Peptidoglycan analysis. PG samples were analyzed as described previously (73). Briefly, 50-ml cultures of D6 endo strain were grown to early/mid exponential phase with or without IPTG (200 mM) for 2 h, harvested, and boiled in 5% sodium dodecyl sulfate (SDS) for 1 h. Sacculi were repeatedly washed by ultracentrifugation (150,000 Â g, 10 min, 20°C) with MilliQ water until SDS was totally removed. Samples were treated with 20 mg proteinase K (1 h, 37°C) for Braun's lipoprotein removal and finally treated with muramidase (100 mg ml 21 ) for 16 h at 37°C. Muramidase digestion was stopped by boiling, and coagulated proteins were removed by centrifugation (22,000 Â g, 10 min). For sample reduction, the pH of the supernatants was adjusted to pH 8.5 to 9.0 with sodium borate buffer, and sodium borohydride was added to a final concentration of 10 mg ml 21 . After incubating for 30 min at room temperature, the pH of the samples was adjusted to pH 3.5 with orthophosphoric acid.
Ultrahigh-performance liquid chromatography (UPLC) analyses of muropeptides were performed on a Waters UPLC system (Waters Corporation, USA) equipped with an Acquity UPLC ethylene bridged hybrid (BEH) C 18 column (130 Å, 1.7 mm, 2.1 mm Â 150 mm) (Waters, USA) and a dual wavelength absorbance detector. Elution of muropeptides was detected at 204 nm. Muropeptides were separated at 45°C using a linear gradient from buffer A (0.1% formic acid in water) to buffer B (0.1% formic acid in acetonitrile) in an 18-min run, with a 0.25-ml/min flow.
Relative total PG amount was calculated by comparison of the total intensities of the chromatograms (total area) from three biological replicas normalized to the same OD 600 and extracted with the same volumes. Muropeptide identity was confirmed by tandem mass spectrometry (MS/MS) analysis, using a Xevo G2-XS quadrupole time of flight (QTof) system (Waters Corporation, USA). Quantification of muropeptides was based on their relative abundances (relative area of the corresponding peak) normalized to their molar ratio.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. MOVIE S1, AVI file, 0.8 MB.