Coarse-grained simulations of bacterial cell wall growth reveal that local coordination alone can be sufficient to maintain rod shape

Initial Model—Remodeler 1.0.

Sacculus growth requires at least three types of enzymes: transglycosylases to synthesize new glycan strands, transpeptidases to crosslink them together, and endopeptidases to cleave existing peptide crosslinks to open space for new material (9, 42, 43). E. coli has other hydrolases that can modify PG, but none of them have been shown to be essential for rod shape maintenance (2, 44, 45). Thus, in our initial simulations, we simply introduced transglycosylases, transpeptidases, and endopeptidases onto the surface of the sacculus and modeled what happened as they diffused around performing their functions. We assumed that transglycosylase and transpeptidase exist as bifunctional enzymes and therefore, tethered them to each other but not to endopeptidase. Large holes, many more than 10 times the average hole size in the starting sacculus, developed almost immediately (Figs. 2A and 3 A and B). In our simulation conditions, the presence of uncoordinated enzymes alone was, therefore, insufficient to maintain rod shape.

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Fig. 2.

Sacculus growth (new strands are shown in green; A) in the initial model; after adding (B) the multienzyme complex, (C) cleaved crosslink capture, (D) bend-induced termination, (E) fixed transglycosylase orientation, (F) crosslink before terminate, (G) tail hydrolysis, (H) paired transpeptidases, (I) peptide maturation, (J) crosslink–dependent processivity, (K) first crosslink always on the same side, (L) hole-dependent processivity, and (M) strands inserted in pairs hypotheses; and (N) the final model.

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Fig. 3.

Structural characterization of grown sacculi. In the initial model, sacculi had many large holes, but they reduced in (A) size and (B) number as hypotheses were added. (C and D) Bulging was obvious in Remodeler 1.6 but reduced gradually toward the final model.

Multi-enzyme Complex Hypothesis—Remodeler 1.1.

Crosslink cleavage clearly must be coupled to the addition of new material for sacculus integrity to be preserved, as argued previously (10, 46). Indirect evidence for multi-enzyme complexes exists (23, 47⇓⇓⇓⇓–52), so that, in the next round of simulations, we tried tethering the transglycosylases to both the transpeptidases and endopeptidases to form trimeric complexes (details in SI Appendix, Fig. S1C). Large holes still formed quickly, leading to loss of sacculus integrity and rod shape (Figs. 2B and 3 A and B). Simply linking the enzymes into complexes was, therefore, insufficient to maintain rod shape.

Cleaved CrossLink Capture Hypothesis—Remodeler 1.2.

Analyzing the dynamics of hole formation, we found that, after a crosslink was cleaved, the two previously crosslinked strands sometimes moved apart because of turgor pressure before the transpeptidase crosslinked them to the new strand (SI Appendix, Fig. S2). Exploring ways this failure mode might be prevented, we increased the transglycosylation rate, so that peptide donors would appear more quickly. Unfortunately, quick transglycosylation created other problems, such as strand bending (discussed in below). We also tried accelerating the peptide loading rate (by increasing the reaction distance), but the problem remained. Implementing a ‘make-before-break’ strategy (described in detail in Final Modifications—Remodeler 1.13) did not solve the problem either, as this strategy only works if multiple strands are inserted concurrently. Finally, hypothesizing that endopeptidases might bind tightly to cleaved crosslinks until being competed off by transpeptidases, we lowered the probability of peptide release after crosslink cleavage to, on average, once every 107 time steps. Transpeptidation then almost always preceded peptide release, greatly reducing formation of large holes (Fig. 3 A and B). However, long bent strands aggregated in a few areas of the sacculus surface (Fig. 2C and SI Appendix, Fig. S3).

Bend-Induced Termination Hypothesis—Remodeler 1.3.

At this stage, elongation and termination of glycan strands were modeled to be purely stochastic and independent of strand conformation. If a growing strand elongated but the distance between the two ends could not increase (for example, because the enzyme could not move forward and the nongrowing end had already been crosslinked), the strand bent (SI Appendix, Fig. S3). In real cells, however, the strand stiffness would affect the affinity of the enzyme, and strand bending would likely pry the tip out of the transglycosylase active site. Indeed, a flap was discovered in the structure of the Aquifex aeolicus transglycosylase PBP1A folding over the active-site cleft, presumably to prevent dissociation of the growing strand (53). Strand bending could pry the flap open, resulting in a higher termination probability. A bend-induced termination hypothesis was, therefore, tested, in which bending increased the probability of termination (details in SI Appendix). As a result, the formation of long bent strands was prevented. New strands, however, still aggregated in a few areas on the sacculus surface, introducing bumps (Fig. 2D).

Fixed Transglycosylase Orientation Hypothesis—Remodeler 1.4.

Each time a transglycosylase terminated and reinitiated a new strand, Brownian motion changed the complex’s orientation, in many cases leading to reversal of synthesis direction and consequently, local aggregation of new material (SI Appendix, Fig. S4). Decreasing the probability of initiation could decrease aggregation, since diffusion would move non-polymerizing enzyme complexes some distance before re-initiating, but this strategy would not prevent reversal of synthesis direction and, therefore, would still result in insertion of incomplete hoops and likely still cause bumps. Some recent experiments (28, 29) suggest that PBPs move processively around cells. Considering that transglycosylase could form a complex with other rod shape-determining proteins, such as MreB/C/D, RodA/Z, and LpoA/B (SI Appendix, Fig. S5) (54), we implemented a fixed transglycosylase orientation hypothesis that resisted deviation of the growing strands’ orientation from the circumferential direction (details in SI Appendix). As a result, new strands no longer aggregated. The enzyme complexes did not reverse direction, instead moving processively around the sacculus, as seen for MreB (28⇓–30). Sacculus growth was smoother, but large holes still developed gradually, giving rise to small distortions and loss of sacculus integrity (Figs. 2E and 3 A and B).

CrossLink Before Terminate Hypothesis—Remodeler 1.5.

At this point, the main cause of hole development was that strand growth sometimes terminated soon after crosslink cleavage. While one of two released peptides became crosslinked to the terminated strand, the other released peptide became crosslinked to the next strand initiated, producing a small hole (SI Appendix, Fig. S5). If this phenomenon happened repeatedly in the same region, the hole enlarged. We hypothesized that there might, therefore, be a feedback mechanism that reduced the probability of transglycosylase termination while a released peptide was not yet crosslinked to the new strand and remained bound to endopeptidase (details in SI Appendix). After this crosslink before terminate hypothesis was added, the biggest problem was no longer hole formation (Fig. 3 A and B) but instead, the blockage of synthetic enzyme complexes by long, uncrosslinked glycan segments (Fig. 2F and SI Appendix, Fig. S6).

Tail Hydrolysis Hypothesis—Remodeler 1.6.

Because of the stochastic nature of the model, transglycosylases sometimes polymerized long initial tails that were never crosslinked to the network (SI Appendix, Fig. S6). These glycan tails did not contribute to the mechanical strength of the sacculus and caused two problems. First, they sometimes drifted above or below and then across existing strands and blocked movement of enzyme complexes (SI Appendix, Fig. S6) (active complexes cannot cross strands in any plane in our simulations, because the complexes presumably extend all the way from the inner to the outer membrane). Second, crosslinks sometimes formed between misoriented tails and new strands being inserted in the sacculus, creating a multilayered and distorted arrangement.

We reasoned that the cell might solve the tail problem in one of two ways: preventing tail formation or removing tails after they formed. The first strategy would require initiating transglycosylases to pause until the first PG units of the growing strand were crosslinked. This mechanism might be possible if transglycosylases were able to sense crosslink formation either directly or by interaction with transpeptidases. The second strategy would involve glycosidases that target PG tails but not crosslinked PG. E. coli does, in fact, have multiple glycosidases, but how they are regulated is not known (2, 45). If they were free to cleave any glycosidic bond, presumably the cell would lyse. Instead, these glycosidases might be tethered to the membranes and therefore, only able to cleave glycan tails that drift above or below the sacculus surface, leaving crosslinked PG intact. Since the first strategy required an unknown sensing mechanism among the enzymes, we decided to test the second strategy, implementing the tail hydrolysis hypothesis (details in SI Appendix). While adding this hypothesis solved the problem of enzyme blockage, as the sacculus grew longer, new glycan strands still sometimes became disordered, giving rise to bulges and loss of rod shape (Figs. 2G and 3 C and D).

Paired-Transpeptidases Hypothesis—Remodeler 1.7.

We found that, sometimes, instead of alternating between the two sides of a growing strand, successive crosslinks formed on the same side (SI Appendix, Fig. S7). As a result, the lengths of the glycan segments forming the opposing edges of a hole were unequal, causing the sacculus to buckle. Because only new peptides can serve as donors (1), crosslink polarity (donor on the left and acceptor on the right or vice versa) alternates along an inserted strand in a well-ordered network. At this point in our model, there was only one transpeptidase in each enzyme complex. Thus, to form all of the expected crosslinks, the transpeptidase would have to flip back and forth across the strand every other tetrasaccharide. This enzyme flip is unreasonable, since the transpeptidase in bifunctional enzymes is rigidly fused to the transglycosylase, which in turn, is clamped around the stiff growing strand.

Considering that a monofunctional transpeptidase, PBP2, is also required for rod shape maintenance (42), we next modeled the complex to have two transpeptidases, one monofunctional and one bifunctional, each restricted to making crosslinks on just one side (SI Appendix, Fig. S8A). Based on the crystal structure of the bifunctional enzyme PBP1B, this enzyme arrangement seemed reasonable, because it appears that the transpeptidase domain is rigidly fixed on one side of the emerging strand (55). The bifunctional transpeptidase was arbitrarily assigned to catalyze crosslinks on the left (looking down the growing strand from above in the direction of its growth), and the monofunctional transpeptidase was arbitrarily assigned to catalyze crosslinks on the right. Except for the assignment of sides, all other properties of the two transpeptidases in the simulation were equivalent. After implementation of this hypothesis, bulging was reduced (Fig. 3 C and D), and overall rod shape was maintained reasonably well through the first length doubling (Fig. 2H). To find out if rod shape could be maintained through additional generations, length-doubled sacculi were divided into halves, polar caps were added to the daughters, and additional growth was simulated. Rod shape deteriorated in the second generation (SI Appendix, Fig. S8B).

Peptide Maturation Hypothesis—Remodeler 1.8.

At this point, most of the defects were caused by the introduction of short strands with just one or two crosslinks. If a terminated strand was attached to the network by only one crosslink, it did not contribute to the mechanical strength of the sacculus. When this loose strand/tail failed to be removed quickly by glycosidases, it was later crosslinked to another new strand and became misoriented (SI Appendix, Fig. S8C).

One solution could be to have instant removal of uncrosslinked PG by glycosidases, a condition that would require colocalization of glycosidases in the synthesis complex and a regulation mechanism that allows them to remove only strands that have already been released by transglycosylases. Cells could solve this problem by at least two other different mechanisms: termination could be prevented until there were several crosslinks (discussed below), or the formation of crosslinks between loose tails and other strands could be prevented. For the second mechanism, either loose tails could be cleaved more quickly or their crosslinking could be delayed to allow more time for them to be cleaved. Considering the abundance of carboxypeptidases that target pentapeptides (56), we added into the model a peptide maturation hypothesis to prevent the loading of pentapeptides onto transpeptidases as acceptors (details in SI Appendix). Addition of this hypothesis solved the problem of loose tails being crosslinked into defective network patterns (Fig. 2I), hole size was well-controlled (Fig. 3 A and B), and bulging was nearly eliminated (Fig. 3 C and D), but other problems arising from quick termination remained.

CrossLink–Dependent Processivity Hypothesis—Remodeler 1.9.

As mentioned above, quick termination sometimes resulted in new strands being incorporated into the sacculus with only two crosslinks. Because of the effect of turgor pressure, these strands were pulled parallel to the long axis of the sacculus, and they sometimes blocked the movement of enzyme complexes (SI Appendix, Fig. S9).

Transglycosylase is processive in vitro (57), perhaps because of the presence of the flap folding over the glycan strand in the active-site cleft (53), but the factors that promote termination in vivo are unknown. Until now, termination had been modeled as a stochastic process with a constant probability at any time step, but this model would not produce the broad length distribution observed experimentally (58). A newly initiated, still–uncrosslinked glycan strand might easily remain in the transglycosylase active site, even as the enzyme experienced random collisions with other macromolecules, but once the nascent strand became crosslinked to the sacculus, thermal motions of the transglycosylase might disrupt its association with the now-immobilized strand.

Based on these considerations, we hypothesized that the enzyme processivity is high when the new strand is uncrosslinked but decreases with increasing numbers of crosslinks (details in SI Appendix). The average lengths of new strands before and after this change were 22 and 25 disaccharides, respectively, within the range of 21–33 disaccharides measured experimentally (58, 59). After addition of the crosslink–dependent processivity hypothesis, the defects caused by quick termination were reduced (Fig. 2J), but rod shape was still lost in the second generation (SI Appendix, Fig. S10).

First CrossLink Always on the Same Side Hypothesis—Remodeler 1.10.

At this point, since the activities of the two transpeptidases in the complex were independent, whether the first crosslink formed on the left or right was random (SI Appendix, Fig. S10). As a result, sometimes the last crosslink of one strand and the first crosslink on the next along a hoop were on the same side, producing a small pucker in the network that became progressively larger. We considered the enzymes’ crystal structures for a clue to how the first crosslink might be formed on a new strand. While the transpeptidase active site must be positioned farther into the periplasm to interact with the sacculus, the transglycosylase active site, being next to the transmembrane region, must be near the inner membrane (55, 60, 61). Thus, newly added disaccharides must move up from the inner membrane to the sacculus before they can be crosslinked, presumably passing directly from the transglycosylase to the transpeptidase active sites (55). We, therefore, reasoned that the first crosslink on a new strand is likely to be formed by the bifunctional enzyme and always be on the same side (Fig. 4A). This rule was introduced as the first crosslink always on the same side hypothesis, with the first crosslink always on the left after the arbitrary assignment made earlier for the bifunctional enzyme.

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Fig. 4.

Addition of the first crosslink always on the same side hypothesis led to sacculus twisting. (A) Schematic depicting the first crosslink always on the same side hypothesis. A bifunctional transglycosylase (orange) transfers the strand to its associated transpeptidase domain (yellow) for transpeptidation (cyan arrow labeled 1st). Thus, the first crosslink is formed by the bifunctional transpeptidase. Only then does the monofunctional transpeptidase participate in transpeptidation (cyan arrow labeled 2nd). The crystal structures shown are of the E. coli bifunctional PBP1B (3FWM) and the Neisseria gonorrheae monofunctional PBP2 (3EQV). (B) Sacculus twisting caused by bias of start crosslinks on one side. (B1) The start sacculus with two markers (red) on the two polar caps. (B2) Before adding the first crosslink always on the same side hypothesis, no sacculus twisting occurred. (B3) Addition of the first crosslink always on the left caused left-handed twisting. (B4) Switching positions of the monofunctional and bifunctional transpeptidases to make first crosslink always on the right caused the sacculus to twist in a right-handed fashion.

The resulting sacculus surface was much smoother (Fig. 2K), but for the first time, the sacculus started twisting during growth (Fig. 4B and Movie S5). The left part of the sacculus always shifted backward and the right always shifted forward with respect to the growth direction of the new strand, resulting in obvious incremental rotation. To confirm that the new rotation was a result of the rule, we switched the positions and functions of the bifunctional and monofunctional transpeptidases, so that the first crosslink was always formed on the right side of the new strand. As expected, the sacculus now twisted in a right-handed fashion during growth (Fig. 4B and Movie S5). Sacculus twisting was also observed experimentally and in simulations where Wang et al. (31) speculated it was caused by the left-handed nature of MreB filaments guiding insertion sites. We speculate that it might also have been caused by biased first crosslinks in their simulations, a point that could be tested by counting the number of first crosslinks formed on the left and the right (Discussion). After introduction of this rule, sacculi grew more smoothly, but some holes still gradually expanded longitudinally (SI Appendix, Fig. S11).

Hole-Dependent Processivity Hypothesis—Remodeler 1.11.

Hole expansion arose from repeated termination in the same location. When an enzyme complex encountered a hole, sometimes it divided the hole by laying a strand across. If, however, the transglycosylase terminated before it crossed the hole (a rare but significant event), the loose tail was subsequently cleaved by glycosidases, and the hole expanded longitudinally (SI Appendix, Fig. S11). To reduce the risk of hole enlargement when a transglycosylase is in a large hole, cells might have a mechanism to increase processivity of the enzyme, reducing termination probability. This mechanism is biophysically plausible, since lipoproteins LpoA and LpoB must protrude through holes in the PG to activate PBP1A and PBP1B (SI Appendix, Fig. S12) (13, 14). Larger holes might allow stronger activation, perhaps by altering a bifunctional enzyme’s orientation or conformation in a way that increases precursor loading rates or reduces termination (3, 14, 54). We, therefore, hypothesized that the probability of transglycosylase loading PG precursors was increased and termination probability decreased, both by a factor of (p/p0)2, when the hole perimeter p was larger than p0=20 nm. Addition of this hypothesis ameliorated the problem of large holes, although rod shape was still lost after a few generations (SI Appendix, Fig. S12).

Strands Inserted in Pairs Hypothesis—Remodeler 1.12.

When a ladder of crosslinks between two strands was cleaved, the opposing acceptor (old) peptide stems were in register, but the donor (new) peptide stems of the new inserting strand alternated sides. The process of crosslinking the new strand into the network, therefore, required that the two sides of the sacculus rotate one disaccharide forward or back with respect to each other to bring the donors and acceptors into register on both sides (SI Appendix, Fig. S13A). As a result, the sacculus twisted gradually as it grew (Fig. 4B and Movie S5). Wherever new strands terminated, small stresses and defects were produced, because the twist could not propagate all of the way around the hoop. While some of these defects were relaxed by later events, others grew, leading to shape loss in subsequent generations.

Based on studies of peptide acceptor:donor ratios, Burman and Park proposed decades ago that new glycan strands are inserted in pairs (10). Two later studies contradicted these results, suggesting instead that new strands are inserted one at a time (62, 63). Later, PBP1A and PBP1B were found to form homodimers in vivo (64, 65), and PBP1B can dimerize at high concentrations and produce pairs of crosslinked strands in vitro (66), again suggesting that new strands might be synthesized in pairs. If new strands were inserted in pairs, all of the new donor peptide stems on both sides would be in register with the old acceptor stems without strand shifting or sacculus twisting (SI Appendix, Fig. S13A).

We, therefore, implemented a strands inserted in pairs mode. A second bifunctional enzyme was added to each complex, so that two new strands could be added simultaneously. The rules governing the processivity of each transglycosylase remained the same (still dependent on crosslink numbers and hole size), and initiation and termination of the two transglycosylases remained stochastic and independent. Insertion of a pair of strands requires crosslinking both between them and on either side. Because our model assumed that the transglycosylases were bifunctional enzymes, as, for example, either PBP1A or PBP1B of E. coli, the positions and orientations of the two associated transpeptidases were assumed to be fixed within the complex (Fixed Transglycosylase Orientation Hypothesis—Remodeler 1.4) and they were expected to form crosslinks of a single polarity. If the two bifunctional enzymes formed their crosslinks on the left, for instance, a third transpeptidase was required to form the remaining row of crosslinks of opposite polarity on the right, forming a triple-transpeptidase complex. Because only the transpeptidase function was needed, we modeled it as a monofunctional transpeptidase, such as PBP2 of E. coli. Because the first crosslink always on the same side hypothesis was now irrelevant, it was removed from the model, allowing all three transpeptidases to act independently (Fig. 5A).

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Fig. 5.

Remodeler 1.13. (A, Upper) Schematic of a synthetic complex in the final model: two bifunctional transglycosylase/transpeptidase enzymes (bi-Gtase/bi-Tpase), a monofunctional transpeptipase (mono-Tpase), and an endopeptidase (Edase). (A, Lower) The projection on the sacculus surface shows how three transpeptidases (visualized as ellipses with donor domains in green and acceptor domains in blue) could be oriented to create all needed crosslinks tethering two new strands (green) to old strands (blue). (B) Plots of (Upper) sacculus straightness and (Lower) surface roughness show that, in the final model, rod shape was maintained well (Left) through multiple growth generations and (Right) with varying sacculus diameter.

Rod shape was now maintained through several rounds of growth and division (SI Appendix, Fig. S13B). To quantitatively characterize rod shape, we calculated sacculus straightness and surface roughness for both the single- and paired-strand insertion modes (SI Appendix, Fig. S13C). While sacculus straightness declined over successive generations in the single-strand insertion mode, it remained constant in the paired-strand insertion mode. Also, the sacculus surface became rougher more quickly in the single-strand insertion mode.

Final Modifications—Remodeler 1.13.

Having implemented many hypotheses in the model that finally resulted in maintenance of rod shape over multiple generations, we wondered whether any of the hypotheses were redundant. As previously noted, the start on left hypothesis was removed in the paired-strand insertion mode. To find out if other hypotheses were required, we removed each from the model and checked if rod shape was still maintained. Repeating this process for all hypotheses, we found two more that were no longer needed. First, introduction of the crosslink–dependent processivity hypothesis prevented defects caused by quick termination, rendering the peptide maturation hypothesis redundant. Second, the crosslink before terminate hypothesis could be removed in the paired-strand insertion mode, because the paired transglycosylases almost never terminated simultaneously. As a result, even when one transglycosylase terminated right after crosslink cleavage, the other continued, crosslinking one of the newly freed peptide stems onto its growing strand and holding the terminated transglycosylase nearby, so that when the terminated transglycosylase reinitiated (a stochastic but usually quick event), it was well positioned to capture the second newly-freed peptide stem before it moved away.

Observing the paired-strand insertion mode, we realized that two other hypotheses, cleaved crosslink capture and bend-induced termination, could be re-implemented with more likely molecular mechanisms. Decades ago, it was shown that, in addition to dimeric crosslinks, trimeric crosslinks can also form (59). A typical dimeric crosslink is formed at the fourth d-Ala residue of the donor and the third A₂pm residue of the acceptor peptide, but the A₂pm residue on the donor peptide can simultaneously serve as an acceptor for another crosslink, resulting in a trimeric crosslink. Based on this possibility, the ‘make-before-break’ strategy (11) was elaborated into a ‘three-for-one’ model, in which formation of a trimeric crosslink hooking three pre-fabricated strands into the sacculus preceded cleavage of the original dimeric crosslink (12). Building on these ideas, we re-implemented the cleaved crosslink capture hypothesis as follows. If endopeptidase bound a peptide crosslink, the exposed A₂pm group on the donor peptide became accessible as an acceptor for transpeptidation. Subsequent formation of a trimeric crosslink then immediately triggered cleavage of the original crosslink, releasing the acceptor peptide. We called this mechanism trimeric crosslink–triggered endopeptidation (SI Appendix, Fig. S14A).

Since the transglycosylase domains of the bifunctional enzymes are located immediately adjacent to the membrane, approximately eight disaccharides are likely added to the strand before it reaches the transpeptidation site (movie 3 in ref. 55). We, therefore, reasoned that, during elongation, when Brownian motion elevated the growing strand, transpeptidase might capture it, clearing the transglycosylase active site for another lipid II precursor and thereby, facilitating translocation in a ratchet-like fashion (SI Appendix, Fig. S13B). We, therefore, replaced the bend-induced termination hypothesis with a transpeptidation-facilitated translocation hypothesis specifying that the probability of translocation was low, once every 2⋅106 steps, if the last-formed peptide stem was not crosslinked, but increased to once every 3⋅104 steps after the stem was crosslinked. The effect, however, might be different on uncrosslinked strands, for example, when transglycosylases synthesize glycan strands in vitro (67) or β-lactam treatment blocks transpeptidation (68). Because uncrosslinked strands can move freely, transpeptidation might not be needed to facilitate translocation. Also note that the effect of crosslinking at the strand tip, which likely draws the strand forward in the active site, is different from the effect of having many crosslinks along the strand, which we propose may reduce transglycosylase processivity (Remodeler 1.9).

With these final changes, rod shape was still maintained well through multiple generations (Fig. 5B and SI Appendix, Fig. S14C). To test the effects of size, we ran simulations on sacculi two and four times larger in diameter (Movie S4). Similar to our previous results, an early model (Remodeler 1.1) quickly lost integrity and rod shape, but the final model (Remodeler 1.13) maintained rod shape well (Fig. 5B and Movie S4), suggesting that size does not fundamentally alter the basic challenges of maintaining integrity and rod shape during growth.

Reviewing the hypotheses in the final model underscores the importance of local spatial and temporal coordination among the enzymes as well as a house-keeping activity. Based on our exploration of rules and parameters, we suggest that the enzymes likely exist in a complex (multi-enzyme complex), in which two bifunctional transglycosylases with fixed orientation (fixed transglycosylase orientation) synthesize two new strands concomitantly (paired-strand insertion), two bifunctional transpeptidases form crosslinks on one side, and one monofunctional transpeptidase forms crosslinks on the other (triple transpeptidase). Next, the enzymes act in a temporally coordinated fashion, as, for instance, if endopeptidation is regulated by formation of trimeric crosslinks (trimeric crosslink–triggered endopeptidation) and transglycosylation is regulated by transpeptidation (transpeptidation-facilitated translocation). Termination of transglycosylase is likely not purely stochastic but rather, regulated, for instance, by crosslinkage of the growing strand (crosslink–dependent processivity) and hole size (hole-dependent processivity). Finally, glycosidases that target uncrosslinked glycan tails may be needed to clean up long tails (tail hydrolysis).