Class A PBPs: It is time to rethink traditional paradigms

Until recently, class A penicillin‐binding proteins (aPBPs) were the only enzymes known to catalyze glycan chain polymerization from lipid II in bacteria. Hence, the discovery of two novel lipid II polymerases, FtsW and RodA, raises new questions and has consequently received a lot of attention from the research community. FtsW and RodA are essential and highly conserved members of the divisome and elongasome, respectively, and work in conjunction with their cognate class B PBPs (bPBPs) to synthesize the division septum and insert new peptidoglycan into the lateral cell wall. The identification of FtsW and RodA as peptidoglycan glycosyltransferases has raised questions regarding the role of aPBPs in peptidoglycan synthesis and fundamentally changed our understanding of the process. Despite their dethronement, aPBPs are essential in most bacteria. So, what is their function? In this review, we discuss recent progress in answering this question and present our own views on the topic.


TA B L E 1 (Continued)
In most bacterial species, pentapeptides from different glycan strands are cross-linked by transpeptidation between the fourth D-alanine residue of the donor chain and the third residue (e.g., Lys or meso-DAP) of the acceptor chain. Alternatively, in some species the stem peptides can be linked together by means of interpeptide bridges, which in the case of S. pneumoniae consists of L-Ser-L-Ala or L-Ala-L-Ala   (Emami et al., 2017;Meeske et al., 2016;Taguchi et al., 2019). These proteins were originally reported to be lipid II flippases (Mohammadi et al., 2011), a function later assigned to MurJ (Sham et al., 2014). However, it remains a possibility that FtsW and RodA have a double role, that is, flippase as well as glycosyltransferase activity (Egan et al., 2020). To synthesize peptidoglycan, FtsW and RodA work in conjunction with monofunctional bPBPs (Emami et al., 2017;Meeske et al., 2016;Sjodt et al., 2020;Taguchi et al., 2019). It is now firmly established that

| THE VARIAB LE E SS ENTIALIT Y OF aPB Ps
aPBPs seem to be present in most peptidoglycan-producing bacteria. Hence, they must perform an important function. However, their numbers vary between bacterial species (Table 1). B. subtilis produces four different aPBPs, S. pneumoniae and E. coli have three, while S. aureus survives and thrives with only one (Sauvage et al., 2008). Interestingly, bacteria producing several different aPBPs, often need only one of them for survival. In S. pneumoniae, single knockouts of pbp1a, pbp2a, and pbp1b can be obtained, but it is not possible to construct a pbp1a/pbp2a double-knockout strain (Paik et al., 1999). The pneumococcus must, therefore, produce
Presumably, PBP3 regulates the extent of cross-linking between glycan strands by limiting the amount of D-alanyl-D-alanine donor groups required for transpeptidation Morlot et al., 2004). The enzyme is distributed across the entire cell surface but is absent from the future division site (Morlot et al., 2004).
Deletion of PBP3 gives rise to severe morphological defects including misplaced division septa Schuster et al., 1990).

| PROPERTIE S OF E . COLI aPB Ps
The peptidoglycan wall of a Gram-negative bacterium, such as E. coli, differs fundamentally from the peptidoglycan wall of Gram-positive bacteria (Egan et al., 2020). It is only one or a couple of layers thick and is surrounded by an outer membrane composed of phospholipids and lipopolysaccharide (Sperandeo et al., 2017;Turner et al., 2013). D,D-endopeptidases cleave the bond between D-Ala and meso-DAP in cross-linked stem peptides, whereas D,D-carboxypetidases remove the terminal residues from stem peptides. In E. coli, none of the class C PBPs are essential for viability. Bacteria lacking all seven of them grow nearly as well as the parental strain and display only modest morphological defects (Denome et al., 1999).
Among the three different aPBPs produced by E. coli, the beststudied and most important are PBP1a and PBP1b. Despite having the same name as two of the pneumococcal class A PBPs, they belong to different subclasses and are not functionally equivalent or closely related to their pneumococcal counterparts. E. coli cells must produce either PBP1a or PBP1b to be viable (Yousif et al., 1985).  Vigouroux and colleagues (2020). In a recent article, they reported that aPBPs have no role in maintaining the cell shape but were crucial for mechanical cell wall integrity (Vigouroux et al., 2020). Hence, aPBPs evidently function to strengthen the cell wall of E. coli. The authors found evidence that this strengthening is due to an adaptive class A-mediated repair mechanism that senses and repairs cell wall defects. In support of this, Lai et al. (2017) found that PBP1bmediated peptidoglycan synthesis increases following overexpression of the space-maker endopeptidase MepS (Spr), an enzyme that makes room for localized insertion of new material during peptidoglycan matrix expansion (Singh et al., 2012). Their findings strongly indicate that aPBPs and their auxiliary proteins detect and fill gaps in the peptidoglycan network. Taken together, available data suggest that aPBPs serve dual roles. They operate in conjunction with the divisome and elongasome during synthesis of the primary cell wall, but in addition function as autonomous entities that maintain and repair the peptidoglycan sacculus.

| THE SYNTHE TIC LE THALIT Y PAR ADOX
As discussed above, aPBPs often form synthetic lethal pairs such as PBP1a/PBP2a in S. pneumoniae and PBP1a/PBP1b in E. coli. Each member of these pairs can be individually deleted, but the bacterium must produce one of them to be viable. Moreover, members of each pair have overlapping as well as nonoverlapping functions, demonstrating that they are semi-redundant. It follows from this that it must be their overlapping function that is essential for cell survival. What could this essential function be? The many studies reporting close interactions between aPBPs and proteins associated with the divisome or elongasome, suggest that aPBPs are intrinsic components of these multiprotein complexes (Bertsche et al., 2006;Boes et al., 2019;Claessen et al., 2008;Fenton et al., 2016;Leclercq et al., 2017;Scheffers & Errington, 2004;Steele et al., 2011). On the contrary, strong recent experimental evidence shows that aPBPs are able to function autonomously (Lai et al., 2017;Straume et al., 2020;Vigouroux et al., 2020). How can this apparent paradox However, it is conceivable that they can perform overlapping repair functions when operating as autonomous entities. We therefore propose that the essential function of class A PBPs is to detect and repair gaps and imperfections in the cell wall peptidoglycan localized outside areas of active peptidoglycan synthesis.

| A T WO -L AYERED CELL WALL IN G R AM -P OS ITIVE BAC TERIA?
In a newly published study by Pasquina-Lemonche and coworkers (2020), atomic force microscopy was used to study the cell wall architecture of the Gram-positives B. subtilis and S. aureus.
Interestingly, the paper reports evidence that nascent septa in both species consist of two different peptidoglycan layers with distinct architectures that indicate two synthesis regimes (Figure 1). The inner layer, in the following referred to as the core or primary cell wall, has a highly ordered structure composed of concentric rings.
The core is sandwiched between the outer layers, which constitute the bulk of the septum thickness. In contrast to the core, the outer layers (the secondary cell walls) are made up of randomly oriented material ( Figure 1). As the core peptidoglycan in nascent septa acts as a scaffold for the outer layers, the core material must be synthesized first. Hence, the authors suggest that the core peptidoglycan is formed at the leading edge of the cross-wall, while the outer layers are subsequently added on top of the core. During cell wall growth and maturation, the internal cytoplasm-facing surface of S. aureus cells remains a disordered mesh with relatively small pores. In contrast, the post-divisional outer surface, which originally had an ordered ring-like architecture, gradually matures into a porous mesh due to the action of various peptidoglycan hydrolases. Similarly, the outer surface of B. subtilis cells reorganizes into a randomly oriented porous network, while the poles retain some of the ring structure originating from the inner core of the split septum. The inner surface of the B. subtilis cell wall cylinder, on the contrary, consists of material that has been deposited in a circumferential orientation ( Figure 1). This is due to the circumferential movement of MreB filaments which guide the movement of the elongasome (Dion et al., 2019;Domínguez-Escobar et al., 2011;Garner et al., 2011;Pasquina-Lemonche et al., 2020). Very recently, a similarly structured two-layered cell wall has also been reported for Staphylococcus warneri (Su et al., 2020).
How are the two different layers of nascent septa synthesized?
A likely solution to this question involves the function of aPBPs.
In the paper by Straume et al. (2020) (Lund et al., 2018), the latter mechanism seems to be the less likely of the two alternatives.
Presumably, the question could be settled by examining the B. subtilis class A-less mutant by atomic force microscopy along the lines described by Pasquina-Lemonche and coworkers (2020).
The fact that B. subtilis is able to grow and multiply without aPBPs shows that the FtsW/PBP2b and RodA/PBP2a/PBPH machineries (Table 1)

F I G U R E 2
Model that depicts possible roles of aPBPs in the construction and maintenance of a two-layered cell wall in S. pneumoniae. SEDS proteins (FtsW and RodA) in conjunction with bPBPs (PBP2x and PBP2b) represent the primary peptidoglycan-synthesizing machineries that build the septum (divisome) and insert new material into the lateral cell wall during cell elongation (elongasome). It has recently been reported that the septal cross-wall of Gram-positive bacteria appears to consist of two peptidoglycan layers with different architectures (Pasquina-Lemonche et al., 2020). Based on this and our own data , we postulate that the FtsW/Pbp2x machinery of the divisome synthesize the highly ordered core layer of the cross-wall (brown) which is matured (light blue) by the action of peptidoglycan (PG) hydrolases, while aPBPs working in conjunction with the divisome synthesize the disordered peptidoglycan layer facing the cytoplasm (green). Furthermore, as illustrated by the aPBP operating outside the midcell region, new evidence suggests that aPBPs can function as autonomous entities that are involved in repair and maintenance of the peptidoglycan sacculus. Illustration by Wendy Chadbourne (inkymousestudios.com) namely to build a thicker cell wall by synthesizing a secondary peptidoglycan layer on top of the primary cell wall made by the FtsW glycosyltransferase and its cognate bPBPs. to synchronize the elongation process between the two layers.
Bacteria produce a large number of peptidoglycan hydrolases which are essential for maintaining the architecture and function of the bacterial cell envelope. They are involved in processes such as cell separation, cell enlargement, recycling of peptidoglycan, and assembly of trans-envelope structures too large to pass through the natural pores of the peptidoglycan sacculus (Scheurwater & Burrows, 2011;Typas et al., 2012;Vollmer, Bernard, et al., 2008). It is not clear how bacteria control the potential suicidal activity of these enzymes, but several different mechanisms are undoubtedly applied. One advantage of evolving a two-layered architecture may be that it enables the cell to better control the activity of peptidoglycan-degrading enzymes in order to avoid autolysis and cell death. Pasquina-Lemonche et al. (2020) reports that in S. aureus, the external side of the wall has significantly larger pores than the internal side, and that the large external pores taper off as they traverse the wall (Figure 1). Perhaps the activity of the enzymes creating these pores is controlled, at least in part, by the architecture of the double-layered cell wall. The same may be the case for potential suicide enzymes involved in cell separation. PcsB, for example, is the peptidoglycan hydrolase that splits the septal cross-wall during pneumococcal daughter cell separation (Bartual et al. 2014). Although PcsB is strictly regulated by the membrane associated FtsEX complex (Alcorlo et al., 2020;Sham et al., 2011;Sham et al., 2013, Rued et al., 2019, its activity may also be controlled by a structurally heterogeneous cell wall. Hence, it is conceivable that PcsB specifically cleaves peptide bridges connecting glycan strands in the circumferentially oriented core layer, while being unable to attack the disordered flanking layers (Figure 2).

| CON CLUS ION
The traditional paradigm of peptidoglycan biogenesis states that aPBPs are intrinsic key components of the divisome and elongasome. However, after it was discovered that the SEDS proteins FtsW and RodA have glycosyltransferase activity and work in conjunction with bPBPs, aPBPs were in principle no longer critical components of these multiprotein complexes. Moreover, aPBPs are not essential for survival in some Gram-positive bacteria, strongly indicating that at least in monoderms, bifunctional PBPs are not directly involved in synthesizing the primary cell wall. It is therefore time to rethink and revise the role of aPBPs. Several recent studies in E. coli present evidence that aPBPs are important for repair and maintenance of the peptidoglycan matrix. Perhaps E. coli produce three different bifunctional PBPs in order to detect and repair different types of damages to the peptidoglycan meshwork. It is likely that aPBPs play a similar repair and maintenance role in Gram-positive bacteria (Figure 2). In addition, we postulate that aPBPs are responsible for synthesizing the cytoplasm-facing section of the two-layered cell wall described for B. subtilis, S. aureus, and S. warneri (see Figure 2) (Pasquina-Lemonche et al., 2020;Su et al., 2020). So far, a two-layered cell wall has only been demonstrated for these species, but there is no reason why it should not be widespread among Gram-positive bacteria.