Identification of the potential active site of the septal peptidoglycan polymerase FtsW.

SEDS (Shape, Elongation, Division and Sporulation) proteins are widely conserved peptidoglycan (PG) glycosyltransferases that form complexes with class B penicillin-binding proteins (bPBPs, with transpeptidase activity) to synthesize PG during bacterial cell growth and division. Because of their crucial roles in bacterial morphogenesis, SEDS proteins are one of the most promising targets for the development of new antibiotics. However, how SEDS proteins recognize their substrate lipid II, the building block of the PG layer, and polymerize it into glycan strands is still not clear. In this study, we isolated and characterized dominant-negative alleles of FtsW, a SEDS protein critical for septal PG synthesis during bacterial cytokinesis. Interestingly, most of the dominant-negative FtsW mutations reside in extracellular loops that are highly conserved in the SEDS family. Moreover, these mutations are scattered around a central cavity in a modeled FtsW structure, which has been proposed to be the active site of SEDS proteins. Consistent with this, we found that these mutations blocked septal PG synthesis but did not affect FtsW localization to the division site, interaction with its partners nor its substrate lipid II. Taken together, these results suggest that the residues corresponding to the dominant-negative mutations likely constitute the active site of FtsW, which may aid in the design of FtsW inhibitors.

The Dual Mode of Antibacterial Action of the Synthetic Small Molecule DCAP Involves Lipid II Binding.

The synthetic small molecule DCAP is a chemically well-characterized compound with antibiotic activity against Gram-positive and Gram-negative bacteria, including drug-resistant pathogens. Until now, its mechanism of action was proposed to rely exclusively on targeting the bacterial membrane, thereby causing membrane depolarization, and increasing membrane permeability (Eun et al. 2012, J. Am. Chem. Soc. 134 (28), 11322-11325; Hurley et al. 2015, ACS Med. Chem. Lett. 6, 466-471). Here, we show that the antibiotic activity of DCAP results from a dual mode of action that is more targeted and multifaceted than previously anticipated. Using microbiological and biochemical assays in combination with fluorescence microscopy, we provide evidence that DCAP interacts with undecaprenyl pyrophosphate-coupled cell envelope precursors, thereby blocking peptidoglycan biosynthesis and impairing cell division site organization. Our work discloses a concise model for the mode of action of DCAP which involves the binding to a specific target molecule to exert pleiotropic effects on cell wall biosynthetic and divisome machineries.

Simulation-Guided Molecular Modeling of Nisin and Lipid II Assembly and Membrane Pore Formation.

The lantibiotic pore-forming peptide nisin is a promising candidate in the fight against multidrug-resistant bacteria due to its unique structure, which allows it to disrupt bacteria in two distinct ways─Lipid II trafficking and transmembrane pore formation. However, exactly how nisin and Lipid II assemble into oligomeric pore structures in the bacterial membrane is not known. Spontaneous peptide assembly into pores is difficult to observe in even the very long-time scale molecular dynamics (MD) simulations. In this study, we adopted an MD-guided modeling approach to investigate the nisin-nisin and nisin-Lipid II associations in the membrane environment. Through extensive microsecond-time scale all-atom MD simulations, we established that nisin monomers dimerize by forming ß-sheets in a POPE:POPG lipid bilayer and oligomerize further to form stable transmembrane channels. We determined that these nisin dimers use Lipid II as a dimer interface to incur enhanced stability. Our results provide a clearer understanding of the self-assembly of nisin monomers within the membrane and insights into the role of Lipid II in the structural integrity of oligomeric structures.

Peptidoglycan synthesis drives an FtsZ-treadmilling-independent step of cytokinesis.

Peptidoglycan is the main component of the bacterial wall and protects cells from the mechanical stress that results from high intracellular turgor. Peptidoglycan biosynthesis is very similar in all bacteria; bacterial shapes are therefore mainly determined by the spatial and temporal regulation of peptidoglycan synthesis rather than by the chemical composition of peptidoglycan. The form of rod-shaped bacteria, such as Bacillus subtilis or Escherichia coli, is generated by the action of two peptidoglycan synthesis machineries that act at the septum and at the lateral wall in processes coordinated by the cytoskeletal proteins FtsZ and MreB, respectively. The tubulin homologue FtsZ is the first protein recruited to the division site, where it assembles in filaments-forming the Z ring-that undergo treadmilling and recruit later divisome proteins. The rate of treadmilling in B. subtilis controls the rates of both peptidoglycan synthesis and cell division. The actin homologue MreB forms discrete patches that move circumferentially around the cell in tracks perpendicular to the long axis of the cell, and organize the insertion of new cell wall during elongation. Cocci such as Staphylococcus aureus possess only one type of peptidoglycan synthesis machinery, which is diverted from the cell periphery to the septum in preparation for division. The molecular cue that coordinates this transition has remained elusive. Here we investigate the localization of S. aureus peptidoglycan biosynthesis proteins and show that the recruitment of the putative lipid II flippase MurJ to the septum, by the DivIB-DivIC-FtsL complex, drives peptidoglycan incorporation to the midcell. MurJ recruitment corresponds to a turning point in cytokinesis, which is slow and dependent on FtsZ treadmilling before MurJ arrival but becomes faster and independent of FtsZ treadmilling after peptidoglycan synthesis activity is directed to the septum, where it provides additional force for cell envelope constriction.

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall.

In oval-shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan syntheses are performed by independent functional complexes: the divisome and the elongasome. Penicillin-binding proteins (PBPs) were long considered the key peptidoglycan-synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with transmembrane glycosyltransferases (FtsW and RodA) from the shape, elongation, division, and sporulation (SEDS) family to make up the core peptidoglycan-synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD that targets the septum of S. pneumoniae cells to show that class A PBPs have an autonomous role during pneumococcal cell wall synthesis. Using assays to specifically inhibit the function of PBP2x and FtsW, we demonstrate that CbpD attacks nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP-mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes recently formed peptidoglycan synthesized by FtsW/PBP2×. Our results support a model in which mature pneumococcal peptidoglycan is synthesized by three functional entities, the divisome, the elongasome, and bifunctional PBPs. The latter modify existing peptidoglycan but are probably not involved in primary peptidoglycan synthesis.

Specific Binding of the α-Component of the Lantibiotic Lichenicidin to the Peptidoglycan Precursor Lipid II Predetermines Its Antimicrobial Activity.

To date, a number of lantibiotics have been shown to use lipid II-a highly conserved peptidoglycan precursor in the cytoplasmic membrane of bacteria-as their molecular target. The α-component (Lchα) of the two-component lantibiotic lichenicidin, previously isolated from the Bacillus licheniformis VK21 strain, seems to contain two putative lipid II binding sites in its N-terminal and C-terminal domains. Using NMR spectroscopy in DPC micelles, we obtained convincing evidence that the C-terminal mersacidin-like site is involved in the interaction with lipid II. These data were confirmed by the MD simulations. The contact area of lipid II includes pyrophosphate and disaccharide residues along with the first isoprene units of bactoprenol. MD also showed the potential for the formation of a stable N-terminal nisin-like complex; however, the conditions necessary for its implementation in vitro remain unknown. Overall, our results clarify the picture of two component lantibiotics mechanism of antimicrobial action.

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.

Structural insights into inhibition of lipid I production in bacterial cell wall synthesis.

Antibiotic-resistant bacterial infection is a serious threat to public health. Peptidoglycan biosynthesis is a well-established target for antibiotic development. MraY (phospho-MurNAc-pentapeptide translocase) catalyses the first and an essential membrane step of peptidoglycan biosynthesis. It is considered a very promising target for the development of new antibiotics, as many naturally occurring nucleoside inhibitors with antibacterial activity target this enzyme. However, antibiotics targeting MraY have not been developed for clinical use, mainly owing to a lack of structural insight into inhibition of this enzyme. Here we present the crystal structure of MraY from Aquifex aeolicus (MraYAA) in complex with its naturally occurring inhibitor, muraymycin D2 (MD2). We show that after binding MD2, MraYAA undergoes remarkably large conformational rearrangements near the active site, which lead to the formation of a nucleoside-binding pocket and a peptide-binding site. MD2 binds the nucleoside-binding pocket like a two-pronged plug inserting into a socket. Further interactions it makes in the adjacent peptide-binding site anchor MD2 to and enhance its affinity for MraYAA. Surprisingly, MD2 does not interact with three acidic residues or the Mg(2+) cofactor required for catalysis, suggesting that MD2 binds to MraYAA in a manner that overlaps with, but is distinct from, its natural substrate, UDP-MurNAc-pentapeptide. We have determined the principles of MD2 binding to MraYAA, including how it avoids the need for pyrophosphate and sugar moieties, which are essential features for substrate binding. The conformational plasticity of MraY could be the reason that it is the target of many structurally distinct inhibitors. These findings can inform the design of new inhibitors targeting MraY as well as its paralogues, WecA and TarO.

Genes encoding lipid II flippase MurJ and peptidoglycan hydrolases are required for chloroplast division in the moss Physcomitrella patens.

KEY MESSAGE: Homologous genes for the peptidoglycan precursor flippase MurJ, and peptidoglycan hydrolases: lytic transglycosylase MltB, and DD-carboxypeptidase VanY are required for chloroplast division in the moss Physcomitrella patens. The moss Physcomitrella patens is used as a model plant to study plastid peptidoglycan biosynthesis. In bacteria, MurJ flippase transports peptidoglycan precursors from the cytoplasm to the periplasm. In this study, we identified a MurJ homolog (PpMurJ) in the P. patens genome. Bacteria employ peptidoglycan degradation and recycling pathways for cell division. We also searched the P. patens genome for genes homologous to bacterial peptidoglycan hydrolases and identified genes homologous for the lytic transglycosylase mltB, N-acetylglucosaminidase nagZ, and LD-carboxypeptidase ldcA in addition to a putative DD-carboxypeptidase vanY reported previously. Moreover, we found a ß-lactamase-like gene (Pplactamase). GFP fusion proteins with either PpMltB or PpVanY were detected in the chloroplasts, whereas fusion proteins with PpNagZ, PpLdcA, or Pplactamase localized in the cytoplasm. Experiments seeking PpMurJ-GFP fusion proteins failed. PpMurJ gene disruption in P. patens resulted in the appearance of macrochloroplasts in protonemal cells. Compared with the numbers of chloroplasts in wild-type plants (38.9 ± 4.9), PpMltB knockout and PpVanY knockout had lower numbers of chloroplasts (14.3 ± 6.7 and 28.1 ± 5.9, respectively). No differences in chloroplast numbers were observed after PpNagZ, PpLdcA, or Pplactamase single-knockout. Chloroplast numbers in PpMltB/PpVanY double-knockout cells were similar to those in PpMltB single-knockout cells. Zymogram analysis of the recombinant PpMltB protein revealed its peptidoglycan hydrolase activity. Our results imply that PpMurJ, PpMltB and PpVanY play a critical role in chloroplast division in the moss P. patens.

CbrA Mediates Colicin M Resistance in Escherichia coli through Modification of Undecaprenyl-Phosphate-Linked Peptidoglycan Precursors.

Colicin M is an enzymatic bacteriocin produced by some Escherichia coli strains which provokes cell lysis of competitor strains by hydrolysis of the cell wall peptidoglycan undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) precursor. The overexpression of a gene, cbrA (formerly yidS), was shown to protect E. coli cells from the deleterious effects of this colicin, but the underlying resistance mechanism was not established. We report here that a major structural modification of the undecaprenyl-phosphate carrier lipid and of its derivatives occurred in membranes of CbrA-overexpressing cells, which explains the acquisition of resistance toward this bacteriocin. Indeed, a main fraction of these lipids, including the lipid II peptidoglycan precursor, now displayed a saturated isoprene unit at the α-position, i.e., the unit closest to the colicin M cleavage site. Only unsaturated forms of these lipids were normally detectable in wild-type cells. In vitro and in vivo assays showed that colicin M did not hydrolyze α-saturated lipid II, clearly identifying this substrate modification as the resistance mechanism. These saturated forms of undecaprenyl-phosphate and lipid II remained substrates of the different enzymes participating in peptidoglycan biosynthesis and carrier lipid recycling, allowing this colicin M-resistance mechanism to occur without affecting this essential pathway.IMPORTANCE Overexpression of the chromosomal cbrA gene allows E. coli to resist colicin M (ColM), a bacteriocin specifically hydrolyzing the undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) peptidoglycan precursor of targeted cells. This resistance results from a CbrA-dependent modification of the precursor structure, i.e., reduction of the α-isoprenyl bond of C55-carrier lipid moiety that is proximal to ColM cleavage site. This modification, observed here for the first time in eubacteria, annihilates the ColM activity without affecting peptidoglycan biogenesis. These data, which further increase our knowledge of the substrate specificity of this colicin, highlight the capability of E. coli to generate reduced forms of C55-carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned.

A Biochemical Lanthanide-Encoding Approach Enables Quantitative Monitoring of the Bacterial Response to Vancomycin Treatment.

A pathogenic bacterium has its own mechanisms for not only pathogenic attack but also exogenous invasion defense, in which the bacterial cell wall is the front line of attack and defense. We developed a biochemical lanthanide-encoding approach to quantify the uncanonical d-amino acid (d-X) that was edited in a small proportion into the terminal acyl-d-Ala-d-X of nascent peptidoglycan UDP-MurNAc-pentapeptides in the bacterial cell wall. This approach overcomes the difficulties regarding quantification and accuracy issues encountered by the popular optical imaging and traditional high-performance liquid chromatography-based methods. Newly synthesized azide-d-Leu and ketone-d-Met were used together with alkynyl-d-Ala for their metabolic assembly and then bioorthogonally encoded by the correspondingly fabricated DBCO-DOTA-Gd, H2NO-DOTA-Eu, and azide-DOTA-Sm tags. This approach allows direct quantification of the d-X in situ in the cell wall using 158Gd, 153Eu, and 154Sm species-unspecific isotope dilution inductively coupled plasma mass spectrometry, avoiding any tedious and complex "cell-broken" pretreatment procedures that might induce racemization of the d-X. The obtained site-specific and accurate in situ information about the d-X enables quantitative monitoring of the bacterial response when Staphylococcus aureus meets vancomycin, showing that the amounts of azide-d-Leu and ketone-d-Met assembled are more important after determining the structure- and composition-dependent bacterial antibiotic resistance mechanisms. In addition, we found that the combined use of vancomycin and d-Ala restores the efficacy of vancomycin and might be a wise and simple way to combat vancomycin intermediate-resistant S. aureus.

Detection of Transport Intermediates in the Peptidoglycan Flippase MurJ Identifies Residues Essential for Conformational Cycling.

Bacterial cell wall synthesis is an essential process in bacteria and one of the best targets for antibiotics. A critical step on this pathway is the export of the lipid-linked cell wall monomer, Lipid II, by its transporter MurJ. The mechanism by which MurJ mediates the transbilayer movement of Lipid II is not understood because intermediate states of this process have not been observed. Here we demonstrate a method to capture and detect interactions between MurJ and its substrate Lipid II by photo-cross-linking and subsequent biotin-tagging. We show that this method can be used to covalently capture intermediate transport states of Lipid II on MurJ in living cells. Using this strategy we probed several lethal arginine mutants and found that they retain appreciable substrate-binding ability despite being defective in Lipid II transport. We propose that Lipid II binding to these residues during transport induces a conformational change in MurJ required to proceed through the Lipid II transport cycle. The methods described to detect intermediate transport states of MurJ will be useful for characterizing mechanisms of inhibitors.

FtsW activity and lipid II synthesis are required for recruitment of MurJ to midcell during cell division in Escherichia coli.

Peptidoglycan (PG) is the unique cell shape-determining component of the bacterial envelope, and is a key target for antibiotics. PG synthesis requires the transmembrane movement of the precursor lipid II, and MurJ has been shown to provide this activity in Escherichia coli. However, how MurJ functions in vivo has not been reported. Here we show that MurJ localizes both in the lateral membrane and at midcell, and is recruited to midcell simultaneously with late-localizing divisome proteins and proteins MraY and MurG. MurJ septal localization is dependent on the presence of a complete and active divisome, lipid II synthesis and PBP3/FtsW activities. Inactivation of MurJ, either directly by mutation or through binding with MTSES, did not affect the midcell localization of MurJ. Our study visualizes MurJ localization in vivo and reveals a possible mechanism of MurJ recruitment during cell division.

Loss of specificity variants of WzxC suggest that substrate recognition is coupled with transporter opening in MOP-family flippases.

Bacteria produce a variety of surface-exposed polysaccharides important for cell integrity, biofilm formation and evasion of the host immune response. Synthesis of these polymers often involves the assembly of monomer oligosaccharide units on the lipid carrier undecaprenyl-phosphate at the inner face of the cytoplasmic membrane. For many polymers, including cell wall peptidoglycan, the lipid-linked precursors must be transported across the membrane by flippases to facilitate polymerization at the membrane surface. Flippase activity for this class of polysaccharides is most often attributed to MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family proteins. Little is known about how this ubiquitous class of transporters identifies and translocates its cognate precursor over the many different types of lipid-linked oligosaccharides produced by a given bacterial cell. To investigate the specificity determinants of MOP proteins, we selected for variants of the WzxC flippase involved in Escherichia coli capsule (colanic acid) synthesis that can substitute for the essential MurJ MOP-family protein and promote transport of cell wall peptidoglycan precursors. Variants with substitutions predicted to destabilize the inward-open conformation of WzxC lost substrate specificity and supported both capsule and peptidoglycan synthesis. Our results thus suggest that specific substrate recognition by a MOP transporter normally destabilizes the inward-open state, promoting transition to the outward-open conformation and concomitant substrate translocation. Furthermore, the ability of WzxC variants to suppress MurJ inactivation provides strong support for the designation of MurJ as the flippase for peptidoglycan precursors, the identity of which has been controversial.

Towards the Native Binding Modes of Antibiotics that Target Lipid†II.

The alarming rise of antimicrobial resistance (AMR) imposes severe burdens on healthcare systems and the economy worldwide, urgently calling for the development of new antibiotics. Antimicrobial peptides could be ideal templates for next-generation antibiotics, due to their low propensity to cause resistance. An especially promising branch of antimicrobial peptides target lipid II, the precursor of the bacterial peptidoglycan network. To develop these peptides into clinically applicable compounds, detailed information on their pharmacologically relevant modes of action is of critical importance. Here we review the binding modes of a selection of peptides that target lipid II and highlight shortcomings in our molecular understanding that, at least partly, relate to the widespread use of artificial membrane mimics for structural studies of membrane-active antibiotics. In particular, with the example of the antimicrobial peptide nisin, we showcase how the native cellular membrane environment can be critical for understanding of the physiologically relevant binding mode.

Unraveling the mechanism of peptidoglycan amidation by the bifunctional enzyme complex GatD/MurT: A comparative structural approach.

The bacterial cell wall provides structural integrity to the cell and protects the cell from internal pressure and the external environment. During the course of the twelve-year funding period of the Collaborative Research Center 766, our work has focused on conducting structure-function studies of enzymes that modify (synthesize or cleave) cell wall components of a range of bacteria including Staphylococcus aureus, Staphylococcus epidermidis, and Nostoc punctiforme. Several of our structures represent promising targets for interference. In this review, we highlight a recent structure-function analysis of an enzyme complex that is responsible for the amidation of Lipid II, a peptidoglycan precursor, in S. aureus.

A Chemical Biology Approach to Understanding Molecular Recognition of Lipid†II by Nisin(1-12): Synthesis and NMR Ensemble Analysis of Nisin(1-12) and Analogues.

Natural products that target lipid II, such as the lantibiotic nisin, are strategically important in the development of new antibacterial agents to combat the rise of antimicrobial resistance. Understanding the structural factors that govern the highly selective molecular recognition of lipid II by the N-terminal region of nisin, nisin(1-12), is a crucial step in exploiting the potential of such compounds. In order to elucidate the relationships between amino acid sequence and conformation of this bicyclic peptide fragment, we have used solid-phase peptide synthesis to prepare two novel analogues of nisin(1-12) in which the dehydro residues have been replaced. We have carried out an NMR ensemble analysis of one of these analogues and of the wild-type nisin(1-12) peptide in order to compare the conformations of these two bicyclic peptides. Our analysis has shown the effects of residue mutation on ring conformation. We have also demonstrated that the individual rings of nisin(1-12) are pre-organised to an extent for binding to the pyrophosphate group of lipid II, with a high degree of flexibility exhibited in the central amide bond joining the two rings.

Molecular Recognition of Lipid II by Lantibiotics: Synthesis and Conformational Studies of Analogues of Nisin and Mutacin Rings A and B.

In response to the growing threat posed by antibiotic-resistant bacterial strains, extensive research is currently focused on developing antimicrobial agents that target lipid II, a vital precursor in the biosynthesis of bacterial cell walls. The lantibiotic nisin and related peptides display unique and highly selective binding to lipid II. A key feature of the nisin-lipid II interaction is the formation of a cage-like complex between the pyrophosphate moiety of lipid II and the two thioether-bridged rings, rings A and B, at the N-terminus of nisin. To understand the important structural factors underlying this highly selective molecular recognition, we have used solid-phase peptide synthesis to prepare individual ring A and B structures from nisin, the related lantibiotic mutacin, and synthetic analogues. Through NMR studies of these rings, we have demonstrated that ring A is preorganized to adopt the correct conformation for binding lipid II in solution and that individual amino acid substitutions in ring A have little effect on the conformation. We have also analyzed the turn structures adopted by these thioether-bridged peptides and show that they do not adopt the tight α-turn or ß-turn structures typically found in proteins.

The Necessity of d-Thr in the New Antibiotic Teixobactin: A Molecular Dynamics Study.

Ever since the discovery of the new antibiotic teixobactin, studies of its structure-activity relationships have never ceased. Here we focus on the chirality of the threonine (Thr) residue, which belongs to the ring motif of teixobactin and plays an important role in the binding with its target, lipid II molecule. We study the structural propensity of the open and closed ring motifs with different chiral Thr residues as well as the teixobactin-lipid II complex with the help of molecular dynamics simulations. Our results suggest that different chiralities lead to different NH orientations of Thr with respect to the ring plane. Only in the closed ring motif with d-Thr is a favored binding cavity achievable with all four NH groups facing the same side of the ring plane. This study develops a deeper understanding of the binding mechanism of teixobactin and lipid II and is expected to be beneficial to new teixobactin-based drug design.

Synthesis of Functionalized N-Acetyl Muramic Acids To Probe Bacterial Cell Wall Recycling and Biosynthesis.

Uridine diphosphate N-acetyl muramic acid (UDP NAM) is a critical intermediate in bacterial peptidoglycan (PG) biosynthesis. As the primary source of muramic acid that shapes the PG backbone, modifications installed at the UDP NAM intermediate can be used to selectively tag and manipulate this polymer via metabolic incorporation. However, synthetic and purification strategies to access large quantities of these PG building blocks, as well as their derivatives, are challenging. A robust chemoenzymatic synthesis was developed using an expanded NAM library to produce a variety of 2 -N-functionalized UDP NAMs. In addition, a synthetic strategy to access bio-orthogonal 3-lactic acid NAM derivatives was developed. The chemoenzymatic UDP synthesis revealed that the bacterial cell wall recycling enzymes MurNAc/GlcNAc anomeric kinase (AmgK) and NAM α-1 phosphate uridylyl transferase (MurU) were permissive to permutations at the two and three positions of the sugar donor. We further explored the utility of these derivatives in the fluorescent labeling of both Gram (-) and Gram (+) PG in whole cells using a variety of bio-orthogonal chemistries including the tetrazine ligation. This report allows for rapid and scalable access to a variety of functionalized NAMs and UDP NAMs, which now can be used in tandem with other complementary bio-orthogonal labeling strategies to address fundamental questions surrounding PG's role in immunology and microbiology.