Peptidoglycan is an essential crosslinked polymer that surrounds bacteria and protects them from osmotic lysis. Beta-lactam antibiotics target the final stages of peptidoglycan biosynthesis by inhibiting the transpeptidases that crosslink glycan strands to complete cell wall assembly. Characterization of transpeptidases and their inhibition by beta-lactams has been hampered by lack of access to substrate. We describe a general approach to accumulate Lipid II in bacteria and to obtain large quantities of this cell wall precursor. We demonstrate utility by isolating Staphylococcus aureus Lipid II and reconstituting the synthesis of crosslinked peptidoglycan by the essential penicillin-binding protein 2, PBP2, which catalyzes both glycan polymerization and transpeptidation. We also show that we can compare the potencies of different beta-lactams by directly monitoring transpeptidase inhibition. The methods reported here will enable a better understanding of cell wall biosynthesis and facilitate studies of next-generation transpeptidase inhibitors.

Penicillin-binding proteins (PBPs) are involved in the synthesis and remodeling of bacterial peptidoglycan (PG). Staphylococcus aureus expresses four PBPs. Genetic studies in S. aureus have implicated PBP4 in the formation of highly cross-linked PG, but biochemical studies have not reached a consensus on its primary enzymatic activity. Using synthetic Lipid II, we show here that PBP4 preferentially acts as a transpeptidase (TP) in vitro. Moreover, it is the PBP primarily responsible for incorporating exogenous d-amino acids into cellular PG, implying that it also has TP activity in vivo. Notably, PBP4 efficiently exchanges d-amino acids not only into PG polymers but also into the PG monomers Lipid I and Lipid II. This is the first demonstration that any TP domain of a PBP can activate the PG monomer building blocks. Exploiting the promiscuous TP activity of PBP4, we developed a simple, highly sensitive assay to detect cellular pools of lipid-linked PG precursors, which are of notoriously low abundance. This method, which addresses a longstanding problem, is useful for assessing how genetic and pharmacological perturbations affect precursor levels, and may facilitate studies to elucidate antibiotic mechanism of action.

The main differences of health-promoting compounds and antioxidant activity of whole and individual edible parts of baby mustard were demonstrated.

Sacculus is a peptidoglycan matrix that protects bacteria from osmotic lysis. In Gram-positive organisms, the sacculus is densely functionalized with glycopolymers important for survival, but how assembly occurs is not known. In Staphylococcus aureus, three LCP family members have been implicated in attaching the major glycopolymer wall teichoic acid (WTA) to peptidoglycan, but ligase activity has not been demonstrated for these or any other LCP proteins. Using WTA and peptidoglycan substrates produced chemoenzymatically, we show that all three proteins can transfer WTA precursors to nascent peptidoglycan, establishing that LCP proteins are peptidoglycan-glycopolymer ligases. Although all S. aureus LCP proteins have the capacity to attach WTA to PG, we show that their cellular functions are not redundant. Strains lacking lcpA have phenotypes similar to WTA null strains, indicating that this is the most important WTA ligase. This work provides a foundation for studying how LCP enzymes participate in cell wall assembly.

Lysobactin, also known as katanosin B, is a potent antibiotic with in vivo efficacy against Staphylococcus aureus and Streptococcus pneumoniae. It was previously shown to inhibit peptidoglycan (PG) biosynthesis, but its molecular mechanism of action has not been established. Using enzyme inhibition assays, we show that lysobactin forms 1:1 complexes with Lipid I, Lipid II, and Lipid IIAWTA, substrates in the PG and wall teichoic acid (WTA) biosynthetic pathways. Therefore, lysobactin, like ramoplanin and teixobactin, recognizes the reducing end of lipid-linked cell wall precursors. We show that despite its ability to bind precursors from different pathways, lysobactin’s cellular mechanism of killing is due exclusively to Lipid II binding, which causes septal defects and catastrophic cell envelope damage.

Staphylococcus aureus is a Gram-positive pathogen with an unusual mode of cell division in that it divides in orthogonal rather than parallel planes. Through selection using moenomycin, an antibiotic proposed to target peptidoglycan glycosyltransferases (PGTs), we have generated resistant mutants containing a single point mutation in the active site of the PGT domain of an essential peptidoglycan (PG) biosynthetic enzyme, PBP2. Using cell free polymerization assays, we show that this mutation alters PGT activity so that much shorter PG chains are made. The same mutation in another S. aureus PGT, SgtB, has a similar effect on glycan chain length. Moenomycin-resistant S. aureus strains containing mutated PGTs that make only short glycan polymers display major cell division defects, implicating peptidoglycan chain length in determining bacterial cell morphology and division site placement.

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Methicillin-resistant Staphylococcus aureus (MRSA) infections are a global public health problem. MRSA strains have acquired a non-native penicillin-binding protein called PBP2a that crosslinks peptidoglycan when the native S. aureus PBPs are inhibited by β-lactams. If assembly of the pentaglycine branch on the cell wall precursor Lipid II is genetically blocked, MRSA strains become susceptible to β-lactams. Therefore, it has been proposed that PBP2a can only crosslink peptidoglycan strands bearing a complete pentaglycine branch. This hypothesis has never been tested because the necessary substrates have not been available. Here, we obtained S. aureus Lipid II variants having shorter glycine branches and have tested whether PBP2a and two other S. aureus transpeptidases, PBP2 and PBP4, can crosslink peptidoglycan strands made from the variants. There are striking differences in enzymatic activity among these enzymes depending on the length of the glycine branch, but we find that PBP2a can, in fact, crosslink glycan strands bearing triglycine. We report experiments in cells that are consistent with our in vitro findings about the crosslinking preferences of these PBPs. In addition to providing insights into the cell wall physiology of a major pathogen, our studies identify the best target for β-lactam potentiators to treat MRSA.