Synthesis of new septal peptidoglycan (sPG) is crucial for bacterial cell division. FtsW, an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a peptidoglycan glycosyltransferase (PGTase). Despite its importance, the septal PGTase activity of FtsW has not been demonstrated in vivo . How its activity is spatiotemporally regulated in vivo has also remained elusive. Here we confirmed FtsW as an essential septum-specific PGTase in vivo using an N -acetylmuramic acid analog incorporation assay. Next, using single-molecule tracking coupled with genetic manipulations, we identified two populations of processively moving FtsW molecules: a fast-moving population correlated with the treadmilling dynamics of the essential cytoskeletal FtsZ protein and a slow-moving population dependent on active sPG synthesis. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving population. Our results suggest a two-track model, in which inactive sPG synthases follow the “Z-track” to be distributed along the septum; FtsN promotes their release from the “Z-track” to become active in sPG synthesis on the slow “sPG-track”. This model provides a mechanistic framework for the spatiotemporal coordination of sPG synthesis in bacterial cell division.

Metabolic glycan probes have emerged as an excellent tool to investigate vital questions in biology. Recently, methodology to incorporate metabolic bacterial glycan probes into the cell wall of a variety of bacterial species has been developed. In order to improve this method, a scalable synthesis of the peptidoglycan precursors is developed here, allowing for access to essential peptidoglycan immunological fragments and cell wall building blocks. The question was asked if masking polar groups of the glycan probe would increase overall incorporation, a common strategy exploited in mammalian glycobiology. Here, we show, through cellular assays, that E. coli do not utilize peracetylated peptidoglycan substrates but do employ methyl esters. The 10-fold improvement of probe utilization indicates that (i) masking the carboxylic acid is favorable for transport and (ii) bacterial esterases are capable of removing the methyl ester for use in peptidoglycan biosynthesis. This investigation advances bacterial cell wall biology, offering a prescription on how to best deliver and utilize bacterial metabolic glycan probes.

Bacterial cells utilize small carbohydrate building blocks to construct peptidoglycan (PG), a highly conserved mesh‐like polymer that serves as a protective coat for the cell. PG production has long been a target for antibiotics, and its breakdown is a source for human immune recognition. A key component of bacterial PG, N‐acetyl muramic acid (NAM), is a vital element in many synthetically derived immunostimulatory compounds. However, the exact molecular details of these structures and how they are generated remain unknown due to a lack of chemical probes surrounding the NAM core. A robust synthetic strategy to generate bioorthogonally tagged NAM carbohydrate units is implemented. These molecules serve as precursors for PG biosynthesis and recycling. Escherichia coli cells are metabolically engineered to incorporate the bioorthogonal NAM probes into their PG network. The probes are subsequently modified using copper‐catalyzed azide‐alkyne cycloaddition to install fluorophores directly into the bacterial PG, as confirmed by super‐resolution microscopy and high‐resolution mass spectrometry. Here, synthetic notes for key elements of this process to generate the sugar probes as well as streamlined user‐friendly metabolic labeling strategies for both microbiology and immunological applications are described. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of peracetylated 2‐azido glucosamine Basic Protocol 2: Synthesis of 2‐azido and 2‐alkyne NAM Basic Protocol 3: Synthesis of 3‐azido NAM methyl ester Basic Protocol 4: Incorporation of NAM probes into bacterial peptidoglycan Basic Protocol 5: Confirmation of bacterial cell wall remodeling by mass spectrometry

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