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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., Competing Interests: The authors have declared that no competing interests exist.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli , septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu 75 -Gln 93 , known as E FtsN) was shown to be essential and sufficient for its functioning in vivo , but what exactly this sequence is doing remained unknown. Here, we show that E FtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with E FtsN, which demonstrates that E FtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the E FtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the Δ ponB - ponA ts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that E FtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Boes et al.)

Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent lipid II) and monitored direct binding between lipid II and interacting proteins (PBP1b, FtsW and MurJ), as well as between lipid II and interacting antibiotics (vancomycin, nisin, ramoplanin and a small molecule). Competition experiments performed using unlabelled lipid II, four lipid II-binding antibiotics and moenomycin demonstrate that the assay can detect compounds interacting with lipid II or the proteins. These results provide a proof-of-concept for the use of this assay in a high-throughput screening of compounds against all these targets. In addition, the assay constitutes a powerful tool in the study of the mode of action of compounds that interfere with these processes. Interestingly, FA assay with lipid II probe has the advantage over moenomycin based probe to potentially identify compounds that interfere with both donor and acceptor sites of the aPBPs GTase as well as compounds that bind to lipid II. In addition, this assay would allow the screening of compounds against SEDS proteins and MurJ which do not interact with moenomycin.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, PG synthesis localizes at midcell under the control of a multiprotein complex, the divisome, allowing the safe formation of two new cell poles and separation of daughter cells. Genetic studies in Escherichia coli pointed out that FtsBLQ and FtsN participate in the regulation of septal PG (sPG) synthesis; however, the underlying molecular mechanisms remained largely unknown. Here we show that FtsBLQ subcomplex directly interacts with the PG synthase PBP1b and with the subcomplex FtsW-PBP3, mainly via FtsW. Strikingly, we discovered that FtsBLQ inhibits the glycosyltransferase activity of PBP1b and that this inhibition was antagonized by the PBP1b activators FtsN and LpoB. The same results were obtained in the presence of FtsW-PBP3. Moreover, using a simple thioester substrate (S2d), we showed that FtsBLQ also inhibits the transpeptidase domain of PBP3 but not of PBP1b. As the glycosyltransferase and transpeptidase activities of PBP1b are coupled and PBP3 activity requires nascent PG substrate, the results suggest that PBP1b inhibition by FtsBLQ will block sPG synthesis by these enzymes, thus maintaining cell division as repressed until the maturation of the divisome is signaled by the accumulation of FtsN, which triggers sPG synthesis and the initiation of cell constriction. These results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which seems to counterbalance the inhibitory effect of FtsBLQ to restore PBP1b activity. IMPORTANCE Bacterial cell division is governed by a multiprotein complex called divisome, which facilitates a precise cell wall synthesis at midcell and daughter cell separation. Protein-protein interactions and activity studies using different combinations of the septum synthesis core of the divisome revealed that the glycosyltransferase activity of PBP1b is repressed by FtsBLQ and that the presence of FtsN or LpoB suppresses this inhibition. Moreover, FtsBLQ also inhibits the PBP3 activity on a thioester substrate. These results provide enzymatic evidence of the regulation of the peptidoglycan synthase PBP1b and PBP3 within the divisome. The results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which functions to relieve the repression on PBP1b by FtsBLQ and to initiate septal peptidoglycan synthesis., (Copyright © 2019 Boes et al.)