FtsN plays an essential role in promoting the inward synthesis of septal peptidoglycan (sPG) by the FtsWI complex during bacterial cell division. How it achieves this role is unclear. Here we use single-molecule tracking to investigate FtsN’s dynamics during sPG synthesis in E. coli. We show that septal FtsN molecules move processively at ~9 nm s−1, the same as FtsWI molecules engaged in sPG synthesis (termed sPG-track), but much slower than the ~30 nm s−1 speed of inactive FtsWI molecules coupled to FtsZ’s treadmilling dynamics (termed FtsZ-track). Importantly, processive movement of FtsN is exclusively coupled to sPG synthesis and is required to maintain active sPG synthesis by FtsWI. Our findings indicate that FtsN is part of the FtsWI sPG synthesis complex, and that while FtsN is often described as a “trigger” for the initiation for cell wall constriction, it must remain part of the processive FtsWI complex to maintain sPG synthesis activity.

Clostridioides (formerly Clostridium) difficile produces two major toxins, TcdA and TcdB, upon entry into stationary phase. Transcription of tcdA and tcdB requires the specialized sigma factor, σ , which also directs RNA Polymerase to transcribe tcdR itself. We fused a gene for a red fluorescent protein to the tcdA promoter to study toxin gene expression at the level of individual C. difficile cells. Surprisingly, only a subset of cells became red fluorescent upon entry into stationary phase. Breaking the positive feedback loop that controls σ production by engineering cells to express tcdR from a tetracycline-inducible promoter resulted in uniform fluorescence across the population. Experiments with two regulators of tcdR expression, σ and CodY, revealed neither is required for bimodal toxin gene expression. However, σ biased cells toward the Toxin-ON state, while CodY biased cells toward the Toxin-OFF state. Finally, toxin gene expression was observed in sporulating cells. We conclude that (i) toxin production is regulated by a bistable switch governed by σ , which only accumulates to high enough levels to trigger toxin gene expression in a subset of cells, and (ii) toxin production and sporulation are not mutually exclusive developmental programs.

Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile σV is required for lysozyme resistance, and σV is activated in response to lysozyme. Once activated, σV, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here we analyze the contribution of individual genes in the σV regulon to lysozyme resistance. Using CRISPR-Cas9 mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation, but loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also use CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficile. Importance: Clostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV operon is required for lysozyme resistance. Here we use CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism in C. difficile. We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation and PdaV, encoded as part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.

Bacillus and Clostridium spores are known to be highly resistant to killing, persisting on environmental and human body surfaces for long periods of time. In favorable environments, these spores may germinate and cause human diseases. It is thus important to identify agents that can be used on both environmental and human skin and mucosal surfaces and that are effective in killing spores. We previously showed that the fatty acid monoester glycerol monolaurate (GML) kills stationary-phase cultures of Bacillus anthracis. Since such cultures are likely to contain spores, it is possible that GML and a human-use-approved GML nonaqueous gel would kill Bacillus and Clostridium spores. The significance of our studies is that we have identified GML, and, to a greater extent, GML solubilized in a nonaqueous gel, as effective in killing spores from both bacterial genera.

The FtsN protein of Escherichia coli and other proteobacteria is an essential and highly conserved bitopic membrane protein that triggers the inward synthesis of septal peptidoglycan (sPG) during cell division. Previous work has shown that the activation of sPG synthesis by FtsN involves a series of interactions of FtsN with other divisome proteins and the cell wall. Precisely how FtsN achieves this role is unclear, but a recent study has shown that FtsN promotes the relocation of the essential sPG synthase FtsWI from an FtsZ-associated track (where FtsWI is inactive) to an sPG-track (where FtsWI engages in sPG synthesis). Whether FtsN works by displacing FtsWI from the Z-track or capturing/retaining FtsWI on the sPG-track is not known. Here we use single-molecule imaging and genetic manipulation to investigate the organization and dynamics of FtsN at the septum and how they are coupled to sPG synthesis activity. We found that FtsN exhibits a spatial organization and dynamics distinct from those of the FtsZ-ring. Single FtsN molecules move processively as a single population with a speed of ~ 9 nm s-1, similar to the speed of active FtsWI molecules on the sPG-track, but significantly different from the ~ 30 nm s-1 speed of inactive FtsWI molecules on the FtsZ-track. Furthermore, the processive movement of FtsN is independent of FtsZ's treadmilling dynamics but driven exclusively by active sPG synthesis. Importantly, only the essential domain of FtsN, a three-helix bundle in the periplasm, is required to maintain the processive complex containing both FtsWI and FtsN on the sPG-track. We conclude that FtsN activates sPG synthesis by forming a processive synthesis complex with FtsWI exclusively on the sPG-track. These findings favor a model in which FtsN captures or retains FtsWI on the sPG-track rather than one in which FtsN actively displaces FtsWI from the Z-track.