Roles for both FtsA and the FtsBLQ subcomplex in FtsN‐stimulated cell constriction in Escherichia coli

Abstract

SUMMARY Escherichia coli FtsN is a bitopic membrane protein that is essential for triggering active cell constriction. A small periplasmic subdomain (EFtsN) is required and sufficient for function, but its mechanism of action is unclear. We isolated extragenic EFtsN*-suppressing mutations that restore division in cells producing otherwise non-functional variants of FtsN. These mapped to the IC domain of FtsA in the cytoplasm and to small subdomains of the FtsB and FtsL proteins in the periplasm. All FtsB and FtsL variants allowed survival without EFtsN, but many then imposed a new requirement for interaction between the cytoplasmic domain of FtsN (NFtsN) with FtsA. Alternatively, variants of FtsA, FtsB or FtsL acted synergistically to allow cell division in the complete absence of FtsN. Strikingly, moreover, substitution of a single residue in FtsB (E56) proved sufficient to rescue ΔftsN cells as well. In FtsN+ cells, EFtsN*-suppressing mutations promoted cell fission at an abnormally small cell size, and caused cell shape and integrity defects under certain conditions. This and additional evidence support a model in which FtsN acts on either side of the membrane to induce a conformational switch in both FtsA and the FtsBLQ subcomplex to derepress septal peptidoglycan synthesis and membrane invagination.

FtsEX acts on FtsA to regulate divisome assembly and activity

“…Because FtsN activates the divisome for constriction, whereas FtsE D162N X allows divisome assembly but not cell constriction, it suggests they have opposing activities in the divisome. Because both the cytoplasmic ( N FtsN) and the essential ( E FtsN) domains of FtsN play important roles in activating the divisome (24), we tested their role in counteracting FtsE D162N X by using FtsN mutants with mutations that inactivate N FtsN (FtsN D5N ) or E FtsN (FtsN WYAA ) (24,25). A Western blot showed that these mutants were expressed at the same level as wild-type FtsN (SI Appendix, Fig.…”

“…Interaction between N FtsN and FtsA brings E FtsN to the division site so that these two signals synergize to derepress the FtsQLB complex and activate septal PG synthesis (Fig. 1B) (24,29,30). As new PG is synthesized, amidases regulated by FtsEX generate denuded glycan chains, resulting in more recruitment of FtsN by binding of S FtsN and forming a positive feedback loop (12).…”

“…The essential function of FtsN ( E FtsN) is located within a helix preceding the SPOR domain (12,24). Although E FtsN does not display a midcell localization when overexpressed and exported to the periplasm, it is sufficient to stimulate septum synthesis (12).…”

“…Insight into the role of E FtsN comes from the isolation of extragenic mutations that allow cell division when E FtsN is inactivated. These mutations are located in FtsA, FtsB, and FtsL, leading to a model in which the divisome is kept inactive by FtsA and the FtsQLB complex (24). Interaction between N FtsN and FtsA brings E FtsN to the division site so that these two signals synergize to derepress the FtsQLB complex and activate septal PG synthesis (Fig.…”

“…First, other than FtsA and FtsZ, the early divisome proteins (ZipA, FtsEX, EnvC, and the Zaps) can be deleted under certain conditions and the divisome can still assemble and function (7,8,(19)(20)(21). Second, FtsA has been reported to interact with many downstream division proteins (22), including FtsN (23)(24)(25). Importantly, FtsA mutants impaired for self-interaction and overexpression of FtsN bypass ZipA, supporting a model in which FtsA monomers recruit downstream division proteins to the Z ring (20,25).…”