Structural and genetic analyses reveal the protein SepF as a new membrane anchor for the Z ring

Abstract

A key step in bacterial cell division is the polymerization of the tubulin homolog FtsZ at midcell. FtsZ polymers are anchored to the cell membrane by FtsA and are required for the assembly of all other cell division proteins. In Gram-positive and cyanobacteria, FtsZ filaments are aligned by the protein SepF, which in vitro polymerizes into large rings that bundle FtsZ filaments. Here we describe the crystal structure of the only globular domain of SepF, located within the C-terminal region. Two-hybrid data revealed that this domain comprises the FtsZ binding site, and EM analyses showed that it is sufficient for ring formation, which is explained by the filaments in the crystals of SepF. Site-directed mutagenesis, gel filtration, and analytical ultracentrifugation indicated that dimers form the basic units of SepF filaments. High-resolution structured illumination microscopy suggested that SepF is membrane associated, and it turned out that purified SepF not only binds to lipid membranes, but also recruits FtsZ. Further genetic and biochemical analyses showed that an amphipathic helix at the N terminus functions as the membrane-binding domain, making SepF a unique membrane anchor for the FtsZ ring. This clarifies why Bacillus subtilis grows without FtsA or the putative membrane anchor EzrA and why bacteria lacking FtsA contain SepF homologs. Both FtsA and SepF use an amphipathic helix for membrane binding. These helices prefer positively curved membranes due to relaxed lipid density; therefore this type of membrane anchor may assist in keeping the Z ring positioned at the strongly curved leading edge of the developing septum.

Roles of the Essential Protein FtsA in Cell Growth and Division in Streptococcus pneumoniae

“…In contrast, FtsZ rapidly delocalizes after depletion of FtsA in S. pneumoniae, indicating that FtsA is required for efficient assembly of Z rings at midcell in this organism. This supports the notion that FtsA is a key membrane anchor for FtsZ in Gram-positive organisms that lack a ZipA homolog (19,67), although EzrA and SepF both bind the membrane and FtsZ and thus also have the potential to be membrane tethers for the Z ring (29)(30)(31). Of these two proteins, EzrA is essential in S. pneumoniae (35; A. J. Perez, unpublished data), whereas SepF is not (60; this work).…”

“…E. coli FtsA variants able to suppress the lack of ZipA support the idea that these proteins have a redundant function in stabilizing the Z ring (12,25,26). However, unlike FtsA, ZipA is absent in Gram-positive bacteria, where the mechanism of FtsZ tethering to the membrane remains to be clarified; other FtsZ-interacting proteins, like SepF (27,28) or EzrA (29,30), may fulfill this function (31).…”

“…Excess FtsA suppresses late septation defects in cells lacking division protein SepF or GpsB. In B. subtilis, SepF has been shown to share some properties with FtsA, including the ability to tether FtsZ to the membrane (27,28,31), and to permit cell division in the absence of FtsA when overproduced (28). Deletion of the gene encoding SepF, previously called ylmF, in S. pneumoniae is not lethal but results in significant defects in septation as well as other morphological abnormalities (60).…”

The Blueprint of a Minimal Cell: MiniBacillus

“…Why FtsA is nonessential in B. subtilis has been enigmatic for a long time. Only the discovery of the unrelated alternative membrane anchor SepF provided the answer (38). Finally, alternative pathways may lead to the same results.…”

Estudo do papel de MinD na ativação de MinC, um regulador chave na divisão bacteriana em Bacillus subtilis

“…Análise da estrutura mostrou uma hélice anfipática na região N-terminal de SepF que funcionaria como domínio de ligação à membrana. Isto esclareceria por que B. subtilis pode crescer sem FtsA (Duman R. et al, 2013) e por que bactérias que não possuem FtsA, como as micobactérias, tem homólogos de SepF, sendo eles parte importante do processo de divisão destes microrganismos (Gola S. et al, 2015).…”