Bacillus subtilis SepF Binds to the C-Terminus of FtsZ

Abstract

Bacterial cell division is mediated by a multi-protein machine known as the “divisome”, which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.

Essential protein SepF of mycobacteria interacts with FtsZ and MurG to regulate cell growth and division

“…We further observed that the conserved lysine residue (190K) of SepF MTB and the C terminus of FtsZ are important for the SepF-FtsZ interaction in mycobacteria. SepF also binds to the C terminus of FtsZ in B. subtilis (Kró l et al, 2012). P372 present in the C terminus of B. subtilis FtsZ is required for interaction with SepF.…”

“…In addition to FtsA, the repertoire of B. subtilis FtsZ-interacting proteins includes SepF, ZapA and ZapB. SepF binds to the C terminus of FtsZ and is required for proper septum formation (Gündogdu et al, 2011;Ishikawa et al, 2006;Kró l et al, 2012). It can complement for the lack of FtsA.…”

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

“…SepF binds directly to the C-terminal domain of FtsZ (6,16). However, the protein has no apparent homology to proteins of known function and the amino acid sequence does not indicate a clear docking site for FtsZ.…”

FtsZ Ring Stability: of Bundles, Tubules, Crosslinks, and Curves

“…In vitro, SepF can bind the CTT of either B. subtilis or E. coli FtsZ, indicating that the SepF-FtsZ interaction is dependent on the secondary and tertiary structure of the FtsZ CTT rather than specific amino acid residues (Fig. 1B) (104). Electron micrographs of purified SepF show large regular ring-like structures (76).…”