SepF Increases the Assembly and Bundling of FtsZ Polymers and Stabilizes FtsZ Protofilaments by Binding along Its Length

Jay Kumar Singh,Ravindra D Makde,Vinay Kumar,Dulal Panda

doi:10.1074/jbc.m805910200

Abstract

SepF (Septum Forming) protein has been recently identified through genetic studies, and it has been suggested to be involved in the division of Bacillus subtilis cells. We have purified functional B. subtilis SepF from the inclusion bodies overexpressed in Escherichia coli. Far-UV circular dichroism and fluorescence spectroscopic analysis involving the extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid suggested that the purified SepF had characteristics of folded proteins. SepF was found to promote the assembly and bundling of FtsZ protofilaments using three complimentary techniques, namely 90 degrees light scattering, sedimentation, and transmission electron microscopy. SepF also decreased the critical concentration of FtsZ assembly, prevented the dilution-induced disassembly of FtsZ protofilaments, and suppressed the GTPase activity of FtsZ. Further, thick bundles of FtsZ protofilaments were observed using fluorescein isothiocyanate-labeled SepF (FITC-SepF). Interestingly, FITC-SepF was found to be uniformly distributed along the length of the FtsZ protofilaments, suggesting that SepF copolymerizes with FtsZ. SepF formed a stable complex with FtsZ, as evident from the gel filtration analysis. Using a C-terminal tail truncated FtsZ (FtsZDelta16) and a C-terminal synthetic peptide of B. subtilis FtsZ (366-382); we provided evidence indicating that SepF binds primarily to the C-terminal tail of FtsZ. The present work in concert with the available in vivo data support a model in which SepF plays an important role in regulating the assembly dynamics of the divisome complex; therefore, it may have an important role in bacterial cell division.

Highlights

SepF formed a stable complex with FtsZ monomers and was found to be recruited in the FtsZ bundles
The results indicated that SepF may play an important role during cytokinesis in B. subtilis
Characterization of the Refolded SepF Protein—The B. subtilis SepF protein was refolded from the inclusion bodies overexpressed in E. coli

Summary

EXPERIMENTAL PROCEDURES

Materials—Pipes, phenylmethylsulfonyl fluoride, bovine serum albumin (BSA), ␤-mercaptoethanol, GTP, FITC, and Sephadex G-100 were purchased from Sigma. Nickel(II)-iminodiacetic acid coupled to Sepharose௡ was purchased from Amersham Biosciences. Cloning and Overexpression of SepF—The genomic DNA of B. subtilis ATCC6633 was used for the PCR amplifications of the desired orf using Pfu Turbo DNA polymerase. The PCR product was cloned into NdeI and BamHI site of pET16b(ϩ) vector (pET16-sepF). The recombinant plasmid was subsequently transformed into E. coli BL21(DE3) for the overexpression of SepF. The deduced amino acid sequence of expressed SepF matched with B. subtilis strain 168 (GenBankTM accession number Z9912) except for two changes (V29E and D86V). The sequence of our construct matched exactly with the genomic sepF of B. subtilis ATCC6633 strain used for cloning. Isolation and Purification of SepF—Recombinant SepF protein of B. subtilis was overexpressed and purified from E. coli BL21(DE3) cells.

The abbreviations used are

RESULTS

DISCUSSION