Free SepF blocks recruitment of late cell division proteins

The cell division protein SepF aligns polymers formed by the key cell division protein FtsZ during synthesis of the (Fts)Z-ring at midcell, the first stage in cytokinesis. In addition, SepF acts as a membrane anchor for the Z-ring. SepF is conserved in Gram-positive and cyanobacteria. Recently, it was shown that SepF overproduction in Mycobacterium smegmatis blocks cell division. Here we investigated this in more detail using the Gram-positive model system Bacillus subtilis. Surprisingly, overproduction of SepF does not interfere with assembly of the Z-ring, but blocks assembly of the late cell division proteins responsible for septum synthesis. Transposon mutagenesis suggested that SepF overproduction inactivates the WalKR two-component system involved in cell division. Indeed, SepF overproduction impairs WalK localization, possibly because septal WalK localization requires late cell division proteins. Unexpectedly, transcriptome analysis showed that WalKR activity was not affected. Another surprise was that the cell division phenotype occurs when SepF does not bind to FtsZ. Further analyses provided an explanation for the contradictory transposon and transcriptome results, and suggested that SepF competes with other cell division proteins for binding to FtsZ. Our data show that an imbalance in early cell division proteins can interfere with recruitment of late cell division proteins. Comparing wild type Bacillus subtilis strain 168 (n=2) with Bacillus subtilis YK240 (Pxyl-sepF, n = 3)