Frederico José Gueiros Filho (IQ-USP); Alexandre Wilson Bisson Filho (IQ-USP)

FtsZ, the prokaryotic homolog of tubulin, self-associates into a ring structure (Z ring) that establishes the place where division will occur. The Z ring represents a scaffold onto which other division proteins will associate to create the new septum. Z ring formation in vivo is affected by a number of proteins that interact with FtsZ and modulate its polymerization properties. The goal of this work was to elucidate how FtsZ interacts with ZapA, a modulator that promotes FtsZ polymerization and Z-ring formation. We created a mutagenized ftsZ plasmid library by error prone PCR, which contained 1.0x10⁵ clones and exhibited a mutation rate of about one substitution per ftsZ copy. To look for mutants within our library that lost the ability to interact with ZapA, we took advantage of the observation that overexpression of a ZapA fusion protein containing a membrane targeting sequence fused to its C-terminus (ZapA-MTS) was lethal to the cell. Using this genetic selection we found 8 different FtsZ mutants that were not killed by ZapA-MTS overexpression. Surprisingly, however, these mutants continued to interact with ZapA, as judged by fluorescence microscopy experiments. Because most substitutions in the ZapA-MTS resistant mutants mapped to the FtsZ-FtsZ interface and GTP binding site, this suggested that resistance was due to alterations in how FtsZ interacted with itself. In line with this, we also found that most of the mutations we isolated were "promiscuous" and caused resistance to the effects of an unrelated FtsZ modulator, the MinCD inhibitor. To directly study the FtsZ polymerization properties of our mutants we performed FRAP experiments. These showed that Z ring turnover in mutants was slightly slower than in the wild type, indicating that the mutations are increasing the stability of the FtsZ polymer. Steady-state measurements of the amount of FtsZ associated with the Z rings of mutants supported the same conclusion. Thus, we have shown that increasing polymer stability makes FtsZ less sensitive to the effect of a positive modulator such as ZapA.