Site-specific mutations of FtsZ--effects on GTPase and in vitro assembly

Abstract

Background: FtsZ, the major cytoskeletal protein in bacterial cytokinesis, assembles in vitro into protofilaments, which can further associate into sheets, bundles or tubes. We have constructed 16 site-directed mutants of E. coli ftsZ, and tested them for GTP hydrolysis and assembly in vitro, and for their ability to complement the temperature sensitive ftsZ84 mutation in E. coli.

Results: The mutants were grouped into three classes. Benign mutants, which mapped mostly to the front and back surface of the protofilament, were able to complement ftsZ84 in vivo and showed normal assembly in vitro. GTP contact mutations had less than 10% of wild type GTPase activity. They could all assemble in vitro, and several of these mutants could complement ftsZ84. A third, and newly discovered, class of mutations mapped to the sides of the protofilaments. These lateral mutants had mostly normal GTPase and assembly in vitro, but none of them complemented ftsZ84. The non-complementing mutants showed greatly reduced expression from the pBS58 vector, suggesting possible dominant negative effects.

Conclusions: Several mutants with greatly reduced GTPase could still complement ftsZ84, suggesting that the high level of GTPase observed in vitro is not essential for in vivo function. All of the lateral mutants failed to complement ftsZ84, which suggests that these surfaces of the protofilaments are important for function in cell division. These lateral surfaces may mediate association of FtsZ protofilaments into pairs or small sheets, although their structure is apparently different from the sheets assembled in DEAE dextran or calcium.

Figure 1

Mapping mutants on the atomic structure of Methanococcus FtsZ [8]. (A) Benign mutations; (B) GTP-contact mutations; (C) Lateral mutations. The amino acid numbers refer to the E. coli sequence, which were mapped to the corresponding amino acid in the Methanococcus sequence. Note that all of these amino acids are conserved in these two sequences as well as in most other FtsZ. Mutants identified in genetic screens are also indicated by their previous name (Z1, Z84). The subunits are oriented as they should be in a protofilament, based on the structure of tubulin viewed from outside the microtubule [9].

Figure 2

FtsZ expression levels in cells containing ftsZ mutants on pBS58 FtsZ levels were determined by quantitative western blotting and normalized to total protein levels to control for cells of varying length or cell density.

Figure 3

Assembly of three FtsZ mutants. (a) FtsZ84 assembled with GTP and 0.6 mg/ml DEAE dextran formed protofilament sheets of exceptional size and regularity (although the quality of the sheets varied for different preparations). Note that wild type FtsZ would have formed exclusively tubes under these conditions. (b) D86K assembled into tubes in MEMK6.5. No DEAE dextran and no GTP or GDP were added. (c) D86K at 0.5 mg/ml in MEMK6.5 plus 2 mM GTP (no DEAE dextran) formed twin protofilaments and short tubes. (d,e) D209A plus 0.6 mg/ml DEAE dextran and 10 mM GTP formed long spirals of protofilament bundles, and some circular hoops. (f) D209A in 2 mM GMPCPP also formed hoops, and these are mixed with tubes typical of assembly in GDP.