New Temperature-Sensitive Alleles of ftsZ in Escherichia coli

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Abstract
We isolated five new temperature-sensitive alleles of the essential cell division gene ftsZ in Escherichia coli , using P1-mediated, localized mutagenesis. The five resulting single amino acid changes (Gly 109 →Ser 109 for ftsZ6460 , Ala 129 →Thr 129 for ftsZ972 , Val 157 →Met 157 for ftsZ2066 , Pro 203 →Leu 203 for ftsZ9124 , and Ala 239 →Val 239 for ftsZ2863 ) are distributed throughout the FtsZ core region, and all confer a lethal cell division block at the nonpermissive temperature of 42°C. In each case the division block is associated with loss of Z-ring formation such that fewer than 2% of cells show Z rings at 42°C. The ftsZ9124 and ftsZ6460 mutations are of particular interest since both result in abnormal Z-ring formation at 30°C and therefore cause significant defects in FtsZ polymerization, even at the permissive temperature. Neither purified FtsZ9124 nor purified FtsZ6460 exhibited polymerization when it was assayed by light scattering or electron microscopy, even in the presence of calcium or DEAE-dextran. Hence, both mutations also cause defects in FtsZ polymerization in vitro. Interestingly, FtsZ9124 has detectable GTPase activity, although the activity is significantly reduced compared to that of the wild-type FtsZ protein. We demonstrate here that unlike expression of ftsZ84 , multicopy expression of the ftsZ6460 , ftsZ972 , and ftsZ9124 alleles does not complement the respective lethalities at the nonpermissive temperature. In addition, all five new mutant FtsZ proteins are stable at 42°C. Therefore, the novel isolates carrying single ftsZ (Ts) point mutations, which are the only such strains obtained since isolation of the classical ftsZ84 mutation, offer significant opportunities for further genetic characterization of FtsZ and its role in cell division.