IMMUNOGOLD LOCALIZATION OF GYRA AND CYRB PROTEINS IN ESCHERICHIA-COLI

Immunogold preparations of Escherichia coli, using anti-GyrA and anti- GyrB antibodies to the subunits of DNA gyrase. showed clear labelling with both secondary antibody and protein A-gold conjugates. Both prote ins were located mainly in the cytoplasm, with typically less than 10% in the nucleoid. This partitioning of gyrase proteins between nucleoi d and cytoplasm was nonrandom and was consistently observed for a rang e of different cell preparations. Total gold particle counts were high ly variable but suggested levels of at least 1000-3000 molecules per c ell for both GyrA and GyrB. Sequential treatment with both anti-GyrA a nd anti-GyrB monoclonal antibodies resulted in simultaneous labelling of both proteins and revealed no clear association between the two gro ups of molecules. Treatment of cells with chloramphenicol caused marke d changes in nucleoid conformation, but no reduction in cytoplasmic la belling of gyrase proteins. On the assumption that gyrase complexes wi thin the nucleoid are not differentially masked from the monoclonal an tibodies, the results obtained in this study suggest that most of the gyrase proteins are not associated with either central nucleoid DNA or cytoplasmic loops of peripheral single-stranded DMA, but are distribu ted randomly throughout the cytoplasm.