ABSTRACT
Yeast two-hybrid system was modified to allow easy detection of prokaryotic protein-protein interactions. Three plasmids (pGBR1, pGBR2, pGBR3) with the ClaI restriction site shifted in the three possible reading frames in fusion with GAL4 activating domain were constructed. The modified plasmids were used for identification of protein partners of FtsZ from Bacillus subtilis. Among partners of FtsZ the FtsA protein and a globular part of the SpoIIE protein were identified. The protein interactions were quantified by measurements of beta-galactosidase activity in yeast cells using 4-methylumbelliferyl beta-D-galactopyranoside as fluorogenic substrate.
Subject(s)
Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Cytoskeletal Proteins , Two-Hybrid System Techniques , Bacterial Proteins/genetics , Genomic Library , Plasmids/genetics , Protein Binding , beta-Galactosidase/metabolismABSTRACT
Mutation of the divIVB locus in Bacillus subtilis causes misplacement of the septum during cell division and allows the formation of anucleate minicells. The divIVB locus contains five open reading frames (ORFs). The last two ORFs (minCD) are homologous to minC and minD of Escherichia coli but a minE homolog is lacking in B. subtilis. There is some similarity between minicell formation and the asymmetric septation that normally occurs during sporulation in terms of polar septum localization. However, it has been proposed that MinCD has no essential role in sporulation septum formation. We have used electron microscopic studies to show septation events during sporulation in some minD strains. We have observed an unusually thin septum at the midcell position in minD and also in minD spoIIE71 mutant cells. Fluorescence microscopy also localized a SpoIIE-green fluorescent protein fusion protein at the midcell site in minD cells. We propose that the MinCD complex plays an important role in asymmetric septum formation during sporulation of B. subtilis cells.
