Fig. 4.

DynA and DynB interact with the cell division machinery. (A) Bacterial two-hybrid analysis of DynA and DynB with SsgA, SsgB, and the three SepF-like proteins. E. coli BTH101 cells carrying plasmids with protein fusion to the T18 and the T25 domain were spotted onto LB agar plates supplemented with Xgal, incubated at 30 °C for 24 h, and imaged. Corresponding β-galactosidase activities of three replicate experiments for each interaction are shown in Fig. S5B. (B) Yeast two-hybrid analysis showing the interaction between SepF and FtsZ. Interaction between proteins fused to the GAL4-activation domain (AD) and the GAL4 DNA-binding domain (BD) allows growth of yeast AH102 on minimal medium, lacking leucine, tryptophan, histidine, and alanine (−LWHA). In parallel, viability of the yeast cells carrying the respective hybrid proteins was confirmed by spotting cells on minimal medium without leucine and tryptophan (−LW). Representative results of three experiments are shown. (C) Colocalization of SepF-mCherry (SS208) and mCherry-SepF2 (SS213) with FtsZ-YPet. Microscopy results are representative of at least two independent experiments. (Scale bar: 2 μm.) (D) The protein interaction wheel between the dynamins and other divisome components based on two-hybrid results. (E) Proposed model of DynA and DynB function in Streptomyces. During sporulation-specific cell division, DynA and DynB form a complex at nascent division sites and interact with the division machinery via binding to SsgB and SepF2. In WT cells, Z-ring formation and cell envelope constriction leads to regular sporulation septum formation, resulting in equally sized, unigenomic spores. In the dynamin mutant, many Z-rings disassemble before completion of division septum synthesis, leading to failed or incomplete and asymmetric cell envelope constrictions and spores of irregular size with variable chromosome number.