Division site selection in B. subtilis and characterization of YpbR, a bacterial dynamin

Cell division in Bacillus subtilis takes place precisely at midcell through the action of Noc, which prevents division from occurring over the nucleoids, and the Min system, which prevents cell division from taking place at the poles. Originally it was thought that the Min system acts directly on FtsZ, preventing the formation of a Z ring, and therefore the formation of a complete cytokinetic ring at the poles. Recently, a new component of the B. subtilis Min system was identified, MinJ, which acts as a bridge between DivIVA and MinCD. By using time lapse microscopy, it is shown here that MinJ moves from the poles to midcell prior to septation, and after this process is completed, moves back to the poles. This indicates that its main site of action is at midcell. Additionally, in the absence of a functional Min system, FtsA remains at the poles instead of being disassembled, which subsequently forms a double ring leading to minicell formation. Late divisome proteins FtsL and PBP-2B are also not disassembled from completed division sites. Previously it was described that FtsL and PBP-2B fail to localize in the absence of MinJ, which seems to lead to the filamentous phenotype of DminJ cells. In this thesis it was shown that this is due to dispersed MinCD, since in a DminCDJ mutant GFP-PBP-2B and GFP-FtsL localize to midcell again, although they are also found at the poles. MinJ mutants were isolated that are able to complement the cell length phenotype of DminJ cells, but lead to an increased production of minicells, showing that in cells expressing these mutants cytokinesis is not impaired but division site selection is. Additionally, it was shown that overexpression of MinD in the absence of MinJ leads to lethal filamentation in B. subtilis, although cytoplasmic components of the cytokinetic ring are able to localize in these cells. In the absence of a functional Min system, cells contain multiple FtsA rings, indicating an impairment in cell division efficiency. Taken together, these results show that i) the Min system is involved in the disassembly of the divisome, rather than preventing its assembly at the poles, which also explains its preferential localization to midcell prior to septation, ii) the failure to disassemble the divisome leads to minicell formation, iii) the Min system is needed for efficient cell division, and iv) MinJ is able to regulate MinCD activity, possibly by restricting its activity to certain sites. Using a bacterial two-hybrid screen, it was found that MinJ interacts with YpbR. YpbR is a large protein containing two GTPase domains and shows homology to the dynamin-like proteins. Although YpbR does not appear to be important for growth, morphology, or sporulation, it was shown that cells deficient in YpbR are less susceptible to antibiotics than wild type. Also, under salt stress, DypbR cells show an altered morphology of septa. This indicates that YpbR probably plays a role in stabilizing the membrane under conditions that impart stress to the lipid membrane. YpbR-GFP is localized to the membrane and forms foci, but when cells are exposed to high concentrations of NaCl, YpbR-GFP forms a uniform structure on the membrane. Also, the first GTPase domain alone (GTPase1) is able to localize in a similar pattern as wild type and forms a uniform structure on the membrane under salt stress, but the second GTPase domain is diffuse throughout the cell. Using mutants that are unable to hydrolyze GTPase with the first GTPase domain (K56A) or the second (K625A), it was shown that the localization to the membrane and subsequent change of localization under salt stress is not dependent on the GTP binding. Under normal conditions, YpbR-GFP and YpbR mutant proteins are found mostly in the membrane fraction but also somewhat in the cytoplasmic fraction, but under salt stress all of the proteins are found mostly in the membrane fraction. It was also shown that YpbR-GFP localizes to spores early in sporulation and its overexpression increases sporulation efficiency. It is postulated that YpbR is mostly involved in stabilizing the membrane or involved in membrane dynamics, but is not essential in B. subtilis