ANALYSIS OF THE CELL DIVISION COMPLEX OF BACILLUS SUBTILIS USING GFP FLUORESCENCE MUCROSCOPY

Guilherme Louzada Silva Meira (USP); Frederico Jose Gueiros-filho (USP)

Bacillus subtilis undergoes binary fission, with the septum forming in the middle of a progenitor cell, resulting in genetically identical daughter cells. Septum formation in bacteria is carried out by a large protein complex known as the divisome. FtsZ (a tubulin homolog) self-assembles in a structure known as "Z ring" in the middle of the cell. The Z ring is the first event in divisome formation and it is responsible to recruit other division proteins to the divisome. The proteins that constitute the divisome complex are well known in the literature, but how the divisome is assembled remains unclear. In this work we have used fluorescence microscopy as a tool to study the kinetics of divisome formation. We created a red fluorescent FtsZ fusion (FtsZ-mCherry) and used it to measure the frequency of co-localization between the Z ring and five divisome proteins (ZapA, EzrA, DivIVA, YpsB, MinC), each individually fused to GFP. By measuring the frequency of co-localization between the Z ring and other division proteins we can establish the sequence of events during divisome assembly. We have found that ZapA, EzrA, YpsB, DivIVA and MinC co-localized with the Z-ring in 97%, 98%, 50%, 42%, 32% of the cells, respectively. The high frequency of co-localization with FtsZ suggests that ZapA and EzrA like to assemble into the divisome immediately after Z ring formation. On the other hand, YpsB, DivIVA and MinC, co-localize much less with the Z ring and, thus, seem to exhibit a delay in their association with the complex. The different co-localization frequencies among proteins suggest that the divisome does not assemble instantly. Instead, it seems to mature in a more gradual way, with each protein arriving at the complex with a different kinetics.