Imaging DivIVA dynamics using photo-convertible and activatable fluorophores in Bacillus subtilis

Abstract

Most rod-shape model organisms such as Escherichia coli or Bacillus subtilis utilize two inhibitory systems for correct positioning of the cell division apparatus. While the nucleoid occlusion system acts in vicinity of the nucleoid, the Min system was thought to protect the cell poles from futile division leading to DNA-free miniature cells. The Min system is composed of an inhibitory protein, MinC, which acts at the level of the FtsZ ring formation. MinC is recruited to the membrane by MinD, a member of the MinD/ParA family of Walker-ATPases. Topological positioning of the MinCD complex depends on MinE in E. coli and MinJ/DivIVA in B. subtilis. While MinE drives an oscillation of MinCD in the E. coli cell with a time-dependent minimal concentration at midcell, the B. subtilis system was thought to be stably tethered to the cell poles by MinJ/DivIVA. Recent developments revealed that the Min system in B. subtilis mainly acts at the site of division, where it seems to prevent reinitiation of the division machinery. Thus, MinCD describe a dynamic behavior in B. subtilis. This is somewhat inconsistent with a stable localization of DivIVA at the cell poles. High resolution imaging of ongoing divisions show that DivIVA also enriches at the site of division. Here we analyze whether polar localized DivIVA is partially mobile and can contribute to septal DivIVA and vice versa. For this purpose we use fusions with green to red photoconvertible fluorophores, Dendra2 and photoactivatable PA-GFP. These techniques have proven very powerful to discriminate protein relocalization in vivo. Our results show that B. subtilis DivIVA is indeed dynamic and moves from the poles to the new septum.

Asymmetric Division and Differential Gene Expression during a Bacterial Developmental Program Requires DivIVA

“…It may, for example, involve the selective degradation of DivIVA on the mother cell face of the polar septum or may involve a transfer of pole-localized DivIVA molecules to the forespore-proximal side of the polar septum which we were unable to measure using our current experimental setup. Such a transfer has indeed been recently reported for vegetative septa, which may occur in 5–20 mins [79]. Curiously, the presence of Min proteins, which are typically recruited by DivIVA, on the forespore-proximal face of the polar septum has been implicated in maintaining the polarity of DNA translocation [80].…”

Dynamics of theBacillus subtilisMin system

“…DivIVA has previously been reported as static [75], however those FRAP experiments were carried out using overexpression strains and a much shorter timeframe than here. Earlier observation from our own lab using a merodiploid strain already suggested that DivIVA is dynamic [62]. Roughly, two thirds of DivIVA molecules were participating in dynamics.…”

“…Since this did not meet the set standards for this study, we turned towards strain 1803 [74], carrying a divIVA-GFP copy with its native promoter in the ectopic amyE locus. While DivIVA-GFP has been shown to not fully complement a ΔdivIVA strain [74, 75], it still localizes correctly and can be used for studies of DivIVA dynamics [62, 75]. Additionally, we performed FRAP on DivIVA-mNeonGreen, which only shows a mild phenotype ( Tab.…”

“…We have recently analyzed DivIVA dynamics in B. subtilis and found that Min proteins redistribute from the cell poles to midcell as soon as a septum is formed [62], which prompted us to reanalyze Min protein dynamics in this organism. To this end, we generated a set of new fusions to DivIVA, MinD and MinJ.…”

Crystal structure of the membrane anchoring domain of mycobacterial Wag31: a dimer-of-dimer suggests how a Wag31 filament might self-assemble

“…Prominent examples of positive curvature sensing proteins include septins and BAR (Bin/amphiphysin/Rvs) domain proteins in eukaryotes and SpoVM in sporulating bacteria (Cannon et al, 2017). In gram positive bacteria, DivIVA recognizes negative, concave membrane curvature, and self-assembles at the cytoplasmic side of the pole and curved region of the cell division septum to form a structural scaffold (Edwards and Errington, 1997;Letek et al, 2008;Lenarcic et al, 2009;Ramamurthi and Losick, 2009;Kaval and Halbedel, 2012;Bach et al, 2014). DivIVA, which is a filamentous, coiled-coil protein, aids in the localizations of various target proteins at the pole for cell-wall growth and a variety of biological functions (Rudner and Losick, 2010;Laloux and Jacobs-Wagner, 2014;Halbedel and Lewis, 2019;Hammond et al, 2019).…”