Cardiolipin-Containing Lipid Membranes Attract the Bacterial Cell Division Protein DivIVA.

DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system during bacterial cell division, DivIVA is involved in chromosome segregation during sporulation, genetic competence, and cell wall synthesis. DivIVA localizes to regions of high membrane curvature, such as the cell poles and cell division site, where it recruits distinct binding partners. Previously, it was suggested that negative curvature sensing is the main mechanism by which DivIVA binds to these specific regions. Here, we show that Clostridioides difficile DivIVA binds preferably to membranes containing negatively charged phospholipids, especially cardiolipin. Strikingly, we observed that upon binding, DivIVA modifies the lipid distribution and induces changes to lipid bilayers containing cardiolipin. Our observations indicate that DivIVA might play a more complex and so far unknown active role during the formation of the cell division septal membrane.

Clostridial DivIVA and MinD interact in the absence of MinJ.

One of the key regulators ensuring proper Z-ring placement in rod-shaped bacteria is the Min system. It does so by creating a concentration gradient of the MinC septation inhibitor along the cell axis. In Escherichia coli, this gradient is established by a MinE-mediated pole-to-pole oscillation of the MinCDE complex. In Bacillus subtilis, the creation of an inhibitory gradient relies on the MinJ and DivIVA pair of topological determinants, which target MinCD to the newly formed cell poles. Introducing the E. coli oscillating Min system into B. subtilis leads to a sporulation defect, suggesting that oscillation is incompatible with sporulation. However, Clostridia, close endospore-forming relatives of Bacilli, do encode oscillating Min homologues in various combinations together with homologues from the less dynamic B. subtilis Min system. Here we address the questions of how these two systems could exist side-by-side and how they influence one another by studying the Clostridium beijerinckii and Clostridium difficile Min systems. The toolbox of genetic manipulations and fluorescent protein fusions in Clostridia is limited, therefore B. subtilis and E. coli were chosen as heterologous systems for studying these proteins. In B. subtilis, MinD and DivIVA interact through MinJ; here, however, we discovered that the MinD and DivIVA proteins of both C. difficile, and C. beijerinckii, interact directly, which is surprising in the latter case, since that organism also encodes a MinJ homologue. We confirm this interaction using both in vitro and in vivo methods. We also show that C. beijerinckii MinJ is probably not in direct contact with DivIVACb and, unlike B. subtilis MinJ, does not mediate the MinDCb and DivIVACb interaction. Our results suggest that the Clostridia Min system uses a new mechanism of function.

Asymmetric cell division during Bacillus subtilis sporulation.

Bacillus subtilis is a rod-shaped bacterium which divides precisely at mid-cell during vegetative growth. Unlike Escherichia coli, another model organism used for studying cell division, B. subtilis can also divide asymmetrically during sporulation, the simplest cell differentiation process. The asymmetrically positioned sporulation septum serves as a morphological foundation for establishing differential gene expression in the smaller forespore and larger mother cell. Both vegetative and sporulation septation events are fine-tuned with cell cycle, and placement of both septa are highly precise. We understand in some detail how this is achieved during vegetative growth but have limited information about how the asymmetric septation site is determined during sporulation.

Oscillating behavior of Clostridium difficile Min proteins in Bacillus subtilis.

In rod-shaped bacteria, the proper placement of the division septum at the midcell relies, at least partially, on the proteins of the Min system as an inhibitor of cell division. The main principle of Min system function involves the formation of an inhibitor gradient along the cell axis; however, the establishment of this gradient differs between two well-studied gram-negative and gram-positive bacteria. While in gram-negative Escherichia coli, the Min system undergoes pole-to-pole oscillation, in gram-positive Bacillus subtilis, proper spatial inhibition is achieved by the preferential attraction of the Min proteins to the cell poles. Nevertheless, when E.coli Min proteins are inserted into B.subtilis cells, they still oscillate, which negatively affects asymmetric septation during sporulation in this organism. Interestingly, homologs of both Min systems were found to be present in various combinations in the genomes of anaerobic and endospore-forming Clostridia, including the pathogenic Clostridium difficile. Here, we have investigated the localization and behavior of C.difficile Min protein homologs and showed that MinDE proteins of C.difficile can oscillate when expressed together in B.subtilis cells. We have also investigated the effects of this oscillation on B.subtilis sporulation, and observed decreased sporulation efficiency in strains harboring the MinDE genes. Additionally, we have evaluated the effects of C.difficile Min protein expression on vegetative division in this heterologous host.