Crucial role of SpoIIE during Bacillus subtilis cell differentiation

 

Z. Chromiková, K. Muchová,, I. Barák

 

Institute of molecular biology, Slovak academy of sciences, Dúbravská cesta 21, 845 51 Bratislava 45, Slovakia

 

One of the characteristics of sporulation process in B. subtilis is asymmetric division of the cell, which leads to formation of two unequally sized compartments. Despite the fact that they contain the same genetic information, these compartments follow separate fates, due to dissimilar gene expression driven by compartment-specific sigma factors. While the smaller compartment, the prespore develops into heat-resistant spore, the bigger compartment, the mother cell nourishes the smaller prespore and finally lyses.

Proper positioning of the asymmetric septum is crucial for the process of sporulation. It is partially triggered by sporulation specific protein, SpoIIE, an exclusive component of the sporulation septum. SpoIIE closely cooperates with tubuline-like protein FtsZ. FtsZ forms higher organized structures, so called Z-rings, which constitute a scaffold for assembly of division machinery. During sporulation, FtsZ forms helical structures emanating from midcell Z ring towards cell poles. SpoIIE, which also forms ring-like structures, most likely stabilizes FtsZ helices at polar positions, thus polar Z-rings are formed [1].

SpoIIE is 827-residue protein, which is thought to consist of three domains. Its N-terminal domain, which contains ten transmembrane helices, anchors the protein into the sporulation septum [2, 3]. The central domain of SpoIIE is conserved only among SpoIIE orthologues. This domain is thought to be the regulation domain of the protein. Central domain is considered to be involved in dimerization of SpoIIE and it is also responsible for interaction of the protein with FtsZ [4]. SpoIIE C-terminal domain is closely related to PP2C domains of eukaryotic Ser/Thr phosphatases, which regulate the stress response [5]. This domain is responsible for activation of the first compartment-specific sigma factor, σF, in the prespore. Activity of σF is regulated by the means of interactions between an anti- sigma factor SpoIIAB and an anti-anti-sigma factor SpoIIAA and SpoIIE phosphatase, resident in the sporulation septum. In the predivisional cell and in the mother cell, σF is held inactive, in the complex with two molecules of SpoIIAB. In the presence of dephosphorylated SpoIIAA, SpoIIAA -SpoIIAB complex is preferably formed thus σF is released and active. Since SpoIIAA is phosphorylated by SpoIIAB kinase and released from this complex, SpoIIE phosphatase activity is critical to maintain the level of dephosphorylated SpoIIAA molecules in the prespore to keep σF active, and able to promote prespore-specific gene expression [8,9]. The structures of the SpoIIA proteins, respectively their domains, have been determined, revealing the phosphorylated and dephosphorylated forms of SpoIIAA [14,15], as well as the interactions of SpoIIAB with σF and SpoIIAA [9,16]. Previously, using a random truncation library approach [17], a set of soluble SpoIIE fragments was identified. Out of these fragments, SpoIIE fragment (590-827) encompassing the PP2C phoshatase domain was over-expressed, characterized, and its crystal structure was determined [18].

Multidomain composition of the protein allows its multiple roles during the process of sporulation. The key regulator SpoIIE has at least three major roles in the process of sporulation. The first role of SpoIIE is to stabilize FtsZ spiral intermediates at polar positions and co-localize with FtsZ in the forming sporulation septum. The second role of SpoIIE lies in its contribution to prespore-specific σF activation, which is an event linked to the completion of sporulation septum. Consequent compartment specific gene expression is arranged by the activity of cell-type specific σ factors, which sequentially activate each other in a cascade-like manner [6]. Dynamic localization studies using SpoIIE-GFP fusions have shown that after completion of sporulation septum, SpoIIE is released from the septum and transiently localizes to all membranes in the forespore compartment. However, after engulfment is initiated, SpoIIE relocalizes to the septal site of the membrane, indicating possible role of SpoIIE in the later stages of sporulation. It was also shown that SpoIIE relocalization to the engulfing septum is dependent on SpoIIQ, the protein which is a part of the channel, connecting prespore and mother cell compartments. Moreover, immunoprecipitation experiments brought the evidence that SpoIIE and SpoIIQ reside in a membrane complex together [7].

Although three roles of SpoIIE are described, not all mechanisms of its actions are fully understood. Moreover, SpoIIE assignment in the stage of engulfment is very little understood. To answer these issues, we gather further information by screening interactions between SpoIIE and proteins taking part in cell division and engulfment by bacterial two hybrid system.

This work was supported by Grant 2/0009/13 from the Slovak Academy of Sciences and by a Grant from the Slovak Research and Development Agency under contract APVV-00335-10.

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