ClpC-Mediated Sporulation Regulation at Engulfment Stage in Bacillus anthracis

Abstract

Bacterial sporulation is a conserved process utilized by members of Bacillus genus and Clostridium in response to stress such as nutrient or temperature. Sporulation initiation is triggered by stress signals perceived by bacterial cell that leads to shutdown of metabolic pathways of bacterial cells. The mechanism of sporulation involves a complex network that is regulated at various checkpoints to form the viable bacterial spore. Engulfment is one such check point that drives the required cellular rearrangement necessary for the spore assembly and is mediated by bacterial proteolytic machinery that involves association of various Clp ATPases and ClpP protease. The present study highlights the importance of degradation of an anti-sigma factor F, SpoIIAB by ClpCP proteolytic machinery playing a crucial role in culmination of engulfment process during the sporulation in Bacillus anthracis.

Keywords: Bacillus anthracis; ClpC; Engulfment; Proteolysis; Sporulation.

Fig. 1

Schematic representation of sporulation stages and mode of action of ClpCP proteolytic machinery in the activation of σF during engulfment stage of sporulation. The different stages of sporulation are represented as: (I) vegetative stage; (II) chain shortening during stress; (III) polar septation; (IV) engulfment of forespore by mother cell; (V) maturation of spore inside mother cell; (VI) mother cell lysis; (VII) release of mature spore. Figure projected within dotted arrows represent the activation of σF in forespore during engulfment stage by proteolysis of anti-sigma factor SpoIIAB by ClpCP proteolytic machinery. SpoIIAB associates with σF at the onset of sporulation leading to its inactivation. Anti-anti-sigma factor SpoIIAA then binds to this complex releasing SpoIIAB and sets σF free. SpoIIAB gets degraded by the ClpCP proteolytic complex while free σF binds to RNA Polymerase and activates the σF regulon resulting in the expression of downstream genes involved in spore engulfment

Fig. 2

Confocal micrographs showing the time course of sporulation in BAS-WT and BAS-ΔclpC strains at different time points (24 h, 48 h, 72 h). Cells of both the strains were diluted to a primary OD (A600nm) of 0.035 and 1 μl of cell suspension was spread on the agarose pads supplemented with sporulation media and FM4-64 membrane staining dye. The agarose pads were incubated at 30 °C and were imaged by Leica SP8 confocal microscope at above mentioned time points. Magnification = ×63; scale bar 10 μm in all the images

Fig. 3

Transmission electron micrographs depicting the ultrastructural details of spores formed by BAS-WT, BAS-ΔclpC, and BAS-ΔclpC::clpC strains. Sporulation was induced as described in the material and method section. BAS-WT spores show intact layers. However, BAS-ΔclpC spore images show a possible defect in engulfment, while this defect was rescued in BAS-ΔclpC::clpC strain. Black arrows represent the different layers of a mature spore: exosporium (ES), spore coat (SC), cortex (C), core wall (CW). MC mother cell, FS forespore. Red arrows denote the engulfment defect in BAS-ΔclpC strain. Magnification = ×15,000; the scale bar is represented on respective images

Fig. 4

Purification and antibody generation against recombinant SpoIIAB and GroEL. a Upper panel shows recombinant SpoIIAB purified by Ni–NTA affinity chromatography resolved on a SDS-PAGE and lower panel shows immunoblot of SpoIIAB using purified recombinant SpoIIAB protein. b Upper panel shows recombinant GroEL purified by Ni–NTA affinity chromatography resolved on a SDS-PAGE and lower panel shows immunoblot of GroEL using purified recombinant GroEL protein

Fig. 5

Expression levels of SpoIIAB in BAS-WT and BAS-ΔclpC strains. a Phase contrast images of sporulating BAS-WT and BAS-ΔclpC strains depicting the stage (t = 25 h) at which cells were harvested for lysate preparation to check the expression of SpoIIAB. The scale bar is represented on the respective image; Magnification = ×100. b Immunoblot representing the expression levels of SpoIIAB protein in both strains. Blots were then stripped and reprobed with anti-GroEL antibody. GroEL here acts as loading control. Representative image is from the one of four independent experiments. c Densitometric analysis of the immunoblots in panel b for the quantification of SpoIIAB expression in BAS-WT and BAS-ΔclpC strains. Each error bar denotes standard deviation where n = 4. Statistical significance was analysed using unpaired t-test and represented above the bars in the form of *. p value was reported to be 0.0008 and considered significant (p < 0.001 denotes***)