A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus.

The human pathogenic bacteria Bacillus cereus, Bacillus anthracis and the entomopathogenic Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in live B. cereus cells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes during B. cereus sporulation. We used these markers to characterize a subpopulation of engulfment-defective B. cereus cells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis, the activity of σK is not strictly linked to the state of forespore development in B. cereus.

The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus.

The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates the interactions of spores with their environment, modulates spore adhesion and germination, and has been implicated in pathogenesis. In Bacillus cereus, the exosporium consists of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, surrounded by a hairy nap composed of glycoproteins. The morphogenetic protein CotE is necessary for the integrity of the B. cereus exosporium, but how CotE directs exosporium assembly remains unknown. Here, we used super-resolution fluorescence microscopy to follow the localization of SNAP-tagged CotE, CotY, and ExsY during B. cereus sporulation and evidenced the interdependencies among these proteins. Complexes of CotE, CotY, and ExsY are present at all sporulation stages, and the three proteins follow similar localization patterns during endospore formation that are reminiscent of the localization pattern of Bacillus subtilis CotE. We show that B. cereus CotE guides the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process, as previously proposed, but occurs through the formation of two caps, as observed during B. subtilis coat morphogenesis, suggesting that a general principle governs the assembly of the spore surface layers of BacillaceaeIMPORTANCE Spores of Bacillaceae are enveloped in an outermost glycoprotein layer. In the B. cereus group, encompassing the Bacillus anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance and separation from the underlying coat by an interspace. In spite of its importance for the environmental interactions of spores, including those with host cells, the mechanism of assembly of the exosporium is poorly understood. We used super-resolution fluorescence microscopy to directly visualize the formation of the exosporium during the sporulation of B. cereus, and we studied the localization and interdependencies of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaffold before a complete exosporium or coat is detectable. We describe how the different proteins localize to the scaffold and how they subsequently assemble around the spore, and we present a model for the assembly of the exosporium.

Post-replicative pairing of sister ter regions in Escherichia coli involves multiple activities of MatP.

The ter region of the bacterial chromosome, where replication terminates, is the last to be segregated before cell division in Escherichia coli. Delayed segregation is controlled by the MatP protein, which binds to specific sites (matS) within ter, and interacts with other proteins such as ZapB. Here, we investigate the role of MatP by combining short-time mobility analyses of the ter locus with biochemical approaches. We find that ter mobility is similar to that of a non ter locus, except when sister ter loci are paired after replication. This effect depends on MatP, the persistence of catenanes, and ZapB. We characterise MatP/DNA complexes and conclude that MatP binds DNA as a tetramer, but bridging matS sites in a DNA-rich environment remains infrequent. We propose that tetramerisation of MatP links matS sites with ZapB and/or with non-specific DNA to promote optimal pairing of sister ter regions until cell division.