The Spore Differentiation Pathway in the Enteric Pathogen Clostridium difficile

Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σ^F in the forespore, and σ^E in the mother cell control early stages of development and are replaced, at later stages, by σ^G and σ^K, respectively. Starting with σ^F, the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σ^E is partially independent of σ^F, that σ^G activity is not dependent on σ^E, and that the activity of σ^K does not require σ^G. We also show that σ^K is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σ^F-to-σ^E, σ^E-to-σ^G, and σ^G-to-σ^K cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.     

Expression of the sigmaF-directed csfB locus prevents premature appearance of sigmaG activity during sporulation of Bacillus subtilis

During sporulation, σ^G becomes active in the prespore upon the completion of engulfment. We show that the inactivation of the σ^F-directed csfB locus resulted in premature activation of σ^G. CsfB exerted control distinct from but overlapping with that exerted by LonA to prevent inappropriate σ^G activation. The artificial induction of csfB severely compromised spore formation.     

Coupling of σG Activation to Completion of Engulfment during Sporulation of Bacillus subtilis Survives Large Perturbations to DNA Translocation and Replication

Spore formation in Bacillus subtilis is characterized by activation of RNA polymerase sigma factors, including the late-expressed σ^G. During spore formation an asymmetric division occurs, yielding the smaller prespore and the larger mother cell. At division, only 30% of the chromosome is in the prespore, and the rest is then translocated into the prespore. Following completion of engulfment of the prespore by the mother cell, σ^G is activated in the prespore. Here we tested the link between engulfment and σ^G activation by perturbing DNA translocation and replication, which are completed before engulfment. One approach was to have large DNA insertions in the chromosome; the second was to have an impaired DNA translocase; the third was to use a strain in which the site of termination of chromosome replication was relocated. Insertion of 2.3 Mb of Synechocystis DNA into the B. subtilis genome had the largest effect, delaying engulfment by at least 90 min. Chromosome translocation was also delayed and was completed shortly before the completion of engulfment. Despite the delay, σ^G became active only after the completion of engulfment. All results are consistent with a strong link between completion of engulfment and σ^G activation. They support a link between completion of chromosome translocation and completion of engulfment.     

A Conserved Cysteine Residue of Bacillus subtilis SpoIIIJ Is Important for Endospore Development

During sporulation in Bacillus subtilis, the onset of activity of the late forespore-specific sigma factor σ^G coincides with completion of forespore engulfment by the mother cell. At this stage, the forespore becomes a free protoplast, surrounded by the mother cell cytoplasm and separated from it by two membranes that derive from the asymmetric division septum. Continued gene expression in the forespore, isolated from the surrounding medium, relies on the SpoIIIA-SpoIIQ secretion system assembled from proteins synthesised both in the mother cell and in the forespore. The membrane protein insertase SpoIIIJ, of the YidC/Oxa1/Alb3 family, is involved in the assembly of the SpoIIIA-SpoIIQ complex. Here we show that SpoIIIJ exists as a mixture of monomers and dimers stabilised by a disulphide bond. We show that residue Cys134 within transmembrane segment 2 (TM2) of SpoIIIJ is important to stabilise the protein in the dimeric form. Labelling of Cys134 with a Cys-reactive reagent could only be achieved under stringent conditions, suggesting a tight association at least in part through TM2, between monomers in the membrane. Substitution of Cys134 by an Ala results in accumulation of the monomer, and reduces SpoIIIJ function in vivo. Therefore, SpoIIIJ activity in vivo appears to require dimer formation.     

A Recombination Directionality Factor Controls the Cell Type-Specific Activation of σK and the Fidelity of Spore Development in Clostridium difficile

The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages of sporulation require the mother cell regulatory protein σ^K. In Bacillus subtilis, the onset of σ^K activity requires both excision of a prophage-like element (skin^Bs) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skin^Bs recombinase is produced specifically in this cell. In C. difficile, σ^K lacks a pro-sequence but a skin^Cd element is present. The product of the skin^Cd gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skin^Cd excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skin^Cd excision is under the control of mother cell regulatory proteins σ^E and SpoIIID. We then demonstrate that σ^E and SpoIIID control the expression of the skin^Cd gene CD1234, and that this gene is required for sporulation and skin^Cd excision. CD1231 and CD1234 appear to interact and both proteins are required for skin^Cd excision while only CD1231 is necessary for skin^Cd integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skin^Cd excision to the terminal mother cell. Finally, while the skin^Cd element is not essential for sporulation, deletion of skin^Cd results in premature activity of σ^K and in spores with altered surface layers. Thus, skin^Cd excision is a key element controlling the onset of σ^K activity and the fidelity of spore development.