The Clostridium sporulation programs: diversity and preservation of endospore differentiation

Abstract

Bacillus and Clostridium organisms initiate the sporulation process when unfavorable conditions are detected. The sporulation process is a carefully orchestrated cascade of events at both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcription factors, proteases, and phosphatases. Like Bacillus genomes, sequenced Clostridium genomes contain genes for all major sporulation-specific transcription and sigma factors (spo0A, sigH, sigF, sigE, sigG, and sigK) that orchestrate the sporulation program. However, recent studies have shown that there are substantial differences in the sporulation programs between the two genera as well as among different Clostridium species. First, in the absence of a Bacillus-like phosphorelay system, activation of Spo0A in Clostridium organisms is carried out by a number of orphan histidine kinases. Second, downstream of Spo0A, the transcriptional and posttranslational regulation of the canonical set of four sporulation-specific sigma factors (σ(F), σ(E), σ(G), and σ(K)) display different patterns, not only compared to Bacillus but also among Clostridium organisms. Finally, recent studies demonstrated that σ(K), the last sigma factor to be activated according to the Bacillus subtilis model, is involved in the very early stages of sporulation in Clostridium acetobutylicum, C. perfringens, and C. botulinum as well as in the very late stages of spore maturation in C. acetobutylicum. Despite profound differences in initiation, propagation, and orchestration of expression of spore morphogenetic components, these findings demonstrate not only the robustness of the endospore sporulation program but also the plasticity of the program to generate different complex phenotypes, some apparently regulated at the epigenetic level.

FIG 1

Formation of spores by endospore-forming bacteria. Upon sensing unfavorable environmental conditions, the cells begin the process of differentiation and spore morphogenesis. The clostridial cell form is seen with its cigar-shaped structure, and granulose vesicles accumulate. The cells then initiate the sporulation process, and asymmetric division occurs, giving rise to the mother cell and prespore compartments. The mother cell then engulfs the prespore; subsequently, the spore cortex begins to develop around the prespore, and pieces of the spore coat form. Once the membranes of the spore are completed, the mother cell lyses, thus releasing the mature endospore. Under favorable conditions, the spore will germinate to give rise to a vegetative cell.

FIG 2

Activation of Spo0A via phosphorylation in B. subtilis (A), C. difficile (B), C. acetobutylicum (C), C. botulinum (D), and C. thermocellum (E). In Bacillus, the phosphorylation process is initiated once the orphan HKs KinA and/or KinB phosphorylates Spo0F, the first component of the phosphorelay system that leads to Spo0A phosphorylation. Based on all Clostridium organisms sequenced so far, there is no evidence that they have a recognizable phosphorelay system. Instead, several orphan HKs were shown to directly transfer a phosphate group to Spo0A, thus activating it. Additionally, it was shown that the orphan HK CAC0437 in C. acetobutylicum has dephosphorylation activity and is thus able to remove the phosphate group from Spo0A, thus rendering it inactive. Clo1313_1973 in C. thermocellum may also have the ability to inactivate Spo0A, although direct evidence of dephosphorylation activity has yet to be obtained.

FIG 3

Comparative summary of the sporulation signaling cascade in Bacillus (A) versus Clostridium (B), involving the master transcriptional regulator Spo0A as well as the major sporulation-specific sigma factors up- and downstream of Spo0A. This model does not apply to C. difficile, as its sporulation program is different from those of the other studied Clostridium organisms, as discussed in the text. Approximate cellular phenotypes for the Clostridium model of differentiation are shown on the right. While similar stages take place in Bacillus, the timing is different (Fig. 5). Several notable differences in the regulation of sporulation between these species have already been discovered. First, in the Bacillus model, Spo0A is phosphorylated (Spo0A∼P) by a phosphorelay system initiated by orphan HKs, mainly KinA and KinB (Fig. 2). Once activated, Bacillus Spo0A∼P initiates the sporulation sigma factor cascade involving four downstream sigma factors (σ^F, σ^E, σ^G, and σ^K). In Clostridium, no phosphorelay system is present. Rather, orphan HKs phosphorylate Spo0A directly (Fig. 2). Second, the last sigma factor in the Bacillus model, σ^K, was shown to play a dual role in Clostridium, one early, upstream of Spo0A, and another late, downstream of σ^G, which is analogous to its role in Bacillus. As the Clostridium model is further refined, additional differences between these species are expected. Green genes indicate confirmed functional roles in B. subtilis. Red genes denote functional roles confirmed by gene inactivation in Clostridium. Blue genes indicate genes with a presumed function in Clostridium, based on homology to B. subtilis, gene organization, and consistent phenotypic evidence. Gray ovals denote suspected protein interactions. A single red question mark denotes suspected transcriptional activity. The dashed arrow for σ^K in the Clostridium model indicates an unknown pathway for early σ^K activity.

FIG 4

Transcriptional control and activation of sigH, spo0A, and the spoIIAA operon in C. difficile. Expression of sigH is initially driven from a σ^A-dependent promoter. Once translated, σ^H regulates the expression of spo0A, the orphan HK CD2492, and the spoIIAA operon, where sigF resides. Once expressed, CD2492, along with other HKs, directly phosphorylates Spo0A, thus activating it. Subsequently, Spo0A∼P augments the expression of sigH and the spoIIAA operon, thus initiating the sigma factor sporulation cascade.

FIG 5

TEM images comparing the various sporulation-specific sigma factor mutants generated in B. subtilis and Clostridium organisms, including C. acetobutylicum, C. difficile, C. perfringens, and C. botulinum. In the sigK deletion mutant of C. acetobutylicum, sporulation was blocked prior to stage II, which is also true for the C. acetobutylicum sigF, spoIIE, and sigE disruption mutants as well as the sigK mutant of C. perfringens. However, the sigK mutant of C. difficile appeared to progress further in sporulation, and a developing forespore appears to be present. On the other hand, the sigF, spoIIE, and sigE disruption mutants of B. subtilis were all able to develop asymmetric septa and exhibited a disporic phenotype. The sigG disruption mutant generated in C. acetobutylicum appeared to progress further into the sporulation pathway than its counterpart in B. subtilis due to the presence of what appears to be a spore coat and cortex, which were not visible in the sigG mutant of B. subtilis. (For an image of a B. subtilis σ^G mutant, see Fig. 2 in reference .) Expression of Spo0A in the sigK deletion mutant of C. acetobutylicum showed that the cells were able to progress further in spore morphogenesis than the sigG mutant; however, they were still unable to form viable spores, and the spore coat and cortex appeared to be ill formed, thus indicating that σ^K is also needed during the last stages of sporulation. (Images of the B. subtilis σ^F mutant, SpoIIE mutant, and σ^E mutant reprinted from reference with permission; image of the C. acetobutylicum σ^F mutant reprinted from reference ; image of the C. acetobutylicum SpoIIE mutant reprinted from reference ; images of the C. acetobutylicum σ^E mutant and σ^G mutant reprinted from reference ; image of the C. acetobutylicum σ^K mutant reprinted from reference ; images of the C. difficile σ^F mutant, σ^E mutant, σ^G mutant, and σ^K mutant reprinted from reference ; images of the C. perfringens σ^E mutant and σ^K mutant reprinted from reference with permission; images of the C. botulinum σ^F mutant, σ^E mutant, and σ^G mutant reprinted from reference with permission.)

FIG 6

Transcriptional and posttranslational control of σ^F, σ^E, and σ^G in C. acetobutylicum. Initial transcriptional expression of the spoIIAA operon is regulated by Spo0A∼P and σ^H. Once translated, σ^F is held inactive by the sequestrating actions of SpoIIAB while also inhibiting SpoIIAA by phosphorylating it. To release σ^F from inhibition, the membrane-bound SpoIIE dephosphorylates SpoIIAA∼P, resulting in its binding to SpoIIAB, thus releasing σ^F from inhibition. Once activated, σ^F autoregulates its own expression as well as those of both sigG and sigE. Initially, σ^E, the mother cell-specific sigma factor, is translated in the inactive form, pro-σ^E, which is subsequently activated by the cleavage of the prosequence by the membrane-bound protease SpoIIGA. In the forespore compartment, σ^G is translated, but its actions are hypothesized to be inhibited by a yet-to-be-determined factor analogous to σ^F inhibitions.

FIG 7

The concept of bistable phenotypes, the basis of bistability (sporulate versus do not sporulate in B. subtilis), and the proposed basis for bistable phenotypes in C. acetobutylicum. (A) The three key elements that lead to bistable phenotypes: a positive feedback circuit with embedded nonlinearities (shown by the red twisting lines) and a good balance in the two arms of the circuit. (B) These key requirements are present in the B. subtilis model of sporulation: on the right, it is the “down” positive but highly nonlinear control of Spo0A phosphorylation through the phosphorylation relay, and on the left, there are several “up” positive feedback circuits, with two resulting from the direct transcriptional stimulation of spo0A and spo0F by activated (phosphorylated) Spo0A. Two other loops are derived from the transcriptional stimulation of kinA expression by phosphorylated Spo0A via two negative feedback loops involving AbrB and σ^H. Additional such positive feedback loops exist in the B. subtilis model. (C) Cartoon displaying the two feedback loops from Spo0A to σ^K, which we propose are the basis for the observed bistable phenotypes in C. acetobutylicum, as discussed in the text.