The SpoIIQ‐SpoIIIAH complex of Clostridium difficile controls forespore engulfment and late stages of gene expression and spore morphogenesis

Abstract

SummaryEngulfment of the forespore by the mother cell is a universal feature of endosporulation. In Bacillus subtilis, the forespore protein SpoIIQ and the mother cell protein SpoIIIAH form a channel, essential for endosporulation, through which the developing spore is nurtured. The two proteins also form a backup system for engulfment. Unlike in B. subtilis, SpoIIQ of Clostridium difficile has intact LytM zinc‐binding motifs. We show that spoIIQ or spoIIIAH deletion mutants of C. difficile result in anomalous engulfment, and that disruption of the SpoIIQ LytM domain via a single amino acid substitution (H120S) impairs engulfment differently. SpoIIQ and SpoIIQH120S interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQH120S, binds Zn2+, and metal absence alters the SpoIIQ‐SpoIIIAH complex in vitro. Possibly, SpoIIQH120S supports normal engulfment in some cells but not a second function of the complex, required following engulfment completion. We show that cells of the spoIIQ or spoIIIAH mutants that complete engulfment are impaired in post‐engulfment, forespore and mother cell‐specific gene expression, suggesting a channel‐like function. Both engulfment and a channel‐like function may be ancestral functions of SpoIIQ‐SpoIIIAH while the requirement for engulfment was alleviated through the emergence of redundant mechanisms in B. subtilis and related organisms.

Differential requirements for conserved peptidoglycan remodeling enzymes during Clostridioides difficile spore formation

“…While our 630∆ erm ∆ spoIIQ strain generated heat‐resistant spores less efficiently than wildtype (~0.05%), loss of IIQ in this strain background has been previously reported to prevent heat‐resistant spore formation (Dembek et al ., ; Serrano et al ., ). This discrepancy is likely due to differences in the sporulation method used (plate‐based vs. liquid), since comparison of the heat resistance of sporulating ∆ IIQ in liquid vs. plate culture revealed a 20‐fold more severe defect during growth in broth with agitation relative to growth on plates (Fig.…”

A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis

“…Interestingly, recent work in the endospore former Clostridium difficile suggests that the A-Q complex may be dispensable for late forespore gene expression under SigG control and therefore may not play a direct role in maintaining transcriptional potential in the C. difficile forespore (8,9). Intriguingly, both studies identified morphological defects in spore differentiation in the absence of the A-Q complex, suggesting that the complex could play a structural role in maintaining forespore development.…”

“…1B) (3). Intriguingly, the mother cell and forespore assemble a multimeric complex spanning these two membranes that bears similarity to specialized secretion systems and is required to maintain forespore development (4)(5)(6)(7)(8)(9). It has been proposed that these proteins constitute a hybrid specialized secretion system with a channel connecting mother cell and forespore.…”

Cell-wall remodeling drives engulfment during Bacillus subtilis sporulation

“…For instance, the SpoIIQ-AH zipper, which is dispensable for engulfment in native conditions (Broder and Pogliano, 2006), might prevent membrane backward movement, and might also help localize the IDC components toward the LE. Interestingly, SpoIIQ-AH interaction is essential for engulfment in Clostridium difficile where the SpoIIQ ortholog posseses endopeptidase activity (Crawshaw et al, 2014; Serrano et al, 2016; Fimlaid et al, 2015). The model also does not consider the impact of the tethering of the LE of the engulfing membrane to the forespore via interactions between the mother cell membrane anchored DMP complex at the LE and forespore synthesized PG.…”