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

Abstract

Engulfment 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 SpoIIQ(H120S) interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQ(H120S) , binds Zn(2+) , and metal absence alters the SpoIIQ-SpoIIIAH complex in vitro. Possibly, SpoIIQ(H120S) 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.

Figure 1

Sporulation and the SpoIIQ‐SpoIIIAH channel. A. Schematic representation of the main stages of sporulation. Asymmetric cell division at one of the poles gives rise to the small daughter cell or forespore, side‐by‐side with the mother cell (I). As sporulation progresses, the mother cell engulfs the forespore within its cytoplasm. In B. subtilis, the DMP protein complex (green) is essential for hydrolysis of PG within the septum during the initial stages of engulfment (II‐IV) (see text for details; see also panel B). In B. subtilis and presumably other sporeformers, SpoIIQ (blue) and SpoIIIAH (yellow), together with additional spoIIIA‐encoded proteins (not represented), are also involved in engulfment and required for sporulation (III‐V). Spore maturation involves the formation of two main protective layers (the cortex PG and the coats) in the intermembrane space (VI). It is not known whether the SpoIIQ‐SpoIIIAH channel is maintained at these late stages in Clostridia and other endosporeformers, but the two proteins are degraded following engulfment completion in B. subtilis (Jiang et al., 2005; Meisner et al., 2008). Finally, the mother cell lyses, releasing the mature spore into the media (VII). B. SpoIIQ‐SpoIIIAH and DMP organization. The proposed organization in B. subtilis, of SpoIIQ (blue) and SpoIIIAH (yellow) multimeric rings in the inner (IFM) and outer (OFM) forespore membranes is shown schematically. The interaction of the two rings is proposed to form a channel that allows communication between the mother cell and the forespore. The DMP complex (SpoIID, D in green; SpoIIP, P in light blue; and SpoIIM, M, in brown) required for PG remodeling during engulfment in B. subtilis is also represented (see text for details). C. Schematic representation of the octacistronic spoIIIA (top) and spoIID‐spoIIQ operons (bottom) of C. difficile. The position of two σ^E‐dependent promoters identified in the spoIIIA operon of B. subtilis is shown by bent arrows. The figure also shows the percentage of sequence similarity between the indicated proteins of C. difficile 630Δerm and their counterparts in B. subtilis. D. LytM motifs in Firmicutes. B. subtilis SpoIIQ, with the LytM domain and the position of the conserved residues in motifs 1 and 2 indicated, with the secondary structure elements observed in the structural models labelled (Meisner et al., 2012; Levdikov et al., 2012) (cylinders: α helices; arrows:β strands). A sequence alignment of SpoIIQ orthologues found in representative Bacilli (top group) and Clostridia (bottom group) (Galperin et al., 2012) when compared to the Hidden Markov Model sequence (HMM) previously described (Crawshaw et al., 2014) shows the relative conservation of the LytM catalytic residues (HxxxD, motif 1 and HxH, motif 2, highlighted). Sequences were retrieved and the HMM sequence created as previously described (Crawshaw et al., 2014). Colors indicate conservation when compared to the HMM sequence.

Figure 2

spoIIQ and spoIIIAH mutants of C. difficile are blocked early in the engulfment sequence. A. Schematic representation of the various alleles used. The ΔspoIIQ deletion removed codons 42 through 195 of the 222‐codons‐long spoIIQ gene, whereas in ΔspoIIIA codons 23–196 of the 218‐codons‐long coding region were deleted. Both are in‐frame deletion mutations, generated by allele‐coupled exchange (ACE); the spoIIQ H120S‐SNAP^Cd allele is expressed from a plasmid; note that the SNAP^Cd moiety is not represented for simplicity. The grey lines represent the region coding for the transmembrane segments of SpoIIQ or SpoIIIAH. B. SR‐SIM images of spoIIQ and spoIIIAH sporangia. The cells were collected from 14 hours SM cultures and stained with FM4‐64. The yellow arrows point to sporangia with bulges or vesicles; the blue arrows point to septa in which bulges form off center; the white arrows indicate septa that curve into the forespore, and the green arrows show cases of asymmetric migration of the engulfing membranes. Scale bar, 2 µm. C. Scoring of the percentage of cells in each of the morphological classes of sporulation (a to f), as seen by phase contrast and fluorescence microscopy (see text for details) for the indicated strains after 14 hours of growth in SM. The percentage of cells in each class is relative to the number of sporulating cells; ‘n’ is the total number of cells scored. Note that class b‐c was divided into sub‐classes b1 (bulges, vesicles and asymmetrically migrating membranes) and b2 (septa curving into the forespore); for sub‐classes b1 and b2, the numbers indicate the percentage of the cells scored as class b‐c showing the indicated phenotypes. D. Schematic representation of the engulfment pathway in WT cells (right) and in DMP mutants of B. subtilis (left).

Figure 3

Localization of SpoIIQ‐SNAP^Cd. The figure illustrates the localization of SpoIIQ‐ and SpoIIQ^H120S‐SNAP^Cd during sporulation, in the presence (A) and in the absence (B) of the WT spoIIQ gene, and of SpoIIQ‐SNAP^Cd in cells of spoIIIAH (C) or sigE (D) mutants. The strains expressing the SpoIIQ‐SNAP^Cd fusions were grown for 14 hours in SM and samples collected for labelling of the SNAP^Cd reporter with TMR‐Star. The labelled cells were viewed by SR‐SIM. The red arrows point to the SNAP^Cd‐TMR‐Star signal, the green arrows to the septa or engulfing membranes (MTG signal) and the yellow arrows to the merged signal. Note that in panels C and D, the SNAP^Cd‐TMR‐Star signal is also found ahead of the engulfing membranes (blue arrows). The numbers in the panels are the percentage of cells found with a similar localization pattern at each of the represented stages of sporulation. At least 50 cells were scored for each stage to derive the indicated percentages. Fluorescence intensity profiles (Fl. Int.; scale in arbitrary units) are shown below panels B, C and D. Scale bar, 2 µm.

Figure 4

Localization of SpoIIIAH‐SNAP^Cd. The figure illustrates the localization of SpoIIIAH‐SNAP^Cd fusion protein in sporulating cells in the presence (A) and in the absence (B) of the WT spoIIIAH allele, and in cells of a spoIIQ deletion mutant (C). The strains were grown for 14 hours in SM and samples collected for labelling of the SNAP^Cd with TMR‐Star, and the labelled cells were imaged by SR‐SIM. The red arrows in panels point to the SNAP^Cd‐TMR‐Star signal, the green arrows to the asymmetric septa or engulfing membranes, and the yellow arrows to the merged signals. The red arrow in panel C indicates the TMR‐Star signal enriched at the septum and the white arrows the signal detected throughout the mother cell membrane. The numbers in the panels indicated the percentage of cells found with a similar localization pattern at each of the represented stages of sporulation. At least 50 cells were scored for each stage to derive the indicated percentages. Fluorescence intensity profiles (Fl. Int.; the scale is in arbitrary units) are shown below panels B and D. Scale bar, 2 µm.

Figure 5

The interaction of SpoIIQ with SpoIIIAH in vitro. A. Quantification of Zn²⁺ content in sSpoIIQ (top) and sSpoIIQ^H120S (bottom). Purified protein samples were analyzed by size exclusion chromatography and the metal content in each fraction determined using ICP‐MS (see Material and Methods for details), whilst protein concentration in each fraction was determined by absorbance at 280 nm. The content of Zn²⁺ was determined for untreated samples (gray trace in both panels), in the presence of fivefold excess of Zn²⁺ (black traces) or 1mM EDTA (red traces) as purified protein samples eluted from the size exclusion column. In order to estimate the metal occupancy in each sample, protein quantification in each sample is also shown (sSpoIIQ, blue trace, top; sSpoIIQ^H120S, purple trace, bottom). In the presence of an excess Zn²⁺, about 80% sSpoIIQ (top panel, blue vs back traces) coordinates the metal, whilst no metal binding is detected for the sSpoIIQ^H120S mutant (bottom panel, purple vs black traces). B. sSpoIIQ interacts with sSpoIIIAH in a zinc‐associated mechanism. Purified sSpoIIQ (blue trace), sSpoIIQ^H120S (purple trace) and sSpoIIIAH (yellow trace) were analyzed by SEC‐MALLS. Traces correspond to light scattering measurements throughout protein elution. All proteins elute as single monomeric species, as indicated by the calculated masses (blue, purple and yellow dotted lines) and confirmed by SDS‐PAGE analysis (bottom, rows 1–3 respectively). Dotted lines correspond to the weight average molecular mass calculated based on refractive index and light scattering measurements. Complex sSpoIIQ‐sSpoIIIAH (1:1 molar ratio, green trace) elutes with a calculated mass of 43.1 kDa (green dotted line). Surprisingly, lack of coordination of Zn²⁺ seems to result in a less stable complex, as the sSpoIIQ^H120S‐sSpoIIIAH (light purple trace) and the WT complex incubated with 1mM EDTA (dashed light green trace) elute with a calculated mass of ∼27 kDa. Fractions (0.5ml) corresponding to elution volumes between 9.5 and 11ml were analyzed by SDS‐PAGE for all chromatography experiments (bottom, dashed gray lines indicate start and end of collection for each fraction).

Figure 6

Split‐SNAP localization. A. The top diagrams depict the predicted topology of SpoIIQ‐SNAP^Cd‐N (Q) and SpoIIIAH‐SNAP^Cd‐C fusions (AH) in the forespore membranes and their interaction based on the B. subtilis model. Assuming that in C. difficile the proteins will form a complex with a similar topology, a fluorescent signal (red star when bound to the reconstructed SNAP domain) will only be observed if the proteins interact. Note that the cells express the fusions (SpoIIQ‐SNAP^Cd‐N or SpoIIQ^H120S‐SNAP^Cd‐N under the control of P_spoIIQ promoter and SpoIIIAH‐SNAP^Cd‐C under P_spoIIIA control) from the same plasmid in an otherwise WT background. Thus, not all the subunits of the putative SpoIIQ‐SNAP^Cd‐N or SpoIIIAH‐SNAP^Cd‐C oligomers may be labelled. For simplicity, only one subunit is shown fused to the reporter. B. The figures show the localization of the fluorescence signal from the reconstituted SNAP^Cd domain (red arrows), in cells expressing the indicated combinations of SNAP^Cd‐N/SNAP^Cd‐C fusions. The cells were grown for 14 hours in SM prior to sampling, labelling with TMR‐Star and SR‐SIM imaging. The bottom series of panels are a magnification of the cells marked with an arrow in the ‘overlay’ panels; the yellow arrows point to foci of SNAP‐TMR‐Star. Scale bar, 2 µm. C. Split‐SNAP accumulation. Whole cell extracts were prepared from sporulating cells (same conditions as in A), producing SpoIIQ‐SNAP^Cd‐N or SpoIIQ^H120S‐SNAP^Cd‐N and SpoIIIAH‐SNAP^Cd‐C (first two lanes), or the SpoIIQ‐SNAP^Cd‐N and SpoIIIAH‐SNAP^Cd‐C proteins alone in an otherwise WT background. Proteins in the extracts were resolved by SDS‐PAGE, subjected to fluoroimaging (top panel) and immunobloting with anti‐SpoIIIAH (middle panel) or anti‐SNAP (bottom) antibodies. The red arrows show the position of SpoIIIAH‐SNAP^Cd‐C, SpoIIIAH or SpoIIQ‐SNAP^Cd‐N. Note that only SpoIIIAH‐SNAP^Cd‐C retains the covalently bound TMR‐Star substrate following SDS‐PAGE. Molecular weight markers (in kDa) are shown to the right side of the panel. D: fluorescence microscopy of cells producing SpoIIIAH‐SNAP^Cd‐C or SpoIIQ‐SNAP^Cd‐N alone and labelled with SNAP‐TMR‐Star. The labelled cells were examined by fluorescence microscopy in the green (auto‐fluorescence) and red channels. Note that the forespore is identified by its reduced auto‐fluorescence signal. Scale bar, 2 µm.

Figure 7

Early cell type‐specific gene expression in spoIIQ and spoIIIAH mutants. A. Cells expressing transcriptional P_gpr‐SNAP^Cd (as a reporter for σ^F activity) or P_spoIIIA‐SNAP^Cd fusions (as a reporter for σ^E activity) in the WT or the congenic spoIIQ or spoIIIAH mutants were collected from SM cultures 14 hours after inoculation, stained with the SNAP substrate TMR‐Star, and examined by phase contrast and fluorescence microscopy. The numbers represent the percentage of cells with the indicated pattern, relative to the total number of sporulating cells. A minimum of 50 sporangia was scored for each panel. Scale bar, 2 µm. B. Quantification of the fluorescence signal for the indicated SNAP^Cd fusions in the WT (in cells that had not completed the engulfment sequence), and in the spoIIQ or spoIIIAH mutants, as indicated. C. The panel illustrates spoIIQ or spoIIIAH sporangia in which loss of compartmentalized activity of the P_gpr‐ and P_spoIIIA‐SNAP^CD fusions is seen; the numbers represent the percentage of sporangia with the indicated pattern relative to the number of cells in A showing expression of either fusion. D. Early cell‐type specific gene expression is unaffected in spoIIQ and spoIIIAH mutants. In the absence of SpoIIQ or of SpoIIIAH (neither protein is represented for simplicity), σ^F and σ^E show essentially normal activity (the active sigma factors are shown inside a star), as seen by labelling of the SNAP reporter with TMR‐Star (stars; red when protein bound).

Figure 8

Late cell type‐specific gene expression in spoIIQ and spoIIIAH mutants. A. Cells expressing transcriptional P_sspA‐SNAP^Cd (as a reporter for σ^G activity) or P_cotE‐SNAP^Cd fusions (as a reporter for σ^K activity) in an otherwise WT background or in congenic spoIIQ or spoIIIAH mutants were collected from SM cultures 14 hours after inoculation, stained with the SNAP substrate TMR‐Star, and examined by phase contrast and fluorescence microscopy. The numbers represent the percentage of cells, relative to the total number of sporulating cells, exhibiting expression of P_sspA‐SNAP^Cd (as a reporter for the activity of σ^G) or P_cotE‐SNAP^Cd (as a reporter for the activity of σ^K). A minimum of 50 sporangia was scored for each panel. Scale bar, 1 µm. B. Quantification of the fluorescence signal for the P_sspA‐SNAP^Cd fusion in the WT (blue) and in the spoIIQ or spoIIIAH mutants (red, as indicated). The two left panels show the fluorescence signal in cells prior to engulfment completion. The right panel shows the fluorescence signal for cells of the spoIIIAH mutant following engulfment completion. C. Lack of spoIIQ or spoIIIAH leads to loss of compartmentalized activity of the P_sspA‐ or P_cotE‐SNAP^CD fusions. The numbers indicate the percentage of sporangia in A showing fluorescence from P_sspA‐ or P_cotE‐SNAP^CD in both the forespore and the mother cell. D. Late cell‐type specific gene expression is reduced in spoIIQ and spoIIIAH mutants. In the absence of SpoIIQ or of SpoIIIAH (not represented), the post‐engulfment activity of both σ^G (forespore) and σ^K (mother cell), as assessed by production and labelling of the SNAP reporter, is severely curtailed (the active σ factors are shown inside a star). In addition, the fraction of cells that display σ^K activity prior to engulfment completion is reduced (light grey star).