σK of Clostridium acetobutylicum is the first known sporulation-specific sigma factor with two developmentally separated roles, one early and one late in sporulation

Abstract

Sporulation in the model endospore-forming organism Bacillus subtilis proceeds via the sequential and stage-specific activation of the sporulation-specific sigma factors, σ(H) (early), σ(F), σ(E), σ(G), and σ(K) (late). Here we show that the Clostridium acetobutylicum σ(K) acts both early, prior to Spo0A expression, and late, past σ(G) activation, thus departing from the B. subtilis model. The C. acetobutylicum sigK deletion (ΔsigK) mutant was unable to sporulate, and solventogenesis, the characteristic stationary-phase phenomenon for this organism, was severely diminished. Transmission electron microscopy demonstrated that the ΔsigK mutant does not develop an asymmetric septum and produces no granulose. Complementation of sigK restored sporulation and solventogenesis to wild-type levels. Spo0A and σ(G) proteins were not detectable by Western analysis, while σ(F) protein levels were significantly reduced in the ΔsigK mutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated in the ΔsigK mutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture. These data show that σ(K) is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in the ΔsigK mutant from a nonnative promoter restored solventogenesis and the production of Spo0A, σ(F), σ(E), and σ(G), but not sporulation, which was blocked past the σ(G) stage of development, thus demonstrating that σ(K) is also necessary in late sporulation. sigK is expressed very early at low levels in exponential phase but is strongly upregulated during the middle to late stationary phase. This is the first sporulation-specific sigma factor shown to have two developmentally separated roles.

FIG 1

Deletion, complementation, and confirmation of double-crossover allelic-exchange mutations at the sigK locus. (A) Wild type; (B) ΔsigK; (C) ΔsigK_um; (D) SigK_comp. The dashed lines in panel A show the approximate regions of homology (∼1 kb each) incorporated in the knockout vector. (E) PCR confirmation of double-crossover sigK deletion and complementation mutants with primers that anneal to the chromosome as indicated. (F) (sqRT-PCR) to confirm the silencing of sigK expression in ΔsigK and the restored expression in the SigK_comp mutant. The WT is also shown. All experiments were done in biological replicates (a and b). MW, λ HindIII digest ladder (NEB); M, 2-log ladder (NEB).

FIG 2

The ΔsigK strain is unable to sporulate after 5 days. (A) Spore and viable-cell counts for the ΔsigK, SigK_comp, ΔsigK p94Spo0A, WT p94Spo0A, and WT strains after 5 days in culture. The ΔsigK and ΔsigK p94Spo0A strains were unable to sporulate, while the SigK_comp strain sporulated successfully. All experiments were done in two biological replicates. Error bars represent SEMs. The horizontal dashed line shows the lower detection limit of the assay. (B) TEM images of ΔsigK and WT strains. No granulose, septum, or forespore structures were identified for the ΔsigK mutant, while the WT strain clearly shows the development of a forespore (I), granulose vesicles (II), and a free spore (III). It is worth noting that the translucent mass on one pole of the ΔsigK strain was also observed in the sigF mutant (FKO1), and it was originally hypothesized to be condensed DNA. However, 4′,6-diamidino-2-phenylindole (DAPI) staining of the FKO1 strain showed a lack of DNA in that region (8). Thus, the composition of that mass remains unknown.

FIG 3

Protein and transcript levels of sporulation and solvent formation genes are severely downregulated in the ΔsigK mutant. Shown are Western blots from biological replicates (a and b) for Spo0A, σ^F, and σ^G in the ΔsigK, WT, and SigK_comp (A) and ΔsigK p94Spo0A (B) strains at 32 h and 45 h. All Western blots were loaded with 30 μg of total protein, except that for the Spo0A WT and ΔsigK p94Spo0A strains, the blots were loaded with 15 μg and 5 μg, respectively. (C) The sigE transcript is detected in the ΔsigK p94Spo0A at 24 h from biological replicates (a and b), while no sigE transcript was detected after 26 h for the ΔsigK strain from biological replicates (a and b). +, reactions in which reverse transcriptase was added; −, reactions in which no reverse transcriptase was added. M, 2-log ladder (NEB). (D) qRT-PCR data showing the relative fold change of the major sporulation-related genes as well as adhE1 in the ΔsigK mutant relative to the WT strain at 6 h, 12 h, 20 h, and 26 h. All experiments were done in two biological replicates. Error bars represent SEMs. Statistical significance was tested with a two-sample t test (*, P < 0.05).

FIG 4

Very early and two-phase expression of sigK in WT C. acetobutylicum and downregulation of cotS and yabG in the ΔsigK mutant. (A) sqRT-PCR showing early expression of sigK at 6 h (A₆₀₀, ∼0.5). Data from two biological replicate experiments (a and b) are shown for both the WT and ΔsigK strains. M, 2-log ladder (NEB). (B) Very early expression of sigK following heat shocking of WT colonies and germination at an OD of ∼0.03. Ten WT cultures were heat shocked and combined into one tube to allow for enough material to extract RNA. +, reactions in which reverse transcriptase was added; −, reactions in which no reverse transcriptase was added. M, 2-log ladder (NEB). (C) qRT-PCR showing the biphasic expression pattern of sigK, low expression early followed by the gradual increase of expression after 24 h. All experiments were done in two biological replicates. Error bars represent SEM. Statistical significance was tested with a two-sample t test (*, P < 0.05; **, P < 0.10). (D) Fold expression of cotS and yabG in ΔsigK. cotS and yabG are both downregulated in the ΔsigK strain relative to the WT up to ∼3.5-fold and ∼7-fold, respectively. Error bars represent SEMs. Statistical significance was tested with a two-sample t test (*, P < 0.05; **, P < 0.10).

FIG 5

TEM images of the ΔsigK p94Spo0A strain after 5 days of culture. The cells appear to be releasing an immature and underdeveloped spore (I). The spore membrane appeared to be crimped and ill formed (II). The strain was able to produce granulose vesicles (III) but showed what appears to be a ruptured membrane (IV) with no identifiable spore coat.

FIG 6

Transcriptional profiling of sigK, spoIVFB, and spoIVB in either EKO1 or GKO1. (A) Expression of sigK in EKO1 is downregulated 84-fold relative to the WT during the transitional phase and ∼4-fold during the stationary phase. (B) Microarray data showing expression fold difference of spoIVFB, spoIVB, and sigK in GKO1 relative to the WT. The fold difference shown is the average of two time points (28 h and 30 h), which correspond to maximum sigG expression in the WT (9). (C) Time course gene expression profiling of spoIVFB and spoIVB in WT cells relative to the first time point. All experiments were done in two biological replicates. The data show a dramatic upregulation of both genes during the late stages of sporulation coinciding with the increased expression of sigK, thus indicating that they may possibly play a role in its processing at the posttranslational level. Error bars represent SEMs. Statistical significance was tested with a two-sample t test (*, P < 0.05).

FIG 7

Proposed sporulation cascade model in C. acetobutylicum. Gray dashed lines indicate a hypothesized interaction between σ^E and σ^G. The figure was constructed from data reported in references , , and .