SpoIIE is necessary for asymmetric division, sporulation, and expression of sigmaF, sigmaE, and sigmaG but does not control solvent production in Clostridium acetobutylicum ATCC 824

Abstract

In order to better characterize the initial stages of sporulation past Spo0A activation and the associated solventogenesis in the important industrial and model organism Clostridium acetobutylicum, the spoIIE gene was successfully disrupted and its expression was silenced. By silencing spoIIE, sporulation was blocked prior to asymmetric division, and no mature spores or any distinguishable morphogenetic changes developed. Upon plasmid-based complementation of spoIIE, sporulation was restored, although the number of spores formed was below that of the plasmid control strain. To investigate the impact of silencing spoIIE on the regulation of sporulation, transcript levels of sigF, sigE, and sigG were examined by semiquantitative reverse transcription-PCR, and the corresponding σF, σE, and σG protein levels were determined by Western analysis. Expression of sigF was significantly reduced in the inactivation strain, and this resulted in very low σF protein levels. Expression of sigE was barely detected, and no sigG transcript was detected at all; consequently, no σE or σG proteins were detected. These data suggest an autostimulatory role for σF in C. acetobutylicum, in contrast to the model organism for endospore formation, Bacillus subtilis, and confirm that high-level expression of σF is required for expression of σE and σG. Unlike the σF and σE inactivation strains, the SpoIIE inactivation strain did not exhibit inoculum-dependent solvent formation and produced good levels of solvents from both exponential- and stationary-phase inocula. Thus, we concluded that SpoIIE does not control solvent formation.

Fig. 1.

Disruption of the spoIIE gene. (A) The pKOSPOIIE vector and the spoIIE locus on the C. acetobutylicum ATCC 824 genome. The two regions of homology are indicated on both the plasmid and the chromosome. (B) Integration of pKOSPOIIE into the genome. The plasmid underwent a single crossover through the 2nd homologous region. The locations of the confirmation primers along with the promoter (P_ptb) (44) and rho-independent terminator (RIT) of the Cm/Th^r cassette are indicated. (C) Schematic of how pKOSPOIIE would have integrated had a single crossover through the 1st homologous region occurred. The locations of the confirmation primers are indicated. (D) PCR results for the confirmation primers used on SPOIIEKO. The sizes demonstrate that pKOSPOIIE integrated by a single crossover through the 2nd region of homology.

Fig. 2.

Southern blot confirmation of pKOSPOIIE integration site. (A) NdeI-digested genomic DNA from C. acetobutylicum WT and SPOIIEKO hybridized with a probe for the 2nd homologous region. Lane 1, NdeI-digested SPOIIEKO DNA; lane 2, BamHI-digested pKOSPOIIE; lane 3, NdeI-digested pKOSPOIIE; lane 4, NdeI-digested WT DNA; lane L, 2-log ladder from NEB. Membrane was exposed for 2 min. Diagrams of expected bands from WT (B), SPOIIEKO (C), and pKOSPOIIE (D) are shown.

Fig. 3.

Expression of spoIIE and sporulation-related transcriptional regulators in C. acetobutylicum WT, SPOIIEKO, and SPOIIEKO(pSPOIIE). (A) Semiquantitative RT-PCR of spoIIE, spo0A, sigF, sigE, and sigG in WT, SPOIIEKO, and SPOIIEKO(pSPOIIE). Transcript expression was tested at 48 h after inoculation. +, reactions in which reverse transcriptase was added to the reaction mixture; −, reactions in which reverse transcriptase was not included in the reaction mixture. Products were imaged on an ethidium bromide-stained 1% agarose gel. (B) Western blot analysis of Spo0A, σ^F, σ^E, and σ^G in WT, SPOIIEKO, and SPOIIEKO(pSPOIIE). The σ^F image with an asterisk to the right was exposed for twice as long as the image above it.

Fig. 4.

Microscopy of C. acetobutylicum SPOIIEKO after 72 h of growth. (A) Phase-contrast microscopy of SPOIIEKO. Only rod-shaped cells with no identifiable differentiation phenotypes are evident. (B) TEM of SPOIIEKO. White, dashed arrows indicate the developing symmetrical septal membrane.

Fig. 5.

Sporulation of complemented strain SPOIIEKO(pSPOIIE). The numbers of CFU of the plasmid control strain 824(pTLH1) and the complemented strain SPOIIEKO(pSPOIIE) are shown. Standard deviations between two biological replicates and two technical replicates (n = 4) are indicated.