Alternative Sigma Factors SigF, SigE, and SigG Are Essential for Sporulation in Clostridium botulinum ATCC 3502

Abstract

Clostridium botulinum produces heat-resistant endospores that may germinate and outgrow into neurotoxic cultures in foods. Sporulation is regulated by the transcription factor Spo0A and the alternative sigma factors SigF, SigE, SigG, and SigK in most spore formers studied to date. We constructed mutants of sigF, sigE, and sigG in C. botulinum ATCC 3502 and used quantitative reverse transcriptase PCR and electron microscopy to assess their expression of the sporulation pathway on transcriptional and morphological levels. In all three mutants the expression of spo0A was disrupted. The sigF and sigE mutants failed to induce sigG and sigK beyond exponential-phase levels and halted sporulation during asymmetric cell division. In the sigG mutant, peak transcription of sigE was delayed and sigK levels remained lower than that in the parent strain. The sigG mutant forespore was engulfed by the mother cell and possessed a spore coat but no peptidoglycan cortex. The findings suggest that SigF and SigE of C. botulinum ATCC 3502 are essential for early sporulation and late-stage induction of sigK, whereas SigG is essential for spore cortex formation but not for coat formation, as opposed to previous observations in B. subtilis sigG mutants. Our findings add to a growing body of evidence that regulation of sporulation in C. botulinum ATCC 3502, and among the clostridia, differs from the B. subtilis model.

The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation

“…Recently, the sigF gene was disrupted in C. botulinum ATCC 3502 (96). As expected, the C. botulinum sigF disruption mutant was unable to form heat-resistant spores, and TEM image analysis revealed that sporulation was blocked at stage II (Fig.…”

“…The sigE gene was also disrupted in C. botulinum ATCC 3502 (96). This sigE mutant was unable to form viable heat-resistant spores, and TEM image analysis showed that sporulation was blocked at stage II, similar to the C. botulinum sigF disruption mutant (Fig.…”

“…5) (96). Additionally, Q-RT-PCR data showed that expression levels of spo0A in the C. botulinum sigF disruption mutant were significantly lower than those in the parent strain and that the cells were also unable to induce the expression of either sigE or sigG, while levels of sigK were also significantly lower in the mutant strain (96).…”

“…5) (96). In addition, Q-RT-PCR analysis showed that expression levels of spo0A, sigF, sigG, and sigK were all lower in the sigE mutant than in the parent strain (96).…”

“…Expression of the promoter fusion P sigG -SNAP Cd was not detected in the C. difficile sigF mutant, indicating that F is necessary for sigG expression (94). Recently, sigG was disrupted in C. botulinum ATCC 3502, and the mutant was unable to form viable heat-resistant spores (96). TEM image analysis revealed that the C. botulinum sigG disruption mutant, like the C. acetobutylicum sigG disruption mutant (26), was able to complete the engulfment stage of spore morphogenesis (stage III) (96).…”