Inactivation of σ ^F in Clostridium acetobutylicum ATCC 824 Blocks Sporulation Prior to Asymmetric Division and Abolishes σ ^E and σ ^G Protein Expression but Does Not Block Solvent Formation

Abstract

Clostridium acetobutylicum is both a model organism for the understanding of sporulation in solventogenic clostridia and its relationship to solvent formation and an industrial organism for anaerobic acetone-butanolethanol (ABE) fermentation. How solvent production is coupled to endospore formation-both stationaryphase events-remains incompletely understood at the molecular level. Specifically, it is unclear how sporulation-specific sigma factors affect solvent formation. Here the sigF gene in C. acetobutylicum was successfully disrupted and silenced. Not only F but also the sigma factors E and G were not detected in the sigF mutant (FKO1), and differentiation was stopped prior to asymmetric division. Since plasmid expression of the spoIIA operon (spoIIAA-spoIIAB-sigF) failed to complement FKO1, the operon was integrated into the FKO1 chromosome to generate strain FKO1-C. In FKO1-C, F expression was restored along with sporulation and E and G protein expression. Quantitative reverse transcription-PCR (RT-PCR) analysis of a select set of genes (csfB, gpr, spoIIP, sigG, lonB, and spoIIR) that could be controlled by F , based on the Bacillus subtilis model, indicated that sigG may be under the control of F , but spoIIR, an important activator of E in B. subtilis, is not, and neither are the rest of the genes investigated. FKO1 produced solvents at a level similar to that of the parent strain, but solvent levels were dependent on the physiological state of the inoculum. Finally, the complementation strain FKO1-C is the first reported instance of purposeful integration of multiple functional genes into a clostridial chromosome-here, the C. acetobutylicum chromosome-with the aim of altering cell metabolism and differentiation.The endospore-forming obligate anaerobe Clostridium acetobutylicum is best known for its acetone, butanol, and ethanol (ABE) fermentation and has recently received renewed attention for of its industrial potential, specifically as a biofuel producer (26,37). Despite this increased attention and potential, many fundamental questions remain about clostridial physiology, differentiation, and metabolism, and the lack of this basic knowledge has limited the success of engineering of C. acetobutylicum for industrial processes (26, 37). Two of these key questions are how clostridial cells regulate differentiation and how differentiation is related to solvent formation (26,37,38). It has been well established that Spo0A is the master regulator of both solventogenesis and sporulation (8,14,41), but the regulation of sporulation downstream of Spo0A and any effect the regulation has on solventogenesis are not yet well understood (37,38).In contrast, sporulation in Bacillus subtilis has been studied extensively, and its regulation is well understood (9,16,48). To initiate sporulation, B. subtilis employs a multicomponent phosphorelay to phosphorylate Spo0A (4, 40), which then stimulates the expression of sigF, the prespore-specific sigma factor, and sigE, the mother cell-specific sigma factor (9, 16), in add...

The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation

“…In addition, the authors of this study were able to show that CPE production is directly regulated by F . The involvement of F in early stages of sporulation was also demonstrated in C. acetobutylicum (88) (Fig. 6).…”

“…The data from sigF inactivation mutants generated in Clostridium organisms to date implicate F in early stages of sporulation (86,88,94). However, there are differences between the sigF mutants generated in Clostridium and those in B. subtilis.…”

“…However, there are differences between the sigF mutants generated in Clostridium and those in B. subtilis. TEM images revealed that the C. acetobutylicum sigF inactivation mutant (88) appeared to have its sporulation blocked prior to stage II, while the B. subtilis sigF mutant did not complete stage III (91) (Fig. 5).…”

“…If cultures of the sigF inactivation mutant were inoculated by using cells from mid-to late exponential phase (as is typical with WT C. acetobutylicum cultures), the final solvent levels were considerably lower than those in the WT culture. On the other hand, if the sigF inactivation mutant culture was inoculated from a stationary-phase culture, normal levels of solvents were produced (88). A potential mechanism for this bistable phenotype is discussed in a separate section, below.…”

Cassava roots: perspectives of a traditional staple for bio-solvents production

“…At this point, the production of solvents extensively increases, especially that of butanol and acetone (Table 3). The emergence of spores in solvent producing anaerobes bacteria is an important indicator of ABE fermentation, since the Clostridium acetobutylicum Spo0A gene encodes both as the regulator of the transcription of the sporulation and solvent formation (Jones et al 2011). On the other side, fermentation carried out in the culture medium M2 showed a high viability of the culture medium neatly prepared from the whole cassava.…”