SpoIIE Regulates Sporulation but Does Not Directly Affect Solventogenesis in Clostridium acetobutylicum ATCC 824

Abstract

Using gene expression reporter vectors, we examined the activity of the spoIIE promoter in wild-type and spo0A-deleted strains of Clostridium acetobutylicum ATCC 824. In wild-type cells, the spoIIE promoter is active in a transient manner during late solventogenesis, but in strain SKO1, where the sporulation initiator spo0A is disrupted, no spoIIE promoter activity is detectable at any stage of growth. Strains 824(pMSpo) and 824(pASspo) were created to overexpress spoIIE and to decrease spoIIE expression via antisense RNA targeted against spoIIE, respectively. Some cultures of strains 824(pMSpo) degenerated during fermentations by losing the pSOL1 megaplasmid and hence did not produce the solvents ethanol, acetone, and butanol. The frequent degeneration event was shown to require an intact copy of spoIIE. Nondegenerate cultures of 824(pMSpo) exhibited normal growth and solvent production. Strain 824(pASspo) exhibited prolonged solventogenesis characterized by increased production of ethanol (225%), acetone (43%), and butanol (110%). Sporulation in strains harboring pASspo was significantly delayed, with sporulating cells exhibiting altered morphology. These results suggest that SpoIIE has no direct effect on the control of solventogenesis and that the changes in solvent production in spoIIE-downregulated cells are mediated by effects on the cell during sporulation.The gram-positive, obligate anaerobe Clostridium acetobutylicum was used for the industrial production of the solvents acetone and butanol for over 60 years in the 20th century. With chemical synthesis of acetone and butanol proving significantly more economic, there are no reported industrial fermentation plants using C. acetobutylicum operational in the world today (11). However, over the last 20 years, the genetics and biochemistry of C. acetobutylicum have been investigated in detail, as we try to understand and improve upon the processes that control the production of solvents.The genes aad, ctfA, ctfB, and adc are required for solvent production and are located on a 192-kb megaplasmid designated pSOL1 (8,27). ctfA and ctfB code for a multifunctional coenzyme A (CoA) transferase, which transfers the CoA moiety from acetoacetyl-CoA to acetate or butyrate (40). Subsequently, acetoacetate is decarboxylated to form acetone, and acetyl-CoA and butyryl-CoA are converted to ethanol and butanol, respectively, via an aldehyde intermediary (26). For a detailed description of clostridial biochemistry, see the paper by Mitchell (26). It has been shown that C. acetobutylicum can lose pSOL1, rendering the cells unable to produce solvents. Cells and strains that have lost pSOL1 are termed "degenerate" (8, 28).Whereas much is known about the biochemistry of C. acetobutylicum metabolism and the genes and proteins that catalyze these processes, relatively little is known about the genetic control of the expression of these genes. Recently, it was shown that a homologue to Bacillus subtilis stage 0 sporulation protein A (Spo0A) controls both the onset of solventogen...

SpoIIE Is Necessary for Asymmetric Division, Sporulation, and Expression of σ F , σ E , and σ G but Does Not Control Solvent Production in Clostridium acetobutylicum ATCC 824

“…This profile generally matches with the promoter activity of spoIIE in B. subtilis, where activity begins during early stationary phase and continues into mid-stationary phase (11). However, when a similar promoter activity study was performed on spoIIE in C. acetobutylicum, activity was not noted until mid-stationary phase, with a peak in expression soon after that (38). In order to investigate the role of spoIIE in sporulation and solventogenesis, the gene was downregulated using antisense RNA (38).…”

“…However, when a similar promoter activity study was performed on spoIIE in C. acetobutylicum, activity was not noted until mid-stationary phase, with a peak in expression soon after that (38). In order to investigate the role of spoIIE in sporulation and solventogenesis, the gene was downregulated using antisense RNA (38). Upon knockdown of spoIIE, solventogenesis in the strain was slightly prolonged, and sporulation was significantly delayed compared to a plasmid control strain (38).…”

“…In order to investigate the role of spoIIE in sporulation and solventogenesis, the gene was downregulated using antisense RNA (38). Upon knockdown of spoIIE, solventogenesis in the strain was slightly prolonged, and sporulation was significantly delayed compared to a plasmid control strain (38).…”

Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism

“…For long-term storage, E. coli strains were cultivated and stored as glycerol stocks at −80°C. C. acetobutylicum strains were stored as lyophilized stocks at room temperature or glycerol stocks at −80°C (Scotcher and Bennett 2005).…”

Transcriptional Program of Early Sporulation and Stationary-Phase Events in Clostridium acetobutylicum

“…In B. subtilis, expression of spoIIE starts approximately 1 h after the onset of sporulation, with the highest expression approximately 1 h later; spoIIE has a temporal expression pattern similar to that of the spoIIA (sigF) and spoIIG (sigE) operons (99). Expression of spoIIE in C. acetobutylicum (CAC3205) has been shown to coincide with solvent formation, and antisense downregulation of the gene has been shown not to affect solvent formation (73). In the experiments presented here, spoIIE expression starts 3 to 5 h following sigF expression and the onset of sporulation and peaks approximately 5 h after the onset of sporulation (2.2-fold higher expression than the initial time point).…”