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.

FIG. 1.

Antisense construct to spoIIE. The spoIIE antisense construct consists of oligonucleotide spoasastop (upper DNA strand) and spoasbtm (lower DNA strand). The single underlined GATC forms the 5′ BamHI “sticky” end. The black-shaded region includes 38 bases of the spoIIE open reading frame followed by 16 bases of the upstream leader sequence, shown shaded in dark gray. The bold region forms the 17-base rho-independent terminator region from an antisense RNA targeted against the glnA gene, found naturally in Clostridium saccharobutylicum NCP262 (10).

FIG. 2.

CAT activities in strains 824(pCATspo) and 824(pCATP). CAT activities are shown in strain 824(pCATspo)(▪) and the control strain 824(pCATP)(×). The solvent concentrations for the 824(pCATspo) fermentations are represented by the following symbols: acetone (▴) and butanol (♦). Data are means ± 1 standard error. For acetone and butanol, n = 6; for 824(pCATP) CAT activity, n = 3; for 824(pCATspo) CAT activity, n = 6.

FIG. 3.

Growth and product formation in fermentations of strains 824(pASsos) and 824(pASspo). The measured quantity for each profile of strain 824(pASsos) (□) and strain 824(pASspo) (▪) is shown above each graph. Data are means ± 1 standard error. For each datum point, n = 4.