Expression of abrB310 and SinR, and effects of decreased abrB310 expression on the transition from acidogenesis to solventogenesis, in Clostridium acetobutylicum ATCC 824

Abstract

The transcription factors sinR and abrB are involved in the control of sporulation initiation in Bacillus subtilis. We identified a single homologue to sinR and three highly similar homologues to abrB, designated abrB310, abrB1941, and abrB3647, in Clostridium acetobutylicum ATCC 824. Using reporter vectors, we showed that the promoters of abrB1941 and abrB3647 were not active under the growth conditions tested. The abrB310 promoter was strongly active throughout growth and exhibited a transient elevation of expression at the onset of solventogenesis. Primer extension assays showed that two transcripts of abrB310 and a single, extremely weak transcript for sinR are expressed. Potential -35 and -10 consensus motifs are readily identifiable surrounding the transcription start sites of abrB310 and sinR, with a single putative 0A box present within the promoter of abrB310. In strains of C. acetobutylicum transformed with plasmids to elevate sinR expression or decrease sinR expression, no significant differences in growth or in acid or solvent production were observed compared to the control strains. In C. acetobutylicum strain 824(pAS310), which expressed an antisense RNA construct targeted against abrB310, the acids acetate and butyrate accumulated to approximately twice the normal concentration. This accumulation corresponded to a delay and decrease in acetone and butanol production. It was also found that sporulation in strain 824(pAS310) was delayed but that the morphology of sporulating cells and spores was normal. Based upon these observations, we propose that abrB310 may act as a regulator at the transition between acidogenic and solventogenic growth.

FIG. 1.

Alignment of the amino acid sequences of AbrB and SinR in B. subtilis and C. acetobutylicum. C.ac310, C.ac1941, and C.ac3647 refer to the three homologues of AbrB identified in C. acetobutylicum as defined by their position in the genome. Alignment was performed using the CLUSTALW tool of the Biology Workbench (San Diego Supercomputer Center). White letters on black background, single, fully conserved residue in all homologues; −, position with no amino acid present (23, 31). (A) Cysteine-56 in the AbrB homologues is indicated by the symbol ▾. (B) The helix-turn-helix DNA-binding domain of sinR in B. subtilis is shown in the first box, with key residues indicated by the symbol ▾ (13). The second boxed region corresponds to the multimerization domain of sinR in B. subtilis (3). B.sub, B. subtilis; C.ac, C. acetobutylicum.

FIG. 2.

CAT activity and solvent production in strain 824(pCAT310). ▪, CAT activity; ▵, acetone; and ◊, butanol. Other products and OD₆₀₀ are omitted for clarity. Data are shown ± 1 standard error; n = 9.

FIG. 3.

Transcription start sites and promoter region sequence of abrB310 and sinR. The transcripts identified by primer extension for abrB310 (panel A) and sinR (panel B) correspond to the transcription start sites shown on the sequences. The sequencing reaction products (lanes A, T, G, and C) were generated with the same primers as those used for the extension reactions. Also identified within the promoter region sequences are putative −35 and −10 consensus motifs, a potential binding site for Spo0A in abrB310 (0A box) and ribosome-binding sites (RBS). The arrows on both sequences indicate the first codon (ATG) of the open reading frame.

FIG. 4.

Transcript levels in strains 824(pMsinR), 824(pASsin) and 824(pAS310). sinR transcript was probed with primer sinext in mRNA extracted from the following strains: lane 1, 824(pMsinR); lane 2, 824(pIMP1); lane 3, 824(pASsos); and lane 4, 824(pASsin). abrB310 transcript was probed with primer 310ext in mRNA extracted from the following strains: lane 5, 824(pAS310); and lane 6, 824(pASsos). Arrows indicate the approximate sizes of transcripts (in bases).

FIG. 5.

Growth and product formation in fermentations of strains 824(pASsos) and 824(pAS310). The measured quantities for each profile of strain 824(pASsos) (□) and strain 824(pAS310) (▪) are shown. Data are shown ± 1 standard error. For each data point, n = 4.