Regulation of nitrogen metabolism, starch utilisation and the β-hbd—adh1 gene cluster in Clostridium acetobutylicum

Abstract

The successful genetic manipulation of Clostridium acetobutylicum for the increased production of solvents will depend on an understanding of gene structure and regulation in the bacterium. The glutamine synthetase (glnA) gene is regulated by antisense RNA, transcribed from a downstream promoter, in the opposite direction to the glnA gene. An open reading frame (ORF) was detected downstream of the glnA gene, which has sequence homology to response regulators with anti-termination activity and may be involved in sensing nitrogen conditions. The expression of the linked beta-hbd, adh1 and fixB genes was investigated throughout the bacterial growth cycle by RNA hybridisation techniques. The adh1 gene was independently expressed as a 2.4-kb transcript which peaked at 12 h, immediately prior to the solventogenic phase. The beta-hbd and fixB genes were transcribed throughout the acidogenic and solventogenic phases. A regulator gene, regA, which complements a Bacillus subtilis ccpA mutant, has been identified and sequenced from C. acetobutylicum P262. The regA gene repressed the degradation of starch by an uncharacterised C. acetobutylicum gene, and may therefore play a role in the utilisation of carbohydrate substrates in this organism.

Small RNAs in the GenusClostridium

“…In C. perfringens , a regulatory RNA molecule (VR-RNA) was found to be responsible for the transcriptional regulation of two toxin genes, the collagenase ( colA ) and alpha-toxin ( plc ) genes (24). In C. saccharobutylicum P262, expression of the glutamine synthetase gene ( glnA ) was found to be regulated by an antisense RNA molecule transcribed from downstream and in the direction opposite that of glnA (25–27). A stand-alone S-box and a T-box riboswitch were shown to regulate a sulfur metabolic operon of C. acetobutylicum based on an antisense-RNA mechanism (28).…”

Co-regulation of the nitrogen-assimilatory gene cluster in Clostridium saccharobutylicum

“…The nitR gene encodes an antiterminator protein, which would allow RNA polymerase to read through the terminator-like structures in the promoter region of the glnA gene (Woods & Reid, 1995). Antisense RNA (AS-RNA), complementary to the 59 end of the glnA mRNA, has been implicated in the post-transcriptional inhibition of glnA translation under nitrogen-rich conditions (Fierro-Monti et al, 1992).…”

“…The C. saccharobutylicum glnA gene, encoding GS, was isolated by functional complementation of the GS-deficient Escherichia coli strain YMC11 (Usdin et al, 1986), and a second gene, nitR, encoding a putative response regulator protein, was subsequently identified downstream (Woods & Reid, 1995). The nitR gene encodes an antiterminator protein, which would allow RNA polymerase to read through the terminator-like structures in the promoter region of the glnA gene (Woods & Reid, 1995).…”

General Biology and Physiology

“…It has also been suggested that the antisense RNA may be involved in regulation of expression of a putative regulatory gene which overlaps the antisense coding region and encodes a product which exhibits homology with both response regulators of two-component systems and transcriptional antiterminators. Expression of the antisense molecule, mediated by activation of P3 in response to a cellular signal reflecting a nitrogen-rich environment, would therefore exert a dual control over glnA expression [262].…”