The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain

Abstract

Degeneration is the process whereby Clostridium acetobutylicum ATCC 824 loses the capacity to produce acetone and butanol after repeated vegetative transfers or in continuous culture. Two degenerate mutants (M5 and DG1) of C. acetobutylicum ATCC 824 do not contain the four genes (ctfA, ctfB, adc, and aad) for acetone and butanol formation. Strain ATCC 824 contains a 210-kb plasmid (pSOL1) which is absent in M5 and DG1. pSOL1 carries the four acetone and butanol formation genes. A restriction map of pSOL1 was constructed by using ApaI, SmaI, SstII, and NarI digestions. M5 and DG1 could be complemented for acetone and butanol formation by expressing the corresponding genes (ctfA, ctfB, and adc for acetone; aad for butanol) on the plasmid. Degeneration of this strain thus appears to be the result of pSOL1 loss.The American Heritage Dictionary defines the term "degenerate" as "having fallen or descended to a state below what is considered normal or desirable." Within the solventogenic clostridial community, the term "degenerate" is used to describe mutants which have one common trait, namely, that they produce more acids (butyrate and acetate) and little or no solvents (butanol, acetone, and ethanol). Degeneracy as applied to solventogenic clostridia appears to have no implications about the nature or origin of the mutation(s).It has long been observed that upon serial subculturing or in continuous culture, solventogenic clostridia undergo a degenerative process which affects both morphological and physiological features and leads to mutant strains with impaired solvent formation capabilities (14). Weizmann's original strain of Clostridium acetobutylicum was found (20) to degenerate after 10 to 20 transfers when the transfers were made during the acidogenic phase of the fermentation. Solvent formation before the 50th transfer dropped to 0.5 to 2.0% of the starch fermented, and the spore formation capability was almost completely abolished. Attempts to recover "normal" solvent-producing strains from degenerate strains by culture with growth factors and various salts and by inducing sporulation were only partially successful. However, when transfers were made following sporulation and heat shocking, a solvent yield of 24.7% was retained after 150 transfers (more than 2 years).Jones et al. observed that certain morphological and cytological changes, which could be correlated with growth and physiological changes, occurred in C. acetobutylicum P262 during solvent production (13). The number of swollen, cigarshaped clostridial forms could be directly correlated with the production of solvents. Initiation of solvent production and clostridial-stage formation are essential for sporulation in different Clostridium species (12, 18). Thus, degenerate mutants would be expected to be asporogenous. However, the existence of an asporogenous strain which is a good solvent producer (17) should be noted, because it demonstrates that degeneracy (assumed here to imply impaired solvent formation ability) is not necessarily equivalent t...

Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum

“…Data generated using these targeted cDNA arrays and these data analysis methods have been shown to be highly reliable. Analysis of a degenerate C. acetobutylicum strain (M5 [9]) missing 178 genes properly classified genes with an accuracy of 99.4% (49). Comparison to Northern blot (49) and quantitative reverse transcription-PCR (50,56) analyses has also shown this method to be accurate in identifying genes as differentially expressed and to be conservative in estimating relative gene expression levels.…”

Metabolite stress and tolerance in the production of biofuels and chemicals: Gene‐expression‐based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum

“…These results suggest a novel mechanism of acetate-induced repression of butyrate formation and vice versa. The pSOL1 megaplasmid (Cornillot et al, 1997) contains a thiolase isozyme (thlB, CAP0078) in a tricistronic operon with a putative repressor (thlR, CAP0079) and a third gene encoding an uncharacterized iron-sulfur protein (thlC', CAP0077; Winzer et al, 2000). Expression of this operon was strongly upregulated by both acetate and butyrate.…”

Degeneration of solventogenic Clostridium strains monitored by Fourier transform infrared spectroscopy of bacterial cells

“…It therefore follows that degeneration results from genetic change ( genetic instability or mutation ) . It is frequently connected with a loss of the ability to sporulate [ 7 ] . In Clostridium acetobutylicum, genes encoding essential enzymes in the solventogenic pathways are located on a megaplasmid whose loss results in a degenerate phenotype [ 6,7,37 ] .…”

“…It is frequently connected with a loss of the ability to sporulate [ 7 ] . In Clostridium acetobutylicum, genes encoding essential enzymes in the solventogenic pathways are located on a megaplasmid whose loss results in a degenerate phenotype [ 6,7,37 ] . In Clostridium saccharoperbutylacetonicum, degenerate mutants were identified with a defect in NADH formation from pyruvate [ 16 ] .…”