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

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.

[1]  E. Papoutsakis,et al.  Expression of plasmid-encoded aad in Clostridium acetobutylicum M5 restores vigorous butanol production , 1994, Journal of bacteriology.

[2]  P. Dürre,et al.  Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum , 1990, Journal of bacteriology.

[3]  M. Mergeay,et al.  Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals , 1985, Journal of bacteriology.

[4]  G. Bennett,et al.  Isolation and Characterization of Mutants of Clostridium acetobutylicum ATCC 824 Deficient in Acetoacetyl-Coenzyme A:Acetate/Butyrate:Coenzyme A-Transferase (EC 2.8.3.9) and in Other Solvent Pathway Enzymes , 1989, Applied and environmental microbiology.

[5]  E. Papoutsakis,et al.  Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824 , 1994, Journal of bacteriology.

[6]  D. T. Jones,et al.  Solvent Production and Morphological Changes in Clostridium acetobutylicum , 1982, Applied and environmental microbiology.

[7]  P. Soucaille,et al.  Solvent-forming genes in clostridia , 1996, Nature.

[8]  M. Olson,et al.  Cosmid mapping of the human chorionic gonadotropin beta subunit genes by field-inversion gel electrophoresis. , 1987, Nucleic acids research.

[9]  E. Papoutsakis,et al.  The effect of pH on nitrogen supply, cell lysis, and solvent production in fermentations of Clostridium acetobutylicum , 1985, Biotechnology and bioengineering.

[10]  D. T. Jones,et al.  Acetone-butanol fermentation revisited. , 1986, Microbiological reviews.

[11]  Z. J. Ordal,et al.  Comparative Metabolism of Vegetative and Sporulating Cultures of Clostridium thermosaccharolyticum , 1970, Journal of bacteriology.

[12]  David T. Jones,et al.  Taxonomy and phylogeny of industrial solvent-producing clostridia. , 1995, International journal of systematic bacteriology.

[13]  K. Baldwin,et al.  Transductional evidence for plasmid linkage of lactose metabolism in streptococcus lactis C2 , 1976, Applied and environmental microbiology.

[14]  M. Young,et al.  Wide diversity of genome size among different strains of Clostridium acetobutylicum , 1993 .

[15]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[16]  N. Minton,et al.  Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation , 1988 .

[17]  S. T. Liu,et al.  Rapid procedure for detection and isolation of large and small plasmids , 1981, Journal of bacteriology.

[18]  D. Dubnau,et al.  Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid from Bacillus subtilis , 1986, Journal of Bacteriology.

[19]  E. Lai,et al.  Pulsed field separation of large supercoiled and open‐circular DNAs and its application to bacterial artificial chromosome cloning , 1995, Electrophoresis.

[20]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[21]  E. Papoutsakis,et al.  Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824. , 1996, Microbiology.

[22]  Hamilton O. Smith,et al.  A simple method for DNA restriction site mapping. , 1976, Nucleic acids research.

[23]  S. Beverley.  Characterization of the 'unusual' mobility of large circular DNAs in pulsed field-gradient electrophoresis. , 1988, Nucleic acids research.

[24]  F. Casse,et al.  Identification and Characterization of Large Plasmids in Rhizobium meliloti using Agarose Gel Electrophoresis , 1979 .

[25]  E. Papoutsakis,et al.  Cloning and expression of Clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli , 1990, Applied and environmental microbiology.

[26]  J. Ramos,et al.  Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways , 1993, Molecular microbiology.

[27]  C. Gaffney,et al.  A RAPID METHOD FOR THE IDENTIFICATION OF DIPHTHERIA BACILLI: ALSO A NEW METHOD FOR IDENTIFICATION OF CARRIERS OF DIPHTHERIA BACILLI. , 1935, Canadian Medical Association journal.

[28]  T. Eckhardt,et al.  A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria. , 1978, Plasmid.

[29]  E. Papoutsakis,et al.  Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon , 1993, Biotechnology and bioengineering.

[30]  P. Dürre,et al.  Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis , 1993, Journal of bacteriology.

[31]  G. Bennett,et al.  Sequence and arrangement of genes encoding enzymes of the acetone-production pathway of Clostridium acetobutylicum ATCC824. , 1993, Gene.