Analysis of Degenerate Variants ofClostridium acetobutylicumATCC 824

Stim-Herndon, Kathleen P.; Nair, Ramesh V.; Papoutsakis, E. T.; Bennett, George N.

doi:10.1006/anae.1996.0002

Cited by 21 publications

(11 citation statements)

References 21 publications

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“…The experiment with the subcultured microorganism (S) presented the best results, 57 IU l -1 of PGA. The same behavior had already been reported by some authors (Moyson and Verachtert, 1993;StimHerndon et al,1996). However, cultures preserved in the form of freezedried cells or frozen spores (cultures L1, L2 and F - Figure 1), which were considered reliable methods for storage (Krasil'nikova and Zakharchuk, 2000), exhibited even smaller PGA productivity.…”

Section: Protease Detectionsupporting

confidence: 85%

Maintenance of penicillin G acylase expression by B. megaterium: preservation methods and activity recovery

Pinotti

Silva

Zangirolami

et al. 2007

Braz. J. Chem. Eng.

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-This work reports the influence of different culture preservation methods on the production of penicillin G acylase (PGA) by Bacillus megaterium ATCC 14945. The initial stock culture, presenting PGA activity of 97 IU l -1 , was preserved for two years using different procedures: monthly subculturing and storage in refrigerator (S), freeze-drying using skim milk 10%, plus inositol 5% as cryoprotectors (L1), freeze-drying using sucrose 7%, plus peptone 7% (L2), and freezing with glycerol 10% (F). After cultivations at standard operational conditions, different values of enzyme activity were obtained: 56 IU l -1 for monthly subculturing (S), 15-41 IU l -1 for freeze-dried cells and frozen spores. All the tested methods have failed in preserving the PGA expression. Among all tested cultures, S presented the highest specific activity, and was used to prepare a standard inoculum in cryovials (-50 o C, frozen spores in a 20% glycerol solution). By cultivation of this inoculum under different conditions, it was found that PGA activity raised to 128 IU l -1 when 0.4 g l -1 of salts was added to the medium.

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Section: Protease Detectionsupporting

confidence: 85%

Maintenance of penicillin G acylase expression by B. megaterium: preservation methods and activity recovery

Pinotti

Silva

Zangirolami

et al. 2007

Braz. J. Chem. Eng.

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“…To investigate the molecular basis of degeneration in C. acetobutylicum ATCC 824, we have examined two distinct (25,36) degenerate mutants of this strain. Neither mutant produces any detectable amounts of butanol or acetone, and both have been phenotypically stable since their isolation several years ago.…”

Section: Resultsmentioning

confidence: 99%

“…The C. acetobutylicum ATCC 824 mutant M5 (4), wherein the three inducible enzymes butyraldehyde dehydrogenase (BYDH), acetoacetate decarboxylase (AADC), and acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (CoAT) are not detectable in vitro (4,29), and consequently butanol and acetone are not produced, was isolated by N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The spontaneous mutant DG1 (25), which lacks in vitro AADC (36) and BYDH activities (25) and does not produce butanol or acetone, was obtained through serial subculturing of strain ATCC 824 for several generations. Mutant M5 has been complemented for acetone (23) and butanol (27) formation by being made to express plasmid-encoded genes for AADC (adc) plus CoAT (ctfA, ctfB) and BYDH (aad), respectively.…”

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confidence: 99%

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

et al. 1997

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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...

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“…Plasmid pPMFH1, provided by Philippe Soucaille (Institut National des Sciences Appliquées, Centre de Bioingénierie G. Durand, Toulouse, France), was used to obtain the hydA gene for PCR amplification and plasmid construction (12). C. acetobutylicum M5 lacks solvent-producing genes, including the region encoding an Fe-Ni hydrogenase (7,29,38).…”

Section: Methodsmentioning

confidence: 99%

2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum

Watrous¹,

Clark

Kutty

et al. 2003

Appl Environ Microbiol

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The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a K m of 152 M. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R 2 ‫؍‬ 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.

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Analysis of Degenerate Variants ofClostridium acetobutylicumATCC 824

Cited by 21 publications

References 21 publications

Maintenance of penicillin G acylase expression by B. megaterium: preservation methods and activity recovery

Maintenance of penicillin G acylase expression by B. megaterium: preservation methods and activity recovery

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

2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum

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