Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation

Oultram, John D.; Loughlin, Michael; Swinfield, T.J.; Brehm, John K.; Thompson, D.E.; Minton, Nigel P.

doi:10.1111/j.1574-6968.1988.tb03154.x

Cited by 111 publications

(33 citation statements)

References 15 publications

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“…We also used a different shuttle vector (pMTL500E) with another origin of replication in an attempt to increase the gene dosage. The pMLT500E plasmid (pAM␤1 origin) has a higher copy number in C. acetobutylicum (19,22) than the plasmids derived from low-copynumber pSOS84 (pIM13 origin) used by Wardwell et al (29). Combined with strong promoters (either P adc or P thl ), our approach resulted in levels of expression of the Cb-acr gene by C. acetobutylicum high enough to lead to 2,3-BD production, despite the fact that the detected specific activity is relatively low.…”

Section: Discussionmentioning

confidence: 99%

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d -2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum

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Kuit

López-Contreras

et al. 2011

Appl Environ Microbiol

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Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions, Clostridium acetobutylicum ATCC 824 (and strains derived from it) generates both D-and L-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under the control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mM D-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of both D-2,3-butanediol and meso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain of C. acetobutylicum.

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Section: Discussionmentioning

confidence: 99%

“…Methylated plasmids were electroporated into C. acetobutylicum by the method of Oultram et al (22). Correct methylation was checked by restriction analysis using Fnu4HI (18).…”

Section: Methodsmentioning

confidence: 99%

d -2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum

Siemerink

Kuit

López-Contreras

et al. 2011

Appl Environ Microbiol

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“…The electroporation technique used was based on published methods, 23 with modifications. After harvesting C. sporogenes, the cells were washed in 10 ml electroporation buffer (10% polyethylene glycol containing 1 mg/ml polyanetholesulfonic acid).…”

Section: Transformation Of C Sporogenesmentioning

confidence: 99%

Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis

Minton²,

et al. 2002

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“…The NCIMB 8052 strain has no indigenous plasmids, but it supports replication of a variety of vectors based on plasmids from Clostridium butyricum, Enterococcus faecalis, and Streptococcus cremoris (41). They may be introduced by either electroporation (46) or intergeneric conjugation with Escherichia coli donors (56). This strain is also susceptible to Tn1545 mutagenesis (58).…”

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Physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome

Wilkinson

Young

1995

J Bacteriol

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A combined physical and genetic map of the single, circular, 6.7-Mbp chromosome of the NCIMB 8052 strain of Clostridium beijerinckii (formerly Clostridium acetobutylicum) has been constructed by using a combination of cloned DNA fragments as hybridization probes and a bank of strains harboring insertions of the conjugative transposon Tn1545. The positions of 81 restriction endonuclease cleavage sites and 32 genes have been determined. Eight genes concerned with solventogenic fermentation are found at three different locations. The chromosome contains at least 13 rrn operons, 11 of which have been located on the map. Their transcriptional orientation diverges from the presumed location of the replication origin.Clostridium beijerinckii (formerly Clostridium acetobutylicum) is an obligately anaerobic spore-forming bacterium that produces acetone, butanol, and small amounts of ethanol when grown on sugary substrates. This process, known as ABE fermentation, has been used extensively for the industrial scale production of acetone and butanol (28). Several different strains are currently under investigation in various laboratories worldwide. They have been separated into three distinct groups on the basis of their biochemical and physiological characteristics, the extent of hybridization between their DNAs, and macrorestriction fragment profiles of their genomes (54, 57). The NCIMB 8052 strain, which belongs to the group that has the largest chromosome, is amenable to genetic manipulation (59, 60). It lacks the active restriction system that is found in the ATCC 824 strain (38). The NCIMB 8052 strain has no indigenous plasmids, but it supports replication of a variety of vectors based on plasmids from Clostridium butyricum, Enterococcus faecalis, and Streptococcus cremoris (41). They may be introduced by either electroporation (46) or intergeneric conjugation with Escherichia coli donors (56). This strain is also susceptible to Tn1545 mutagenesis (58). Homologous recombination between incoming sequences and their counterparts in the bacterial chromosome has recently been demonstrated (55).As an additional aid to genetic analysis of this organism, we have constructed a physical map of the 6.7-Mbp chromosome using pulsed-field gel electrophoresis (PFGE) (50). The application of PFGE to the analysis of bacterial genomes has revealed that the majority of bacteria have single circular chromosomes. However, several exceptions to this general rule have been described. Some bacteria, such as Borrelia burgdorferi and Rhodococcus fascians, have linear chromosomes (4,20,24), whereas Streptomyces lividans and Streptomyces coelicolor have linear chromosomes that may become circular as a result of deletions spanning both telomeres (34,35). Still others, such as Brucella melitensis and Leptospira interrogans, have segmented genomes (40, 63). The segmented genome of Agrobacterium tumefaciens has one circular and one linear component (1). The results reported here indicate that the NCIMB 8052 strain of C. beijerinckii, like most othe...

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Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation

Cited by 111 publications

References 15 publications

d -2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum

d -2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum

Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis

Physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome

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