Yasuhiko Takeda

Isolation of a tetracycline resistance plasmid from a glutamate-producing corynebacterium, Corynebacterium melassecola

Abstract A tetracycline resistance plasmid pAG1 (20.4 kb) and a cryptic plasmid pAG3 (4.5 kb) have been isolated from a non-pathogenic glutamate-producing corynebacterium, Corynebacterium melassecola . A 3.1-kb Bam HI/ Bgl II fragment carrying the tetracycline resistance gene of pAG1 was isolated and ligated into the Bam HI site of pAG3. A resultant small tetracycline resistance plasmid pAG50 (7.6 kb) was obtained. This tetracycline resistance marker is good enough to allow direct selection in transformation experiments.

Cloning and sequencing of the gene encoding cytochrome c-553 (CO) from Gluconobacter suboxydans

Abstract The gene encoding cytochrome c -553 (CO) from Gluconobacter suboxydans was cloned by using a synthetic oligonucleotide probe. The complete nucleotide sequence of the cytochrome c -553 (CO) gene including 5′ and 3′ flanking regions has been determined. The sequence predicts a 478 amino acid residue protein product which contains three Cys-X-Y-Cys-His heme c binding sites. The predicted amino acid sequence of the cytochrome c -553 (CO) gene was found to have considerable similarity (60%) to that of the alcohol dehydrogenase (ADH) cytochrome subunit from Acetobacter aceti . It is suggested that the cytochrome c -553 (CO) may act as an ADH cytochrome subunit for ethanol oxidation and be related to the KCN-insensitive and non-energy-generating bypath in the sugar-oxidizing respiratory chain in G. suboxydans .

Expression of cytochrome c-553(CO) gene that complements the second subunit deficiency of membrane-bound alcohol dehydrogenase in Gluconobacter suboxydans subsp. α

Cytochrome c -553 (CO) gene was subcloned onto a shuttle vector pGEA1 able to replicate in Gluconobacter suboxydans and Escherichia coli , forming pGEAC1. pGEAC1 transformed G. suboxydans subsp. α which has low ethanol oxidation activity because of a second subunit deficiency of membrane-bound alcohol dehydrogenase (ADH). The transformants harboring pGEAC1 expressed ethanol oxidation activity and produced material cross-reactive with anti-cytochrome c -553 (CO) antibody in the membrane. Furthermore, expression of cytochrome c -553 (CO) gene resulted in elevated levels of heme c and CO-reactive cytochrome c , complemented the deficiency of the second ADH subunit, and restored ethanol oxidase activity. We previously reported that cytochrome c -553 (CO) has significant amino acid sequence homology (60%) to the cytochrome subunit of ADH from Acetobacter aceti (Takeda, Y. et al. , J. Ferment. Bioeng., 72, 1, 1991). Hence, cytochrome c -553 (CO) is the second subunit of ADH.

Role of cytochrome c-553(CO), the second subunit of alcohol dehydrogenase, in the azide-insensitive respiratory chain and in oxidative fermentation of Gluconobacter species

Abstract Gluconobacter suboxydans subsp. α, a variant species of G. suboxydans hardly capable of oxidizing ethanol, is known to be defective in the second subunit of alcohol dehydrogenase (ADH) so that the membranes of the strain exhibit neither ADH nor ethanol oxidase activity. These enzyme activities have been shown to be restored by reconstituting the second subunit to the membranes. The second subunit of ADH has been shown to be a cytochrome c -553(CO) (Takeda, Y. et al. , J. Ferment. Bioeng., 73, 89, 1992). A plasmid, pGEAC1, carrying the cytochrome c -553(CO) gene was introduced in G. suboxydans subsp. α and could increase dehydrogenase activities for d -glucose, d -sorbitol and glycerol in the membranes. Oxidase activities for d -glucose, d -sorbitol and glycerol were also increased in both the membrane and the cells of G. suboxydans subsp. α haboring pGEAC1. Furthermore, pGEAC1 could restore azide insensitivity in the respiratory chain of G. suboxydans subsp. α which lacks insensitivity against azide or cyanide. The azide insensitivity was observed not only in the membranes but also in the intact cells of G. suboxydans subsp. α (pGEAC1). Thus, cytochrome c -553(CO) seems to be indispensable in the pathway of the azide-insensitive respiratory chain bypass of G. suboxydans . In addition, the increase of oxidative ability in the practical oxidative fermentation was observed in the intact cells of G. suboxydans subsp. α (pGEAC1) which increased the azide insensitivity in the respiratory chain.

Cloning and expression in Escherichia coli of the glutamate dehydrogenase gene, gdh, from Corynebacterium melassecola

Abstract A hybrid plasmid containing a fragment of the Corynebacterium melassecola chromosome cloned into pBR325 restored growth of glutamate auxotrophs of Escherichia coli strains that have mutations in the genes for glutamate dehydrogenase and glutamate synthase. A 3.1-kilobase pair region was shown by complementation analysis and enzyme measurements to carry the glutamate dehydrogenase gene, gdh. Glutamate dehydrogenase encoded by gdh carried on recombinant plasmids was elevated over 100-fold in E. coli cells. The gdh promoter was located by in vitro fusion to a promoter-deficient galK gene.