Molecular cloning of the leuB gene from Bacteroides fragilis by functional complementation in Escherichia coli.
Clones containing the Bacteroides fragilis leuB-complementing gene were isolated by screening of a B. fragilis genomic library constructed in Escherichia coli. One recombinant clone, designated pOT865, with the smallest DNA insert (4.5 kb) could complement three independent leuB mutations in E. coli and the leuB-complementing determinant in pOT865 was localized to a region of 1.5-kb DNA. The results of Southern blot analysis suggested that a single copy of the cloned gene was present in the B. fragilis genome. The cloned fragment appeared to contain a sequence that could function as promoter in E. coli and direct the synthesis of a 42-kDa protein. These results suggest that the cloned segment contains the structural gene for beta-isopropylmalate dehydrogenase (leuB).
Sarker
M R
MR
Department of Bacteriology, School of Medicine, University of Tokushima, Japan.
Akimoto
S
S
Ono
T
T
Kinouchi
T
T
Ohnishi
Y
Y
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D001441:Bacteroides fragilis / Q000201:enzymology / Q000235:genetics*; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D005816:Genetic Complementation Test; D015698:Genomic Library; D007930:Leucine / Q000096:biosynthesis; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping
D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; D007930:Leucine