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Molecular Cloning of the leuB Gene from Bacteroides fragilis by Functional Complementation in Escherichia coli
Author(s) -
Sarker Mahfuzur R.,
Akimoto Shigeru,
Ono Tsuneko,
Kinouchi Takemi,
Ohnishi Yoshinari
Publication year - 1995
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.1995.tb02163.x
Subject(s) - biology , bacteroides fragilis , complementation , escherichia coli , microbiology and biotechnology , southern blot , gene , genomic library , molecular cloning , cloning (programming) , genetics , clone (java method) , peptide sequence , mutant , bacteria , computer science , programming language
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 a 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 β‐isopropylmalate dehydrogenase ( leuB ).