Open AccessCloning and Sequencing of the Gene which Encodes the Highly Inducible Acetamidase of Mycobacterium Smegmatis
Author(s) -
Eshwar Mahenthiralingam,
P. Draper,
Davis Eo,
Colston Mj
Publication year - 1993
Publication title -
journal of general microbiology/journal of general microbiology
Language(s) - English
Resource type - Journals
eISSN - 2059-9323
pISSN - 0022-1287
DOI - 10.1099/00221287-139-3-575
Subject(s) - gene , biology , mycobacterium smegmatis , amidase , nucleic acid sequence , ribosomal binding site , microbiology and biotechnology , start codon , open reading frame , genetics , oligonucleotide , peptide sequence , cloning (programming) , mycobacterium tuberculosis , ribosome , nucleotide , bacteria , rna , medicine , tuberculosis , pathology , computer science , programming language
The acetamidase of Mycobacterium smegmatis NCTC 8159 was purified, and the sequences of its amino-terminus and of two peptides obtained by proteolysis of the protein were obtained. A DNA fragment including the amidase structural gene was cloned in Escherichia coli, using oligonucleotide probes designed on the basis of the peptide sequences and a codon usage table calculated from published sequences of nine protein-antigen-encoding genes of the Mycobacterium tuberculosis complex. Sequence analysis of the cloned DNA revealed that the amidase gene encoded 406 amino acid residues. The nucleotide sequence close to and upstream of the amidase gene contained a probable ribosome-binding site but no identifiable promoter sequences. Three additional potential open-reading frames were found upstream of and very close to the amidase gene, with consensus '-35' and '-10' promoter sites between the first and second of these. It is hoped that the highly inducible expression of the acetamidase gene can be exploited to allow regulated expression of other genes cloned in mycobacteria.