Cloning and sequencing of p-aminobenzoic acid synthetase genes from Lactococcus lactis subsp. lactis NCDO496 and Streptomyces lividans 1326.
Date
1991
Authors
Arhin, Francis Felix.
Journal Title
Journal ISSN
Volume Title
Publisher
Dalhousie University
Abstract
Description
Genes involved in the biosynthesis of p-aminobenzoic acid (PABA) were cloned from Lactococcus lactis subsp. lactis NCDO496 and Streptomyces lividans 1326. The L. lactis subsp. lactis gene was cloned by complementation of a pab mutant of S. lividans using pIJ41 as a vector. A deletion of about 1.2-kb from the vector sequence was required to allow transcriptional readthrough from a vector promoter. Attempts to complement pab mutants of Escherichia coli with the cloned L. lactis subsp. lactis fragment resulted in complementation of the pabB but not the pabA mutation. The nucleotide sequence of the cloned fragment contained a single open reading frame (ORF) similar to pabB genes from other organisms. A putative ribosome binding site (RBS) was also located. There was no consensus promoter sequence within the sequenced region. The S. lividans PABA synthetase genes were cloned as a single fragment that complemented the pabA and pabB mutations of E. coli. The cloned genes were expressed in E. coli when present in opposite orientations in the vector, indicating that an S. lividans promoter on the cloned fragment was being used. Attempts to express the cloned gene in pab mutants of S. lividans led to integration into the host chromosome. Use of a 2.7-kb pab-complementing fragment of the cloned gene as a probe at high stringency showed a hybridizing sequence in S. lividans strains 1326 and M252. At lower stringency, these S. lividans strains, as well as other Streptomyces, showed multiple hybridizing sequences. The nucleotide sequence of the 2.7-kb fragment revealed two ORFs similar to pabA and pabB genes from other organisms. Putative RBSs were located immediately upstream of each ORF. A putative promoter sequence was also located upstream of the pabB ORF. Comparison of codon usage in the PABA synthetase genes of L. lactis subsp. lactis, S. lividans and related genes from other organisms showed a bias in the direction of the G + C content of the organism.
Thesis (Ph.D.)--Dalhousie University (Canada), 1991.
Thesis (Ph.D.)--Dalhousie University (Canada), 1991.
Keywords
Biology, Molecular.