Open AccessVector for regulated expression of cloned genes in a wide range of gram-negative bacteria
Author(s) -
Nicolas Mermod,
Juan L. Ramos,
P. R. Lehrbach,
Kenneth N. Timmis
Publication year - 1986
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.167.2.447-454.1986
Subject(s) - biology , plasmid , gene , promoter , bacteria , pseudomonas putida , cloning (programming) , vector (molecular biology) , cloning vector , expression vector , multiple cloning site , restriction enzyme , ecori , gene expression , genetics , molecular cloning , microbiology and biotechnology , recombinant dna , computer science , programming language
A pKT231-based broad-host-range plasmid vector was constructed which enabled regulation of expression of cloned genes in a wide range of gram-negative bacteria. This vector, pNM185, contained upstream of its EcoRI, SstI, and SstII cloning sites the positively activated pm twin promoters of the TOL plasmid and xylS, the gene of the positive regulator of these promoters. Expression of cloned genes was induced with micromolar quantities of benzoate or m-toluate, the inexpensive coinducers of the pm promoters. Expression of a test gene, xylE, which specifies catechol 2,3-dioxygenase, cloned in this vector was tested in representative strains of a variety of gram-negative bacteria. Regulated expression of xylE was observed in most strains examined, and induced levels of enzyme representing up to 5% of total cellular protein and ratios of induced:noninduced levels of enzyme up to a factor of 600 were observed. The level of xylE gene expression in different bacteria tended to be correlated with their phylogenetic distance from Pseudomonas putida.