Pm promoter expression mutants and their use in broad-host-range RK2 plasmid vectors.
By coupling the Pm/xylS promoter system to minimal replicons of the broad-host-range plasmid RK2 we recently showed that such vectors are useful for both high- and low-level inducible expression of cloned genes in gram-negative bacteria. In this report, we extend this potential by identifying point mutations in or near the -10 transcriptional region of Pm. Point mutations leading to gene-independent enhancements of expression levels of the induced state or reduced background expression levels were identified using Escherichia coli as a host. By combining these mutations an additive effect in expression levels from the constructed Pm was observed. The highest induced expression level was obtained by inserting an E. coli consensus sigma70 - 10 recognition region. Most of the remaining activities in the reduced-background mutations appeared to originate from a transcriptional start site other than Pm. The effects of some of these mutations were also analyzed in Pseudomonas aeruginosa and were found to act similarly, but less pronounced in this host.
UNIGEN Center for Molecular Biology and Department of Biotechnology, Norwegian University of Science and Technology, Trondheim.
H C
HC
Winther-Larsen
J M
JM
Blatny
B
B
Valand
T
T
Brautaset
S
S
Valla
D001426:Bacterial Proteins; D001483:Base Sequence; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D015534:Trans-Activators; C078720:XylS protein, Pseudomonas putida