Transcription reporters that shuttle cloned DNA between high-copy Escherichia coli plasmids and low-copy broad-host-range plasmids.
We describe and apply lacZ transcription reporter plasmids designed for both biochemical analyses requiring high DNA yield and physiological studies requiring low gene dosage. Standard DNA ligations are performed at seven unique restriction sites 5' to the lacZ gene on high-copy ColE1 plasmids suitable for double- or single-strand DNA sequencing. A divergent gusA transcription reporter is included and serves as an internal control. Rec(+) Escherichia coli cells readily shuttle DNA placed between gusA and lacZ by allelic exchange with pRK290-based plasmids that subsequently conjugate and replicate in most gram-negative bacteria. We applied this system to study Caulobacter crescentus cell cycle promoters directed by the CtrA response-regulator protein. Synthetic oligonucleotides were ligated to create altered CtrA binding sites and corresponding promoters with varied transcription strength. We also document the phenomenon of long-range promoter interference. A strong promoter can repress up to twofold the transcription from a divergent promoter located 100 bp away. However, the cell cycle timing of both promoters is not changed. Additional applications of our system and theoretical aspects of promoter organization are discussed.
Ouimet
M C
MC
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, H3A 2B4, Canada.
Marczynski
G T
GT
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D001483:Base Sequence; D016935:Caulobacter crescentus / Q000235:genetics*; D002453:Cell Cycle; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D017930:Genes, Reporter; D005822:Genetic Vectors; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000032:analysis; D015183:Restriction Mapping; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C489570:FliQ protein, E coli; D008565:Membrane Proteins; D011994:Recombinant Proteins; C074891:FliQ protein, Bacteria; D001616:beta-Galactosidase