Construction and use of a new broad-host-range lacZ transcriptional fusion vector, pHRP309, for gram- bacteria

A new lacZ transcriptional fusion vector, pHRP309, based on the IncQ plasmid RSF1010, was constructed and shown to be easily mobilized into a variety of Gram- eubacteria. We also developed a two-step cloning procedure to facilitate the cloning of small promoter fragments into the fusion vector. A set of 'cohort' vectors was constructed which allowed directed cloning of fragments downstream from an omega streptomycin/spectinomycin-resistance cassette while maintaining multiple flanking restriction sites. The omega cassette provides a selectable antibiotic-resistance marker for cloning promoters into the fusion vector and makes mapping to determine fragment orientation unnecessary. The presence of the omega cassette also decreases background beta-galactosidase activity by decreasing readthrough transcription from plasmid sequences. The fusion vector carries a gentamicin-resistance-encoding gene as the selectable marker and can therefore be used in Tn5 (kanamycin-resistant) and Tn10 (tetracycline-resistant) mutant strains. Since pHRP309 is a member of the IncQ incompatibility group, it is compatible with IncP cloning vectors and can be used in strains carrying cloned regulatory genes. Using this system, we cloned the positively regulated Pseudomonas putida pcaI promoter and studied its regulation.