An enhanced broad-host-range vector for gram-negative bacteria: avoiding tetracycline phototoxicity during the growth...

A mobilizable, broad-host-range (bhr) plasmid was derived from the widely used IncP1 vector pRK415. The new vector, pRKD418, contains an additional resistance gene and an enlarged multiple cloning site (MCS) region. The optimal growth of pRK415-containing bacteria under photosynthetic conditions generally requires the use of optical filters to protect the selective antibiotic tetracycline (Tc) from photooxidation with the resulting production of toxic photoproducts; pRK415 is not stably maintained in the absence of selective pressure. The addition of a trimethoprim-resistant dihydrofolate reductase-encoding gene provided for optimal photosynthetic growth in the presence of a selective antibiotic without any special apparatus. The presence of an antibiotic marker not found in commonly used cloning vectors in many cases facilitates the subcloning of inserts into the bhr plasmid. The new MCS region provides further cloning flexibility with at least sixteen available restriction sites. Easily constructed derivative plasmids, exemplified by pRKD418KmE, provide a convenient screening procedure for the detection of recombinants during subcloning.