pTIM3, a plasmid delivery vector for a transposon-based inducible marker gene system in gram-negative bacteria

pTIM3 is a suicide plasmid vector for the delivery of a transposition defective derivative of Tn5, expressing inducible mercury resistance (HgR) and catechol 2,3-dioxygenase (C23O) activity, to a range of gram-negative microorganisms. pTIM3 was constructed by a four-stage process from pNMM1, a derivative of pSUP5011 containing a modified Tn5 where antibiotic-resistance determinants have been replaced by the mer operon of Tn501 and tdnC (encoding a C23O). In pTIM3, tdnC is fused to merD, to bring C23O expression under the control of the mer operon. pTIM3 was introduced into Acinetobacter calcoaceticus, Pseudomonas putida, Burkholderia cepacia, and Alcaligenes sp., and isolates were examined for loss of the vector, which is not stably maintained outside the Enterobacteriaceae, but retention of the Tn5 derivative. Transposition results from the action of a vector-encoded Tn5 transposase (Tnp) on the ends of IS50L and IS50R retained in the Tn5 construct. Between 10 and 20% of the isolates contained a single copy of the defective transposon in the chromosome. A single-copy isolate of each species was assayed for inducibility of C23O activity using HgCl2 as inducer. The increase in C230 activity was linear with increasing HgCl2 concentration and ranged between 10- and 20-fold at 20 micrograms/ml. HgR and C23O+ phenotypes were found to be stably maintained in each of the isolates following 40 generations of nonselective growth. A novel aspect of this marker gene system is that isolates can be recovered on nonselective medium and then sprayed with a catechol/HgCl2 mixture for rapid colorimetric detection. The system can be applied to the tracking of genetically modified microorganisms in the environment and has the advantage that their detection may be achieved cheaply and without the use of sophisticated equipment.