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 ( Hg-R) and catechol 2,3-dioxygenase (C230) activity, to a range of gram -negative microorganisms. pTIM3 was constructed by a four-stage proces s from pNMM1, a derivative of pSUP5011 containing a modified Tn5 where antibiotic-resistance determinants have been replaced by the mer oper on of Tn501 and tdnC (encoding a C230). In pTIM3, tdnC is fused to mer D, to bring C230 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 Ent erobacteriaceae, but retention of the Tn5 derivative. Transposition re sults 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 transpos on in the chromosome. A single-copy isolate of each species was assaye d for inducibility of C230 activity using HgCl2 as inducer. The increa se in C230 activity was Linear with increasing HgCl2 concentration and ranged between 10- and 20-fold at 20 mu g/ml. Hg-R and C23O(+) phenot ypes were found to be stably maintained in each of the isolates follow ing 40 generations of nonselective growth. A novel aspect of this mark er gene system is that isolates can be recovered on nonselective mediu m and then sprayed with a catechol/HgCl2, mixture for rapid colorimetr ic 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 sophis ticated equipment. (C) 1995 Academic Press, Inc.