Analysis of Pseudomonas gene products using lacIq/Ptrp-lac plasmids and transposons that confer conditional phenotypes

Abstract

Novel transposon and plasmid-based broad-host-range expression systems have been developed to facilitate the genetic analysis of gene products of Pseudomonas and related Gram- bacteria. The properties of lacIq/Ptrp-lac were used to construct mini-Tn5 expression vector transposons and RSF1010-derived plasmids for controlled expression and generation of conditional phenotypes. These plasmids were used to hyper-express the XylS regulator of the meta operon of the TOL plasmid of P. putida or the bphB and bphC genes of the polychlorobiphenyl-degrading pathway of Pseudomonas sp. LB400 in different strains of Pseudomonas instead of in Escherichia coli. Specific activity of 2.3 dihydroxybiphenyl dioxygenase (bphC gene product) was increased tenfold when hyperproduced in its native host as compared to E. coli, but under the same in vivo conditions, the XylS regulator formed protein aggregates. The other lacIq/Ptrp-lac-based expression vector presented here, transposon mini-Tn5 lacIq/Ptrc, facilitates the insertion of genetic cassettes containing heterologous genes under the control of lac inducers in the chromosome of target bacteria, as shown by monitoring expression of a lacZ reporter cloned in mini-Tn5 lacIq/Ptrc and inserted in the chromosome of P. putida.