Broad host range plasmids carrying the Escherichia coli lactose and galactose operons

Abstract

1. SUMMARY We have developed a number of broad-host-range plasmids that allow the expression of the Escherichia coli lac operon from any cloned promoter , and the creation of 'in phase" fusions between lacZ and other cloned genes. In a second series of constructions, the E. coli gal operon has been cloned into the broad-host-range vector and a plasmid carrying both the E. coli gal and lac genes is described. These plasmids have been transferred into Pseudomonas aen¢gbzosa and Zy-mmnonas mobilis and their effects on the utilisa-tion of lactose and galactose have been investigated. 2. INTRODUCTION Broad-host-range plasmids facilitate the transfer of genes to a wide range of genetic back-Com'slnmdence to: S. Busby, School of BiochemistD'. University of Birmingham, P.O. Box 363, Birmingham BI5 2TT, UK. grounds [1]. We recently described the development of a new broad-host-range promoter-probe plasmid carrying the Escherichia coli lac genes. without a promoter, cloned downstream from a polylinker [2]. This plasmid has now been sent to several dozen different laboratories and has been exploited to assay cloned promoters in a variety of situations, (see for example refs. 3,4). Here we describe a number of improvements to pRW2, the construction of derivatives carrying the E. coli gal operon and the effects of these derivatives on the growth of Pseudomonas aeruginosa and Zy-momonas mobilis. 3. MATERIALS AND METHODS E. coli M182 and P. aeruginosa PAOI were grown and transformed as described previously [2]. Plasmids were maintained by the addition of tetracycline at 100/.tg/ml for P. aeruginosa or 35 /.tg/ml for E. coli. For growth of P. aeruginosa and E. coli in minimal medium, we used the chemically defined medium described by Brown et al. [5] supplemented with different sugars. Z.