Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing

Abstract

Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacI q - lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alpha- and gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZ α. In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10- to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens , a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation.