VTR EXPRESSION CASSETTES FOR ENGINEERING CONDITIONAL PHENOTYPES IN PSEUDOMONAS - ACTIVITY OF THE PU PROMOTER OF THE TOL PLASMID UNDER LIMITING CONCENTRATIONS OF THE XYLR ACTIVATOR PROTEIN

A simplified procedure to construct recombinant Pseudomonas putida (Pp ) and related bacteria, which transcribe conditionally specific genes inserted into their chromosome in response to lac inducers such as IPT G, has been developed. The method is based on the so-called VTR expres sion cassettes. These are three small (1.98-kb) DNA segments engineere d as NotI restriction fragments that include a lacI(q) gene along with the hybrid trp/lac promoter, Ptrc, followed by an optimised translati on initiation region with a leading ATG and a multiple cloning site in each of the three reading frames. This arrangement allows the chromos omal insertion of the conditionally expressed genes of interest throug h its transfer to any of the mini-Tn5 transposon vectors available. VT R cassettes permit construction of specialized strains that are instru mental to address, by genetic means, otherwise intractable regulatory problems observed in biodegradative pathways of Pp. In this context, t he VTR system was employed to examine the effect of the intracellular concentration of XylR, the main regulator of the TOL (toluene biodegra dation) plasmid pWW0, on the exponential silencing of the promoter of the upper operon, Pu. Increasing concentrations of XylR resulted in mo re intense induction of the system that, however, remained silent duri ng fast cell growth regardless of activator levels.