isopropyl-thiogalactoside and 3-xylene

The sigma 54-dependent Pu promoter of the TOL plasmid pWW0 of Pseudomonas putida becomes activated by the prokaryotic enhancer-binding XyIR protein when cells encounter m-xylene in the medium. However, even in the presence of the aromatic inducer, Pu activity is silenced in vivo during rapid exponential growth of the cells in rich medium. Various elements known to be involved in the control of the transcriptional activity of the promoter were examined to ascertain the mechanism by which expression of Pu is limited during the exponential phase of growth. A truncated and fully constitutive XyIR derivative deleted of its signal-reception N-terminal domain was found to be subjected to the same exponential silencing as the wild-type XyIR when exposed to m-xylene. This indicated that the phenomenon is not due to a late activation of XyIR by the aromatic effector. A Pu variant in which the integration host factor (IHF)-binding site had been functionally replaced by a statically curved DNA segment showed the same induction pattern, thus ruling out variations in the intracellular levels of IHF changes during growth as the element responsible for the inactivity of Pu in rapidly growing cells. On the contrary, overproduction of the sigma 54 factor allowed Pu expression during exponential phase. As sigma 54 protein levels remained approximately constant during growth, the exponential silencing of Pu could be caused ultimately by changes in the activity of the factor itself. This effect may not be exclusive to Pu, but could be a general co-regulation mechanism in sigma 54-dependent promoters that connects transcription of a specific set of genes with the general physiological status of the cells.

Topics: Bacterial Proteins; beta-Galactosidase; Blotting, Western; Cell Division; DNA-Binding Proteins; DNA-Directed RNA Polymerases; Enhancer Elements, Genetic; Escherichia coli; Gene Expression Regulation, Bacterial; Integration Host Factors; Isopropyl Thiogalactoside; Lac Operon; Plasmids; Promoter Regions, Genetic; Pseudomonas putida; Sigma Factor; Transcription Factors; Transcription, Genetic; Transformation, Genetic; Xylenes

The mechanism under which the signal-reception amino-terminal portion (A domain) of the prokaryotic enhancer-binding protein XylR controls the activity of the regulator has been investigated through complementation tests in vivo, in which the various protein segments were produced as independent polypeptides. Separate expression of the A domain repressed the otherwise constitutive activity of a truncated derivative of XylR deleted of its A domain (XylR delta A). Such inhibition was not released by m-xylene, the natural inducer of the system. Repression caused by the A domain was specific for XylR because it did not affect activation of the sigma 54 promoter PnifH by a derivative of its cognate regulator, NifA, deleted of its own A domain. The A domain was also unable to repress the activity of a NifA-XylR hybrid protein resulting from fusing two-thirds of the central domain of NifA to the carboxyl-terminal third of XylR, which includes its DNA-binding domain. The inhibitory effect caused by the A domain of XylR on XylR delta A seems, therefore, to result from specific interactions in trans between the two truncated proteins and not from mere hindering of an activating surface.

Topics: Bacterial Proteins; Binding Sites; DNA-Binding Proteins; Enhancer Elements, Genetic; Escherichia coli; Isopropyl Thiogalactoside; Models, Structural; Peptide Fragments; Promoter Regions, Genetic; Recombinant Fusion Proteins; Repressor Proteins; Transcription Factors; Xylenes