In vivo and in vitro effects of (p)ppGpp on the sigma(54) promoter Pu of the TOL plasmid of Pseudomonas putida

The connection between the physiological control of the sigma(54)-dependent Pa promoter of the TOL plasmid pWW0 of Pseudomonas putida and the stringen t response mediated by the alarmone (p)ppGpp has been examined in vivo an i n vitro. To this end, the key regulatory elements of the system were faithf ully reproduced in an Escherichia coli strain and assayed as lacZ fusions i n various genetic backgrounds lacking (p)ppGpp or overexpressing reLA. Neit her the responsiveness of Pu to S-methyl benzylalcohol mediated by its cogn ate activator XylR nor the down-regulation of the promoter by rapid growth were affected in relA/spoT strains to an extent which could account for the known physiological control that governs this promoter. Overexpression of the relA gene [predicted to increase intracellullar (p)ppGpp levels] did, h owever, cause a significant gain in Pu activity. Since such a gain might be the result of indirect effects, we resorted to an in vitro transcription s ystem to assay directly the effect of ppGpp on the transcriptional machiner y. Although we did observe a significant increase in Pu performance through a range of sigma(54)-RNAP concentrations, such an increase never exceeded twofold. The difference between these results and the behavior of the relat ed Po promoter of the phenol degradation plasmid pVI150 could be traced to the different promoter sequences, which may dictate the type of metabolic s ignals recruited for the physiological control of sigma(54)-systems.