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 Pu promoter of the TOL plasmid pWW0 of Pseudomonas putida and the stringent response mediated by the alarmone (p)ppGpp has been examined in vivo an in vitro. To this end, the key regulatory elements of the system were faithfully reproduced in an Escherichia coli strain and assayed as lacZ fusions in various genetic backgrounds lacking (p)ppGpp or overexpressing relA. Neither the responsiveness of Pu to 3-methyl benzylalcohol mediated by its cognate 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, however, 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 system to assay directly the effect of ppGpp on the transcriptional machinery. 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 related Po promoter of the phenol degradation plasmid pVI150 could be traced to the different promoter sequences, which may dictate the type of metabolic signals recruited for the physiological control of sigma(54)-systems.
Carmona
M
M
Department of Environment, Universidad Europea CEES, Villaviciosa de Odón, 28670 Madrid, Spain.
Rodríguez
M J
MJ
Martínez-Costa
O
O
De Lorenzo
V
V
D000596:Amino Acids / Q000494:pharmacology; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D006158:Guanosine Pentaphosphate / Q000235:genetics / Q000378:metabolism*; D007763:Lac Operon; D008025:Ligases / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics* / Q000502:physiology; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000235:genetics*; D014158:Transcription, Genetic
D000596:Amino Acids; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012808:Sigma Factor; C017721:casamino acids; C497751:rpoN protein, E coli; D006158:Guanosine Pentaphosphate; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases