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Journal of Bacteriology, September 2000, p. 4711-4718, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

In Vivo and In Vitro Effects of (p)ppGpp on the sigma 54 Promoter Pu of the TOL Plasmid of Pseudomonas putida

Manuel Carmona,1 Maria J. Rodríguez,2 Óscar Martínez-Costa,2 and Víctor de Lorenzo2,*

Department of Environment, Universidad Europea CEES, Villaviciosa de Odón, 28670 Madrid,1 and Department of Microbial Biotechnology, Centro Nacional de Biotecnología CSIC, 28049 Madrid,2 Spain

Received 13 March 2000/Accepted 28 May 2000

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.

* Corresponding author. Mailing address: Centro Nacional de Biotecnología del CSIC, 28049 Madrid, Spain. Phone: 34 91 585 4536. Fax: 43 91 585 4506. E-mail: vdlorenzo{at}cnb.uam.es.

Journal of Bacteriology, September 2000, p. 4711-4718, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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