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J Bacteriol, June 1998, p. 2889-2894, Vol. 180, No. 11
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Activation and Repression of Transcription at the Double Tandem Divergent Promoters for the xylR and xylS Genes of the TOL Plasmid of Pseudomonas putida

Silvia Marqués,1 María-Trinidad Gallegos,1 Maximino Manzanera,1 Andreas Holtel,2 Kenneth N. Timmis,2 and Juan L. Ramos1,*

Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Department of Biochemistry, E-18008 Granada, Spain,1 and GBF, D-38124 Braunschweig, Germany2

Received 9 January 1998/Accepted 23 March 1998

The xylR and xylS genes are divergent and control transcription of the TOL plasmid catabolic pathways for toluene metabolism. Four promoters are found in the 300-bp intergenic region: Pr1 and Pr2 are constitutive sigma 70-dependent tandem promoters that drive expression of xylR, while expression of the xylS gene is driven from Ps2, a constitutive sigma 70-dependent promoter, and by the regulatable sigma 54 class Ps1 promoter. In Ps1 the XylR targets (upstream activator sequences [UASs]) overlap the Pr promoters, and two sites for integration host factor (IHF) binding are located at the region from positions -2 to -30 (-2/-30 region) and the -137/-156 region, the latter overlapping the Pr promoters. When the XylR protein binds to the UASs in the absence of effector, it represses expression from Pr promoters. In the XylR-plus background and in the absence of an effector, the level of expression from Ps1 is low, although detectable, whereas Ps2 is active. In this background and in the presence of an effector, XylR increases autorepression. In a sigma 54-deficient Pseudomonas putida background, no expression occurred from Ps1 regardless of the presence of an effector. However, in the presence of an effector, the amount of RNA produced from Pr promoters was almost undetectable. This finding suggests that when no transcription occurred at the Ps1 promoter, clearance of XylR from the UASs was almost negligible. In this background, expression from Ps2 was very high regardless of the presence of an effector; this finding suggests that RNA polymerase containing sigma 54 modulates expression from the downstream Ps2 sigma 70-dependent promoter. In a P. putida IHF-minus background and in the presence of effector, Ps1 expression was the highest found; in contrast, the basal levels of this promoter were the lowest observed. This finding suggests that IHF acts in vivo as a repressor of the sigma 54-dependent Ps1 promoter. In an IHF-deficient host background, expression from Ps2 in the presence of effector was negligible. Thus, binding of RNA polymerase containing sigma 54 at the upstream promoter may modulate expression from the Ps2 promoter.

* Corresponding author. Mailing address: CSIC-Estación Experimental del Zaidín, Apdo Correos 419, E-18008 Granada, Spain. Phone: 34-58-121011. Fax: 34-58-129600. E-mail: jlramos{at}eez.csic.es.

J Bacteriol, June 1998, p. 2889-2894, Vol. 180, No. 11
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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