Transcriptional wiring of the TOL plasmid regulatory network to its host involves the submission of the sigma54-promoter Pu to the response regulator PprA.
Implantation of the regulatory circuit of the degradation pathway of TOL plasmid pWW0 in the native transcriptional network of the host Pseudomonas putida involves interplay between plasmid- and chromosome-encoded factors. We have employed a reverse genetics approach to investigate such a molecular wiring by identifying host proteins that form stable complexes with Pu, the sigma(54)-dependent promoter of the upper TOL operon of pWW0. This approach revealed that the Pu upstream activating sequences (UAS), the target sites of the cognate activator XylR, form a specific complex with a host protein which, following DNA affinity purification and mass spectrometry analysis, was identified as the LytTR-type two-component response regulator PprA. Directed inactivation of pprA resulted in the upregulation of the Pu promoter in vivo, while expression of the same gene from a plasmid vector strongly repressed Pu activity. Such a downregulation of Pu by PprA could be faithfully reproduced both in vitro with purified components and in an in vivo reporter system assembled in Escherichia coli. The overlap of the PprA and XylR binding sites suggested that the basis for the inhibitory effect on Pu was a mutual exclusion mechanism between the two proteins to bind the UAS. We argue that the binding of the response regulator PprA to Pu (a case without precedents in sigma(54)-dependent transcription) helps to anchor the TOL regulatory subnetwork to the wider context of the host transcriptome, thereby allowing the entry of physiological signals that modulate the outcome of promoter activity.
Vitale
Elena
E
Department of Biomolecular Sciences and Biotechnology, University of Milan, Milan, Italy.
Milani
Andrea
A
Renzi
Francesco
F
Galli
Enrica
E
Rescalli
Emanuela
E
de Lorenzo
Víctor
V
Bertoni
Giovanni
G
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D001673:Biodegradation, Environmental; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D015964:Gene Expression Regulation, Bacterial; D009876:Operon; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012808:Sigma Factor / Q000378:metabolism; D014050:Toluene / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C074322:XylR protein, Pseudomonas; C104566:XylW protein, Pseudomonas putida; D014050:Toluene