Identification of genes regulated by the MvaT-like paralogues TurA and TurB of Pseudomonas putida KT2440.

The genome of the Pseudomonas putida strain KT2440 contains five paralogous proteins (TurA, TurB, TurC, TurD and TurE) of the H-NS-like MvaT class of transcription regulators. TurA and TurB belong to groups I and II, respectively, both containing orthologous MvaT proteins that are present in all Pseudomonadaceae species. On the contrary, TurC, TurD and TurE belong to group III, which contains species-specific paralogous MvaT proteins. We analysed the global effects on the P. putida KT2440 transcriptome of eliminating the conserved TurA and TurB proteins, which had been identified in our previous studies aimed to search for novel specific co-regulators of the upper TOL operon for toluene biodegradation. While the loss of TurA de-repressed the expression of many genes covering a broad range of functional classes in both mid-exponential and early stationary phases, the absence of TurB brought about a very different outcome. Although the loss of TurB affected also very different functions, the number of genes that changed in the turB mutant was fivefold smaller than that of TurA. Furthermore, TurB does not act generally as repressor. Interestingly, the degree of overlap between their mutual regulons is very limited. A closer examination of one case where such overlap clearly occurs (a gene cluster for biosynthesis of lipodepsinonapeptide phytotoxins) revealed that TurA and TurB can act in concert, perhaps by forming a heterodimer. In addition, our results indicate that TurA is the master regulator of TurB as well as of the other paralogues, TurD and TurE.

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.

Novel physiological modulation of the Pu promoter of TOL plasmid: negative regulatory role of the TurA protein of Pseudomonas putida in the response to suboptimal growth temperatures.

From crude protein extracts of Pseudomonas putida KT2440, we identified a small protein, TurA, able to bind to DNA fragments bearing the entire Pu promoter sequence of the TOL plasmid. The knock-out inactivation of the turA gene resulted in enhanced transcription initiation from the Pu promoter, initially suggesting a negative regulatory role of TurA on Pu expression. Ectopic expression of TurA both in P. putida and in Escherichia coli reporter strains and transcription in vitro of the Pu promoter in the presence of purified TurA confirmed the TurA repressor role on Pu activity. turA gene inactivation did not significantly alter two well characterized physiological regulations of the Pu expression in routine conditions of cultivation, exponential silencing, and carbon-mediated repression, respectively. However, the growth at suboptimal temperatures resulted in a TurA-dependent increase of Pu repression. These results strongly suggest that a physiological significance of the negative role of TurA on Pu activity could be limitation of the expression of the toluene-degrading enzymes at suboptimal growth temperatures. Therefore, the identification of TurA as Pu-binding protein revealed a novel physiological modulation of Pu promoter that is different from those strictly nutritional described previously.