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.

Novel auto-inducing expression systems for the development of whole-cell biocatalysts.

Novel expression systems for the development of whole-cell biocatalysts were generated. Their novelty consists both in the host, Pseudomonas putida, and in the ability to auto-induce the expression of genes of interest at the exhaustion of the carbon source used for the biomass growth. The auto-induction relies on new expression vectors developed in this study and based on the activator TouR from Pseudomonas sp. OX1, which was shown to mediate the activation of target promoters in an effector-independent growth-phase-dependent manner when the carbon source is exhausted at the onset of the stationary phase. We validated the suitability of these expression systems through the production of (S)-styrene oxide by the styrene monooxygenase from Pseudomonas fluorescens ST. The yields of epoxides produced by these biocatalysts in flask experiments showed to be as efficient as those currently available based on inducible Escherichia coli systems. In addition, a larger scale of biomass production showed no reduction of biocatalysis efficiency. Therefore, the systems developed in this study constitute a valid alternative to current expression systems to use in bioconversion processes.

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.

Styrene lower catabolic pathway in Pseudomonas fluorescens ST: identification and characterization of genes for phenylacetic acid degradation.

Pseudomonas fluorescens ST is a styrene degrading microorganism that, by the sequential oxidation of the vinyl side chain, converts styrene to phenylacetic acid. The cluster of styrene upper pathway catabolic genes (sty genes) has been previously localized on a chromosomal region. This report describes the isolation, sequencing and analysis of a new chromosomal fragment deriving from the ST strain genomic bank that contains the styrene lower degradative pathway genes (paa genes), involved in the metabolism of phenylacetic acid. Analysis of the paa gene cluster led to the description of 14 putative genes: a gene encoding a phenylacetyl-CoA ligase (paaF), the enzyme required for the activation of phenylacetic acid; five ORFs encoding the subunits of a ring hydroxylation multienzymatic system (paaGHIJK); the gene paaW encoding a membrane protein of unknown function; five genes for a beta-oxidation-like system (paaABCDE), involved in the steps following the aromatic ring cleavage; a gene encoding a putative permease (paaL) and a gene (paaN) probably involved in the aromatic ring cleavage. The function of some of the isolated genes has been proved by means of biotransformation experiments.

TouR-mediated effector-independent growth phase-dependent activation of the sigma54 Ptou promoter of Pseudomonas stutzeri OX1.

Transcription of the catabolic touABCDEF operon, encoding the toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1, is driven by the sigma(54)-dependent Ptou promoter, whose activity is controlled by the phenol-responsive NtrC-like activator TouR. In this paper we describe for the first time a peculiar characteristic of this system, namely, that Ptou transcription is activated in a growth phase-dependent manner in the absence of genuine effectors of the cognate TouR regulator. This phenomenon, which we named gratuitous activation, was observed in the native strain P. stutzeri OX1, as well as in a Pseudomonas putida PaW340 host harboring the reconstructed tou regulatory circuit. Regulator-promoter swapping experiments demonstrated that the presence of TouR is necessary and sufficient for imposing gratuitous activation on the Ptou promoter, as well as on other sigma(54)-dependent catabolic promoters, whereas the highly similar phenol-responsive activator DmpR is unable to activate the Ptou promoter in the absence of effectors. We show that this phenomenon is specifically triggered by carbon source exhaustion but not by nitrogen starvation. An updated model of the tou regulatory circuit is presented.