QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa.

The opportunistic pathogenic bacterium Pseudomonas aeruginosa uses quorum-sensing signaling systems as global regulators of virulence genes. There are two quorum-sensing signal receptor and signal generator pairs, LasR-LasI and RhlR-RhlI. The recently completed P. aeruginosa genome-sequencing project revealed a gene coding for a homolog of the signal receptors, LasR and RhlR. Here we describe a role for this gene, which we call qscR. The qscR gene product governs the timing of quorum-sensing-controlled gene expression and it dampens virulence in an insect model. We present evidence that suggests the primary role of QscR is repression of lasI. A qscR mutant produces the LasI-generated signal prematurely, and this results in premature transcription of a number of quorum-sensing-regulated genes. When fed to Drosophila melanogaster, the qscR mutant kills the animals more rapidly than the parental P. aeruginosa. The repression of lasI by QscR could serve to ensure that quorum-sensing-controlled genes are not activated in environments where they are not useful.

Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme.

The ortho-cleavage pathways of catechol and 3-chlorocatechol are central catabolic pathways of Pseudomonas putida that convert aromatic and chloroaromatic compounds to tricarboxylic acid (TCA)-cycle intermediates. They are encoded by the evolutionarily related catBCA and clcABD operons, respectively. Expression of the cat and clc operons requires the LysR-type transcriptional activators CatR and ClcR, and the inducer molecules cis,cis-muconate and 2-chloro-cis,cis-muconate. In addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR mutants. DNase-I footprinting, DNA bending and in vitro transcription analyses with RNA polymerase mutants indicate that CatR and ClcR activate transcription via a similar mechanism which involves interaction with the C-terminal domain of the alpha-subunit (alpha-CTD) of RNA polymerase. In vitro transcription assays with different regions of the clc promoter indicate that the ClcR dimer bound to the promoter proximal site (the activation binding site) interacts with the alpha-CTD. Gel shift assays and DNase-I footprinting have demonstrated that CatR occupies two adjacent sites proximal to the catBCA promoter in the presence of inducer and an additional binding site within the catB structural gene called the internal binding site (IBS). CatR binds the IBS with low intrinsic affinity that is increased by cooperativity in presence of the two promoter binding sites. Site-directed mutations in the IBS indicate a probable cis-acting repressor function for the IBS. The location of the IBS within the catB structural gene, the cooperativity observed in footprinting studies and phasing studies suggest that the IBS participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA. Although the core transcriptional activation mechanisms of CatR and ClcR have been conserved, nature has provided some flexibility to respond to different environmental signals in addition to the presence of inducer. Transcriptional fusion studies demonstrate that the expression from the clc promoter is repressed when the cells are grown on succinate, citrate or fumarate and that this repression is ClcR-dependent and occurs at the transcriptional level. The presence of these organic acids did not affect the expression from the cat promoter. In vitro transcription assays demonstrate that the TCA-cycle intermediate, fumarate, directly and specifically inhibits the formation of the clcA transcript. No such inhibition was observed when CatR was used as activator on either the cat or clc template. Since both the catechol and the chlorocatechol pathways feed into the TCA cycle, but only the chlorocatechol pathway is inhibited by fumarate, there is a subtle difference in the regulation of these two pathways where intracellular sensing of a TCA-cycle intermediate leads to a reduction of chloroaromatic degradation.

Transcriptional repression mediated by LysR-type regulator CatR bound at multiple binding sites.

The catBCA operon of Pseudomonas putida encodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to the catBCA promoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of the catBCA promoter within the catB structural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured beta-galactosidase activity of catB-lacZ transcriptional fusions in vivo. Our results suggest a probable cis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within the catB structural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.

Activation of the catBCA promoter: probing the interaction of CatR and RNA polymerase through in vitro transcription.

The soil bacterium Pseudomonas putida is capable of degrading many aromatic compounds, including benzoate, through catechol as an intermediate. The catabolism of catechol is mediated by the catBCA operon, whose induction requires the pathway intermediate cis,cis-muconate as an inducer and the regulatory protein, CatR. CatR also regulates the plasmid-borne pheBA operon of P. putida PaW85, which is involved in phenol catabolism. We have used an in vitro transcription system to study the roles of CatR, cis,cis-muconate, Escherichia coli RNA polymerase, and promoter sequences in expression of the cat and phe operons. The assay confirmed the requirement of both CatR and cis,cis-muconate for transcript formation. We also examined the in vitro transcription of three site-directed mutants of the catBCA promoter; the results obtained compared favorably with previous in vivo data. The requirement of the alpha subunit of RNA polymerase for expression of the catBCA and the pheBA transcripts was also examined. The C-terminal region of the alpha subunit of RNA polymerase has been implicated in direct protein-protein contact with transcriptional regulatory proteins and/or direct contact with the DNA. We show that the carboxyl terminus of the alpha subunit is required for the expression of the catBCA and the pheBA operons because RNA polymerases with truncated alpha subunits were deficient in activation. Further experiments demonstrated the arginine at position 265 and the asparagine at position 268 of the alpha subunit as possible amino acids involved in activation. On the basis of these and previous results, we propose a model to explain the interaction of the different regulatory components leading to CatR-dependent activation of the catBCA operon.

Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis.

We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2 and algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild-type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane-associated fractions of P. aeruginosa. The cytosolic Ndk was non-specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction events within the cell and in the production of GDP-mannose, an essential alginate precursor, clearly indicates the importance of Ndk in cellular processes as well as in alginate synthesis.

Characterization of nucleoside-diphosphate kinase from Pseudomonas aeruginosa: complex formation with succinyl-CoA synthetase.

The enzyme nucleoside-diphosphate kinase (Ndk), responsible for the conversion of (deoxy)ribonucleoside diphosphates to their corresponding triphosphates, has been purified from Pseudomonas aeruginosa. The N-terminal 12 amino acid sequence of P. aeruginosa Ndk shows significant homology with that of Myxococcus xanthus and that of Escherichia coli. Ndk enzyme activity is also associated with succinyl-CoA synthetase activity in P. aeruginosa, whose alpha and beta subunits show extensive sequence homology with those of E. coli and Dictyostelium discoideum. The 33-kDa alpha subunit of succinyl-CoA synthetase of P. aeruginosa appears to undergo autophosphorylation in the presence of either ATP or GTP, although the presence of small amounts of Ndk activity may influence the level of such phosphorylation.