RESULTS:

The opportunistic bacterial pathogen Pseudomonas aeruginosa causes acute and chronic infections that are notoriously difficult to treat. In people with cystic fibrosis P. aeruginosa can cause lifelong lung infections and isolation of mucoid P. aeruginosa resulting from the overproduction of alginate is associated with chronic infection. The histone-like protein AlgP has previously been implicated in the control of alginate gene expression in mucoid strains but this regulation is unclear. To explore AlgP in further detail we deleted algP in mucoid strains and demonstrated that the deletion of algP did not result in a nonmucoid phenotype or a decrease in alginate production. We showed that the algP promoter is expressed by both the nonmucoid strain PAO1 and the isogenic mucoid strain PDO300 suggesting that there may be genes that are differentially regulated between these strains. In support of this using RNA sequencing we identified a small AlgP regulon that has no significant overlap between PAO1 and PDO300 and established that alginate genes were not differentially regulated by the deletion of algP. Of note we found that deleting algP in PAO1 increased expression of the nitric oxide operon norCBD and the nitrous oxide reductase genes nosRZ and subsequently promoted growth of PAO1 under anaerobic conditions. Altogether we have defined a narrow regulon of genes controlled by AlgP and provided evidence that alginate production is not greatly affected by AlgP countering the long-standing premise in the field.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes infections in the lungs of individuals with the genetic disease cystic fibrosis. Density-dependent production of toxic factors regulated by the Pseudomonas quinolone signal (2-heptyl-3-hydroxy-4-quinolone; PQS) have been proposed to be involved in P. aeruginosa virulence. PQS biosynthesis requires conversion of the central metabolite chorismate to anthranilate by anthranilate synthase. This reaction is also the first step in tryptophan biosynthesis. P. aeruginosa possesses two functional anthranilate synthases TrpEG and PhnAB and these enzymes are not functionally redundant as trpEG mutants are tryptophan auxotrophs but produce PQS while mutants in phnAB are tryptophan prototrophs but do not produce PQS in minimal media. The goal of the work described in this paper was to determine the mechanism for this lack of functional complementation of TrpEG and PhnAB. Our results reveal that overexpression of either enzyme compensates for tryptophan auxotrophy and PQS production in the trpEG and phnAB mutants respectively leading to the hypothesis that differential regulation of these genes is responsible for the lack of functional complementation. In support of this hypothesis trpEG was shown to be expressed primarily during low-density growth while phnAB was expressed primarily at high density. Furthermore dysregulation of phnAB expression eliminated tryptophan auxotrophy in the P. aeruginosa trpEG mutant. Based on these data we propose a model for anthranilate sequestration by differential transcriptional regulation of the two P. aeruginosa anthranilate synthase enzymes.

The human opportunistic pathogen Pseudomonas aeruginosa is the major cause of morbidity and mortality of cystic fibrosis patients and is responsible for a variety of infections in compromised hosts. Using PCR-based signature-tagged mutagenesis we identified a P. aeruginosa STM5437 mutant with an insertion into the PA5437 gene (called pycR for putative pyruvate carboxylase regulator). PycR inactivation results in 100 000-fold attenuation of virulence in the rat lung in vivo. PycR has the signature of a transcriptional regulator with a predicted helix–turn–helix motif binding to a typical LysR DNA binding site in the PA5436 (pycA)–PA5437 (pycR) intercistronic region. Two pyruvate carboxylase subunits (pycA and pycB) are divergently transcribed upstream of pycR. Transcriptional start sites of pycR and pycA are located at −127 and −88 bp upstream of their initiation codons with Shine–Dalgarno and putative promoter sequences containing −10 and −35 sequences. The DNA binding of PycR was confirmed by DNA mobility shift assay. Genome-wide transcriptional profiling and quantitative real-time PCR (qRT-PCR) indicated that the genes differentially regulated by PycR include two pyruvate carboxylase genes and genes necessary for lipid metabolism lipolytic activity anaerobic respiration and biofilm formation. PycR is a regulator with pleiotropic effects on virulence factors such as lipase and esterase expression and biofilm formation which are important for maintenance of P. aeruginosa in chronic lung infection.