Expression analysis of the Pseudomonas aeruginosa AlgZR two-component regulatory system.

Pseudomonas aeruginosa virulence components are subject to complex regulatory control primarily through two-component regulatory systems that allow for sensing and responding to environmental stimuli. In this study, the expression and regulation of the P. aeruginosa AlgZR two-component regulatory system were examined. Primer extension and S1 nuclease protection assays were used to identify two transcriptional initiation sites for algR within the algZ coding region, and two additional start sites were identified upstream of the algZ coding region. The two algR transcriptional start sites, RT1 and RT2, are directly regulated by AlgU, consistent with previous reports of increased algR expression in mucoid backgrounds, and RpoS additionally plays a role in algR transcription. The expression of the first algZ promoter, ZT1, is entirely dependent upon Vfr for expression, whereas Vfr, RpoS, or AlgU does not regulate the second algZ promoter, ZT2. Western blot, real-time quantitative PCR (RT-qPCR), and transcriptional fusion analyses show that algZR expression is Vfr dependent. The algZ and algR genes also are cotranscribed in both nonmucoid and mucoid backgrounds. Furthermore, algZR was found to be cotranscribed with hemCD by RT-PCR. RT-qPCR confirmed that hemC transcription in the PAO1 ΔalgZ mutant was 40% of the level of the wild-type strain. Taken together, these results indicate that algZR transcription involves multiple factors at multiple start sites that control individual gene expression as well as coexpression of this two-component system with heme biosynthetic genes.

Pseudomonas aeruginosa AlgR controls cyanide production in an AlgZ-dependent manner.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infections in individuals suffering from the genetic disorder cystic fibrosis. In P. aeruginosa, the transcriptional regulator AlgR controls a variety of virulence factors, including alginate production, twitching motility, biofilm formation, quorum sensing, and hydrogen cyanide (HCN) production. In this study, the regulation of HCN production was examined. Strains lacking AlgR or the putative AlgR sensor AlgZ produced significantly less HCN than did a nonmucoid isogenic parent. In contrast, algR and algZ mutants showed increased HCN production in an alginate-producing (mucoid) background. HCN production was optimal in a 5% O2 environment. In addition, cyanide production was elevated in bacteria grown on an agar surface compared to bacteria grown in planktonic culture. A conserved AlgR phosphorylation site (aspartate at amino acid position 54), which is required for surface-dependent twitching motility but not alginate production, was found to be critical for cyanide production. Nuclease protection mapping of the hcnA promoter identified a new transcriptional start site required for HCN production. A subset of clinical isolates that lack this start site produced small amounts of cyanide. Taken together, these data show that the P. aeruginosa hcnA promoter contains three transcriptional start sites and that HCN production is regulated by AlgZ and AlgR and is maximal under microaerobic conditions when the organism is surface attached.

Pseudomonas aeruginosa AlgR represses the Rhl quorum-sensing system in a biofilm-specific manner.

AlgR controls numerous virulence factors in Pseudomonas aeruginosa, including alginate, hydrogen cyanide production, and type IV pilus-mediated twitching motility. In this study, the role of AlgR in biofilms was examined in continuous-flow and static biofilm assays. Strain PSL317 (DeltaalgR) produced one-third the biofilm biomass of wild-type strain PAO1. Complementation with algR, but not fimTU-pilVWXY1Y2E, restored PSL317 to the wild-type biofilm phenotype. Comparisons of the transcriptional profiles of biofilm-grown PAO1 and PSL317 revealed that a number of quorum-sensing genes were upregulated in the algR deletion strain. Measurement of rhlA::lacZ and rhlI::lacZ promoter fusions confirmed the transcriptional profiling data when PSL317 was grown as a biofilm, but not planktonically. Increased amounts of rhamnolipids and N-butyryl homoserine lactone were detected in the biofilm effluent but not the planktonic supernatants of the algR mutant. Additionally, AlgR specifically bound to the rhlA and rhlI promoters in mobility shift assays. Moreover, PAO1 containing a chromosomal mutated AlgR binding site in its rhlI promoter formed biofilms and produced increased amounts of rhamnolipids similarly to the algR deletion strain. These observations indicate that AlgR specifically represses the Rhl quorum-sensing system during biofilm growth and that such repression is necessary for normal biofilm development. These data also suggest that AlgR may control transcription in a contact-dependent or biofilm-specific manner.

Identification of AlgR-regulated genes in Pseudomonas aeruginosa by use of microarray analysis.

The Pseudomonas aeruginosa transcriptional regulator AlgR controls a variety of different processes, including alginate production, type IV pilus function, and virulence, indicating that AlgR plays a pivotal role in the regulation of gene expression. In order to characterize the AlgR regulon, Pseudomonas Affymetrix GeneChips were used to generate the transcriptional profiles of (i) P. aeruginosa PAO1 versus its algR mutant in mid-logarithmic phase, (ii) P. aeruginosa PAO1 versus its algR mutant in stationary growth phase, and (iii) PAO1 versus PAO1 harboring an algR overexpression plasmid. Expression analysis revealed that, during mid-logarithmic growth, AlgR activated the expression of 58 genes while it repressed the expression of 37 others, while during stationary phase, it activated expression of 45 genes and repression of 14 genes. Confirmatory experiments were performed on two genes found to be AlgR repressed (hcnA and PA1557) and one AlgR-activated operon (fimU-pilVWXY1Y2). An S1 nuclease protection assay demonstrated that AlgR repressed both known hcnA promoters in PAO1. Additionally, direct measurement of hydrogen cyanide (HCN) production showed that P. aeruginosa PAO1 produced threefold-less HCN than did its algR deletion strain. AlgR also repressed transcription of two promoters of the uncharacterized open reading frame PA1557. Further, the twitching motility defect of an algR mutant was complemented by the fimTU-pilVWXY1Y2E operon, thus identifying the AlgR-controlled genes responsible for this defect in an algR mutant. This study identified four new roles for AlgR: (i) AlgR can repress gene transcription, (ii) AlgR activates the fimTU-pilVWXY1Y2E operon, (iii) AlgR regulates HCN production, and (iv) AlgR controls transcription of the putative cbb3-type cytochrome PA1557.

Transcriptome analysis of Pseudomonas aeruginosa after interaction with human airway epithelial cells.

The transcriptional profile of Pseudomonas aeruginosa after interactions with primary normal human airway epithelial cells was determined using Affymetrix GeneChip technology. Gene expression profiles indicated that various genes involved in phosphate acquisition and iron scavenging were differentially regulated.

The transcriptional regulator AlgR controls cyanide production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis (CF) patients. One characteristic of P. aeruginosa CF isolates is the overproduction of the exopolysaccharide alginate, controlled by AlgR. Transcriptional profiling analyses comparing mucoid P. aeruginosa strains to their isogenic algR deletion strains showed that the transcription of cyanide-synthesizing genes (hcnAB) was approximately 3-fold lower in the algR mutants. S1 nuclease protection assays corroborated these findings, indicating that AlgR activates hcnA transcription in mucoid P. aeruginosa. Quantification of hydrogen cyanide (HCN) production from laboratory isolates revealed that mucoid laboratory strains made sevenfold more HCN than their nonmucoid parental strains. In addition, comparison of laboratory and clinically derived nonmucoid strains revealed that HCN was fivefold higher in the nonmucoid CF isolates. Moreover, the average amount of cyanide produced by mucoid clinical isolates was 4.7 +/- 0.85 micromol of HCN/mg of protein versus 2.4 +/- 0.40 micromol of HCN/mg of protein for nonmucoid strains from a survey conducted with 41 P. aeruginosa CF isolates from 24 patients. Our data indicate that (i) mucoid P. aeruginosa regardless of their origin (laboratory or clinically derived) produce more cyanide than their nonmucoid counterparts, (ii) AlgR regulates HCN production in P. aeruginosa, and (iii) P. aeruginosa CF isolates are more hypercyanogenic than nonmucoid laboratory strains. Taken together, cyanide production may be a relevant virulence factor in CF lung disease, the production of which is regulated, in part, by AlgR.