Transcriptional regulation of the flavohemoglobin gene for aerobic nitric oxide detoxification by the second nitric oxide-responsive regulator of Pseudomonas aeruginosa

Abstract

The regulatory gene for a sigma54-dependent-type transcriptional regulator, fhpR, is located upstream of the fhp gene for flavohemoglobin in Pseudomonas aeruginosa. Transcription of fhp was induced by nitrate, nitrite, nitric oxide (NO), and NO-generating reagents. Analysis of the fhp promoter activity in mutant strains deficient in the denitrification enzymes indicated that the promoter was regulated by NO or related reactive nitrogen species. The NO-responsive regulation was operative in a mutant strain deficient in DNR (dissimilatory nitrate respiration regulator), which is the NO-responsive regulator required for expression of the denitrification genes. A binding motif for sigma54 was found in the promoter region of fhp, but an FNR (fumarate nitrate reductase regulator) box was not. The fhp promoter was inactive in the fhpR or rpoN mutant strain, suggesting that the NO-sensing regulation of the fhp promoter was mediated by FhpR. The DNR-dependent denitrification promoters (nirS, norC, and nosR) were active in the fhpR or rpoN mutants. These results indicated that P. aeruginosa has at least two independent NO-responsive regulatory systems. The fhp or fhpR mutant strains showed sensitivity to NO-generating reagents under aerobic conditions but not under anaerobic conditions. These mutants also showed significantly low aerobic NO consumption activity, indicating that the physiological role of flavohemoglobin in P. aeruginosa is detoxification of NO under aerobic conditions.

FIG. 1.

Transcriptional start points and structure of the fhp and fhpR promoters. A and B. The transcriptional start points of fhp (A) and fhpR (B) were determined by primer extension analysis. Lanes RT, the primer extension products from strain PAO1 grown anaerobically in the presence of sodium nitrite; lanes A, T, G, and C, sequence ladders generated with the same primers. C. Structure of the intergenic promoter region between fhpR and fhp. Transcriptional start points are indicated by boldface and small arrows. A motif for binding of σ⁵⁴ is shown in boldface, and consensus −24 and −12 motifs are boxed. Consensus NorR-binding motifs are boxed. The IHF-binding motif is double underlined. Sequences of the primers used for the primer extension analysis are overlined by arrows. Putative ribosome binding sites are underlined.

FIG. 2.

Study of the NO resistance of wild-type and mutant strains. Strain PAO1 (wild type), PDM2664 (fhp), PFM4302 (fhpR), and RM495 (norCBD) were grown aerobically in minimal medium (A) or anaerobically in LB medium containing 40 mM NaNO₃ (B). Open symbols and filled symbols indicate growth without and with 5 mM GSNO, respectively. The growth curves are representative of at least two independent cultures.

FIG. 3.

Molecular phylogenetic tree for the NorR/FhpR-type transcriptional regulators. Multiple sequence alignment was done using ClustalW. Tree topology and evolutionary distance estimations were done by the neighbor-joining method (Phylip 3.5). XylR of P. putida was used as an outgroup. The numbers indicated at the nodes are bootstrap values calculated from 100 replications using the Seqboot, Protdist, Neighbor, and Consense programs of the Phylip 3.5 program package. The genes for NO-metabolizing enzymes located adjacent to the regulatory genes are indicated on the right side of the tree.