Transcriptional activity of Pseudomonas aeruginosa fhp promoter is dependent on two regulators in addition to FhpR

The regulation of flavohemoglobin expression is complex and depending on its host organism requires a wide variety of different transcriptional regulators. In Pseudomonas aeruginosa, the flavohemoglobin (Fhp) and its cognate regulator FhpR form an NO-sensing and detoxifying system regulated by their common bidirectional promoter P _fhp/P _fhpR. The intergenic fhp-fhpR region of P. aeruginosa PAO1 was used as a bait to isolate proteins affecting the transcription of fhp and fhpR. In addition to the FhpR, we identified two previously uncharacterized P. aeruginosa proteins, PA0779 and PA3697. Both PA0779 and PA3697 were found to be essential for NO₃ ^- and NO₂ ^- induced P _fhp activity under aerobic and low-oxygen conditions, and needed for the full function of P _fhp/P _fhpR as NO responsive regulatory circuit under aerobic conditions. In addition, we show that the transcriptional activity of P _fhpR is highly inducible upon addition of SNP under aerobic conditions, but not by NO₃ ^-, NO₂ ^- or under low-oxygen conditions, supporting the findings that FhpR is not the only factor affecting flavohemoglobin expression in P. aeruginosa.