Discovery of a Novel Nitric Oxide Binding Protein and Nitric-Oxide-Responsive Signaling Pathway in Pseudomonas aeruginosa

Abstract

Nitric oxide (NO) is a radical diatomic gas molecule that, at low concentrations, plays important signaling roles in both eukaryotes and bacteria. In recent years, it has become evident that bacteria respond to low levels of NO in order to modulate their group behavior. Many bacteria respond via NO ligation to a well-established NO sensor called H-NOX (heme-nitric oxide/oxygen binding domain). Many others, such as Pseudomonas aeruginosa, lack an annotated hnoX gene in their genome yet are able to respond to low levels of NO to disperse their biofilms. This suggests the existence of a previously uncharacterized NO sensor. In this study, we describe the discovery of a novel nitric oxide binding protein (NosP; NO-sensing protein), which is much more widely conserved in bacteria than H-NOX, as well as a novel NO-responsive pathway in P. aeruginosa. We demonstrate that biofilms of a P. aeruginosa mutant lacking components of the NosP pathway lose the ability to disperse in response to NO. Upon cloning, expressing, and purifying NosP, we find it binds heme and ligates to NO with a dissociation rate constant that is comparable to that of other well-established NO-sensing proteins. Moreover, we show that NO-bound NosP is able to regulate the phosphorelay activity of a hybrid histidine kinase that is involved in biofilm regulation in P. aeruginosa. Thus, here, we present evidence of a novel NO-responsive pathway that regulates biofilm in P. aeruginosa.

Discovery of a Nitric Oxide Responsive Quorum Sensing Circuit in Vibrio cholerae

“…This rate constant is independent of CO concentration as well as dithionite at all concentrations tested (300 μ M, 3 mM, 30 mM, and 100 mM dithionite). The VcNosP NO dissociation rate constant is comparable to both the rate constant reported for the dissociation of NO from P. aeruginosa NosP (1.8 × 10 −4 s −1 ) 11 and the reported NO dissociation rate constant for sGC (3.6 × 10 −4 s −1 ; sGC is a mammalian member of the H-NOX family). 21 Interestingly, the NO dissociation rate constant for VcNosP is much slower than the NO dissociation rate constant reported for VcH-NOX (0.3 s −1 ).…”

“…The data were fit as described previously. 11 The measured rate of 4.6 × 10 −4 is independent of CO and dithionite at all concentrations tested (300 μ M, 3 mM, 30 mM, and 100 mM dithionite). (B) Absorbance difference spectrum where the spectrum at time = 0 min is subtracted from all subsequent spectra collected over time as the Fe II −CO complex is formed.…”

“…Phosphotransfer assays between WT or DA VpsS with LuxU were conducted as previously described. 11 Phosphotransfer assays which included NO-bound NosP were started by incubating a solution of VcFe II −NO and VpsS with ATP so that the final ATP concentration was 2.5 μ M with 10 μ Ci [ γ − 32 P]-ATP per reaction. Incubation of VcFe II −NO and VpsS with ATP was allowed to proceed for 15 min, after which LuxU was added.…”

“…Indeed, our laboratory recently discovered an additional NO sensing protein, NosP, which is broadly conserved in bacterial genomes. 11 Like H-NOX, NosP coordinates NO via ligation to the iron atom of a heme cofactor. We first characterized NosP from the opportunistic pathogen Pseudomonas aeruginosa ( P. aeruginosa does not encode an hnoX gene) and demonstrated that NosP causes biofilm dispersal in the presence of NO through regulation of an associated histidine kinase signaling pathway.…”

Spectral Characterization of a Novel NO Sensing Protein in Bacteria: NosP

“…NO-bound Pa NosP has been shown to regulate the activity of a co-cistronic histidine kinase that is required for NO-dependent biofilm reduction. 11 NO-bound Vc NosP has been demonstrated to regulate the autophosphorylation activity 18 of the quorum sensing kinase VpsS, 19–21 and Lpg NosP modulates a co-cistronic kinase (Fischer, et al unpublished data) whose cognate response regulator has been shown to result in a hyper-biofilm phenotype when deleted. 21, 22 …”