Nitric oxide signaling and transcriptional control of denitrification genes in Pseudomonas stutzeri

Abstract

The expression of denitrification by a facultatively anaerobic bacterium requires as exogenous signals a low oxygen tension concomitant with an N oxide. We have studied the role of nitric oxide (NO), nitrous oxide (N2O), and nitrite as signal molecules for the expression of the denitrification apparatus of Pseudomonas stutzeri. Transcriptional kinetics of structural genes were monitored by Northern blot analysis in a 60-min time frame after cells were exposed to an N oxide signal. To differentiate the inducer role of NO from that of nitrite, mRNA kinetics were monitored under anoxic conditions in a nirF strain, where NO generation from nitrite is prevented because of a defect in heme D(1) biosynthesis. NO-triggered responses were monitored from the nirSTB operon (encoding cytochrome cd(1) nitrite reductase), the norCB operon (encoding NO reductase), nosZ (encoding nitrous oxide reductase), and nosR (encoding a putative regulator). Transcription of nirSTB and norCB was activated by 5 to 50 nM NO, whereas the nosZ promoter required about 250 nM. Nitrite at 5 to 50 nM elicited no response. At a threshold concentration of 650 nM N2O, we observed in the anoxic cell the transient appearance of nosZ and nosR transcripts. Constant levels of transcripts of both genes were observed in an anoxic cell sparged with N2O. NO at 250 nM stimulated in this cell type the expression of nos genes severalfold. The transcription factor DnrD, a member of the FNR-CRP family, was found to be part of the NO-triggered signal transduction pathway. However, overexpression of dnrD in an engineered strain did not result in NirS synthesis, indicating a need for activation of DnrD. NO modified the transcriptional pattern of the dnrD operon by inducing the transcription of dnrN and dnrO, located upstream of dnrD. Insertional mutagenesis of dnrN altered the kinetic response of the nirSTB operon towards nitrite. Our data establish NO and DnrD as key elements in the regulatory network of denitrification in P. stutzeri. The NO response adds to the previously identified nitrate-nitrite response mediated by the NarXL two-component system for the expression of respiratory nitrate reductase encoded by the narGHJI operon.

FIG. 1

NO is required for the transcription of the nirSTB and norCB operons. Northern blot analysis was used to monitor anaerobic cells (labeled −O₂) of mutant MK220 (nirF::Gm^r) for the appearance of transcripts from both operons in response to the addition of NO or nitrite. NO gas or deoxygenated solutions of nitrite were added to cells under an argon atmosphere to make the cell suspension 5 or 50 nM in the respective N oxide. The specificity of the NO response was asserted by the addition of deoxygenated hemoglobin (+Hb; 5 μM). The kinetics of nirSTB and norCB transcription were monitored during 1 h. The fdxA signal is shown as a control to show equal gel loadings and mRNA integrity by monitoring a constitutive mRNA species.

FIG. 2

NO as a signal molecule for transcription is effective in the low nanomolar range. (Top panels) Concentration dependency on NO of transcripts from the nirSTB operon; (bottom panels) the same for transcripts from the norCB operon. Anaerobically incubated cells of MK220 (labeled −O₂) were analyzed during 1 h for mRNA transcripts from the nirSTB and norCB operons by Northern blotting in response to increasing amounts of NO. NO gas was added to cell suspensions to obtain concentrations from 0.5 to 500 nM in solution. The shift to denitrifying conditions consisted of 3 h of incubation of aerobic cells at a shaking speed of 120 rpm (thereby generating O₂-limited conditions), followed by 30 min under an Ar atmosphere. The zero time point was taken to be at the end of this period.

FIG. 3

Both NO and N₂O are required as signal molecules for high-level transcription of nosZ. (Top panels) The NO response of nosZ is dependent on DnrD and NosR. Cells were placed in AC medium in an N₂O atmosphere and saturated for 3 h with the gas by sparging (+N₂O) (see Materials and Methods). Transcripts were monitored at the indicated time intervals after the addition of 250 nM NO. (Middle panels) Expression of nosR is under NO-dependent control of DnrD. Growth conditions were as described for the top panels. (Bottom panels) A pulse of N₂O elicits the transient transcription of nosZ and nosR. Cells were grown under O₂-limited conditions in AC medium (−O₂), pulsed with 650 nM N₂O, and probed by Northern blot analysis. RNA for Northern hybridization was prepared from strains MK220 (nirF::Km^r) in panels without further specifications, MRD235 (dnrD::Km^r), and MK418 (nosR::Tn5).

FIG. 4

Multicistronic dnrD operon of P. stutzeri. (A) Physical map and transcriptional organization of the dnrD operon. Wavy lines indicate the observed transcripts; open bars represent sizes and locations of probes used in Northern hybridization. Arrowheads point to putative FNR boxes. P, dnrP gene. (B) Transcripts as detected by Northern hybridization with the gene probes indicated in panel A. Total RNA was prepared from aerobically cultivated MK21 without nitrate (lane 1), from cells shifted to O₂-limited growth conditions for 15 min (shaking speed, 120 rpm) (lane 2), and from O₂-limited cells incubated for 15 min with 0.1% sodium nitrate (denitrifying conditions) (lane 3). The dnrP probe overlapped a few nucleotides with dnrD, hence the presence of a 0.9-kb signal upon hybridization.

FIG. 5

Expression of dnrD is amplified in response to NO, but not nitrite, by activating the promoter of dnrN. The appearance of a 2.4-kb transcript on addition of NO is indicative of the operon structure comprising dnrN, dnrO, dnrD, and dnrP. Anaerobically incubated cells (labeled −O₂) were monitored for dnrD transcripts in response to NO or nitrite by Northern hybridization. NO trapping by hemoglobin (Hb) and use of mutant MK220 (nirF::Gm^r) ensured the specificity of the N oxide signal. Growth conditions were identical to those described in the legend to Fig. 2.

FIG. 6

Disruption of dnrN alters the transcriptional response of the nirSTB operon. Cells were grown for 3 h aerobically (240 rpm) in AC medium and then shifted to nitrite-denitrifying conditions by adding sodium nitrite to a 0.05% final concentration and decreasing simultaneously the shaker speed to 120 rpm. RNA was prepared from MK21, representing wild-type traits (□), and MRD236 (dnrN::Km^r) (○). The zero time point represents nitrite addition. Signals from Northern blot analysis detecting nirSTB mRNA were quantified densitometrically.

FIG. 7

Overexpression of dnrD is insufficient for NirS synthesis. (A) Expression vector for dnrD derived from pUCP20 (49). REP, replication locus. (B) Northern blot analysis for dnrD expression. O₂-limited cells of MK21 (wild type [WT]) and of the dnrD mutant MRD235c (dnrD⁺) complemented with the vector pUCP146BK were analyzed (10 μg of total RNA). The sample for RNA extraction was drawn after 15 min. For the cell sample in the second lane, the medium was made 0.1% in sodium nitrate to generate denitrifying conditions. (C) Northern blot analysis for nirSTB expression. Aerobically grown cells (first lane) where shifted to O₂ limitation (second lane) or nitrate-denitrifying conditions (third lane). (D) Western blot analysis for cytochrome cd₁ nitrite reductase by a polyclonal anti-NirS antiserum. Cultures were incubated overnight under conditions of O₂ limitation (120 rpm) in the absence (first lane) or presence (second lane) of 0.1% sodium nitrate. Equal amounts of cell extract (15 μg) were separated by electrophoresis (36).

FIG. 8

N oxide signaling, components, and hierarchies in the regulation of denitrification in P. stutzeri. Boxed nar, nir, nor, and nos designations represent as regulatory targets the narGHJI operon (nitrate reductase), the nirSTB operon (nitrite reductase), the norCB operon (NO reductase), and nosZ (N₂O reductase), respectively. The substrates and products of each N oxide-reducing system are shown. The way NO interacts with DnrD is an open issue. The modulating role of DnrD on the expression of nosR and/or nosZ is shown by a double-headed arrow. Whether N₂O acts via NosR is unknown. For further discussion, see the text.