Two-pronged survival strategy for the major cystic fibrosis pathogen, Pseudomonas aeruginosa, lacking the capacity to degrade nitric oxide during anaerobic respiration

Abstract

Protection from NO gas, a toxic byproduct of anaerobic respiration in Pseudomonas aeruginosa, is mediated by nitric oxide (NO) reductase (NOR), the norCB gene product. Nevertheless, a norCB mutant that accumulated approximately 13.6 microM NO paradoxically survived anaerobic growth. Transcription of genes encoding nitrate and nitrite reductases, the enzymes responsible for NO production, was reduced >50- and 2.5-fold in the norCB mutant. This was due, in part, to a predicted compromise of the [4Fe-4S](2+) cluster in the anaerobic regulator ANR by physiological NO levels, resulting in an inability to bind to its cognate promoter DNA sequences. Remarkably, two O(2)-dependent dioxygenases, homogentisate-1,2-dioxygenase (HmgA) and 4-hydroxyphenylpyruvate dioxygenase (Hpd), were derepressed in the norCB mutant. Electron paramagnetic resonance studies showed that HmgA and Hpd bound NO avidly, and helped protect the norCB mutant in anaerobic biofilms. These data suggest that protection of a P. aeruginosa norCB mutant against anaerobic NO toxicity occurs by both control of NO supply and reassignment of metabolic enzymes to the task of NO sequestration.

Figure 1

A PA norCB mutant maintains anaerobic viability despite high in vivo NO levels. (A) Anaerobic respiratory (denitrification) pathway. Enzymes involved in each reduction step are termed nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR) and nitrous oxide reductase (NOS), respectively. (B) NO tracings of wild type, norCB, nirS and anr mutants during anaerobic culture. Aerobic, stationary phase precultures were diluted 30-fold in 3 ml of LBN and placed in the NO electrode chamber. Before inoculation, the NO signal baseline was stabilized for at least 30 min. Note the different time scale between PAO1 and the mutant strains. (C) Anaerobic growth curve of wild-type and norCB mutant grown in LBN. Aerobic starter cultures were diluted 100- and 10-fold for wild-type and norCB mutant bacteria, respectively. Because of this, the initial turbidity was higher in the culture of the norCB mutant to demonstrate that anaerobic growth of the norCB mutant is virtually ceased. A 10-fold more initial bacterial load in norCB mutant, but OD₆₀₀ remained constant. The inset indicates anaerobic wild-type and norCB mutant bacteria colony forming units in presence versus absence of 10 mM C-PTIO when grown in medium containing 10 mM KNO₃ at 3, 6 and 12 h. Because the C-PTIO is stoichiometrically exhausted after 12 h, the final viable cell count was enumerated at this time point. Gray line (PAO1), white line (norcB mutant+C-PTIO), black line (norCB mutant). (D) Confocal images of anaerobic biofilms of wild-type PAO1 and the norCB mutant. Bacteria were grown anaerobically for 24 h in LBN and stained for viability assessment using the BacLite^® live/dead stain. Green and red organisms represent those that are either live or dead, respectively.

Figure 2

The norCB mutant has dramatically reduced NAR/NIR activities and nar/nir gene transcription relative to wild-type bacteria. (A) Cell extracts of wild-type and norCB mutant bacteria were prepared after anaerobic growth in LBN for 24 h. Wild-type PAO1 and norCB mutant bacteria harboring single-copy narK1-lacZ and nirS-lacZ fusions were assayed for β-galactosidase reporter activity in triplicate and mean±s.e.m. is presented. C-PTIO at 10 mM was added for biochemical complementation purposes for the lacZ. (B) Nitrate (NAR) and nitrite (NIR) reductase activity of cell extracts of wild-type and norCB mutant bacteria after anaerobic growth in LBN for 24 h. Each assay was performed in triplicate and the mean±s.e.m. is shown.

Figure 3

Purification of PA ANR and inactivation by NO. (A) SDS–PAGE of purified ANR. Lane 1, molecular weight standard (kDa); lane 2, 1 μg of purified ANR. (B) Absorption spectra of anaerobic ANR (1.2 mg/ml), air-treated and NO-treated ANR. The latter treatment is a 1% gaseous NO (balance) argon exposure for 1 h. The ratio of 1% NO (19 μM NO in solution) to 1.2 mg/ml ANR protein is 1:1 on a molar molar basis. To obtain spectra of anaerobic and NO-treated ANR, samples (200 μl) were placed in sealed cuvettes in an anaerobic chambe and scanned. (C) The 4.2 K Mössbauer spectra of anaerobically prepared ANR. The solid line is a doublet for [4Fe–4S]²⁺ simulated with E_Q=1.2 mm/s and δ=0.43 mm/s. (D) HinF1 restriction protection assay. The narK1 promoter sequence, in which an ANR box overlaps a HinF1 recognition site, was digested with HinF1 in the absence (lane 3) or presence of ANR (lanes 4, 5 and 6). Before digestion, the promoter sequence was incubated with anaerobic (lane 4), air-treated (lane 5) or NO-treated (lane 6) ANR for 30 min. DNA fragments after HinF1 digestion were separated on a 1.5% agarose gel and photographed. Lane 1, DNA ladder (New England Biolabs Inc.); lane 2, DNA only.

Figure 4

Upregulation of two oxygen-dependent enzymes in the anaerobic norCB mutant. (A) A portion of the norCB 2-D gel that contains spots for HmgA and Hpd proteins was compared with the same area of a 2-D gel from wild-type bacteria. A 60 μg weight of whole-cell extracts was separated in the 2-D gel system. The norCB mutant suspension was diluted 10-fold in the main anaerobic culture to obtain comparable amounts of cells with wild-type PAO1. Bacteria were grown in LBN under anaerobic conditions for 24 h. The identity of HmgA and Hpd was confirmed by MALDI-TOF mass spectrometric analysis. For the full 2-D gel images and the complete list of identified proteins, refer to the Supplementary data. (B) Measurements of transcript levels of hmgA and hpd in wild-type PAO1 (black bar), norCB mutant (gray bar) and norCB mutant bacteria +10 mM C-PTIO (white bars) under anaerobic conditions. Cultures were assayed for β-galactosidase activity in triplicate as described in the Materials and Methods and mean±s.e.m. is shown.

Figure 5

Binding of NO by HmgA and Hpd helps protect norCB mutant bacteria in anaerobic biofilms. (A) EPR spectra of 13 μM HmgA and Hpd in the absence or presence of NO. To establish that the Fe(II)–NO complex forms specifically at the catalytic center of these enzymes, spectra with the addition of stoichiometric levels of substrate (homogentisate for HmgA (HmgA-S-NO) and 4-hydroxyphenylpyruvate for Hpd (Hpd-S-NO)) are also shown. EPR conditions are as follows: temperature, 2 K; microwave frequency, 9.64 GHz; microwave power, 50 μW; modulation frequency 100 kHz; modulation amplitude, 10 G. Purity of HmgA or Hpd (lane 2) is shown by SDS–PAGE with molecular weight standards in kDa (lane 1). (B) NO tracings of norCB (line 1) and norCB hmgA hpd triple mutants (line 3) and the triple mutant+hmgA and hpd (line 2) during anaerobic culture. Experimental conditions were identical to those in Figure 1B. (C) Confocal images of anaerobic biofilms of wild-type PAO1 (panel 1), hmgA hpd (panel 2), norCB (panel 3), norCB hmgA hpd (panel 4), norCB hmgA hpd+hmgA and hpd in attB site (panel 5).

Figure 6

norCB and anr mutants show similar anaerobic phenotypes with regard to hmgA and hpd regulation. (A, B) Measurements of transcript levels of hmgA and hpd in wild-type PAO1 (black bar), norCB mutant (gray bar) and anr mutant (hatched bar) under both aerobic (A) and anaerobic (B) conditions. Cultures were assayed for β-galactosidase activity in triplicate as described in Materials and Methods.

Figure 7

Summary of the protective mechanisms used by a PA norCB mutant during anaerobic respiration. (1) NO is produced anaerobically in the periplasmic space by NIR and can either exit the cell or enter the cytoplasm; (2) Once in the cytoplasm, NO can inactivate dimeric ANR, causing release of iron from the [4Fe–4S]²⁺, causing conversion to a putative [2Fe–2S]²⁺ cluster, thus rendering it incapable of transcriptional activation of genes under its control (3), this is indicated by the ‘no' sign (e.g., ). In contrast, genes that are anaerobically repressed, including hmgA and hpd, become derepressed, and function as NO scavengers; (4) because ANR is required for transcription of dnr, DNR is not produced; (5) thus, narK1, nirS, nirQ and oprE transcripts are either absent or extremely low (denoted by red X); (6) ANR, and perhaps DNR, denoted as a ‘?', however, loses it activity as a repressor. Thus, genes that would be repressed anaerobically, such as hmgA and hpd, are now derepressed (denoted by green circle). Because of the inherent NO-binding properties of HmgA and Hpd (refer to Figure 5A and B), elevated levels of these enzymes served to help protect the anaerobic norCB mutant against NO-mediated toxicity. OM, outer membrane; PP, periplasmic space; IM, inner membrane; NarK1/2, putative NO₂⁻ extrusion pump; CytC (red), reduced cytochrome c; Azurin, periplasmic protein donating electrons to NIR; e⁻, electron flow; NADH, electron donor for NAR.