Response to Gaseous NO2 Air Pollutant of P. fluorescens Airborne Strain MFAF76a and Clinical Strain MFN1032

Abstract

Human exposure to nitrogen dioxide (NO2), an air pollutant of increasing interest in biology, results in several toxic effects to human health and also to the air microbiota. The aim of this study was to investigate the bacterial response to gaseous NO2. Two Pseudomonas fluorescens strains, namely the airborne strain MFAF76a and the clinical strain MFN1032 were exposed to 0.1, 5, or 45 ppm concentrations of NO2, and their effects on bacteria were evaluated in terms of motility, biofilm formation, antibiotic resistance, as well as expression of several chosen target genes. While 0.1 and 5 ppm of NO2did not lead to any detectable modification in the studied phenotypes of the two bacteria, several alterations were observed when the bacteria were exposed to 45 ppm of gaseous NO2. We thus chose to focus on this high concentration. NO2-exposed P. fluorescens strains showed reduced swimming motility, and decreased swarming in case of the strain MFN1032. Biofilm formed by NO2-treated airborne strain MFAF76a showed increased maximum thickness compared to non-treated cells, while NO2 had no apparent effect on the clinical MFN1032 biofilm structure. It is well known that biofilm and motility are inversely regulated by intracellular c-di-GMP level. The c-di-GMP level was however not affected in response to NO2 treatment. Finally, NO2-exposed P. fluorescens strains were found to be more resistant to ciprofloxacin and chloramphenicol. Accordingly, the resistance nodulation cell division (RND) MexEF-OprN efflux pump encoding genes were highly upregulated in the two P. fluorescens strains. Noticeably, similar phenotypes had been previously observed following a NO treatment. Interestingly, an hmp-homolog gene in P. fluorescens strains MFAF76a and MFN1032 encodes a NO dioxygenase that is involved in NO detoxification into nitrites. Its expression was upregulated in response to NO2, suggesting a possible common pathway between NO and NO2 detoxification. Taken together, our study provides evidences for the bacterial response to NO2 toxicity.

Keywords: Pseudomonas fluorescens; air pollution; airborne; antibiotic sensitivity; biofilm; motility; nitrogen dioxide.

Figure 1

Schematic representation of NO₂ gas delivery system. Bacterial NO₂ exposure was done in gas phase for 2 h. Two exposure chambers (one for the NO₂ exposure, the second one for the control—synthetic air exposure) were used. The gases, including NO₂, N₂ and O₂ were mixed together to obtain pre-calculated concentrations of NO₂ and maintain the O₂/N₂ ratio at 2/8 (v/v). NO₂ concentrations, temperature and relative humidity were controlled.

Figure 2

NO₂ effect on P. fluorescens biofilm and intracellular c-di-GMP level. (A) Airborne MFAF76a and (B) clinical MFN1032 P. fluorescens strains were exposed in triplicate to 45 ppm of NO₂. Biofilm formation was analyzed in static conditions after 24 h development using confocal laser scanning microscope. The biofilm biomass and the maximum thickness were estimated from 6 fields on 3 independent experiments using COMSTAT software. Intracellular c-di-GMP concentrations (C) were measured in triplicate by LC-MS/MS for control () and 45 ppm of NO₂ treated () MFAF76a and MFN1032. Obtained results are presented as average values ± SEM. Statistical significance was calculated by the non-parametric Mann-Whitney-Test. n.s., non-significant.

Figure 3

NO₂ decreases P. fluorescens motility. Airborne MFAF76a and clinical MFN1032 P. fluorescens strains were exposed in triplicate to 45 ppm of NO₂(). Swimming (A) and swarming (B) motilities were assayed on DMB-swim/swarm plates after 24 h incubation. The motile bacterial movement was evaluated in three independent experiments with three replicates. The data were compared with control exposed to synthetic air (). Obtained results are presented as average values ± SEM. Statistical significance was calculated by the non-parametric Mann-Whitney-Test p < 0.05 (^*) and < 0.001 (^***).

Figure 4

NO₂ effect on MexEF-OprN and MexXY efflux pump gene transcription. The nucleotide sequences of the mexEF-, oprN- and mexXY-homolog genes were obtained using the non-annotated genome drafts of airborne MFAF76a () and clinical MFN1032 () P. fluorescens. The GenBank accession numbers of nucleotide sequences are listed in Table S2. Quantification of mRNA level was assayed using qRT-PCR on RNAs extracted from NO₂- and synthetic air- exposed P. fluorescens. The PCR reactions were performed in triplicate and the standard deviations were lower than 0.15 Ct. Statistical analysis used pairwise strain comparisons (t-test) p < 0.01 (^**) and < 0.001 (^***). Dotted line shows the gene expression in synthetic air- exposed control.

Figure 5

NO₂ protects Pseudomonas fluorescens from chloramphenicol toxicity. After 2 h exposure to 45 ppm of NO₂, growth of airborne MFAF76a (A) and clinical MFN1032 (B) P. fluorescens with ciprofloxacin () and chloramphenicol () was assayed. Growth curves were performed with ciprofloxacin (3.125 μg/mL for MFAF76a and 1.156 μg/mL for MFN1032) and chloramphenicol (25 and 100 μg/mL respectively), and A₅₈₀ was recorded at the indicated time points. The control sample was bacteria exposed to synthetic air, and grown in presence of antibiotics in indicated concentrations. The data are shown as percentages of growth relative to synthetic air-exposed control. Pooled data from three independent experiments in duplicate ± SEM are reported. Statistical significance was calculated by the non-parametric Mann-Whitney-Test p < 0.05 (^*); n.s., non-significant. Dotted line shows the control (100%).

Figure 6

NO₂ exposure affects Pseudomonas fluorescens growth with aminoglycosides. After 2 h exposure to 45 ppm of NO₂, growth of airborne MFAF76a (A) and clinical MFN1032 (B) in presence of tobramycin (1.55 μg/mL; ) and kanamycin (3.1 μg/mL; ) was tested. A₅₈₀ was recorded at indicated time points. The control sample was bacteria exposed to synthetic air, and grown in presence of antibiotics in indicated concentrations. The data are presented as percentages of growth relative to air-exposed control. Pooled data from three independent experiments in duplicate ± SEM are reported. Statistical significance was calculated by the non-parametric Mann-Whitney-Test p < 0.05 (^*), < 0.01 (^**); n.s. non-significant. Dotted line shows the control (100%).

Figure 7

Scheme of Hmp-mediated NO detoxification and NO₂ reduction pathways in Pseudomonas spp. (A) Flavohemoglobin (Hmp) is involved in NO detoxification acting as an NO dioxygenase to transform NO to NO${}_3^{-}$. The NO₂ reduction is performed by nitrite reductase enzymes, including the respiratory cytochrome cd1 nitrite reductase, NIR (B) and the assimilatory nitrite reductase NirBD (C). The respiratory NIR is involved in NO${}_2^{-}$ reduction to NO in anaerobic conditions. NirBD takes a part of the nitrate assimilatory pathway, and reduces nitrite to ammonia.

Figure 8

Transcription of hmp is increased in response to NO₂ exposure. The nucleotide sequences of the hmp-homolog gene in P. fluorescens strains were obtained using the non-annotated genome drafts of airborne P. fluorescens MFAF76a () and clinical MFN1032 (). The GenBank accession numbers of hmp nucleotide sequences are listed in Table S2. Quantification of mRNA level was assayed using qRT-PCR on RNAs extracted from NO${}_2^{-}$ and synthetic air-exposed P. fluorescens. The PCR reactions were performed in triplicate and the standard deviations were lower than 0.15 Ct. Statistical analysis used pairwise strain comparisons (t-test) p < 0.01 (^**). Dotted line shows the gene expression in air-exposed control.