Detoxification Response of Pseudomonas fluorescens MFAF76a to Gaseous Pollutants NO2 and NO

Abstract

Bacteria are often exposed to nitrosative stress from their environment, from atmospheric pollution or from the defense mechanisms of other organisms. Reactive nitrogen species (RNS), which mediate nitrosative stress, are notably involved in the mammalian immune response through the production of nitric oxide (NO) by the inducible NO synthase iNOS. RNS are highly reactive and can alter various biomolecules such as lipids, proteins and DNA, making them toxic for biological organisms. Resistance to RNS is therefore important for the survival of bacteria in various environments, and notably to successfully infect their host. The fuel combustion processes used in industries and transports are responsible for the emission of important quantities of two major RNS, NO and the more toxic nitrogen dioxide (NO₂). Human exposure to NO₂ is notably linked to increases in lung infections. While the response of bacteria to NO in liquid medium is well-studied, few data are available on their exposure to gaseous NO and NO₂. This study showed that NO₂ is much more toxic than NO at similar concentrations for the airborne bacterial strain Pseudomonas fluorescens MFAF76a. The response to NO₂ involves a wide array of effectors, while the response to NO seemingly focuses on the Hmp flavohemoprotein. Results showed that NO₂ induces the production of other RNS, unlike NO, which could explain the differences between the effects of these two molecules.

Keywords: NO2; P. fluorescens; cell morphology; membrane; membrane integrity.

Figure 1

Physiological modifications of P. fluorescens MFAF76a after exposure to NO₂ or NO. (a) Cultivability of bacteria after exposure to NO₂ (in orange) or NO (in green) compared to cells exposed to synthetic air. (b) Membrane integrity assessed by live-dead flow cytometry assays using Live/Dead BacLight kit (L-7012, ThermoFisher) according to the manufacturer’s recommendations. (c) Expression of genes encoding proteins involved in RNS and ROS detoxification. Graphs represent means ± SEM. ns = p > 0.05; * = p < 0.05; ** = p < 0.01; *** = p < 0.001, N ≥ 3.

Figure 2

Impact of hmp deletion on the physiological modifications of P. fluorescens MFAF76a after exposure to NO₂ or NO. (a) Cultivability of bacteria after exposure to NO₂ or NO compared to cells exposed to synthetic air. (b) Membrane integrity of P. fluorescens MFAF76a and MFAF76a Δhmp assessed by the Live/Dead BacLight kit (L-7012, ThermoFisher) flow cytometry assays after exposure to 45 ppm of NO₂. (c) Membrane integrity of P. fluorescens MFAF76a and MFAF76a Δhmp assessed by live-dead flow cytometry assays after exposure to 45 ppm of NO. Graphs represent means ± SEM. ns = p > 0.05; * = p < 0.05; *** = p < 0.001, N ≥ 3. In cytometry experiments, black stars represent the significance of overall cell membrane alterations, while orange stars represent the difference between partially damaged membranes between the wild-type and the Δhmp mutant.

Figure 3

Observation of NOx formation after NO₂ and NO exposure in the bacterial supernatant. (a) Kinetics of DAFFM-DA fluorescence in response to NO after exposure to NO and NO₂. (b) Kinetics of DHR fluorescence in response to ONOO⁻ after exposure to NO and NO₂. (c) Optical density at 520 nm of Griess reagent in response to NO₂⁻. N = 3.

Figure 4

Physiological modifications of P. fluorescens MFAF76a after exposure to various concentrations of NO₂. (a) Cultivability of bacteria after exposure to NO₂ compared to cells exposed to synthetic air. (b) Membrane integrity assessed by live-dead flow cytometry assays using Live/Dead BacLight kit (L-7012, ThermoFisher) according to the manufacturer’s recommendations. (c) Expression of genes coding for proteins involved in RNS and ROS detoxification. Graphs represent means ± SEM. ns = p > 0.05; * = p < 0.05; *** = p < 0.001, N ≥ 3.

Figure 5

Proteomic profile of P. fluorescens MFAF76a after NO₂ exposure. ROS/RNS: reactive oxygen/nitrogen species; ISC: iron-sulfur cluster. The identification of proteins was performed using P. fluorescens A506 as reference (N = 4).