Metabolic modulations of Pseudomonas graminis in response to H2O2 in cloud water

Abstract

In cloud water, microorganisms are exposed to very strong stresses especially related to the presence of reactive oxygen species including H₂O₂ and radicals, which are the driving force of cloud chemistry. In order to understand how the bacterium Pseudomonas graminis isolated from cloud water respond to this oxidative stress, it was incubated in microcosms containing a synthetic solution of cloud water in the presence or in the absence of H₂O₂. P. graminis metabolome was examined by LC-MS and NMR after 50 min and after 24 hours of incubation. After 50 min, the cells were metabolizing H₂O₂ while this compound was still present in the medium, and it was completely biodegraded after 24 hours. Cells exposed to H₂O₂ had a distinct metabolome as compared to unexposed cells, revealing modulations of certain metabolic pathways in response to oxidative stress. These data indicated that the regulations observed mainly involved carbohydrate, glutathione, energy, lipid, peptides and amino-acids metabolisms. When cells had detoxified H₂O₂ from the medium, their metabolome was not distinguishable anymore from unexposed cells, highlighting the capacity of resilience of this bacterium. This work illustrates the interactions existing between the cloud microbial metabolome and cloud chemistry.

Figure 1

(a) Temporal evolution of H₂O₂ concentration in the presence (circles) or absence of P. graminis 13b-3 (triangles). (b) Evolution of ATP concentration in P. graminis 13b-3 incubated with H₂O₂ (triangles) or without H₂O₂ (squares). Error bars are standard errors of the means of three replicates. Where error bars are not visible, they are smaller than the symbol.

Figure 2

PLS-DA analyses (UV scaling). (a) NMR and (b) LC-MS example of the positive ionization mode. Blue circles correspond to the samples incubated in the presence of H₂O₂ and extracted at 50 min; red circles correspond to the samples incubated in the absence of H₂O₂ and extracted at 50 min; green circles correspond to the samples extracted at 24 hours with and without H₂O₂.

Figure 3

PLS-DA analyses. Score plots (UV scaling) obtained for bacteria metabolomes extracted at 50 min of incubation in the presence (blue circles) or in the absence (red circles) of H₂O₂: (a) NMR and (b) LC-MS (example of the positive ionization mode).

Figure 4

Carbohydrate metabolism showing metabolites over-produced in the presence of hydrogen peroxide (red boxes) and those under-produced (purple boxes). Likely metabolic intermediates are indicated in white boxes with dotted frame.

Figure 5

Central metabolism: Citrate cycle, lipids, amino acids and peptides. The metabolites, which are over-produced in the presence of hydrogen peroxide, are indicated in red boxes, while those which are under-produced are indicated in purple boxes. Putative metabolic intermediates are indicated in white boxes with dotted frame.

Figure 6

Glutathione metabolism. The metabolites, which are over-produced in the presence of hydrogen peroxide are indicated in red boxes, while those, which are underproduced, are indicated in purple boxes. Putative metabolic intermediates are indicated in white boxes with dotted frame.

Figure 7

Simplified scheme of the interactions of H₂O₂ with the bacterium metabolome and the organic matter (OM) present in the cloud droplet. The modulation of the metabolome by H₂O₂, in particular, amino acid and carboxylic acid pathways, could have an impact on cloud chemistry by changing the amino acid and carboxylic acid concentrations in the cloud droplet. Carbox acid: carboxylic acids. Dotted lines indicate unknown pathways (transport, reactions…).