Arginine Biosynthesis Modulates Pyoverdine Production and Release in Pseudomonas putida as Part of the Mechanism of Adaptation to Oxidative Stress

Abstract

Iron is essential for most life forms. Under iron-limiting conditions, many bacteria produce and release siderophores-molecules with high affinity for iron-which are then transported into the cell in their iron-bound form, allowing incorporation of the metal into a wide range of cellular processes. However, free iron can also be a source of reactive oxygen species that cause DNA, protein, and lipid damage. Not surprisingly, iron capture is finely regulated and linked to oxidative-stress responses. Here, we provide evidence indicating that in the plant-beneficial bacterium Pseudomonas putida KT2440, the amino acid l-arginine is a metabolic connector between iron capture and oxidative stress. Mutants defective in arginine biosynthesis show reduced production and release of the siderophore pyoverdine and altered expression of certain pyoverdine-related genes, resulting in higher sensitivity to iron limitation. Although the amino acid is not part of the siderophore side chain, addition of exogenous l-arginine restores pyoverdine release in the mutants, and increased pyoverdine production is observed in the presence of polyamines (agmatine and spermidine), of which arginine is a precursor. Spermidine also has a protective role against hydrogen peroxide in P. putida, whereas defects in arginine and pyoverdine synthesis result in increased production of reactive oxygen species.IMPORTANCE The results of this study show a previously unidentified connection between arginine metabolism, siderophore turnover, and oxidative stress in Pseudomonas putida Although the precise molecular mechanisms involved have yet to be characterized in full detail, our data are consistent with a model in which arginine biosynthesis and the derived pathway leading to polyamine production function as a homeostasis mechanism that helps maintain the balance between iron uptake and oxidative-stress response systems.

Keywords: amino acid biosynthesis; iron acquisition; iron regulation; oxidative stress; siderophores.

FIG 1

Analysis of pyoverdine production and release in KT2440, ΔargG, ΔargH, and pvdD (negative-control) strains. (A) Fluorescence detection in liquid cultures grown in King’s B medium in the absence (control) or presence of 5 mM l-arginine. wt, wild type. (B) CAS assay detection of pyoverdine release (orange halo) in the absence or presence of different compounds related to arginine metabolism added at a final concentration of 5 mM. The ΔargG and ΔargH mutants complemented with plasmids carrying each wild-type gene (pME1088 and pME0184, respectively) were also included. Experiments were repeated three times, with three replicates each; representative images are shown. GABA, gamma-aminobutyric acid.

FIG 2

Analysis of pyoverdine production and release in different mutants in the arginine biosynthesis pathway. CAS assays similar to those shown in Fig. 1 were performed in the presence or absence of 5 mM l-arginine (top), and the area of the halo produced by each strain was calculated after subtracting the area covered by the culture growth patch (bottom). The data correspond to averages and standard deviations from three independent experiments with three replicates each. Statistically significant differences from the wild type are indicated by asterisks (Student's t test; P < 0.05).

FIG 3

(A) Quantification of residual iron in King’s B medium after overnight growth of cultures of KT2440, ΔargG, ΔargH, and pvdD strains. The data were corrected for growth (OD₆₆₀), and average relative values and standard deviations are presented, corresponding to two biological replicates with three technical replicates each. Differences between the wild type and the three mutants were statistically significant (Student's t test; P < 0.01). (B) Effect of iron chelation on growth of KT2440 and the ΔargG and ΔargH mutants. Cultures were inoculated at an initial OD₆₆₀ of 0.05 in liquid LB medium with increasing concentrations of 2,2′-bipyridyl, in duplicate. Growth was analyzed after 24 h by measuring the turbidity (OD₆₆₀) of the cultures. The data correspond to averages and standard deviations of three independent experiments.

FIG 4

Expression levels of pyoverdine-related genes in ΔargG and ΔargH mutants with respect to the wild type, analyzed by qRT-PCR after growth in King’s B medium. The data are averages and standard deviations from three biological replicates with three technical replicates each. The asterisks indicate relevant changes based on statistically significant differences from the wild type (Student's t test; P < 0.05) and considering a ±1.5-fold change as the cutoff value (dashed lines).

FIG 5

Quantification of extracellular and intracellular pyoverdine fractions in liquid cultures grown in King’s B medium in the absence (A) or presence (B) of 200 μM 2,2′-bipyridyl. Fluorescence was measured using a Varioskan Lux microplate reader by recording emission at 455 nm upon excitation at 398 nm. Fluorescence readings were corrected for growth (OD₆₆₀), and average relative values and standard deviations are presented, corresponding to three biological replicates with three technical replicates each. Statistically significant differences with respect to the corresponding value in the wild type are indicated by asterisks (Student's t test; P < 0.01).

FIG 6

Quantification of fluorescence resulting from extracellular pyoverdine in KT2440 and the ΔargG and ΔargH mutants after 24 h of growth in King’s B medium supplemented with different polyamines, using a Varioskan Lux fluorimeter. Fluorescence readings were normalized for growth (OD₆₆₀). Average relative values and standard deviations are presented, corresponding to three biological replicates with three technical replicates each. The asterisks indicate statistically significant differences from the control without addition (Student's t test; P < 0.01).

FIG 7

Growth of P. putida KT2440 in the absence (open symbols) and presence (closed symbols) of 5 mM H₂O₂, without (circles) or with (triangles) 5 mM spermidine. Cultures were grown as described in Materials and Methods. The results are averages and standard errors from two independent assays with 4 replicates.

FIG 8

Fluorescence-based measurement of reactive oxygen species in KT2440 and the pvdD, ΔargG, and ΔargH mutants, using CellROX Deep Red reagent. The data correspond to fluorescence readings normalized with respect to the OD₆₆₀ at each time point and are the averages and standard errors from two independent assays with 6 replicates.

FIG 9

Schematic summary of the proposed connections between arginine/polyamines, iron uptake, and oxidative stress. (A) In the wild-type strain, low iron activates pyoverdine (PVD) production and maturation (dark-green pentagons) to restore intracellular iron pools. The siderophore sequesters extracellular and intracellular iron, while polyamines protect against ROS that can derive from iron chemistry. (B) When polyamine production is disfavored, such as in the arginine biosynthesis mutants, protection against ROS is hampered. This leads to reduced pyoverdine release and therefore limited iron capture, as a proposed mechanism to control intracellular free iron and minimize ROS production. (C) In a pyoverdine-deficient mutant, polyamine synthesis would be intact but insufficient for full protection against ROS due to the lack of siderophore available to sequester free iron.