Siderophore-mediated iron acquisition influences motility and is required for full virulence of the xylem-dwelling bacterial phytopathogen Pantoea stewartii subsp. stewartii

Abstract

Iron is a key micronutrient for microbial growth but is often present in low concentrations or in biologically unavailable forms. Many microorganisms overcome this challenge by producing siderophores, which are ferric-iron chelating compounds that enable the solubilization and acquisition of iron in a bioactive form. Pantoea stewartii subsp. stewartii, the causal agent of Stewart's wilt of sweet corn, produces a siderophore under iron-limiting conditions. The proteins involved in the biosynthesis and export of this siderophore are encoded by the iucABCD-iutA operon, which is homologous to the aerobactin biosynthetic gene cluster found in a number of enteric pathogens. Mutations in iucA and iutA resulted in a decrease in surface-based motility that P. stewartii utilizes during the early stages of biofilm formation, indicating that active iron acquisition impacts surface motility for P. stewartii. Furthermore, bacterial movement in planta is also dependent on a functional siderophore biosynthesis and uptake pathway. Most notably, siderophore-mediated iron acquisition is required for full virulence in the sweet corn host, indicating that active iron acquisition is essential for pathogenic fitness for this important xylem-dwelling bacterial pathogen.

FIG 1

Genomic orientation of the Pantoea stewartii iucABCD-iutA gene cluster. Filled arrows indicate gene deletions created in the present study. The Fur binding sequence is indicated upstream of the iucA ORF.

FIG 2

Siderophore-mediated iron acquisition requires iucA and iutA. The production of siderophores was detected using colorimetric CAS agar plates (32). (A) For each strain, inoculum was taken from a single colony grown on nutrient agar and streaked down the middle of the CAS plates, followed by incubation for 10 days at 28°C. The radius of the zone of color change was measured at its widest point from the edge of colony growth. (B) The graph represents the mean halo radius from 15 individual plates for each strain. Bars with different letters indicate significance based on a Student t test using P ≤ 0.05. Error bars indicate standard errors of the mean.

FIG 3

Siderophore-mediated iron acquisition is required for surface motility. Portions (2 μl) containing ∼10⁷ cells were placed in the centers of motility plates, followed by incubation at 28°C. Images were taken of the same plates after 24 h of incubation. Experiments were performed with at least three biological replicates, with five technical replicates each (total 15 plates per strain). Error bars indicate the standard errors of the mean.

FIG 4

Siderophore mutants exhibit wild-type growth rates in vitro. Strains were grown in 3 ml of liquid LB medium (A) and MM9 medium (B). The OD₆₀₀ was recorded over the course of 24 h. The graphs represent the mean of at least three biological replicates plus or minus the standard errors of the mean.

FIG 5

Iron limitation inhibits swarming motility. Portions (2 μl) containing ∼10⁷ cells of WT DC283 were placed in the centers of motility plates with or without 200 μM 2,2-dipyridyl, followed by incubation at 28°C. Images were taken of the same plates after 16, 30, and 48 h of incubation. Experiments were conducted with at least three biological replicates, with five technical replicates each (a total 15 plates per strain).

FIG 6

Iron supplementation restores motility in ΔiucA and ΔiutA mutants. Portions (2 μl) containing ∼10⁷ WT DC283, ΔiucA, or ΔiutA cells were placed in the centers of motility plates with or without 20 μM FeSO₄, followed by incubation at 28°C. Photographs were taken after 24 h of incubation. Experiments were conducted with at least three biological replicates, with five technical replicates each (a total of fifteen plates per strain).

FIG 7

Siderophore production is negatively regulated by Fur. The production of siderophores was detected using colorimetric CAS agar plates (32). For each strain, inoculum was taken from a single colony grown on nutrient agar and streaked down the middle of the CAS plates, followed by incubation for 10 days at 28°C. The halo radius was measured at its widest point from the edge of the colony growth. The graph represents the mean halo radius from 15 plates. Bars with different letters indicate significance based on a Student t test using P ≤ 0.05. Error bars indicate the standard errors of the mean.

FIG 8

Fur regulation influences surface motility. Portions (2 μl) containing ∼10⁷ cells were placed in the centers of swarm plates, followed by incubation at 28°C. Images were taken of the same plates after 12 and 30 h of incubation. Experiments were performed with at least three biological replicates, with five technical replicates each (a total of fifteen plates per strain).

FIG 9

Δfur mutants accumulate intracellular iron. (A) Growth of streptonigrin-treated cells. (B) Growth of untreated cells. Strains were grown overnight in 2 ml of LB medium, diluted 1:20 in fresh LB medium, and divided into untreated and treated samples. Streptonigrin was added to the treated samples at a final concentration of 1 μM. Cell growth was measured as the OD₆₀₀ over a period of 24 h, while incubating at 28°C with shaking at 180 rpm. Error bars represent the standard deviations between replicates of each strain.

FIG 10

Siderophore production and uptake are necessary for bacterial movement in planta. DC283, ΔiucA, ΔiutA, ΔiucA/iucA⁺, and ΔiutA/iutA⁺ strains constitutively expressing GFP from plasmid pHC60 were used to inoculate sweet corn seedlings (cv. Jubilee) in a partial incision on the leaf surface. Plants were inoculated with 2 μl of bacterial suspension containing ∼10⁷ cells in 1× PBS-T. After 72 h, the distance of movement was quantified by measuring the length of GFP fluorescence in the basipetal direction using a Leica MZ FLIII fluorescence equipped stereoscope (Leica Microsystems). Fluorescent microsphere beads (FluoSpheres) were used as a negative control to correct for passive movement. The results are expressed as the mean distance from the inoculation point for at least 10 replicates for each strain ± the standard errors of the mean.

FIG 11

Siderophore production and uptake are required for full virulence. Ten-day-old sweet corn seedlings were inoculated with either a WT DC283, ΔiucA, or ΔiucA/iucA⁺ strain or 1× PBS buffer (A) or a WT DC283, ΔiutA, or ΔiutA/iutA⁺ strain or 1× PBS buffer (B). Plants were observed for symptom development daily, and disease severity was assessed based on an arbitrary rating system between 0 and 4. The graph represents the mean of three biological replicates with five technical replicates each (a total 15 plants per strain). Experiments were repeated at least three times with similar results. Error bars indicate the standard errors of the mean.