Production of siderophores increases resistance to fusaric acid in Pseudomonas protegens Pf-5

Abstract

Fusaric acid is produced by pathogenic fungi of the genus Fusarium, and is toxic to plants and rhizobacteria. Many fluorescent pseudomonads can prevent wilt diseases caused by these fungi. This study was undertaken to evaluate the effect of fusaric acid on P. protegens Pf-5 and elucidate the mechanisms that enable the bacterium to survive in the presence of the mycotoxin. The results confirm that fusaric acid negatively affects growth and motility of P. protegens. Moreover, a notable increase in secretion of the siderophore pyoverdine was observed when P. protegens was grown in the presence of fusaric acid. Concomitantly, levels of enzymes involved in the biosynthesis of pyoverdine and enantio-pyochelin, the second siderophore encoded by P. protegens, increased markedly. Moreover, while similar levels of resistance to fusaric acid were observed for P. protegens mutants unable to synthesize either pyoverdine or enanto-pyochelin and the wild type strain, a double mutant unable to synthesize both kinds of siderophores showed a dramatically reduced resistance to this compound. This reduced resistance was not observed when this mutant was grown under conditions of iron excess. Spectrophotometric titrations revealed that fusaric acid binds not only Fe2+ and Fe3+, but also Zn2+, Mn2+ and Cu2+, with high affinity. Our results demonstrate that iron sequestration accounts at least in part for the deleterious effect of the mycotoxin on P. protegens.

Figure 1. Effect of FA on growth of P. protegens Pf-5.

Cultures of P. protegens Pf-5 were grown aerobically in E₂ glucose minimal medium supplemented or not with the indicated concentrations of FA. Growth was monitored by measurement of the OD₆₀₀ at the indicated times after inoculation. OD₆₀₀ levels are mean values for three independently grown cultures, and error bars depict standard deviations of the mean. The asterisk (*) denotes significant differences from the culture grown without FA (P<0.05) using ANOVA.

Figure 2. Effect of FA on cell motility and biofilm formation by P. protegens Pf-5.

A. Cultures of P. protegens Pf-5 (wild type) were grown aerobically overnight to OD = 3 in KMB medium. An aliquot was spotted onto the centre of plates containing 20 fold diluted KMB medium supplemented or not with FA and solidified with various concentrations of Bacto-Agar, depending on which motility was to be evaluated. After 24 h of incubation at room temperature, the distance of the migration front from the point of inoculation was measured. B. Cultures of P. protegens Pf-5 were grown in 96-well polystyrene plates containing E₂ glucose minimal medium supplemented or not with FA. After 24 h of incubation, biofilm formation was assessed by the determining the proportion of attached and non-attached bacteria (A₅₉₅/OD₆₀₀). In all cases, error bars indicate the standard deviation of the mean. The asterisk (*) denotes significant differences (P<0.05) using ANOVA.

Figure 3. SDS-PAGE of the membrane fraction of P. protegens Pf-5 grown in presence and absence of FA.

Triplicate cultures of P. protegens Pf-5 were grown aerobically for 12 h in E₂ glucose minimal medium with (1, 2 and 3) or without (4, 5 and 6) the addition of 2 mM FA. Aliquots containing 100 μg of protein obtained from the total membrane fraction were loaded onto the gel. Boxed bands mark proteins whose levels differ in cells grown in the presence and absence of FA and were identified by peptide fingerprint analysis (a: Pyochelin synthetase F, b: Pyochelin synthetase E, c: ABC transporter ATP-binding protein, d: L-ornithine 5-monooxygenase PvdA).

Figure 4. Correlation between siderophore production in P. protegens and resistance to FA.

P. protegens Pf-5, P. protegens Pf-21, P. protegens Pf-4 and P. protegens Pf-21.10 were cultured aerobically during 16 h in 96-well polystyrene plates containing E₂ glucose minimal medium supplemented with increasing concentrations of FA (A). Cultures of P. protegens Pf-5 (wild type) and P. protegens Pf-21.10 (ΔpvdF, ΔpchF) were grown under the same conditions with or without FeCl₃ supplementation at a final concentration of 100 μM (B). Cultures of P. protegens Pf-5 (wild type) and P. protegens Pf-21.10 (ΔpvdF, ΔpchF) were grown under the same conditions with or without EPch supplementation at a final concentration of 1.3 μM (C). Values of maximal cell densities (OD₆₀₀) are shown. The experiments were performed three times and error bars represent the standard deviation of the mean.

Figure 5. Effect of FA and iron supplementation on pyoverdine production.

P. protegens Pf-5 was cultured aerobically during 16 h in 96-well polystyrene plates containing E₂ glucose minimal medium with or without increasing concentrations of FA and with or without FeCl₃ supplementation at a final concentration of 100 μM. The fluorescence intensity of the supernatant was measured with a fluorimeter and growth was estimated by determination of the OD₆₀₀. The fluorescence intensity obtained was normalized to the OD₆₀₀, and is thus expressed in relative fluorescence units (RFU). The experiments were performed three times and error bars represent the standard deviation of the mean.