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Involvement of type VI secretion system in secretion of iron chelator pyoverdine in Pseudomonas taiwanensis

View Article: PubMed Central - PubMed

ABSTRACT

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive rice diseases worldwide. Therefore, in addition to breeding disease-resistant rice cultivars, it is desirable to develop effective biocontrol agents against Xoo. Here, we report that a soil bacterium Pseudomonas taiwanensis displayed strong antagonistic activity against Xoo. Using matrix-assisted laser desorption/ionization imaging mass spectrometry, we identified an iron chelator, pyoverdine, secreted by P. taiwanensis that could inhibit the growth of Xoo. Through Tn5 mutagenesis of P. taiwanensis, we showed that mutations in genes that encode components of the type VI secretion system (T6SS) as well as biosynthesis and maturation of pyoverdine resulted in reduced toxicity against Xoo. Our results indicated that T6SS is involved in the secretion of endogenous pyoverdine. Mutations in T6SS component genes affected the secretion of mature pyoverdine from the periplasmic space into the extracellular medium after pyoverdine precursor is transferred to the periplasm by the inner membrane transporter PvdE. In addition, we also showed that other export systems, i.e., the PvdRT-OpmQ and MexAB-OprM efflux systems (for which there have been previous suggestions of involvement) and the type II secretion system (T2SS), are not involved in pyoverdine secretion.

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Related in: MedlinePlus

Characterization of the pyoverdine secretion system.(a) Zone of inhibition assay for anti-Xoo activity was tested in 1/2 TSB medium which contained Xoo. Whole culture (top a) and cell-free culture supernatant of WT, clpV::Tn5, pvdL::Tn5, and pvdE::Tn5, which were collected after incubation for 24 h in LP media, were injected into the hole to test toxicity against Xoo (bottom a). (b) MALDI-IMS imaging of pyoverdine from incubation of wild-type and mutant P. taiwanensis with Xoo. Competition plates of P. taiwanensis and mutants co-cultured with Xoo for IMS (top b). The MALDI-IMS image shows an ion of m/z 1044 [M + H]+, displaying the highest level surrounding the hole between the wild-type and mutant treatment (bottom b). Scale bar, 2 mm. Intensity gradients for pyoverdine are illustrated by color histograms (maximum, white; minimum, black). (c) LC-MS quantification of pyoverdine in cell free culture supernatants of WT and clpV::Tn5 after 24-h incubation in iron-limited media. (d) HPLC quantification of pyoverdine (m/z 1044) and culture supernatant from wild-type and mutants. Fluorescent pyoverdine was monitored at 400 nm under the UV detector. Pyoverdine was purified using Cu-sepharose.
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f2: Characterization of the pyoverdine secretion system.(a) Zone of inhibition assay for anti-Xoo activity was tested in 1/2 TSB medium which contained Xoo. Whole culture (top a) and cell-free culture supernatant of WT, clpV::Tn5, pvdL::Tn5, and pvdE::Tn5, which were collected after incubation for 24 h in LP media, were injected into the hole to test toxicity against Xoo (bottom a). (b) MALDI-IMS imaging of pyoverdine from incubation of wild-type and mutant P. taiwanensis with Xoo. Competition plates of P. taiwanensis and mutants co-cultured with Xoo for IMS (top b). The MALDI-IMS image shows an ion of m/z 1044 [M + H]+, displaying the highest level surrounding the hole between the wild-type and mutant treatment (bottom b). Scale bar, 2 mm. Intensity gradients for pyoverdine are illustrated by color histograms (maximum, white; minimum, black). (c) LC-MS quantification of pyoverdine in cell free culture supernatants of WT and clpV::Tn5 after 24-h incubation in iron-limited media. (d) HPLC quantification of pyoverdine (m/z 1044) and culture supernatant from wild-type and mutants. Fluorescent pyoverdine was monitored at 400 nm under the UV detector. Pyoverdine was purified using Cu-sepharose.

Mentions: To identify the factors that affect the antagonistic activity of P. taiwanensis against Xoo, we generated Tn5 transposon-insertion mutant pools of P. taiwanensis and screened for mutants with attenuated antagonistic activities against Xoo. A total of 5000 colonies screened, three mutants were isolated and the location of Tn5 insertion was determined using thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and sequencing analyses to have Tn5 insertion sites in genes that encode T6SS (clpV, GenBank AC no. KM061430), pyoverdine synthetase (pvdL, GenBank AC no. KM036007) and pyoverdine translocation and maturation (pvdE, GenBank AC no. KM036029). ATPase ClpV is an important core component of T6SS and contributes to VipA/VipB tubule remodeling, which is required for T6SS activity20. PvdL is a nonribosomal peptide synthetase (NRPS) that is involved in the biosynthesis of pyoverdine chromophore8. PvdE is a cell membrane protein that is involved in transportation of pyoverdine precursors to the periplasm21. In antagonistic assays, whole culture or cell-free culture supernatants of wild-type P. taiwanensis showed substantial toxicity towards Xoo (Fig. 2a) suggesting that soluble compounds were secreted by P. taiwanensis to inhibit the growth of Xoo. In contrast, whole culture or cell-free culture supernatants of clpV::Tn5 mutant showed a lower toxicity than those of the wild-type, and those of pvdL::Tn5 and pvdE::Tn5 mutants exhibited no toxicity towards Xoo. No significant differences in the growth rates of the wild-type (WT), clpV::Tn5, pvdL::Tn5 and pvdE::Tn5 mutants were detected from 4 h (lag phase) to 72 h (stationary phase) under iron-limited LP broth cultures (Supplementary Fig. S1), indicating that the reduction of anti-Xoo activities of these mutants is not due to reduced viability.


Involvement of type VI secretion system in secretion of iron chelator pyoverdine in Pseudomonas taiwanensis
Characterization of the pyoverdine secretion system.(a) Zone of inhibition assay for anti-Xoo activity was tested in 1/2 TSB medium which contained Xoo. Whole culture (top a) and cell-free culture supernatant of WT, clpV::Tn5, pvdL::Tn5, and pvdE::Tn5, which were collected after incubation for 24 h in LP media, were injected into the hole to test toxicity against Xoo (bottom a). (b) MALDI-IMS imaging of pyoverdine from incubation of wild-type and mutant P. taiwanensis with Xoo. Competition plates of P. taiwanensis and mutants co-cultured with Xoo for IMS (top b). The MALDI-IMS image shows an ion of m/z 1044 [M + H]+, displaying the highest level surrounding the hole between the wild-type and mutant treatment (bottom b). Scale bar, 2 mm. Intensity gradients for pyoverdine are illustrated by color histograms (maximum, white; minimum, black). (c) LC-MS quantification of pyoverdine in cell free culture supernatants of WT and clpV::Tn5 after 24-h incubation in iron-limited media. (d) HPLC quantification of pyoverdine (m/z 1044) and culture supernatant from wild-type and mutants. Fluorescent pyoverdine was monitored at 400 nm under the UV detector. Pyoverdine was purified using Cu-sepharose.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f2: Characterization of the pyoverdine secretion system.(a) Zone of inhibition assay for anti-Xoo activity was tested in 1/2 TSB medium which contained Xoo. Whole culture (top a) and cell-free culture supernatant of WT, clpV::Tn5, pvdL::Tn5, and pvdE::Tn5, which were collected after incubation for 24 h in LP media, were injected into the hole to test toxicity against Xoo (bottom a). (b) MALDI-IMS imaging of pyoverdine from incubation of wild-type and mutant P. taiwanensis with Xoo. Competition plates of P. taiwanensis and mutants co-cultured with Xoo for IMS (top b). The MALDI-IMS image shows an ion of m/z 1044 [M + H]+, displaying the highest level surrounding the hole between the wild-type and mutant treatment (bottom b). Scale bar, 2 mm. Intensity gradients for pyoverdine are illustrated by color histograms (maximum, white; minimum, black). (c) LC-MS quantification of pyoverdine in cell free culture supernatants of WT and clpV::Tn5 after 24-h incubation in iron-limited media. (d) HPLC quantification of pyoverdine (m/z 1044) and culture supernatant from wild-type and mutants. Fluorescent pyoverdine was monitored at 400 nm under the UV detector. Pyoverdine was purified using Cu-sepharose.
Mentions: To identify the factors that affect the antagonistic activity of P. taiwanensis against Xoo, we generated Tn5 transposon-insertion mutant pools of P. taiwanensis and screened for mutants with attenuated antagonistic activities against Xoo. A total of 5000 colonies screened, three mutants were isolated and the location of Tn5 insertion was determined using thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and sequencing analyses to have Tn5 insertion sites in genes that encode T6SS (clpV, GenBank AC no. KM061430), pyoverdine synthetase (pvdL, GenBank AC no. KM036007) and pyoverdine translocation and maturation (pvdE, GenBank AC no. KM036029). ATPase ClpV is an important core component of T6SS and contributes to VipA/VipB tubule remodeling, which is required for T6SS activity20. PvdL is a nonribosomal peptide synthetase (NRPS) that is involved in the biosynthesis of pyoverdine chromophore8. PvdE is a cell membrane protein that is involved in transportation of pyoverdine precursors to the periplasm21. In antagonistic assays, whole culture or cell-free culture supernatants of wild-type P. taiwanensis showed substantial toxicity towards Xoo (Fig. 2a) suggesting that soluble compounds were secreted by P. taiwanensis to inhibit the growth of Xoo. In contrast, whole culture or cell-free culture supernatants of clpV::Tn5 mutant showed a lower toxicity than those of the wild-type, and those of pvdL::Tn5 and pvdE::Tn5 mutants exhibited no toxicity towards Xoo. No significant differences in the growth rates of the wild-type (WT), clpV::Tn5, pvdL::Tn5 and pvdE::Tn5 mutants were detected from 4 h (lag phase) to 72 h (stationary phase) under iron-limited LP broth cultures (Supplementary Fig. S1), indicating that the reduction of anti-Xoo activities of these mutants is not due to reduced viability.

View Article: PubMed Central - PubMed

ABSTRACT

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive rice diseases worldwide. Therefore, in addition to breeding disease-resistant rice cultivars, it is desirable to develop effective biocontrol agents against Xoo. Here, we report that a soil bacterium Pseudomonas taiwanensis displayed strong antagonistic activity against Xoo. Using matrix-assisted laser desorption/ionization imaging mass spectrometry, we identified an iron chelator, pyoverdine, secreted by P. taiwanensis that could inhibit the growth of Xoo. Through Tn5 mutagenesis of P. taiwanensis, we showed that mutations in genes that encode components of the type VI secretion system (T6SS) as well as biosynthesis and maturation of pyoverdine resulted in reduced toxicity against Xoo. Our results indicated that T6SS is involved in the secretion of endogenous pyoverdine. Mutations in T6SS component genes affected the secretion of mature pyoverdine from the periplasmic space into the extracellular medium after pyoverdine precursor is transferred to the periplasm by the inner membrane transporter PvdE. In addition, we also showed that other export systems, i.e., the PvdRT-OpmQ and MexAB-OprM efflux systems (for which there have been previous suggestions of involvement) and the type II secretion system (T2SS), are not involved in pyoverdine secretion.

No MeSH data available.


Related in: MedlinePlus