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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.

No MeSH data available.


Subcellular localization and quantification of mature fluorescence pyoverdine in the periplasm and cytoplasm.(a) Localization of mature fluorescence pyoverdine was detected in wild-type, clpV::Tn5, ΔicmF, tssC::Tn5, pvdL::Tn5, and pvdE::Tn5 mutants through a fluorescence microscope with a DAPI filter. (Scale bar = 2 μm). (b) Quantification of periplasmic and cytoplasmic pyoverdine was conducted by measuring fluorescence at excitation 405 nm and emission 460 nm. Pyoverdine values were normalized against the cell optical densities (Ex405, Em 460/OD600), and relative values were determined by comparing each value to the periplasmic mean value of the wild-type. Values are mean ± SD of three independent experiments. P. taiwanensis and mutants (clpV::Tn5, ΔicmF, tssC::Tn5, pvdL::Tn5 and pvdE::Tn5) were grown on the iron-limited media for 12 h at 28 °C.
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f5: Subcellular localization and quantification of mature fluorescence pyoverdine in the periplasm and cytoplasm.(a) Localization of mature fluorescence pyoverdine was detected in wild-type, clpV::Tn5, ΔicmF, tssC::Tn5, pvdL::Tn5, and pvdE::Tn5 mutants through a fluorescence microscope with a DAPI filter. (Scale bar = 2 μm). (b) Quantification of periplasmic and cytoplasmic pyoverdine was conducted by measuring fluorescence at excitation 405 nm and emission 460 nm. Pyoverdine values were normalized against the cell optical densities (Ex405, Em 460/OD600), and relative values were determined by comparing each value to the periplasmic mean value of the wild-type. Values are mean ± SD of three independent experiments. P. taiwanensis and mutants (clpV::Tn5, ΔicmF, tssC::Tn5, pvdL::Tn5 and pvdE::Tn5) were grown on the iron-limited media for 12 h at 28 °C.

Mentions: Next, the other component mutants of T6SS were used to confirm the involvement of T6SS in pyoverdine secretion. After screening 11,600 mutants, only one additional T6SS component mutant tssC::Tn5 (GenBank AC no. KU668948) was isolated. We further generated the third component mutant ΔicmF (GenBank AC no. KU668949) by site-specific recombination using the lambda-Red system. TssC is a T6SS core component that forms a contractile tail sheath28. IcmF is responsible for T6SS-mediated Hcp tube secretion29. We used live cell imaging to evaluate the involvement of T6SS in the secretion of mature pyoverdines from the periplasm to the extracellular medium. The DAPI filter set is appropriate for detection of the fluorescent pyoverdine. Interestingly, fluorescent pyoverdine was accumulated more in the periplasm of T6SS mutants (clpV::Tn5, tssC::Tn5 and ΔicmF) compared to WT under fluorescence microscopy with DAPI filter (Fig. 5a). Wild-type only displayed slight fluorescence in the periplasm. In the pvdL::Tn5 and pvdE::Tn5 mutants no fluorescent pyoverdine could be visualized under fluorescence microscopy with DAPI filter (Fig. 5a). The maxima fluorescence wavelength ranges of pyoverdine were confirmed at 360–410 nm of excitation and 450–480 nm of emission (Supplementary Fig. S6a). Pyoverdine mutant strain pvdL::Tn5 displayed no florescence intensity of emission (Supplementary Fig. S6b). Together with ultraviolet (UV) light detection (Supplementary Fig. S6), these results show that the only florescent substance produced in P. taiwanensis is a pyoverdine. Next, we quantified mature pyoverdine (fluorescent pyoverdine) in the periplasm and cytoplasm of the wild-type and the four mutants defective in anti-Xoo activity by measuring fluorescence at Ex/Em = 405/460 nm (Fig. 5b). In the three T6SS mutants (clpV::Tn5, tssC::Tn5 and ΔicmF), the amounts of mature pyoverdine in the periplasm were higher than that of wild-type. Mature pyoverdine was not detected in the cytoplasm of negative controls (pvdL::Tn5 and pvdE::Tn5 mutants) (Fig. 5b). Our data showed that T6SS is involved in the secretion of de novo synthesized pyoverdine. However, all T6SS mutants did have significant, albeit low, levels of pyoverdine in the extracellular media or agar plates (Fig. 2b–d, and Fig. 6) suggesting that an additional pathway might be involved in pyoverdine secretion.


Involvement of type VI secretion system in secretion of iron chelator pyoverdine in Pseudomonas taiwanensis
Subcellular localization and quantification of mature fluorescence pyoverdine in the periplasm and cytoplasm.(a) Localization of mature fluorescence pyoverdine was detected in wild-type, clpV::Tn5, ΔicmF, tssC::Tn5, pvdL::Tn5, and pvdE::Tn5 mutants through a fluorescence microscope with a DAPI filter. (Scale bar = 2 μm). (b) Quantification of periplasmic and cytoplasmic pyoverdine was conducted by measuring fluorescence at excitation 405 nm and emission 460 nm. Pyoverdine values were normalized against the cell optical densities (Ex405, Em 460/OD600), and relative values were determined by comparing each value to the periplasmic mean value of the wild-type. Values are mean ± SD of three independent experiments. P. taiwanensis and mutants (clpV::Tn5, ΔicmF, tssC::Tn5, pvdL::Tn5 and pvdE::Tn5) were grown on the iron-limited media for 12 h at 28 °C.
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f5: Subcellular localization and quantification of mature fluorescence pyoverdine in the periplasm and cytoplasm.(a) Localization of mature fluorescence pyoverdine was detected in wild-type, clpV::Tn5, ΔicmF, tssC::Tn5, pvdL::Tn5, and pvdE::Tn5 mutants through a fluorescence microscope with a DAPI filter. (Scale bar = 2 μm). (b) Quantification of periplasmic and cytoplasmic pyoverdine was conducted by measuring fluorescence at excitation 405 nm and emission 460 nm. Pyoverdine values were normalized against the cell optical densities (Ex405, Em 460/OD600), and relative values were determined by comparing each value to the periplasmic mean value of the wild-type. Values are mean ± SD of three independent experiments. P. taiwanensis and mutants (clpV::Tn5, ΔicmF, tssC::Tn5, pvdL::Tn5 and pvdE::Tn5) were grown on the iron-limited media for 12 h at 28 °C.
Mentions: Next, the other component mutants of T6SS were used to confirm the involvement of T6SS in pyoverdine secretion. After screening 11,600 mutants, only one additional T6SS component mutant tssC::Tn5 (GenBank AC no. KU668948) was isolated. We further generated the third component mutant ΔicmF (GenBank AC no. KU668949) by site-specific recombination using the lambda-Red system. TssC is a T6SS core component that forms a contractile tail sheath28. IcmF is responsible for T6SS-mediated Hcp tube secretion29. We used live cell imaging to evaluate the involvement of T6SS in the secretion of mature pyoverdines from the periplasm to the extracellular medium. The DAPI filter set is appropriate for detection of the fluorescent pyoverdine. Interestingly, fluorescent pyoverdine was accumulated more in the periplasm of T6SS mutants (clpV::Tn5, tssC::Tn5 and ΔicmF) compared to WT under fluorescence microscopy with DAPI filter (Fig. 5a). Wild-type only displayed slight fluorescence in the periplasm. In the pvdL::Tn5 and pvdE::Tn5 mutants no fluorescent pyoverdine could be visualized under fluorescence microscopy with DAPI filter (Fig. 5a). The maxima fluorescence wavelength ranges of pyoverdine were confirmed at 360–410 nm of excitation and 450–480 nm of emission (Supplementary Fig. S6a). Pyoverdine mutant strain pvdL::Tn5 displayed no florescence intensity of emission (Supplementary Fig. S6b). Together with ultraviolet (UV) light detection (Supplementary Fig. S6), these results show that the only florescent substance produced in P. taiwanensis is a pyoverdine. Next, we quantified mature pyoverdine (fluorescent pyoverdine) in the periplasm and cytoplasm of the wild-type and the four mutants defective in anti-Xoo activity by measuring fluorescence at Ex/Em = 405/460 nm (Fig. 5b). In the three T6SS mutants (clpV::Tn5, tssC::Tn5 and ΔicmF), the amounts of mature pyoverdine in the periplasm were higher than that of wild-type. Mature pyoverdine was not detected in the cytoplasm of negative controls (pvdL::Tn5 and pvdE::Tn5 mutants) (Fig. 5b). Our data showed that T6SS is involved in the secretion of de novo synthesized pyoverdine. However, all T6SS mutants did have significant, albeit low, levels of pyoverdine in the extracellular media or agar plates (Fig. 2b–d, and Fig. 6) suggesting that an additional pathway might be involved in pyoverdine secretion.

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.