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The monofunctional catalase KatE of Xanthomonas axonopodis pv. citri is required for full virulence in citrus plants.

Tondo ML, Petrocelli S, Ottado J, Orellano EG - PLoS ONE (2010)

Bottom Line: Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides.The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process.Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Division, Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT

Background: Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase).

Methodology/principal findings: Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H(2)O(2). A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation.

Conclusions: Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

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Related in: MedlinePlus

Catalase activity in Xac as influenced by the growth phase.(A) Xac cultures were grown aerobically in SB medium to early exponential (EE, 4 h), mid-exponential (ME, 8 h), stationary (S, 24 h) and late stationary (LS, 48 h) phases, and soluble extracts were prepared as described in Materials and Methods. Total catalase activity was assayed as described by Beers and Sizer [18] with 10 mM H2O2 at 25°C. (B) Equal amounts of protein (25 µg) were separated by 8% non-denaturing PAGE and stained for catalase activity by the method of Scandalios [19]. A simultaneously run Coomassie-stained gel (not shown) indicated equal protein loadings between samples. The positions of the electrophoretically discernible catalase species Kat1, Kat2, and Kat3 are indicated.
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pone-0010803-g001: Catalase activity in Xac as influenced by the growth phase.(A) Xac cultures were grown aerobically in SB medium to early exponential (EE, 4 h), mid-exponential (ME, 8 h), stationary (S, 24 h) and late stationary (LS, 48 h) phases, and soluble extracts were prepared as described in Materials and Methods. Total catalase activity was assayed as described by Beers and Sizer [18] with 10 mM H2O2 at 25°C. (B) Equal amounts of protein (25 µg) were separated by 8% non-denaturing PAGE and stained for catalase activity by the method of Scandalios [19]. A simultaneously run Coomassie-stained gel (not shown) indicated equal protein loadings between samples. The positions of the electrophoretically discernible catalase species Kat1, Kat2, and Kat3 are indicated.

Mentions: We investigated the growth phase-dependent pattern of catalase activity in Xac by conducting activity assays on soluble extracts from cultures at different growth stages. A typical growth curve of Xac in SB medium is depicted in Figure S1. As shown in Figure 1A, the highest levels of catalase activity were observed in the stationary and late stationary phases (with similar values of ∼7 µmol min−1 mg−1), being approximately 2.5-fold higher than those determined for the cultures in exponential growth.


The monofunctional catalase KatE of Xanthomonas axonopodis pv. citri is required for full virulence in citrus plants.

Tondo ML, Petrocelli S, Ottado J, Orellano EG - PLoS ONE (2010)

Catalase activity in Xac as influenced by the growth phase.(A) Xac cultures were grown aerobically in SB medium to early exponential (EE, 4 h), mid-exponential (ME, 8 h), stationary (S, 24 h) and late stationary (LS, 48 h) phases, and soluble extracts were prepared as described in Materials and Methods. Total catalase activity was assayed as described by Beers and Sizer [18] with 10 mM H2O2 at 25°C. (B) Equal amounts of protein (25 µg) were separated by 8% non-denaturing PAGE and stained for catalase activity by the method of Scandalios [19]. A simultaneously run Coomassie-stained gel (not shown) indicated equal protein loadings between samples. The positions of the electrophoretically discernible catalase species Kat1, Kat2, and Kat3 are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875408&req=5

pone-0010803-g001: Catalase activity in Xac as influenced by the growth phase.(A) Xac cultures were grown aerobically in SB medium to early exponential (EE, 4 h), mid-exponential (ME, 8 h), stationary (S, 24 h) and late stationary (LS, 48 h) phases, and soluble extracts were prepared as described in Materials and Methods. Total catalase activity was assayed as described by Beers and Sizer [18] with 10 mM H2O2 at 25°C. (B) Equal amounts of protein (25 µg) were separated by 8% non-denaturing PAGE and stained for catalase activity by the method of Scandalios [19]. A simultaneously run Coomassie-stained gel (not shown) indicated equal protein loadings between samples. The positions of the electrophoretically discernible catalase species Kat1, Kat2, and Kat3 are indicated.
Mentions: We investigated the growth phase-dependent pattern of catalase activity in Xac by conducting activity assays on soluble extracts from cultures at different growth stages. A typical growth curve of Xac in SB medium is depicted in Figure S1. As shown in Figure 1A, the highest levels of catalase activity were observed in the stationary and late stationary phases (with similar values of ∼7 µmol min−1 mg−1), being approximately 2.5-fold higher than those determined for the cultures in exponential growth.

Bottom Line: Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides.The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process.Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Division, Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT

Background: Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase).

Methodology/principal findings: Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H(2)O(2). A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation.

Conclusions: Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.

Show MeSH
Related in: MedlinePlus