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A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization.

Kraiselburd I, Alet AI, Tondo ML, Petrocelli S, Daurelio LD, Monzón J, Ruiz OA, Losi A, Orellano EG - PLoS ONE (2012)

Bottom Line: We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation.Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type.Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process.

View Article: PubMed Central - PubMed

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

ABSTRACT
Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.

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Biofilm formation by X. axonopodis pv. citri strains.(A) Green fluorescent protein (GFP)-labeled bacteria were grown on chambered cover slides and visualized under confocal laser scanning microscopy (CLSM) after two (i) and five (ii) days of bacterial growth. For each time period, the upper panels show the biofilms developed at the bottom of the chambered cover slides with a magnification of 400X, and the bottom panels show a 2X zoom of the regions marked in the previous panels. Scale bars, 50 µm. In this experiment Δlov-plov corresponds to the Δlov strain complemented with pBBR-plov2 (Δlov-plov’). (B) X. axonopodis pv. citri strains were statically grown on glass tubes for two weeks at 28°C. Biofilms were observed on the air-liquid interface. In each case, bacteria were grown under light and dark conditions.
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pone-0038226-g006: Biofilm formation by X. axonopodis pv. citri strains.(A) Green fluorescent protein (GFP)-labeled bacteria were grown on chambered cover slides and visualized under confocal laser scanning microscopy (CLSM) after two (i) and five (ii) days of bacterial growth. For each time period, the upper panels show the biofilms developed at the bottom of the chambered cover slides with a magnification of 400X, and the bottom panels show a 2X zoom of the regions marked in the previous panels. Scale bars, 50 µm. In this experiment Δlov-plov corresponds to the Δlov strain complemented with pBBR-plov2 (Δlov-plov’). (B) X. axonopodis pv. citri strains were statically grown on glass tubes for two weeks at 28°C. Biofilms were observed on the air-liquid interface. In each case, bacteria were grown under light and dark conditions.

Mentions: Biofilm formation is associated with the production of exopolysaccharide and is important for the virulence of some pathogenic bacteria because it can promote their survival against the action of antimicrobial compounds derived from host organisms [46]. We used confocal laser scanning microscopy (CLSM) to analyze the morphology of bacterial biofilms developed by green fluorescent protein (GFP)-labeled strains of X. axonopodis pv. citri on chambered cover glass slides over different periods of time. The X. axonopodis pv. citri WT, Δlov and complemented strains were able to develop complex structures consisting of clustered bacteria in close contact with each other. However, as shown in Figure 6A, we observed different patterns of bacterial aggregation between the different strains. After two days of incubation under light conditions, the three strains generated isolated microcolonies in which bacteria were densely packed and organized, maintaining lateral interactions with each other, however these structures were larger for the WT and complemented strains. After five days, the WT and complemented strains generated aggregates that extended over the entire surface, while the microcolonies developed by the Δlov strain remained disperse. In dark conditions, after two days of incubation, the pattern of cell aggregation of the WT and complemented strains was similar to that observed for Δlov under light conditions. However, the Δlov strain generated even smaller structures that were considerably less organized than those generated under light conditions. After five days, cell aggregation patterns were rather similar for the three strains with the formation of a cellular matrix that filled the entire surface of the chamber bottom.


A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization.

Kraiselburd I, Alet AI, Tondo ML, Petrocelli S, Daurelio LD, Monzón J, Ruiz OA, Losi A, Orellano EG - PLoS ONE (2012)

Biofilm formation by X. axonopodis pv. citri strains.(A) Green fluorescent protein (GFP)-labeled bacteria were grown on chambered cover slides and visualized under confocal laser scanning microscopy (CLSM) after two (i) and five (ii) days of bacterial growth. For each time period, the upper panels show the biofilms developed at the bottom of the chambered cover slides with a magnification of 400X, and the bottom panels show a 2X zoom of the regions marked in the previous panels. Scale bars, 50 µm. In this experiment Δlov-plov corresponds to the Δlov strain complemented with pBBR-plov2 (Δlov-plov’). (B) X. axonopodis pv. citri strains were statically grown on glass tubes for two weeks at 28°C. Biofilms were observed on the air-liquid interface. In each case, bacteria were grown under light and dark conditions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366940&req=5

pone-0038226-g006: Biofilm formation by X. axonopodis pv. citri strains.(A) Green fluorescent protein (GFP)-labeled bacteria were grown on chambered cover slides and visualized under confocal laser scanning microscopy (CLSM) after two (i) and five (ii) days of bacterial growth. For each time period, the upper panels show the biofilms developed at the bottom of the chambered cover slides with a magnification of 400X, and the bottom panels show a 2X zoom of the regions marked in the previous panels. Scale bars, 50 µm. In this experiment Δlov-plov corresponds to the Δlov strain complemented with pBBR-plov2 (Δlov-plov’). (B) X. axonopodis pv. citri strains were statically grown on glass tubes for two weeks at 28°C. Biofilms were observed on the air-liquid interface. In each case, bacteria were grown under light and dark conditions.
Mentions: Biofilm formation is associated with the production of exopolysaccharide and is important for the virulence of some pathogenic bacteria because it can promote their survival against the action of antimicrobial compounds derived from host organisms [46]. We used confocal laser scanning microscopy (CLSM) to analyze the morphology of bacterial biofilms developed by green fluorescent protein (GFP)-labeled strains of X. axonopodis pv. citri on chambered cover glass slides over different periods of time. The X. axonopodis pv. citri WT, Δlov and complemented strains were able to develop complex structures consisting of clustered bacteria in close contact with each other. However, as shown in Figure 6A, we observed different patterns of bacterial aggregation between the different strains. After two days of incubation under light conditions, the three strains generated isolated microcolonies in which bacteria were densely packed and organized, maintaining lateral interactions with each other, however these structures were larger for the WT and complemented strains. After five days, the WT and complemented strains generated aggregates that extended over the entire surface, while the microcolonies developed by the Δlov strain remained disperse. In dark conditions, after two days of incubation, the pattern of cell aggregation of the WT and complemented strains was similar to that observed for Δlov under light conditions. However, the Δlov strain generated even smaller structures that were considerably less organized than those generated under light conditions. After five days, cell aggregation patterns were rather similar for the three strains with the formation of a cellular matrix that filled the entire surface of the chamber bottom.

Bottom Line: We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation.Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type.Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process.

View Article: PubMed Central - PubMed

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

ABSTRACT
Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.

Show MeSH
Related in: MedlinePlus