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Insights into xanthomonas axonopodis pv. citri biofilm through proteomics.

Zimaro T, Thomas L, Marondedze C, Garavaglia BS, Gehring C, Ottado J, Gottig N - BMC Microbiol. (2013)

Bottom Line: Among them, several porins and TonB-dependent receptor were differentially regulated in the biofilm compared to the planktonic cells, indicating that these proteins may serve in maintaining specific membrane-associated functions including signaling and cellular homeostasis.Firstly, proteins that are down-regulated in X. a. pv. citri biofilms are enriched for the gene ontology (GO) terms 'generation of precursor metabolites and energy' and secondly, the biofilm proteome mainly changes in 'outer membrane and receptor or transport'.We argue that the differentially expressed proteins have a critical role in maintaining a functional external structure as well as enabling appropriate flow of nutrients and signals specific to the biofilm lifestyle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas (IBR-CONICET), Ocampo y Esmeralda, Rosario, Santa Fe, Argentina.

ABSTRACT

Background: Xanthomonas axonopodis pv. citri (X. a. pv. citri) causes citrus canker that can result in defoliation and premature fruit drop with significant production losses worldwide. Biofilm formation is an important process in bacterial pathogens and several lines of evidence suggest that in X. a. pv. citri this process is a requirement to achieve maximal virulence since it has a major role in host interactions. In this study, proteomics was used to gain further insights into the functions of biofilms.

Results: In order to identify differentially expressed proteins, a comparative proteomic study using 2D difference gel electrophoresis was carried out on X. a. pv. citri mature biofilm and planktonic cells. The biofilm proteome showed major variations in the composition of outer membrane proteins and receptor or transport proteins. Among them, several porins and TonB-dependent receptor were differentially regulated in the biofilm compared to the planktonic cells, indicating that these proteins may serve in maintaining specific membrane-associated functions including signaling and cellular homeostasis. In biofilms, UDP-glucose dehydrogenase with a major role in exopolysaccharide production and the non-fimbrial adhesin YapH involved in adherence were over-expressed, while a polynucleotide phosphorylase that was demonstrated to negatively control biofilm formation in E. coli was down-regulated. In addition, several proteins involved in protein synthesis, folding and stabilization were up-regulated in biofilms. Interestingly, some proteins related to energy production, such as ATP-synthase were down-regulated in biofilms. Moreover, a number of enzymes of the tricarboxylic acid cycle were differentially expressed. In addition, X. a. pv. citri biofilms also showed down-regulation of several antioxidant enzymes. The respective gene expression patterns of several identified proteins in both X. a. pv. citri mature biofilm and planktonic cells were evaluated by quantitative real-time PCR and shown to consistently correlate with those deduced from the proteomic study.

Conclusions: Differentially expressed proteins are enriched in functional categories. Firstly, proteins that are down-regulated in X. a. pv. citri biofilms are enriched for the gene ontology (GO) terms 'generation of precursor metabolites and energy' and secondly, the biofilm proteome mainly changes in 'outer membrane and receptor or transport'. We argue that the differentially expressed proteins have a critical role in maintaining a functional external structure as well as enabling appropriate flow of nutrients and signals specific to the biofilm lifestyle.

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Confocal laser scanning microscopy analysisX. a.pv.citri in vitrobiofilms. Representative photographs of laser scanning confocal analysis of GFP-expressing X. a. pv. citri cells cultured in static liquid XVM2 in 24-well PVC plates for one, three and seven days (upper panels). Serial images were taken at 0.5 μm distances (z-stack). White arrows point to cell aggregations and dotted white arrows point to network channels. Scale bars: 30 μm. For a better visualization, the lower panels are images of biofilm channels and cell aggregates at 7 days.
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Figure 1: Confocal laser scanning microscopy analysisX. a.pv.citri in vitrobiofilms. Representative photographs of laser scanning confocal analysis of GFP-expressing X. a. pv. citri cells cultured in static liquid XVM2 in 24-well PVC plates for one, three and seven days (upper panels). Serial images were taken at 0.5 μm distances (z-stack). White arrows point to cell aggregations and dotted white arrows point to network channels. Scale bars: 30 μm. For a better visualization, the lower panels are images of biofilm channels and cell aggregates at 7 days.

Mentions: Biofilm formation generally requires a number of different processes including the initial surface attachment of cells, cell multiplication to form micro-colonies and maturation of the biofilm [20]. For a better understanding of the dynamics of this process in X. a. pv. citri, biofilm structure of a GFP-expressing X. a. pv. citri strain (Xac-GFP) was observed at different growth stages by confocal laser scanning microscopy. To this end, Xac-GFP was cultured in static liquid XVM2 medium, a minimal medium that mimics the nutritional conditions found in plant tissues [21]. As previously described, biofilms are important for X. a. pv. citri virulence, and thus XVM2 medium was used to analyze bacterial biofilm formation in a plant-like environment. After one day of growth, some cells began to attach to the surface of the PVC plate wells, however, the majority of cells remained dispersed in the culture medium (Figure 1). After three days of growth, cells initiated accumulation and formation of a biofilm (Figure 1), and after seven days, Xac-GFP cells formed a distinctly structured and dense biofilm consisting of large cell aggregations separated by a network of large channels (Figure 1) that ensured appropriate micronutrient and oxygen fluxes [22]. We also evaluated the population size of these biofilms and observed that at day seven of growth the biofilms reached a maximum population size of 1 x 109 cfu/ml. In a planktonic culture in XVM2 medium, a similar maximal population size is reached in early stationary phase. Therefore, these two conditions of growth were used to identify differentially expressed proteins between the two lifestyles at their respective maximum population sizes and prior to the occurrence of noticeable cell death.


Insights into xanthomonas axonopodis pv. citri biofilm through proteomics.

Zimaro T, Thomas L, Marondedze C, Garavaglia BS, Gehring C, Ottado J, Gottig N - BMC Microbiol. (2013)

Confocal laser scanning microscopy analysisX. a.pv.citri in vitrobiofilms. Representative photographs of laser scanning confocal analysis of GFP-expressing X. a. pv. citri cells cultured in static liquid XVM2 in 24-well PVC plates for one, three and seven days (upper panels). Serial images were taken at 0.5 μm distances (z-stack). White arrows point to cell aggregations and dotted white arrows point to network channels. Scale bars: 30 μm. For a better visualization, the lower panels are images of biofilm channels and cell aggregates at 7 days.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750573&req=5

Figure 1: Confocal laser scanning microscopy analysisX. a.pv.citri in vitrobiofilms. Representative photographs of laser scanning confocal analysis of GFP-expressing X. a. pv. citri cells cultured in static liquid XVM2 in 24-well PVC plates for one, three and seven days (upper panels). Serial images were taken at 0.5 μm distances (z-stack). White arrows point to cell aggregations and dotted white arrows point to network channels. Scale bars: 30 μm. For a better visualization, the lower panels are images of biofilm channels and cell aggregates at 7 days.
Mentions: Biofilm formation generally requires a number of different processes including the initial surface attachment of cells, cell multiplication to form micro-colonies and maturation of the biofilm [20]. For a better understanding of the dynamics of this process in X. a. pv. citri, biofilm structure of a GFP-expressing X. a. pv. citri strain (Xac-GFP) was observed at different growth stages by confocal laser scanning microscopy. To this end, Xac-GFP was cultured in static liquid XVM2 medium, a minimal medium that mimics the nutritional conditions found in plant tissues [21]. As previously described, biofilms are important for X. a. pv. citri virulence, and thus XVM2 medium was used to analyze bacterial biofilm formation in a plant-like environment. After one day of growth, some cells began to attach to the surface of the PVC plate wells, however, the majority of cells remained dispersed in the culture medium (Figure 1). After three days of growth, cells initiated accumulation and formation of a biofilm (Figure 1), and after seven days, Xac-GFP cells formed a distinctly structured and dense biofilm consisting of large cell aggregations separated by a network of large channels (Figure 1) that ensured appropriate micronutrient and oxygen fluxes [22]. We also evaluated the population size of these biofilms and observed that at day seven of growth the biofilms reached a maximum population size of 1 x 109 cfu/ml. In a planktonic culture in XVM2 medium, a similar maximal population size is reached in early stationary phase. Therefore, these two conditions of growth were used to identify differentially expressed proteins between the two lifestyles at their respective maximum population sizes and prior to the occurrence of noticeable cell death.

Bottom Line: Among them, several porins and TonB-dependent receptor were differentially regulated in the biofilm compared to the planktonic cells, indicating that these proteins may serve in maintaining specific membrane-associated functions including signaling and cellular homeostasis.Firstly, proteins that are down-regulated in X. a. pv. citri biofilms are enriched for the gene ontology (GO) terms 'generation of precursor metabolites and energy' and secondly, the biofilm proteome mainly changes in 'outer membrane and receptor or transport'.We argue that the differentially expressed proteins have a critical role in maintaining a functional external structure as well as enabling appropriate flow of nutrients and signals specific to the biofilm lifestyle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas (IBR-CONICET), Ocampo y Esmeralda, Rosario, Santa Fe, Argentina.

ABSTRACT

Background: Xanthomonas axonopodis pv. citri (X. a. pv. citri) causes citrus canker that can result in defoliation and premature fruit drop with significant production losses worldwide. Biofilm formation is an important process in bacterial pathogens and several lines of evidence suggest that in X. a. pv. citri this process is a requirement to achieve maximal virulence since it has a major role in host interactions. In this study, proteomics was used to gain further insights into the functions of biofilms.

Results: In order to identify differentially expressed proteins, a comparative proteomic study using 2D difference gel electrophoresis was carried out on X. a. pv. citri mature biofilm and planktonic cells. The biofilm proteome showed major variations in the composition of outer membrane proteins and receptor or transport proteins. Among them, several porins and TonB-dependent receptor were differentially regulated in the biofilm compared to the planktonic cells, indicating that these proteins may serve in maintaining specific membrane-associated functions including signaling and cellular homeostasis. In biofilms, UDP-glucose dehydrogenase with a major role in exopolysaccharide production and the non-fimbrial adhesin YapH involved in adherence were over-expressed, while a polynucleotide phosphorylase that was demonstrated to negatively control biofilm formation in E. coli was down-regulated. In addition, several proteins involved in protein synthesis, folding and stabilization were up-regulated in biofilms. Interestingly, some proteins related to energy production, such as ATP-synthase were down-regulated in biofilms. Moreover, a number of enzymes of the tricarboxylic acid cycle were differentially expressed. In addition, X. a. pv. citri biofilms also showed down-regulation of several antioxidant enzymes. The respective gene expression patterns of several identified proteins in both X. a. pv. citri mature biofilm and planktonic cells were evaluated by quantitative real-time PCR and shown to consistently correlate with those deduced from the proteomic study.

Conclusions: Differentially expressed proteins are enriched in functional categories. Firstly, proteins that are down-regulated in X. a. pv. citri biofilms are enriched for the gene ontology (GO) terms 'generation of precursor metabolites and energy' and secondly, the biofilm proteome mainly changes in 'outer membrane and receptor or transport'. We argue that the differentially expressed proteins have a critical role in maintaining a functional external structure as well as enabling appropriate flow of nutrients and signals specific to the biofilm lifestyle.

Show MeSH
Related in: MedlinePlus