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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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Proteome profiles of X. a. pv. citri biofilms and planktonic cultures. Proteins extracts (approximately 50 μg) from X. a. pv. citri biofilms (left gel) and planktonic cultures (right gel) were separated by 2D gel electrophoresis using 7-cm IPG strips pH range 4–7 and 12% SDS-PAGE. Proteome profiles of the cultures were compared using the Delta-2D (Decodon, Greifswald, Germany) analysis software.
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Figure 2: Proteome profiles of X. a. pv. citri biofilms and planktonic cultures. Proteins extracts (approximately 50 μg) from X. a. pv. citri biofilms (left gel) and planktonic cultures (right gel) were separated by 2D gel electrophoresis using 7-cm IPG strips pH range 4–7 and 12% SDS-PAGE. Proteome profiles of the cultures were compared using the Delta-2D (Decodon, Greifswald, Germany) analysis software.

Mentions: Since proteomics is a powerful method to obtain systems information on the physiology of bacterial cells, we aimed at analyzing and characterizing mature biofilms of X. a. pv. citri, and compare the proteome to that of planktonic X. a. pv. citri cells. Total proteins of these cultures were extracted and separated by two-dimensional gel electrophoresis (2-DE) (see “Methods” section). Protein extractions were performed from three independent biological samples, and two technical replicate gels for each cell type were compared. A total of 46 protein spots were differentially regulated (Figure 2), excised and processed for analysis by mass spectrometry. Forty-one spots were identified, corresponding to a total of 53 proteins (Additional file 1: Table S1), while five spots (spots 250, 382, 348, 352 and 357) remained unidentified, probably due to the very low protein concentration in these spots. Some protein spots were assigned to more than one protein, possibly because the proteins co-migrated as a result of having the same pI and molecular weight. This pattern of co-migration is not uncommon in proteomic studies and was reported previously [23,24]. The 31 up- and 22 down-regulated X. a. pv. citri biofilm proteins were classified into different categories based on their functions [25] (Additional file 1: Table S1). The protein spot displaying the strongest up-regulation was 50S ribosomal protein L4 (XAC0973; +5.1 fold; spot 79), followed by TonB-dependent receptor (XAC3489; +4.9 fold; spot 168), while the protein spot with the most pronounced down-regulation was an ATP synthase beta chain (XAC3649; -10.7 fold; spot 76). Here we focus on interpreting a subset (see Table 1) of the differentially expressed biofilm proteins.


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)

Proteome profiles of X. a. pv. citri biofilms and planktonic cultures. Proteins extracts (approximately 50 μg) from X. a. pv. citri biofilms (left gel) and planktonic cultures (right gel) were separated by 2D gel electrophoresis using 7-cm IPG strips pH range 4–7 and 12% SDS-PAGE. Proteome profiles of the cultures were compared using the Delta-2D (Decodon, Greifswald, Germany) analysis software.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Proteome profiles of X. a. pv. citri biofilms and planktonic cultures. Proteins extracts (approximately 50 μg) from X. a. pv. citri biofilms (left gel) and planktonic cultures (right gel) were separated by 2D gel electrophoresis using 7-cm IPG strips pH range 4–7 and 12% SDS-PAGE. Proteome profiles of the cultures were compared using the Delta-2D (Decodon, Greifswald, Germany) analysis software.
Mentions: Since proteomics is a powerful method to obtain systems information on the physiology of bacterial cells, we aimed at analyzing and characterizing mature biofilms of X. a. pv. citri, and compare the proteome to that of planktonic X. a. pv. citri cells. Total proteins of these cultures were extracted and separated by two-dimensional gel electrophoresis (2-DE) (see “Methods” section). Protein extractions were performed from three independent biological samples, and two technical replicate gels for each cell type were compared. A total of 46 protein spots were differentially regulated (Figure 2), excised and processed for analysis by mass spectrometry. Forty-one spots were identified, corresponding to a total of 53 proteins (Additional file 1: Table S1), while five spots (spots 250, 382, 348, 352 and 357) remained unidentified, probably due to the very low protein concentration in these spots. Some protein spots were assigned to more than one protein, possibly because the proteins co-migrated as a result of having the same pI and molecular weight. This pattern of co-migration is not uncommon in proteomic studies and was reported previously [23,24]. The 31 up- and 22 down-regulated X. a. pv. citri biofilm proteins were classified into different categories based on their functions [25] (Additional file 1: Table S1). The protein spot displaying the strongest up-regulation was 50S ribosomal protein L4 (XAC0973; +5.1 fold; spot 79), followed by TonB-dependent receptor (XAC3489; +4.9 fold; spot 168), while the protein spot with the most pronounced down-regulation was an ATP synthase beta chain (XAC3649; -10.7 fold; spot 76). Here we focus on interpreting a subset (see Table 1) of the differentially expressed biofilm proteins.

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