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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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Analysis of the expression of selected genes encoding differentially expressed proteins. A significant difference in expression was detected by qRT-PCR between planktonic and biofilm conditions for selected genes confirming their expression during X. a. pv. citri biofilm formation. Black bars indicate the expression levels of X. a. pv. citri transcripts in biofilm compared to a reference planktonic growth (white bars). As a reference gene, a fragment of 16S rRNA was amplified. Values represent the means of four independent experiments. Error bars indicate standard deviations. Data were statistically analyzed using one-way ANOVA (p < 0.05) and Student t-test (p < 0.05).
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Figure 4: Analysis of the expression of selected genes encoding differentially expressed proteins. A significant difference in expression was detected by qRT-PCR between planktonic and biofilm conditions for selected genes confirming their expression during X. a. pv. citri biofilm formation. Black bars indicate the expression levels of X. a. pv. citri transcripts in biofilm compared to a reference planktonic growth (white bars). As a reference gene, a fragment of 16S rRNA was amplified. Values represent the means of four independent experiments. Error bars indicate standard deviations. Data were statistically analyzed using one-way ANOVA (p < 0.05) and Student t-test (p < 0.05).

Mentions: We selected some of these genes for further validation by quantitative real-time PCR (qRT-PCR). Total RNA was extracted from X. a. pv. citri mature biofilms and from planktonic cells, both grown as for the proteomic study. Bacterial cDNA was obtained from 1 μg of total RNA in both growth conditions. The assay was performed with specific primers for the following X. a. pv. citri genes: XAC3581 (UDP-glucose dehydrogenase), XAC0973 (50S ribosomal protein L4), XAC0957 (EfTu), XAC2504 (RpfN), XAC3489 (TonB-dependent receptor), XAC2151 (YapH), XAC3664 (OmpW) and XAC1522 (DnaK). We noted that the changes in transcript levels of theses genes mirrored the changes observed in the proteomics analysis (p < 0.05) (Figure 4).


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)

Analysis of the expression of selected genes encoding differentially expressed proteins. A significant difference in expression was detected by qRT-PCR between planktonic and biofilm conditions for selected genes confirming their expression during X. a. pv. citri biofilm formation. Black bars indicate the expression levels of X. a. pv. citri transcripts in biofilm compared to a reference planktonic growth (white bars). As a reference gene, a fragment of 16S rRNA was amplified. Values represent the means of four independent experiments. Error bars indicate standard deviations. Data were statistically analyzed using one-way ANOVA (p < 0.05) and Student t-test (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Analysis of the expression of selected genes encoding differentially expressed proteins. A significant difference in expression was detected by qRT-PCR between planktonic and biofilm conditions for selected genes confirming their expression during X. a. pv. citri biofilm formation. Black bars indicate the expression levels of X. a. pv. citri transcripts in biofilm compared to a reference planktonic growth (white bars). As a reference gene, a fragment of 16S rRNA was amplified. Values represent the means of four independent experiments. Error bars indicate standard deviations. Data were statistically analyzed using one-way ANOVA (p < 0.05) and Student t-test (p < 0.05).
Mentions: We selected some of these genes for further validation by quantitative real-time PCR (qRT-PCR). Total RNA was extracted from X. a. pv. citri mature biofilms and from planktonic cells, both grown as for the proteomic study. Bacterial cDNA was obtained from 1 μg of total RNA in both growth conditions. The assay was performed with specific primers for the following X. a. pv. citri genes: XAC3581 (UDP-glucose dehydrogenase), XAC0973 (50S ribosomal protein L4), XAC0957 (EfTu), XAC2504 (RpfN), XAC3489 (TonB-dependent receptor), XAC2151 (YapH), XAC3664 (OmpW) and XAC1522 (DnaK). We noted that the changes in transcript levels of theses genes mirrored the changes observed in the proteomics analysis (p < 0.05) (Figure 4).

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