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Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence.

An SQ, Caly DL, McCarthy Y, Murdoch SL, Ward J, Febrer M, Dow JM, Ryan RP - PLoS Pathog. (2014)

Bottom Line: Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation.Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence.The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, United Kingdom.

ABSTRACT
Bis-(3',5') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d)∼2 µM). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

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Transcriptome and phenotypic characterisation of an XC_3703 deletion mutant reveals roles in biofilm formation and virulence in Xcc.(A) The virulence of the Xcc strains was tested by measurement of the lesion length after bacteria were introduced into the vascular system of Chinese Radish by leaf clipping. (i) Representative virulence assays for (from left to right) Xcc wild-type (8004), XC_3703 deletion mutant and negative control (H2O). (ii) Re-integration of the gene encoding XC_3703 into a neutral site in the mutant chromosome (indicated as XC_3703 (cXC_3703)) restored the reduced virulence of the mutant towards wild-type. Values given are the mean and standard deviation of triplicate measurements each comprising of 30 leaves. Asterisks indicate values that are significantly different from the wild-type (p<0.01, two-tailed Student's t-test). (B) Deletion of XC_3703 also led to decreased biofilm and cell adhesion on a glass surface when assessed by crystal violet staining. Biofilm biomass is measured as a ratio of absorbance at 550 and 600 nm. Re-integration of the gene encoding XC_3703 into the chromosome (indicated as XC_3703 (c3703)) restores the phenotypes towards wild-type. Values given are the mean and standard deviation of triplicate measurements. Asterisks indicate values that are significantly different from the wild-type (p<0.01, two-tailed Student's t-test). (C) Differential expression of selected genes implicated in virulence or biofilm formation in the XC_3703 deletion mutant and wild-type as determined by RNA-Seq (dark grey) and qRT–PCR (light grey). Mutation of XC_3703 affected transcript levels of XC_0026 (cellulase), XC_0027 (endoglucanase), XC_1058 (pilin), XC_1165 (TonB receptor) XC_1732 (glycosyltransferase), XC_2013 (MASE1 domain-containing protein), XC_2724 (methyltransferase), and XC_3591 (pectate lyase). The qRT–PCR data were normalised to 16S rRNA and is presented as the fold change with respect to the wild-type for each gene. Data (means ± standard deviation) are representative of four independent biological experiments. The complete RNA-Seq data set is detailed in Table S4.
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ppat-1004429-g002: Transcriptome and phenotypic characterisation of an XC_3703 deletion mutant reveals roles in biofilm formation and virulence in Xcc.(A) The virulence of the Xcc strains was tested by measurement of the lesion length after bacteria were introduced into the vascular system of Chinese Radish by leaf clipping. (i) Representative virulence assays for (from left to right) Xcc wild-type (8004), XC_3703 deletion mutant and negative control (H2O). (ii) Re-integration of the gene encoding XC_3703 into a neutral site in the mutant chromosome (indicated as XC_3703 (cXC_3703)) restored the reduced virulence of the mutant towards wild-type. Values given are the mean and standard deviation of triplicate measurements each comprising of 30 leaves. Asterisks indicate values that are significantly different from the wild-type (p<0.01, two-tailed Student's t-test). (B) Deletion of XC_3703 also led to decreased biofilm and cell adhesion on a glass surface when assessed by crystal violet staining. Biofilm biomass is measured as a ratio of absorbance at 550 and 600 nm. Re-integration of the gene encoding XC_3703 into the chromosome (indicated as XC_3703 (c3703)) restores the phenotypes towards wild-type. Values given are the mean and standard deviation of triplicate measurements. Asterisks indicate values that are significantly different from the wild-type (p<0.01, two-tailed Student's t-test). (C) Differential expression of selected genes implicated in virulence or biofilm formation in the XC_3703 deletion mutant and wild-type as determined by RNA-Seq (dark grey) and qRT–PCR (light grey). Mutation of XC_3703 affected transcript levels of XC_0026 (cellulase), XC_0027 (endoglucanase), XC_1058 (pilin), XC_1165 (TonB receptor) XC_1732 (glycosyltransferase), XC_2013 (MASE1 domain-containing protein), XC_2724 (methyltransferase), and XC_3591 (pectate lyase). The qRT–PCR data were normalised to 16S rRNA and is presented as the fold change with respect to the wild-type for each gene. Data (means ± standard deviation) are representative of four independent biological experiments. The complete RNA-Seq data set is detailed in Table S4.

Mentions: Cyclic di-GMP is known to have a broad regulatory action in Xcc that includes influences on the synthesis of virulence factors and the formation of biofilms [7], [8]. The possible role of XC_3703 in these diverse regulatory actions was investigated by comparative phenotypic and transcriptomic analyses of the wild-type and XC_3703 deletion mutant. The XC_3703 mutant showed reduced virulence to Chinese Radish (Figure 2A) when tested by leaf clipping (see Materials and Methods) and reduced biofilm biomass when grown in complex medium (Figure 2B). Complementation restored these phenotypes towards wild-type.


Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence.

An SQ, Caly DL, McCarthy Y, Murdoch SL, Ward J, Febrer M, Dow JM, Ryan RP - PLoS Pathog. (2014)

Transcriptome and phenotypic characterisation of an XC_3703 deletion mutant reveals roles in biofilm formation and virulence in Xcc.(A) The virulence of the Xcc strains was tested by measurement of the lesion length after bacteria were introduced into the vascular system of Chinese Radish by leaf clipping. (i) Representative virulence assays for (from left to right) Xcc wild-type (8004), XC_3703 deletion mutant and negative control (H2O). (ii) Re-integration of the gene encoding XC_3703 into a neutral site in the mutant chromosome (indicated as XC_3703 (cXC_3703)) restored the reduced virulence of the mutant towards wild-type. Values given are the mean and standard deviation of triplicate measurements each comprising of 30 leaves. Asterisks indicate values that are significantly different from the wild-type (p<0.01, two-tailed Student's t-test). (B) Deletion of XC_3703 also led to decreased biofilm and cell adhesion on a glass surface when assessed by crystal violet staining. Biofilm biomass is measured as a ratio of absorbance at 550 and 600 nm. Re-integration of the gene encoding XC_3703 into the chromosome (indicated as XC_3703 (c3703)) restores the phenotypes towards wild-type. Values given are the mean and standard deviation of triplicate measurements. Asterisks indicate values that are significantly different from the wild-type (p<0.01, two-tailed Student's t-test). (C) Differential expression of selected genes implicated in virulence or biofilm formation in the XC_3703 deletion mutant and wild-type as determined by RNA-Seq (dark grey) and qRT–PCR (light grey). Mutation of XC_3703 affected transcript levels of XC_0026 (cellulase), XC_0027 (endoglucanase), XC_1058 (pilin), XC_1165 (TonB receptor) XC_1732 (glycosyltransferase), XC_2013 (MASE1 domain-containing protein), XC_2724 (methyltransferase), and XC_3591 (pectate lyase). The qRT–PCR data were normalised to 16S rRNA and is presented as the fold change with respect to the wild-type for each gene. Data (means ± standard deviation) are representative of four independent biological experiments. The complete RNA-Seq data set is detailed in Table S4.
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Related In: Results  -  Collection

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ppat-1004429-g002: Transcriptome and phenotypic characterisation of an XC_3703 deletion mutant reveals roles in biofilm formation and virulence in Xcc.(A) The virulence of the Xcc strains was tested by measurement of the lesion length after bacteria were introduced into the vascular system of Chinese Radish by leaf clipping. (i) Representative virulence assays for (from left to right) Xcc wild-type (8004), XC_3703 deletion mutant and negative control (H2O). (ii) Re-integration of the gene encoding XC_3703 into a neutral site in the mutant chromosome (indicated as XC_3703 (cXC_3703)) restored the reduced virulence of the mutant towards wild-type. Values given are the mean and standard deviation of triplicate measurements each comprising of 30 leaves. Asterisks indicate values that are significantly different from the wild-type (p<0.01, two-tailed Student's t-test). (B) Deletion of XC_3703 also led to decreased biofilm and cell adhesion on a glass surface when assessed by crystal violet staining. Biofilm biomass is measured as a ratio of absorbance at 550 and 600 nm. Re-integration of the gene encoding XC_3703 into the chromosome (indicated as XC_3703 (c3703)) restores the phenotypes towards wild-type. Values given are the mean and standard deviation of triplicate measurements. Asterisks indicate values that are significantly different from the wild-type (p<0.01, two-tailed Student's t-test). (C) Differential expression of selected genes implicated in virulence or biofilm formation in the XC_3703 deletion mutant and wild-type as determined by RNA-Seq (dark grey) and qRT–PCR (light grey). Mutation of XC_3703 affected transcript levels of XC_0026 (cellulase), XC_0027 (endoglucanase), XC_1058 (pilin), XC_1165 (TonB receptor) XC_1732 (glycosyltransferase), XC_2013 (MASE1 domain-containing protein), XC_2724 (methyltransferase), and XC_3591 (pectate lyase). The qRT–PCR data were normalised to 16S rRNA and is presented as the fold change with respect to the wild-type for each gene. Data (means ± standard deviation) are representative of four independent biological experiments. The complete RNA-Seq data set is detailed in Table S4.
Mentions: Cyclic di-GMP is known to have a broad regulatory action in Xcc that includes influences on the synthesis of virulence factors and the formation of biofilms [7], [8]. The possible role of XC_3703 in these diverse regulatory actions was investigated by comparative phenotypic and transcriptomic analyses of the wild-type and XC_3703 deletion mutant. The XC_3703 mutant showed reduced virulence to Chinese Radish (Figure 2A) when tested by leaf clipping (see Materials and Methods) and reduced biofilm biomass when grown in complex medium (Figure 2B). Complementation restored these phenotypes towards wild-type.

Bottom Line: Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation.Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence.The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, United Kingdom.

ABSTRACT
Bis-(3',5') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d)∼2 µM). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

Show MeSH
Related in: MedlinePlus