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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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Binding of XC_2801//XC_3703 to the flhBA promoter is modulated by cyclic di-GMP.(A). The impact of various concentrations of cyclic di-GMP on the binding of XC_2801//XC_3703 to the flhBA promoter was assessed by the use of electromobility shift assay (EMSA). The DIG-labelled promoter fragment was incubated with purified XC_2801 and XC_3703 proteins and cyclic di-GMP. Concentrations of proteins and nucleotide are indicated above each well. (B) No binding of XC_2801 to the flhBA promoter region was seen in the presence of various concentrations of cyclic di-GMP as assessed by the use of EMSA. The DIG-labelled promoter fragment was incubated with purified XC_2801 and cyclic di-GMP. Concentrations of protein and nucleotide are indicated above each well.
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ppat-1004429-g005: Binding of XC_2801//XC_3703 to the flhBA promoter is modulated by cyclic di-GMP.(A). The impact of various concentrations of cyclic di-GMP on the binding of XC_2801//XC_3703 to the flhBA promoter was assessed by the use of electromobility shift assay (EMSA). The DIG-labelled promoter fragment was incubated with purified XC_2801 and XC_3703 proteins and cyclic di-GMP. Concentrations of proteins and nucleotide are indicated above each well. (B) No binding of XC_2801 to the flhBA promoter region was seen in the presence of various concentrations of cyclic di-GMP as assessed by the use of EMSA. The DIG-labelled promoter fragment was incubated with purified XC_2801 and cyclic di-GMP. Concentrations of protein and nucleotide are indicated above each well.

Mentions: As outlined above, XC_3703 preferentially binds cyclic di-GMP with a high affinity. This raised the question as to whether cyclic di-GMP could influence the binding of the XC_2801//XC_3703 complex to target promoters. To investigate this, the EMSA assay for the binding of XC_3703//XC_2801 to the flhBA promoter was repeated in the presence of added cyclic di-GMP at different concentrations. Addition of cyclic di-GMP at 1 µM had no apparent effect, but at 10 µM completely prevented DNA binding (Figure 5A). Other nucleotides did not have the same effect (Figure S4). XC_2801 alone did not bind to the flhBA promoter (as shown above) and addition of cyclic di-GMP did not alter this outcome (Figure 5B). Furthermore, XC_2801 appeared to have no affinity for the nucleotide as measured by ITC (Figure S5). These results suggest that cyclic di-GMP inhibits the interaction of the XC_3703//XC_2801 complex with DNA by binding to XC_3703, thus preventing the transcription of flhBA.


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)

Binding of XC_2801//XC_3703 to the flhBA promoter is modulated by cyclic di-GMP.(A). The impact of various concentrations of cyclic di-GMP on the binding of XC_2801//XC_3703 to the flhBA promoter was assessed by the use of electromobility shift assay (EMSA). The DIG-labelled promoter fragment was incubated with purified XC_2801 and XC_3703 proteins and cyclic di-GMP. Concentrations of proteins and nucleotide are indicated above each well. (B) No binding of XC_2801 to the flhBA promoter region was seen in the presence of various concentrations of cyclic di-GMP as assessed by the use of EMSA. The DIG-labelled promoter fragment was incubated with purified XC_2801 and cyclic di-GMP. Concentrations of protein and nucleotide are indicated above each well.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199771&req=5

ppat-1004429-g005: Binding of XC_2801//XC_3703 to the flhBA promoter is modulated by cyclic di-GMP.(A). The impact of various concentrations of cyclic di-GMP on the binding of XC_2801//XC_3703 to the flhBA promoter was assessed by the use of electromobility shift assay (EMSA). The DIG-labelled promoter fragment was incubated with purified XC_2801 and XC_3703 proteins and cyclic di-GMP. Concentrations of proteins and nucleotide are indicated above each well. (B) No binding of XC_2801 to the flhBA promoter region was seen in the presence of various concentrations of cyclic di-GMP as assessed by the use of EMSA. The DIG-labelled promoter fragment was incubated with purified XC_2801 and cyclic di-GMP. Concentrations of protein and nucleotide are indicated above each well.
Mentions: As outlined above, XC_3703 preferentially binds cyclic di-GMP with a high affinity. This raised the question as to whether cyclic di-GMP could influence the binding of the XC_2801//XC_3703 complex to target promoters. To investigate this, the EMSA assay for the binding of XC_3703//XC_2801 to the flhBA promoter was repeated in the presence of added cyclic di-GMP at different concentrations. Addition of cyclic di-GMP at 1 µM had no apparent effect, but at 10 µM completely prevented DNA binding (Figure 5A). Other nucleotides did not have the same effect (Figure S4). XC_2801 alone did not bind to the flhBA promoter (as shown above) and addition of cyclic di-GMP did not alter this outcome (Figure 5B). Furthermore, XC_2801 appeared to have no affinity for the nucleotide as measured by ITC (Figure S5). These results suggest that cyclic di-GMP inhibits the interaction of the XC_3703//XC_2801 complex with DNA by binding to XC_3703, thus preventing the transcription of flhBA.

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