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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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Identification of Xanthomonas campestris XC_3703 as a cyclic di-GMP- binding protein.(A) SDS-Polyacrylamide gel separation of Xcc proteins retained on cyclic di-GMP-coupled beads. Lanes from left: Molecular weight markers; Total crude extract of Xcc soluble proteins; Proteins retained by the cyclic di-GMP-coupled beads. The protein band indicated by the asterisk was identified by mass spectrometry of tryptic peptides as XC_3703. (B) Isothermal titration calorimetry analysis of binding of cyclic di-GMP and cyclic GMP by XC_3703. Lower panels show the integrated data obtained from the raw data, after subtracting the heat of dilution. Injection of cyclic di-GMP yielded an endothermic binding isotherm. Experimental data were fitted using the MicroCal ORIGIN version 7.0 software and a Kd for cyclic di-GMP was calculated as 2 µM. No binding of cyclic GMP to XC_3703 was detected.
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ppat-1004429-g001: Identification of Xanthomonas campestris XC_3703 as a cyclic di-GMP- binding protein.(A) SDS-Polyacrylamide gel separation of Xcc proteins retained on cyclic di-GMP-coupled beads. Lanes from left: Molecular weight markers; Total crude extract of Xcc soluble proteins; Proteins retained by the cyclic di-GMP-coupled beads. The protein band indicated by the asterisk was identified by mass spectrometry of tryptic peptides as XC_3703. (B) Isothermal titration calorimetry analysis of binding of cyclic di-GMP and cyclic GMP by XC_3703. Lower panels show the integrated data obtained from the raw data, after subtracting the heat of dilution. Injection of cyclic di-GMP yielded an endothermic binding isotherm. Experimental data were fitted using the MicroCal ORIGIN version 7.0 software and a Kd for cyclic di-GMP was calculated as 2 µM. No binding of cyclic GMP to XC_3703 was detected.

Mentions: To identify cyclic di-GMP receptor proteins, we performed an affinity pull-down assay using cyclic di-GMP–coupled magnetic beads and soluble protein extracts derived from the Xcc wild-type strain 8004. The selectively bound proteins were separated by SDS-polyacrylamide gel electrophoresis (Figure 1A) and were identified by peptide mass fingerprinting. Overall, 7 putative cyclic di-GMP binding proteins were identified from three cyclic di-GMP pull down experiments on Xcc 8004 lysates (Table S1). Three of these proteins were previously characterized cyclic di-GMP binding proteins from Xcc: the two PilZ domain-containing proteins XC_0965 and XC_3221 and the transcriptional regulator Clp, which is XC_0486. In addition, the analysis identified XC_1036, a protein containing a GGDEF domain with a predicted I-site (allosteric) cyclic di-GMP binding motif. Of particular interest to the work here was XC_3703 (MASCOT score 1156) a member of the highly conserved YajQ family of bacterial proteins. XC_3703 is related to YajQ of Escherichia coli (BLASTP E value is 10−20), a protein of unknown function that has motifs characteristic of nucleotide-binding proteins (Figure S1). YajQ family proteins are encoded by many bacterial genomes, to include both Gram-negative and Gram-positive bacteria. An amino acid sequence alignment of XC_3703 with sequences from a selected range of organisms is shown in Figure S1.


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)

Identification of Xanthomonas campestris XC_3703 as a cyclic di-GMP- binding protein.(A) SDS-Polyacrylamide gel separation of Xcc proteins retained on cyclic di-GMP-coupled beads. Lanes from left: Molecular weight markers; Total crude extract of Xcc soluble proteins; Proteins retained by the cyclic di-GMP-coupled beads. The protein band indicated by the asterisk was identified by mass spectrometry of tryptic peptides as XC_3703. (B) Isothermal titration calorimetry analysis of binding of cyclic di-GMP and cyclic GMP by XC_3703. Lower panels show the integrated data obtained from the raw data, after subtracting the heat of dilution. Injection of cyclic di-GMP yielded an endothermic binding isotherm. Experimental data were fitted using the MicroCal ORIGIN version 7.0 software and a Kd for cyclic di-GMP was calculated as 2 µM. No binding of cyclic GMP to XC_3703 was detected.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4199771&req=5

ppat-1004429-g001: Identification of Xanthomonas campestris XC_3703 as a cyclic di-GMP- binding protein.(A) SDS-Polyacrylamide gel separation of Xcc proteins retained on cyclic di-GMP-coupled beads. Lanes from left: Molecular weight markers; Total crude extract of Xcc soluble proteins; Proteins retained by the cyclic di-GMP-coupled beads. The protein band indicated by the asterisk was identified by mass spectrometry of tryptic peptides as XC_3703. (B) Isothermal titration calorimetry analysis of binding of cyclic di-GMP and cyclic GMP by XC_3703. Lower panels show the integrated data obtained from the raw data, after subtracting the heat of dilution. Injection of cyclic di-GMP yielded an endothermic binding isotherm. Experimental data were fitted using the MicroCal ORIGIN version 7.0 software and a Kd for cyclic di-GMP was calculated as 2 µM. No binding of cyclic GMP to XC_3703 was detected.
Mentions: To identify cyclic di-GMP receptor proteins, we performed an affinity pull-down assay using cyclic di-GMP–coupled magnetic beads and soluble protein extracts derived from the Xcc wild-type strain 8004. The selectively bound proteins were separated by SDS-polyacrylamide gel electrophoresis (Figure 1A) and were identified by peptide mass fingerprinting. Overall, 7 putative cyclic di-GMP binding proteins were identified from three cyclic di-GMP pull down experiments on Xcc 8004 lysates (Table S1). Three of these proteins were previously characterized cyclic di-GMP binding proteins from Xcc: the two PilZ domain-containing proteins XC_0965 and XC_3221 and the transcriptional regulator Clp, which is XC_0486. In addition, the analysis identified XC_1036, a protein containing a GGDEF domain with a predicted I-site (allosteric) cyclic di-GMP binding motif. Of particular interest to the work here was XC_3703 (MASCOT score 1156) a member of the highly conserved YajQ family of bacterial proteins. XC_3703 is related to YajQ of Escherichia coli (BLASTP E value is 10−20), a protein of unknown function that has motifs characteristic of nucleotide-binding proteins (Figure S1). YajQ family proteins are encoded by many bacterial genomes, to include both Gram-negative and Gram-positive bacteria. An amino acid sequence alignment of XC_3703 with sequences from a selected range of organisms is shown in Figure S1.

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