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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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XC_2801 binds to specific promoters and only in the presence of XC_3703.(A) Promoter-fusion analysis of the upstream regions flhB, aaeB, flgG and fliL revealed significantly decrease in promoter activity in the XC_2801 mutant strain. Values given are the mean and standard deviation of triplicate measurements. Values for promoter activity in the XC_2801 mutant strain are significantly different from the wild-type at p<0.01 by two-tailed Student's t-test. The decrease in promoter activity was consistently observed in three independent experiments each consisting of three biological replicates. (B) Binding of XC_2801 to the promoter regions of flhB, aaeB, flgG and fliL in the absence (i) or presence (ii) of XC_3703 assessed by the use of electromobility shift assay (EMSA). Each lane contained 1.5 nM DIG-labelled Probe DNA, and in addition nanomolar concentrations of purified His6-tag XC_2801 protein as indicated below each well. (C) Bioinformatics reveals a putative LysR family regulator-binding site (TCCCGAATCCCCGA) located 80-67 nucleotides upstream of the predicted translational start site of flhB. (D) Although a shift was observed with the flhB upstream (promoter) region, no shift was observed with an overlapping DNA fragment that lacks the putative XC_2801 binding site (indicated as truncated flhB). The nanomolar concentration of His-tagged XC_2801 used in each assay is indicated below each well. DIG-labelled DNA at 1.5 nM was used.
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ppat-1004429-g004: XC_2801 binds to specific promoters and only in the presence of XC_3703.(A) Promoter-fusion analysis of the upstream regions flhB, aaeB, flgG and fliL revealed significantly decrease in promoter activity in the XC_2801 mutant strain. Values given are the mean and standard deviation of triplicate measurements. Values for promoter activity in the XC_2801 mutant strain are significantly different from the wild-type at p<0.01 by two-tailed Student's t-test. The decrease in promoter activity was consistently observed in three independent experiments each consisting of three biological replicates. (B) Binding of XC_2801 to the promoter regions of flhB, aaeB, flgG and fliL in the absence (i) or presence (ii) of XC_3703 assessed by the use of electromobility shift assay (EMSA). Each lane contained 1.5 nM DIG-labelled Probe DNA, and in addition nanomolar concentrations of purified His6-tag XC_2801 protein as indicated below each well. (C) Bioinformatics reveals a putative LysR family regulator-binding site (TCCCGAATCCCCGA) located 80-67 nucleotides upstream of the predicted translational start site of flhB. (D) Although a shift was observed with the flhB upstream (promoter) region, no shift was observed with an overlapping DNA fragment that lacks the putative XC_2801 binding site (indicated as truncated flhB). The nanomolar concentration of His-tagged XC_2801 used in each assay is indicated below each well. DIG-labelled DNA at 1.5 nM was used.

Mentions: The transcriptional analyses outlined above showed that levels of transcripts of the flhB, aaeA, fliL and flgG genes were decreased approximately fivefold in both the XC_3703 and XC_2801 mutant compared to the wild-type. The regulation of these genes by XC_2801 was also examined by the use of promoter fusions to gusA (see Materials and Methods). Differences in the level of GusA between the XC_2801 mutant and wild-type were seen with all four fusions (Figure 4A), demonstrating that XC_2801 (directly or indirectly) regulates the expression of flhB, aaeA, fliL and flgG.


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)

XC_2801 binds to specific promoters and only in the presence of XC_3703.(A) Promoter-fusion analysis of the upstream regions flhB, aaeB, flgG and fliL revealed significantly decrease in promoter activity in the XC_2801 mutant strain. Values given are the mean and standard deviation of triplicate measurements. Values for promoter activity in the XC_2801 mutant strain are significantly different from the wild-type at p<0.01 by two-tailed Student's t-test. The decrease in promoter activity was consistently observed in three independent experiments each consisting of three biological replicates. (B) Binding of XC_2801 to the promoter regions of flhB, aaeB, flgG and fliL in the absence (i) or presence (ii) of XC_3703 assessed by the use of electromobility shift assay (EMSA). Each lane contained 1.5 nM DIG-labelled Probe DNA, and in addition nanomolar concentrations of purified His6-tag XC_2801 protein as indicated below each well. (C) Bioinformatics reveals a putative LysR family regulator-binding site (TCCCGAATCCCCGA) located 80-67 nucleotides upstream of the predicted translational start site of flhB. (D) Although a shift was observed with the flhB upstream (promoter) region, no shift was observed with an overlapping DNA fragment that lacks the putative XC_2801 binding site (indicated as truncated flhB). The nanomolar concentration of His-tagged XC_2801 used in each assay is indicated below each well. DIG-labelled DNA at 1.5 nM was used.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199771&req=5

ppat-1004429-g004: XC_2801 binds to specific promoters and only in the presence of XC_3703.(A) Promoter-fusion analysis of the upstream regions flhB, aaeB, flgG and fliL revealed significantly decrease in promoter activity in the XC_2801 mutant strain. Values given are the mean and standard deviation of triplicate measurements. Values for promoter activity in the XC_2801 mutant strain are significantly different from the wild-type at p<0.01 by two-tailed Student's t-test. The decrease in promoter activity was consistently observed in three independent experiments each consisting of three biological replicates. (B) Binding of XC_2801 to the promoter regions of flhB, aaeB, flgG and fliL in the absence (i) or presence (ii) of XC_3703 assessed by the use of electromobility shift assay (EMSA). Each lane contained 1.5 nM DIG-labelled Probe DNA, and in addition nanomolar concentrations of purified His6-tag XC_2801 protein as indicated below each well. (C) Bioinformatics reveals a putative LysR family regulator-binding site (TCCCGAATCCCCGA) located 80-67 nucleotides upstream of the predicted translational start site of flhB. (D) Although a shift was observed with the flhB upstream (promoter) region, no shift was observed with an overlapping DNA fragment that lacks the putative XC_2801 binding site (indicated as truncated flhB). The nanomolar concentration of His-tagged XC_2801 used in each assay is indicated below each well. DIG-labelled DNA at 1.5 nM was used.
Mentions: The transcriptional analyses outlined above showed that levels of transcripts of the flhB, aaeA, fliL and flgG genes were decreased approximately fivefold in both the XC_3703 and XC_2801 mutant compared to the wild-type. The regulation of these genes by XC_2801 was also examined by the use of promoter fusions to gusA (see Materials and Methods). Differences in the level of GusA between the XC_2801 mutant and wild-type were seen with all four fusions (Figure 4A), demonstrating that XC_2801 (directly or indirectly) regulates the expression of flhB, aaeA, fliL and flgG.

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