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Rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae produces multiple DSF-family signals in regulation of virulence factor production.

He YW, Wu J, Cha JS, Zhang LH - BMC Microbiol. (2010)

Bottom Line: Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence.All the three DSF-family signals promote EPS production and xylanase activity in Xoo, but CDSF is less active than its analogues DSF and BDSF.The composition and ratio of the three DSF-family signals produced by Xoo are influenced by the composition of culture media.

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Affiliation: Institute of Molecular and Cell Biology, Singapore. yawen@imcb.a-star.edu.sg

ABSTRACT

Background: Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease. Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence. Genetic and genomics evidence suggest that Xoo might use the diffusible signal factor (DSF) type quorum sensing (QS) system to regulate the virulence factor production. However, little is known about the chemical structure of the DSF-like signal(s) produced by Xoo and the factors influencing the signal production.

Results: Xoo genome harbours an rpf cluster comprising rpfB, rpfF, rpfC and rpfG. The proteins encoded by these genes are highly homologous to their counterparts in X. campestris pv. campestris (Xcc), suggesting that Xcc and Xoo might use similar mechanisms for DSF biosynthesis and autoregulation. Consistent with in silico analysis, the rpfF mutant was DSF-deficient and the rpfC mutant produced about 25 times higher DSF-like activity than the wild type Xoo strain KACC10331. From the supernatants of rpfC mutant, we purified three compounds showing strong DSF-like activity. Mass spectrometry and NMR analysis revealed that two of them were the previously characterized DSF and BDSF; the third one was a novel unsaturated fatty acid with 2 double bonds and was designated as CDSF in this study. Further analysis showed that all the three DSF-family signals were synthesized via the enzyme RpfF encoded by Xoo2868. DSF and BDSF at a final concentration of 3 microM to the rpfF mutant could fully restore its extracellular xylanase activity and EPS production to the wild type level, but CDSF was less active than DSF and BDSF in induction of EPS and xylanase. DSF and CDSF shared a similar cell density-dependent production time course with the maximum production being detected at 42 h after inoculation, whereas the maximum production of BDSF was observed at 36 h after inoculation. When grown in a rich medium such as YEB, LB, PSA, and NYG, Xoo produced all the three signals with the majority being DSF. Whereas in nutritionally poor XOLN medium Xoo only produced BDSF and DSF but the majority was BDSF.

Conclusions: This study demonstrates that Xoo and Xcc share the conserved mechanisms for DSF biosynthesis and autoregulation. Xoo produces DSF, BDSF and CDSF signals in rich media and CDSF is a novel signal in DSF-family with two double bonds. All the three DSF-family signals promote EPS production and xylanase activity in Xoo, but CDSF is less active than its analogues DSF and BDSF. The composition and ratio of the three DSF-family signals produced by Xoo are influenced by the composition of culture media.

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Xoo and Xcc share conserved mechanisms for DSF biosynthesis autoregulation. (A) Physical map of the part of the rpf gene cluster from rpfB to rpfG in Xoo strain KACC10331 and Xcc strain ATCC33913. The organization of ORFs predicted by sequence analysis together with predicted directions of transcription are indicated by the broad arrows. (B) DSF production of Xoo strain KACC10331 and derivatives.
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Figure 1: Xoo and Xcc share conserved mechanisms for DSF biosynthesis autoregulation. (A) Physical map of the part of the rpf gene cluster from rpfB to rpfG in Xoo strain KACC10331 and Xcc strain ATCC33913. The organization of ORFs predicted by sequence analysis together with predicted directions of transcription are indicated by the broad arrows. (B) DSF production of Xoo strain KACC10331 and derivatives.

Mentions: In Xcc, the rpf cluster is involved in DSF biosynthesis, signal sensing and response. RpfF, a putative enoyl-CoA hydratase, is a key enzyme involved in DSF biosynthesis and mutation of rpfF abolishes DSF production [4]. RpfC negatively controls DSF biosynthesis by binding to RpfF at low cell density [10], and disruption of rpfC results in a 16-fold higher DSF accumulation than the wild-type Xcc [5,11]. The genomes of three sequenced Xoo strains (KACC10331, MAFF311018 and PX099A) contain the rpf cluster comprising rpfB, rpfF, rpfG and rpfC, but not rpfH [26-28]. These rpf homologous from Xcc and Xoo share more than 86% identify at the amino acids level (Fig. 1A), suggesting the conserved mechanism in DSF biosynthesis and in DSF signalling. To confirm this possibility, the rpfF, rpfC and rpfG mutants of Xoo strain KACC 10331, which were described previously [25], were assayed for DSF production. The results showed that the rpfF mutant is DSF-deficient while the rpfC mutant produced DSF signal around 25 times higher than its wild type parental strain did (Fig. 1B). The DSF production patterns of rpfC, rpfF and rpfG mutants of Xoo were very similar to those of Xcc [5,10,11], which indicates that, similar to XC1, Xoo also uses the RpfC-RpfF protein-protein interaction mechanism to autoregulate the biosynthesis of DSF-like signals.


Rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae produces multiple DSF-family signals in regulation of virulence factor production.

He YW, Wu J, Cha JS, Zhang LH - BMC Microbiol. (2010)

Xoo and Xcc share conserved mechanisms for DSF biosynthesis autoregulation. (A) Physical map of the part of the rpf gene cluster from rpfB to rpfG in Xoo strain KACC10331 and Xcc strain ATCC33913. The organization of ORFs predicted by sequence analysis together with predicted directions of transcription are indicated by the broad arrows. (B) DSF production of Xoo strain KACC10331 and derivatives.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2909994&req=5

Figure 1: Xoo and Xcc share conserved mechanisms for DSF biosynthesis autoregulation. (A) Physical map of the part of the rpf gene cluster from rpfB to rpfG in Xoo strain KACC10331 and Xcc strain ATCC33913. The organization of ORFs predicted by sequence analysis together with predicted directions of transcription are indicated by the broad arrows. (B) DSF production of Xoo strain KACC10331 and derivatives.
Mentions: In Xcc, the rpf cluster is involved in DSF biosynthesis, signal sensing and response. RpfF, a putative enoyl-CoA hydratase, is a key enzyme involved in DSF biosynthesis and mutation of rpfF abolishes DSF production [4]. RpfC negatively controls DSF biosynthesis by binding to RpfF at low cell density [10], and disruption of rpfC results in a 16-fold higher DSF accumulation than the wild-type Xcc [5,11]. The genomes of three sequenced Xoo strains (KACC10331, MAFF311018 and PX099A) contain the rpf cluster comprising rpfB, rpfF, rpfG and rpfC, but not rpfH [26-28]. These rpf homologous from Xcc and Xoo share more than 86% identify at the amino acids level (Fig. 1A), suggesting the conserved mechanism in DSF biosynthesis and in DSF signalling. To confirm this possibility, the rpfF, rpfC and rpfG mutants of Xoo strain KACC 10331, which were described previously [25], were assayed for DSF production. The results showed that the rpfF mutant is DSF-deficient while the rpfC mutant produced DSF signal around 25 times higher than its wild type parental strain did (Fig. 1B). The DSF production patterns of rpfC, rpfF and rpfG mutants of Xoo were very similar to those of Xcc [5,10,11], which indicates that, similar to XC1, Xoo also uses the RpfC-RpfF protein-protein interaction mechanism to autoregulate the biosynthesis of DSF-like signals.

Bottom Line: Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence.All the three DSF-family signals promote EPS production and xylanase activity in Xoo, but CDSF is less active than its analogues DSF and BDSF.The composition and ratio of the three DSF-family signals produced by Xoo are influenced by the composition of culture media.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular and Cell Biology, Singapore. yawen@imcb.a-star.edu.sg

ABSTRACT

Background: Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease. Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence. Genetic and genomics evidence suggest that Xoo might use the diffusible signal factor (DSF) type quorum sensing (QS) system to regulate the virulence factor production. However, little is known about the chemical structure of the DSF-like signal(s) produced by Xoo and the factors influencing the signal production.

Results: Xoo genome harbours an rpf cluster comprising rpfB, rpfF, rpfC and rpfG. The proteins encoded by these genes are highly homologous to their counterparts in X. campestris pv. campestris (Xcc), suggesting that Xcc and Xoo might use similar mechanisms for DSF biosynthesis and autoregulation. Consistent with in silico analysis, the rpfF mutant was DSF-deficient and the rpfC mutant produced about 25 times higher DSF-like activity than the wild type Xoo strain KACC10331. From the supernatants of rpfC mutant, we purified three compounds showing strong DSF-like activity. Mass spectrometry and NMR analysis revealed that two of them were the previously characterized DSF and BDSF; the third one was a novel unsaturated fatty acid with 2 double bonds and was designated as CDSF in this study. Further analysis showed that all the three DSF-family signals were synthesized via the enzyme RpfF encoded by Xoo2868. DSF and BDSF at a final concentration of 3 microM to the rpfF mutant could fully restore its extracellular xylanase activity and EPS production to the wild type level, but CDSF was less active than DSF and BDSF in induction of EPS and xylanase. DSF and CDSF shared a similar cell density-dependent production time course with the maximum production being detected at 42 h after inoculation, whereas the maximum production of BDSF was observed at 36 h after inoculation. When grown in a rich medium such as YEB, LB, PSA, and NYG, Xoo produced all the three signals with the majority being DSF. Whereas in nutritionally poor XOLN medium Xoo only produced BDSF and DSF but the majority was BDSF.

Conclusions: This study demonstrates that Xoo and Xcc share the conserved mechanisms for DSF biosynthesis and autoregulation. Xoo produces DSF, BDSF and CDSF signals in rich media and CDSF is a novel signal in DSF-family with two double bonds. All the three DSF-family signals promote EPS production and xylanase activity in Xoo, but CDSF is less active than its analogues DSF and BDSF. The composition and ratio of the three DSF-family signals produced by Xoo are influenced by the composition of culture media.

Show MeSH
Related in: MedlinePlus