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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.

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.

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Time course of DSF, BDSF and CDSF production in Xoo during growth. (A) Time course of the bacterial growth in YEB medium. (B) Time course of DSF production. (C) Time course of BDSF and CDSF production. Units of DSF, BDSF and CDSF were determined by peak area in HPLC elute as indicated in Materials and Methods.
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Figure 5: Time course of DSF, BDSF and CDSF production in Xoo during growth. (A) Time course of the bacterial growth in YEB medium. (B) Time course of DSF production. (C) Time course of BDSF and CDSF production. Units of DSF, BDSF and CDSF were determined by peak area in HPLC elute as indicated in Materials and Methods.

Mentions: The maximal DSF production in Xcc was found to be at the late stationary phase using a bioassay approach [5]. In this study, a more sensitive HPLC method was used to determine the production profiles of the DSF-family signals in Xoo. The bacterial strain was grown in the same medium for 48 h as described for Xcc [5], and the bacterial cell density and the levels of DSF, BDSF, and CDSF in the supernatants were monitored every 6 hours. The results showed that Xoo strains grew relatively slow during the first 30 h and then multiplied exponentially at about 36 h after inoculation (Fig. 5A). In agreement with this trend, the DSF level remained relatively low before 36 h after inoculation and a substantial increase was observed at 42 h after inoculation (Fig. 5B). The CDSF shared a similar production pattern as DSF except that the CDSF level in the supernatants was around 10 times lower than that of DSF at 42 h after inoculation (Fig. 5C). In contrast, the BDSF level in the supernatants increased stably from 18 h after inoculation and the maximal BDSF production occurred at 36 h after inoculation (Fig. 5C). A substantial decrease in BDSF production was observed 42 h after inoculation (Fig. 5C). At 36 h after inoculation, the BDSF level in the supernatants was around 2 times lower than that of DSF (Fig. 5C).


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)

Time course of DSF, BDSF and CDSF production in Xoo during growth. (A) Time course of the bacterial growth in YEB medium. (B) Time course of DSF production. (C) Time course of BDSF and CDSF production. Units of DSF, BDSF and CDSF were determined by peak area in HPLC elute as indicated in Materials and Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Time course of DSF, BDSF and CDSF production in Xoo during growth. (A) Time course of the bacterial growth in YEB medium. (B) Time course of DSF production. (C) Time course of BDSF and CDSF production. Units of DSF, BDSF and CDSF were determined by peak area in HPLC elute as indicated in Materials and Methods.
Mentions: The maximal DSF production in Xcc was found to be at the late stationary phase using a bioassay approach [5]. In this study, a more sensitive HPLC method was used to determine the production profiles of the DSF-family signals in Xoo. The bacterial strain was grown in the same medium for 48 h as described for Xcc [5], and the bacterial cell density and the levels of DSF, BDSF, and CDSF in the supernatants were monitored every 6 hours. The results showed that Xoo strains grew relatively slow during the first 30 h and then multiplied exponentially at about 36 h after inoculation (Fig. 5A). In agreement with this trend, the DSF level remained relatively low before 36 h after inoculation and a substantial increase was observed at 42 h after inoculation (Fig. 5B). The CDSF shared a similar production pattern as DSF except that the CDSF level in the supernatants was around 10 times lower than that of DSF at 42 h after inoculation (Fig. 5C). In contrast, the BDSF level in the supernatants increased stably from 18 h after inoculation and the maximal BDSF production occurred at 36 h after inoculation (Fig. 5C). A substantial decrease in BDSF production was observed 42 h after inoculation (Fig. 5C). At 36 h after inoculation, the BDSF level in the supernatants was around 2 times lower than that of DSF (Fig. 5C).

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