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The Multiple DSF-family QS Signals are Synthesized from Carbohydrate and Branched-chain Amino Acids via the FAS Elongation Cycle.

Zhou L, Yu Y, Chen X, Diab AA, Ruan L, He J, Wang H, He YW - Sci Rep (2015)

Bottom Line: Furthermore, our biochemical analyses show that the key DSF synthase RpfF has both thioesterase and dehydratase activities, and uses 3-hydroxydedecanoyl-ACP as a substrate to produce BDSF.Finally, our results show that the classic fatty acid synthesis elongation cycle is required for the biosynthesis of DSF-family signals.Taken all together, these findings establish a general biosynthetic pathway for the DSF-family quorum sensing signals.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.

ABSTRACT
Members of the diffusible signal factor (DSF) family are a novel class of quorum sensing (QS) signals in diverse Gram-negative bacteria. Although previous studies have identified RpfF as a key enzyme for the biosynthesis of DSF family signals, many questions in their biosynthesis remain to be addressed. In this study with the phytopathogen Xanthomonas campestris pv. campestris (Xcc), we show that Xcc produces four DSF-family signals (DSF, BDSF, CDSF and IDSF) during cell culture, and that IDSF is a new functional signal characterized as cis-10-methyl-2-dodecenoic acid. Using a range of defined media, we further demonstrate that Xcc mainly produces BDSF in the presence of carbohydrates; leucine and valine are the primary precursor for DSF biosynthesis; isoleucine is the primary precursor for IDSF biosynthesis. Furthermore, our biochemical analyses show that the key DSF synthase RpfF has both thioesterase and dehydratase activities, and uses 3-hydroxydedecanoyl-ACP as a substrate to produce BDSF. Finally, our results show that the classic fatty acid synthesis elongation cycle is required for the biosynthesis of DSF-family signals. Taken all together, these findings establish a general biosynthetic pathway for the DSF-family quorum sensing signals.

No MeSH data available.


Related in: MedlinePlus

The effects of various carbohydrates on BDSF biosynthesis.(a) DSF and BDSF production of strain ΔrpfC in media XY, XYS, XYT, XYG and XYF. XY medium contains 0.7 g/L K2HPO4, 0.2 g/L KH2PO4, 1.0 g/L (NH4)2SO4, 0.1 g/L MgCl2, 0.01 g/L FeSO4, 0.001 g/L MnCl2, 0.2 g/L yeast extract, pH7.0. XYS medium: XY medium supplemented 2.0 g/L sucrose; XYT: XY medium supplemented with 2.0 g/L starch; XYG: XY medium supplemented with 2.0 g/L glucose; XYF: XY medium supplemented with 2 g/L fructose. (b) DSF and BDSF production time courses in XYS medium. Data are means ± one standard deviation of three independent assays. Different letters indicate significant differences between treatments (LSD at P = 0.05).
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f2: The effects of various carbohydrates on BDSF biosynthesis.(a) DSF and BDSF production of strain ΔrpfC in media XY, XYS, XYT, XYG and XYF. XY medium contains 0.7 g/L K2HPO4, 0.2 g/L KH2PO4, 1.0 g/L (NH4)2SO4, 0.1 g/L MgCl2, 0.01 g/L FeSO4, 0.001 g/L MnCl2, 0.2 g/L yeast extract, pH7.0. XYS medium: XY medium supplemented 2.0 g/L sucrose; XYT: XY medium supplemented with 2.0 g/L starch; XYG: XY medium supplemented with 2.0 g/L glucose; XYF: XY medium supplemented with 2 g/L fructose. (b) DSF and BDSF production time courses in XYS medium. Data are means ± one standard deviation of three independent assays. Different letters indicate significant differences between treatments (LSD at P = 0.05).

Mentions: In order to understand how medium composition influences the production of DSF-family signals, a range of media were developed based on the salt composition of XOLN medium (Supplementary Table 2)34. Medium XY contains XOLN salts, 0.2 g/L yeast extract, and was used as a base medium for analyzing DSF-family signal production. In XY medium, the ΔrpfC strain grew poorly with a maximum OD600 of 0.3 and produced very low levels of DSF-family signals (Fig. 2a). Addition of 2.0 g/L sucrose to XY medium (XYS) significantly improved Xcc growth to an OD600 maximum of 0.8. In XYS medium after 24 h of growth, the ΔrpfC strain produced two DSF-family signals BDSF (1.10 μM or 3.7 × 103 μmol/109 cells) and DSF (0.19 μM or 0.63 × 10−3 μmol/109 cells) (Fig. 2b). An extremely low level of IDSF (0.005–0.008 μM) and no CDSF were detected in the supernatant of ΔrpfC cultured in XYS medium. The addition of starch (medium XYT), glucose (medium XYG) or fructose (medium XYF) to medium XY at 2 g/L had effects similar to those of sucrose on bacterial growth. In media XYT, XYG and XYF, more than 80.0% of the DSF-family signals produced by the ΔrpfC strain were BDSF and the DSF levels are relatively stable (Fig. 2a).


The Multiple DSF-family QS Signals are Synthesized from Carbohydrate and Branched-chain Amino Acids via the FAS Elongation Cycle.

Zhou L, Yu Y, Chen X, Diab AA, Ruan L, He J, Wang H, He YW - Sci Rep (2015)

The effects of various carbohydrates on BDSF biosynthesis.(a) DSF and BDSF production of strain ΔrpfC in media XY, XYS, XYT, XYG and XYF. XY medium contains 0.7 g/L K2HPO4, 0.2 g/L KH2PO4, 1.0 g/L (NH4)2SO4, 0.1 g/L MgCl2, 0.01 g/L FeSO4, 0.001 g/L MnCl2, 0.2 g/L yeast extract, pH7.0. XYS medium: XY medium supplemented 2.0 g/L sucrose; XYT: XY medium supplemented with 2.0 g/L starch; XYG: XY medium supplemented with 2.0 g/L glucose; XYF: XY medium supplemented with 2 g/L fructose. (b) DSF and BDSF production time courses in XYS medium. Data are means ± one standard deviation of three independent assays. Different letters indicate significant differences between treatments (LSD at P = 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4542539&req=5

f2: The effects of various carbohydrates on BDSF biosynthesis.(a) DSF and BDSF production of strain ΔrpfC in media XY, XYS, XYT, XYG and XYF. XY medium contains 0.7 g/L K2HPO4, 0.2 g/L KH2PO4, 1.0 g/L (NH4)2SO4, 0.1 g/L MgCl2, 0.01 g/L FeSO4, 0.001 g/L MnCl2, 0.2 g/L yeast extract, pH7.0. XYS medium: XY medium supplemented 2.0 g/L sucrose; XYT: XY medium supplemented with 2.0 g/L starch; XYG: XY medium supplemented with 2.0 g/L glucose; XYF: XY medium supplemented with 2 g/L fructose. (b) DSF and BDSF production time courses in XYS medium. Data are means ± one standard deviation of three independent assays. Different letters indicate significant differences between treatments (LSD at P = 0.05).
Mentions: In order to understand how medium composition influences the production of DSF-family signals, a range of media were developed based on the salt composition of XOLN medium (Supplementary Table 2)34. Medium XY contains XOLN salts, 0.2 g/L yeast extract, and was used as a base medium for analyzing DSF-family signal production. In XY medium, the ΔrpfC strain grew poorly with a maximum OD600 of 0.3 and produced very low levels of DSF-family signals (Fig. 2a). Addition of 2.0 g/L sucrose to XY medium (XYS) significantly improved Xcc growth to an OD600 maximum of 0.8. In XYS medium after 24 h of growth, the ΔrpfC strain produced two DSF-family signals BDSF (1.10 μM or 3.7 × 103 μmol/109 cells) and DSF (0.19 μM or 0.63 × 10−3 μmol/109 cells) (Fig. 2b). An extremely low level of IDSF (0.005–0.008 μM) and no CDSF were detected in the supernatant of ΔrpfC cultured in XYS medium. The addition of starch (medium XYT), glucose (medium XYG) or fructose (medium XYF) to medium XY at 2 g/L had effects similar to those of sucrose on bacterial growth. In media XYT, XYG and XYF, more than 80.0% of the DSF-family signals produced by the ΔrpfC strain were BDSF and the DSF levels are relatively stable (Fig. 2a).

Bottom Line: Furthermore, our biochemical analyses show that the key DSF synthase RpfF has both thioesterase and dehydratase activities, and uses 3-hydroxydedecanoyl-ACP as a substrate to produce BDSF.Finally, our results show that the classic fatty acid synthesis elongation cycle is required for the biosynthesis of DSF-family signals.Taken all together, these findings establish a general biosynthetic pathway for the DSF-family quorum sensing signals.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.

ABSTRACT
Members of the diffusible signal factor (DSF) family are a novel class of quorum sensing (QS) signals in diverse Gram-negative bacteria. Although previous studies have identified RpfF as a key enzyme for the biosynthesis of DSF family signals, many questions in their biosynthesis remain to be addressed. In this study with the phytopathogen Xanthomonas campestris pv. campestris (Xcc), we show that Xcc produces four DSF-family signals (DSF, BDSF, CDSF and IDSF) during cell culture, and that IDSF is a new functional signal characterized as cis-10-methyl-2-dodecenoic acid. Using a range of defined media, we further demonstrate that Xcc mainly produces BDSF in the presence of carbohydrates; leucine and valine are the primary precursor for DSF biosynthesis; isoleucine is the primary precursor for IDSF biosynthesis. Furthermore, our biochemical analyses show that the key DSF synthase RpfF has both thioesterase and dehydratase activities, and uses 3-hydroxydedecanoyl-ACP as a substrate to produce BDSF. Finally, our results show that the classic fatty acid synthesis elongation cycle is required for the biosynthesis of DSF-family signals. Taken all together, these findings establish a general biosynthetic pathway for the DSF-family quorum sensing signals.

No MeSH data available.


Related in: MedlinePlus