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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.


The effect of RpfF on acyl-ACP thioesters.(a) SDS-PAGE electrophoresis of purified RpfF protein. (b) RpfF reaction with acyl-ACPs, showing that RpfF cleaves acyl-ACP thioester bonds to release holo-ACP (lanes 2, 4, 6, 8); lane 9 contains only holo-ACP as a control. C8:0-ACP, octanoyl-ACP; C10:0-ACP, decanoyl-ACP; C12:0-ACP, dodecanoyl-ACP; C14:0-ACP, tetradencanoyl-ACP. (c) RpfF reaction with 3-hydroxydodecanoyl-ACP (3-OH-C12:0-ACP). Acyl-ACPs were first prepared as described in Materials and Methods. (d) RpfF reactions with 3-hydroxyacyl-ACPs. The cis-2-acyl-ACPs formed are indicated by arrows (lanes 2, 5, 7). Acyl-ACPs were prepared as described in Materials and Methods. (e) HPLC analysis of DSF extracts in the reaction mixture containing RpfF and 3-OH-C12:0-ACP (lane 5). LC-MS analysis showed that the active product in HPLC elute is BDSF.
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f5: The effect of RpfF on acyl-ACP thioesters.(a) SDS-PAGE electrophoresis of purified RpfF protein. (b) RpfF reaction with acyl-ACPs, showing that RpfF cleaves acyl-ACP thioester bonds to release holo-ACP (lanes 2, 4, 6, 8); lane 9 contains only holo-ACP as a control. C8:0-ACP, octanoyl-ACP; C10:0-ACP, decanoyl-ACP; C12:0-ACP, dodecanoyl-ACP; C14:0-ACP, tetradencanoyl-ACP. (c) RpfF reaction with 3-hydroxydodecanoyl-ACP (3-OH-C12:0-ACP). Acyl-ACPs were first prepared as described in Materials and Methods. (d) RpfF reactions with 3-hydroxyacyl-ACPs. The cis-2-acyl-ACPs formed are indicated by arrows (lanes 2, 5, 7). Acyl-ACPs were prepared as described in Materials and Methods. (e) HPLC analysis of DSF extracts in the reaction mixture containing RpfF and 3-OH-C12:0-ACP (lane 5). LC-MS analysis showed that the active product in HPLC elute is BDSF.

Mentions: B. cepacia BDSF synthase Bcam0581 was shown to have both acyl-ACP thioesterase and dehydratase activity17. This finding inspired us to further investigate the enzymatic activity of RpfF in Xcc. We tagged RpfF with hexa-histidine and purified it by affinity chromatography (Fig. 5a). To assay the thioesterase activity, RpfF and the acyl-ACP substrates were incubated in a reaction mixture containing 20 mM Tris-HCl and 2 mM β-mercaptoethanol at 37 °C for 30 min. RpfF converted the acyl-ACP substrates into holo-ACP, the unacylated species, indicating the presence of thioesterase activity (Fig. 5b, lanes 2, 4, 6, 8). Among the acyl-ACP substrates tested, decanoyl-ACP (C10:0-ACP) and dodecanoyl-ACP (C12:0-ACP) were found to be optimum for RpfF activity, and almost all the substrates were converted into free fatty acids and holo-ACPs (Fig. 5b, lanes 4 and 6). RpfF could also use 3-hydroxydodecanoyl-ACP (3-OH-C12:0-ACP) as a substrate and effectively cleave acyl-ACP thioester bonds to release holo-ACP (Fig. 5c).


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 effect of RpfF on acyl-ACP thioesters.(a) SDS-PAGE electrophoresis of purified RpfF protein. (b) RpfF reaction with acyl-ACPs, showing that RpfF cleaves acyl-ACP thioester bonds to release holo-ACP (lanes 2, 4, 6, 8); lane 9 contains only holo-ACP as a control. C8:0-ACP, octanoyl-ACP; C10:0-ACP, decanoyl-ACP; C12:0-ACP, dodecanoyl-ACP; C14:0-ACP, tetradencanoyl-ACP. (c) RpfF reaction with 3-hydroxydodecanoyl-ACP (3-OH-C12:0-ACP). Acyl-ACPs were first prepared as described in Materials and Methods. (d) RpfF reactions with 3-hydroxyacyl-ACPs. The cis-2-acyl-ACPs formed are indicated by arrows (lanes 2, 5, 7). Acyl-ACPs were prepared as described in Materials and Methods. (e) HPLC analysis of DSF extracts in the reaction mixture containing RpfF and 3-OH-C12:0-ACP (lane 5). LC-MS analysis showed that the active product in HPLC elute is BDSF.
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Related In: Results  -  Collection

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f5: The effect of RpfF on acyl-ACP thioesters.(a) SDS-PAGE electrophoresis of purified RpfF protein. (b) RpfF reaction with acyl-ACPs, showing that RpfF cleaves acyl-ACP thioester bonds to release holo-ACP (lanes 2, 4, 6, 8); lane 9 contains only holo-ACP as a control. C8:0-ACP, octanoyl-ACP; C10:0-ACP, decanoyl-ACP; C12:0-ACP, dodecanoyl-ACP; C14:0-ACP, tetradencanoyl-ACP. (c) RpfF reaction with 3-hydroxydodecanoyl-ACP (3-OH-C12:0-ACP). Acyl-ACPs were first prepared as described in Materials and Methods. (d) RpfF reactions with 3-hydroxyacyl-ACPs. The cis-2-acyl-ACPs formed are indicated by arrows (lanes 2, 5, 7). Acyl-ACPs were prepared as described in Materials and Methods. (e) HPLC analysis of DSF extracts in the reaction mixture containing RpfF and 3-OH-C12:0-ACP (lane 5). LC-MS analysis showed that the active product in HPLC elute is BDSF.
Mentions: B. cepacia BDSF synthase Bcam0581 was shown to have both acyl-ACP thioesterase and dehydratase activity17. This finding inspired us to further investigate the enzymatic activity of RpfF in Xcc. We tagged RpfF with hexa-histidine and purified it by affinity chromatography (Fig. 5a). To assay the thioesterase activity, RpfF and the acyl-ACP substrates were incubated in a reaction mixture containing 20 mM Tris-HCl and 2 mM β-mercaptoethanol at 37 °C for 30 min. RpfF converted the acyl-ACP substrates into holo-ACP, the unacylated species, indicating the presence of thioesterase activity (Fig. 5b, lanes 2, 4, 6, 8). Among the acyl-ACP substrates tested, decanoyl-ACP (C10:0-ACP) and dodecanoyl-ACP (C12:0-ACP) were found to be optimum for RpfF activity, and almost all the substrates were converted into free fatty acids and holo-ACPs (Fig. 5b, lanes 4 and 6). RpfF could also use 3-hydroxydodecanoyl-ACP (3-OH-C12:0-ACP) as a substrate and effectively cleave acyl-ACP thioester bonds to release holo-ACP (Fig. 5c).

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.