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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.


A schematic model for the general biosynthetic pathway of DSF, BDSF and IDSF in Xanthomonas.BCAA: branched-chain amino acids; BCKA: branched-chain α-ketoacids. ACC: acetyl-CoA carboxylase. KIC: 2-keto-isocaproic acid. KMV: 2-keto-β-methylvaleric acid.
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f8: A schematic model for the general biosynthetic pathway of DSF, BDSF and IDSF in Xanthomonas.BCAA: branched-chain amino acids; BCKA: branched-chain α-ketoacids. ACC: acetyl-CoA carboxylase. KIC: 2-keto-isocaproic acid. KMV: 2-keto-β-methylvaleric acid.

Mentions: The present study shows that DSF-family signals are synthesized via the essential FAS elongation cycle (Figs 6 and 7). On the basis of previous and present findings, a general biosynthetic pathway for DSF-family signals is proposed (Fig. 8). In the presence of carbohydrates, acetyl-CoA is produced via aerobic cellular respiration and is further converted into malonyl-CoA by acetyl-CoA carboxylase (ACC). Malonyl-ACP is synthesized from ACP and malonyl-CoA by FabD (Xcc1016), which is then condensed with acetyl-CoA by FabH (Xcc1017) to form 3-keto-butyl-ACP for the initial step of the FAS elongation cycle. The intermediate 3-hydroxydodecanoyl-ACP is formed via the FAS elongation cycle. RpfF catalyzes the synthesis of BDSF by using 3-hydroxydodecanoyl-ACP as a substrate. In the presence of carbohydrates, leucine and isoleucine (such as in the rich media YEB or NA), the branched-chain amino acid aminotransferase IlvE encoded by Xcc0850 catalyzes the deamination of leucine and isoleucine to form 2-keto-isocaproic acid (KIC) and 2-keto-β-methylvaleric acid (KMV) respectively, which are then further acted upon by α-ketoacid dehydrogenase (BCKA) to form iso-butyryl-CoA and 2-methylbutyryl-CoA respectively. Malonyl-ACP is then condensed with these acyl-CoAs to form 3-keto-butyl-ACP, iso-3-keto-hexanoyl-ACP and anteiso-3-keto-hexanoyl-ACP for the initial step of the FAS cycle. The intermediates 3-hydroxydodecanoyl-ACP, 11-methyl-cis-2-dodecenoyl-ACP and 10-methyl-cis-2-dodecenoyl-ACP are formed via the FAS elongation cycle. These acyl-ACP intermediates compete for binding to RpfF. The relative proportion of DSF, BDSF and IDSF formed is determined by the relative concentrations of acyl-ACP intermediates and their affinities for RpfF. Since 11-methyl-3-hydroxydodecanoyl-ACP is more hydrophobic than 3-hydroxydodecanoyl-ACP, and the DSF biosynthetic pocket in RpfF is highly hydrophobic16, 11-methyl-3-hydroxydodecanoyl-ACP gains easier access to the biosynthetic pocket than 3-hydroxydodecanoyl-ACP. This might explain why more DSF, rather than BDSF, is produced in the rich media YEB or LB and why BDSF production was reduced in the leucine-rich XYSL medium or the isoleucine-rich XYSV medium (Figs 3 and 4). Further insights into this proposed pathway will be gained from the experimental analysis of FabD, FabH, ACC, and BCKA in Xcc.


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)

A schematic model for the general biosynthetic pathway of DSF, BDSF and IDSF in Xanthomonas.BCAA: branched-chain amino acids; BCKA: branched-chain α-ketoacids. ACC: acetyl-CoA carboxylase. KIC: 2-keto-isocaproic acid. KMV: 2-keto-β-methylvaleric acid.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: A schematic model for the general biosynthetic pathway of DSF, BDSF and IDSF in Xanthomonas.BCAA: branched-chain amino acids; BCKA: branched-chain α-ketoacids. ACC: acetyl-CoA carboxylase. KIC: 2-keto-isocaproic acid. KMV: 2-keto-β-methylvaleric acid.
Mentions: The present study shows that DSF-family signals are synthesized via the essential FAS elongation cycle (Figs 6 and 7). On the basis of previous and present findings, a general biosynthetic pathway for DSF-family signals is proposed (Fig. 8). In the presence of carbohydrates, acetyl-CoA is produced via aerobic cellular respiration and is further converted into malonyl-CoA by acetyl-CoA carboxylase (ACC). Malonyl-ACP is synthesized from ACP and malonyl-CoA by FabD (Xcc1016), which is then condensed with acetyl-CoA by FabH (Xcc1017) to form 3-keto-butyl-ACP for the initial step of the FAS elongation cycle. The intermediate 3-hydroxydodecanoyl-ACP is formed via the FAS elongation cycle. RpfF catalyzes the synthesis of BDSF by using 3-hydroxydodecanoyl-ACP as a substrate. In the presence of carbohydrates, leucine and isoleucine (such as in the rich media YEB or NA), the branched-chain amino acid aminotransferase IlvE encoded by Xcc0850 catalyzes the deamination of leucine and isoleucine to form 2-keto-isocaproic acid (KIC) and 2-keto-β-methylvaleric acid (KMV) respectively, which are then further acted upon by α-ketoacid dehydrogenase (BCKA) to form iso-butyryl-CoA and 2-methylbutyryl-CoA respectively. Malonyl-ACP is then condensed with these acyl-CoAs to form 3-keto-butyl-ACP, iso-3-keto-hexanoyl-ACP and anteiso-3-keto-hexanoyl-ACP for the initial step of the FAS cycle. The intermediates 3-hydroxydodecanoyl-ACP, 11-methyl-cis-2-dodecenoyl-ACP and 10-methyl-cis-2-dodecenoyl-ACP are formed via the FAS elongation cycle. These acyl-ACP intermediates compete for binding to RpfF. The relative proportion of DSF, BDSF and IDSF formed is determined by the relative concentrations of acyl-ACP intermediates and their affinities for RpfF. Since 11-methyl-3-hydroxydodecanoyl-ACP is more hydrophobic than 3-hydroxydodecanoyl-ACP, and the DSF biosynthetic pocket in RpfF is highly hydrophobic16, 11-methyl-3-hydroxydodecanoyl-ACP gains easier access to the biosynthetic pocket than 3-hydroxydodecanoyl-ACP. This might explain why more DSF, rather than BDSF, is produced in the rich media YEB or LB and why BDSF production was reduced in the leucine-rich XYSL medium or the isoleucine-rich XYSV medium (Figs 3 and 4). Further insights into this proposed pathway will be gained from the experimental analysis of FabD, FabH, ACC, and BCKA in Xcc.

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.