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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 FabG-encoding gene Xcc1018 is involved in DSF, BDSF, CDSF and IDSF biosynthesis in Xcc.(a) The proposed FAS elongation cycle in Xcc strain ATCC33913. The enzymes composing the FAS elongation cycle (FabA, FabB, FabF, FabG, FabZ) were proposed based on BLASTP analysis of their counterparts in E. coli. (b) The effect of overexpression of Xcc1018 via the expression vector pBBR-1-MCS2 on DSF-family biosynthesis 24 h after inoculation. The data are the means ± one standard deviation of three independent assays. Different letters indicate significant differences between treatments (LSD at P = 0.05).
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f6: The FabG-encoding gene Xcc1018 is involved in DSF, BDSF, CDSF and IDSF biosynthesis in Xcc.(a) The proposed FAS elongation cycle in Xcc strain ATCC33913. The enzymes composing the FAS elongation cycle (FabA, FabB, FabF, FabG, FabZ) were proposed based on BLASTP analysis of their counterparts in E. coli. (b) The effect of overexpression of Xcc1018 via the expression vector pBBR-1-MCS2 on DSF-family biosynthesis 24 h after inoculation. The data are the means ± one standard deviation of three independent assays. Different letters indicate significant differences between treatments (LSD at P = 0.05).

Mentions: In bacteria, the intermediate 3-OH-acyl ACPs are usually derived from the FAS elongation cycle17. Xcc contains all of the genes required for the FAS elongation cycle, including Xcc0582 (FabA), Xcc0581 (FabB), Xcc1020 (FabF), Xcc1018 (FabG), Xcc0115 (FabV) and Xcc1362 (FabZ) (Fig. 6a). This led us to further investigate whether DSF-family signals are synthesized via the FAS elongation cycle in Xcc. Since the key enzyme β-ketoacyl-ACP reductase (FabG) is directly responsible for the synthesis of 3-OH-acyl ACPs in the FAS elongation cycle (Fig. 6a)22, we first attempted to delete the FabG-encoding gene Xcc1018. However, these experiments were not successful probably due to the fact that Xcc1018 is an essential gene. As an alternative approach, we overexpressed Xcc1018 in the ΔrpfC strain, and BDSF and DSF levels were examined. The results showed that overexpression of Xcc1018 in strain ΔrpfC had no significant effect on Xcc growth in NA medium, but led to a significant increase in the production of DSF, BDSF, CDSF and IDSF at 24 h (Fig. 6b).


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 FabG-encoding gene Xcc1018 is involved in DSF, BDSF, CDSF and IDSF biosynthesis in Xcc.(a) The proposed FAS elongation cycle in Xcc strain ATCC33913. The enzymes composing the FAS elongation cycle (FabA, FabB, FabF, FabG, FabZ) were proposed based on BLASTP analysis of their counterparts in E. coli. (b) The effect of overexpression of Xcc1018 via the expression vector pBBR-1-MCS2 on DSF-family biosynthesis 24 h after inoculation. The data are the 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

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

f6: The FabG-encoding gene Xcc1018 is involved in DSF, BDSF, CDSF and IDSF biosynthesis in Xcc.(a) The proposed FAS elongation cycle in Xcc strain ATCC33913. The enzymes composing the FAS elongation cycle (FabA, FabB, FabF, FabG, FabZ) were proposed based on BLASTP analysis of their counterparts in E. coli. (b) The effect of overexpression of Xcc1018 via the expression vector pBBR-1-MCS2 on DSF-family biosynthesis 24 h after inoculation. The data are the means ± one standard deviation of three independent assays. Different letters indicate significant differences between treatments (LSD at P = 0.05).
Mentions: In bacteria, the intermediate 3-OH-acyl ACPs are usually derived from the FAS elongation cycle17. Xcc contains all of the genes required for the FAS elongation cycle, including Xcc0582 (FabA), Xcc0581 (FabB), Xcc1020 (FabF), Xcc1018 (FabG), Xcc0115 (FabV) and Xcc1362 (FabZ) (Fig. 6a). This led us to further investigate whether DSF-family signals are synthesized via the FAS elongation cycle in Xcc. Since the key enzyme β-ketoacyl-ACP reductase (FabG) is directly responsible for the synthesis of 3-OH-acyl ACPs in the FAS elongation cycle (Fig. 6a)22, we first attempted to delete the FabG-encoding gene Xcc1018. However, these experiments were not successful probably due to the fact that Xcc1018 is an essential gene. As an alternative approach, we overexpressed Xcc1018 in the ΔrpfC strain, and BDSF and DSF levels were examined. The results showed that overexpression of Xcc1018 in strain ΔrpfC had no significant effect on Xcc growth in NA medium, but led to a significant increase in the production of DSF, BDSF, CDSF and IDSF at 24 h (Fig. 6b).

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