Abstract
We describe an original, short, and convenient chemical synthesis of enantiopure (S)-4,5-dihydroxy-2,3-pentanedione (DPD), starting from commercial methyl (S)-(-)-2,2-dimethyl-1,3-dioxolane-4-carboxylate. DPD is the precursor of autoinducer (AI)-2, the proposed signal for bacterial interspecies communication. AI-2 is synthesized by many bacterial species in three enzymatic steps. The last step, a LuxS-catalyzed reaction, leads to the formation of DPD, which spontaneously cyclizes into AI-2. AI-2-like activity of the synthesized molecule was ascertained by the Vibrio harveyi bioassay. To further validate the biological activity of synthetic DPD and to explore its potential in studying DPD (AI-2)-mediated signaling, a Salmonella typhimurium luxS mutant was constructed. Expression of the AI-2 regulated lsr operon can be rescued in this luxS mutant by addition of synthetic DPD or genetic complementation. Biofilm formation by S. typhimurium has been reported to be defective in a luxS mutant, and this was confirmed in this study to test DPD for chemical complementation. However, biofilm formation of the luxS mutant cannot be restored by addition of DPD. In contrast, introduction of luxS under control of its own promoter complemented biofilm formation. Further results demonstrated that biofilm formation of the luxS mutant cannot be restored with luxS under control of the strong nptII promoter. This indicates that altering the intrinsic promoter activity of luxS affects Salmonella biofilm formation. Conclusively, we synthesized biologically active DPD. Using this chemical compound in combination with genetic approaches opens new avenues in studying AI-2-mediated signaling.
Highlights
Bacteria possess an arsenal of chemical signal molecules that enable them to communicate within and between species
Biofilm formation by S. typhimurium has been reported to be defective in a luxS mutant, and this was confirmed in this study to test DPD for chemical complementation
To prove that synthetic DPD is sufficient for lsr regulation, we investigated whether exogenously supplied synthetic DPD could induce the lsrA::lux fusion in S. typhimurium containing a null mutation in luxS
Summary
The synthesis route to DPD, starting from methyl (S)-(Ϫ)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (Fluka), is depicted in Fig. 1A [2]. The key step is the ozonolysis (iv) of (S)-1,2-dihydroxy-4-methyl-4penten-3-one (see Fig. 1A, 5). A stream of 0.75 g/h (15.7 mmol/h) O3 in O2 was bubbled through the solution for 0.5 min with a total flow of 10.8 dm3/h. The excess of ozone was removed from the solution with a stream of nitrogen gas and dimethyl sulfide was added (5–10 eq). After the addition of 3 ml of water, the volatiles (methanol, dimethyl sulfide) were removed by evaporation. NMR showed the signals of DPD and its anomers, accompanied by minor traces of remaining formaldehyde hydrate, which is split off during ozonolysis, methanol, dimethyl sulfide, and Me2SO or dimethyl sulfone.
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