Chemical Synthesis of (S)-4,5-Dihydroxy-2,3-pentanedione, a Bacterial Signal Molecule Precursor, and Validation of Its Activity in Salmonella typhimurium

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.

Functional Analysis ofluxSinStaphylococcus aureusReveals a Role in Metabolism but Not Quorum Sensing

“…Significantly, none of the above-mentioned studies attempted to use a defined preparation of AI-2 to restore the biofilm characteristics of the wild type. Very recently, however, De Keersmaecker et al (15) demonstrated that AI-2 derived from synthetic DPD could not restore biofilm formation by an S. enterica serovar Typhimurium luxS mutant, whereas introduction of luxS under the control of its own promoter complemented the defect. Similarly, addition of synthetic AI-2 failed to restore type III secretion and motility defects in enterohemorrhagic E. coli (60), phenotypes previously attributed to luxS/AI-2-based quorum sensing by Sperandio et al (57,58,59).…”

“…For S. enterica serovar Typhimurium, De Keersmaecker et al (15) showed that the addition of neither cysteine, methionine, SAM, nor DPD restored biofilm formation by a luxS mutant. Thus, it is unlikely that a lack of sulfur amino acids or extracellular DPD was responsible for this phenotype.…”

Functional Analysis ofluxSin the Probiotic StrainLactobacillus rhamnosusGG Reveals a Central Metabolic Role Important for Growth and Biofilm Formation

“…A method for assaying biofilm formation of L. rhamnosus GG in vitro was optimized based on methods described previously by De Keersmaecker et al (15), with minor modifications. Briefly, biofilm formation on polystyrene pegs hanging into microtiter plate wells was assayed.…”

Attachment and Biofilm Formation by Salmonella in Food Processing Environments

“…The Lsr ABC transporter is known to be involved in the detection and transport of AI-2 into the cell (Taga et al, 2001), while the rbs transporter has recently been suggested as an alternative AI-2 uptake system (Jesudhasan et al, 2010). A S. Typhimurium luxS deletion mutant was impaired in biofilm formation on polystyrene (De Keersmaecker et al, 2005;Jesudhasan et al, 2010). However, this phenotype could not be complemented by extracellular addition of QS signal molecules, suggesting that AI-2 is not the actual signal involved in Salmonella biofilm formation (De Keersmaecker et al, 2005).…”

“…A S. Typhimurium luxS deletion mutant was impaired in biofilm formation on polystyrene (De Keersmaecker et al, 2005;Jesudhasan et al, 2010). However, this phenotype could not be complemented by extracellular addition of QS signal molecules, suggesting that AI-2 is not the actual signal involved in Salmonella biofilm formation (De Keersmaecker et al, 2005). To this direction, Kint et al (2010) analyzed additional luxS mutants for their biofilm phenotype.…”