Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jun 18;8:98.
doi: 10.1186/1471-2180-8-98.

LuxS-independent Formation of AI-2 From ribulose-5-phosphate

Affiliations
Free PMC article

LuxS-independent Formation of AI-2 From ribulose-5-phosphate

Timothy J Tavender et al. BMC Microbiol. .
Free PMC article

Abstract

Background: In many bacteria, the signal molecule AI-2 is generated from its precursor S-ribosyl-L-homocysteine in a reaction catalysed by the enzyme LuxS. However, generation of AI-2-like activity has also been reported for organisms lacking the luxS gene and the existence of alternative pathways for AI-2 formation in Escherichia coli has recently been predicted by stochastic modelling. Here, we investigate the possibility that spontaneous conversion of ribulose-5-phosphate could be responsible for AI-2 generation in the absence of luxS.

Results: Buffered solutions of ribulose-5-phosphate, but not ribose-5-phosphate, were found to contain high levels of AI-2 activity following incubation at concentrations similar to those reported in vivo. To test whether this process contributes to AI-2 formation by bacterial cells in vivo, an improved Vibrio harveyi bioassay was used. In agreement with previous studies, culture supernatants of E. coli and Staphylococcus aureus luxS mutants were found not to contain detectable levels of AI-2 activity. However, low activities were detected in an E. coli pgi-eda-edd-luxS mutant, a strain which degrades glucose entirely via the oxidative pentose phosphate pathway, with ribulose-5-phosphate as an obligatory intermediate.

Conclusion: Our results suggest that LuxS-independent formation of AI-2, via spontaneous conversion of ribulose-5-phosphate, may indeed occur in vivo. It does not contribute to AI-2 formation in wildtype E. coli and S. aureus under the conditions tested, but may be responsible for the AI-2-like activities reported for other organisms lacking the luxS gene.

Figures

Figure 1
Figure 1
Pathways of DPD and AI-2 formation. The schematic integrates pathways described for production of 4,5-dihydroxy-2,3-pentanedione (DPD) from D-ribulose-5-phosphate [13] and S-ribosylhomocysteine (SRH) [4] with the subsequent formation of AI-2 molecules (yellow box) detected by V. harveyi and S. enterica serovar Typhimurium [10]. Ribulose-5-phosphate is formed enzymatically from other sugar phosphates and its reactive open-chain carbonyl form in aqueous solution facilitates DPD generation. DPD cyclisation leads to several products forming via 2,4-dihydroxy-2-methylhydrofuran-3-one intermediates, including two distinct autoinducer molecules, S-THMF-borate ((2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran-borate; detected by V. harveyi) and R-THMF ((2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran; detected by S. enterica serovar Typhimurium) as well as MHF (4-hydroxy-5-methyl-3(2H)-furanone). Intermediates or side products shown are: S-THMF, (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran; R-DHMF, (2R,4S)-2,4-dihydroxy-2-methyldihydrofuran-3-one; S-DHMF: (2S,4S)-2,4-dihydroxy-2-methyldihydrofuran-3-one; Hcy, homocysteine.
Figure 2
Figure 2
Spontaneous generation of AI-2 activity in ribulose-5-phosphate solutions. (A) AI-2 activity in pentose phosphate solutions. Reaction buffer (10 mM sodium phosphate, pH 7.7) was incubated alone (control) or containing either 5 mM MHF, 5 mM ribose-5-phosphate (Rib-5-P), 5 mM xylulose-5-phosphate (Xyl-5-P), or 5 mM ribulose-5-phosphate (Rul-5-P). After incubation for 24 h at 37°C, the solutions were analysed for the presence of bioluminescence-inducing activity using the V. harveyi BB170 and BB886 bioassays. V. harveyi BB170 (white bars) is specifically activated by AI-2 and BB886 (grey bars) by AI-1. (B) AI-2 activity in 5 mM ribose-5-phosphate solutions incubated with phosphoriboisomerase (10 U/ml). Samples were taken immediately after the start of the experiment (white bars, 0 h) or after 2 h incubation at 37°C (grey bars). Controls contained reaction buffer only (buffer), no enzyme (buffer + Rib-5-P), no substrate (buffer + enzyme), or heat-inactivated enzyme (buffer + Rib-5-P + enzyme (inactive)). (C) Kinetics of AI-2 formation in Rul-5-P solutions. 0.5 mM Rul-5-P in reaction buffer was incubated at 37°C and samples removed and snap-frozen in liquid nitrogen at the times indicated. The samples were then analysed for AI-2 activity using V. harveyi BB170. Results represent the mean (± SD) of three independent experiments. AI-2 activity is determined by the fold-induction of light emission relative to that of the negative reaction buffer control. For comparison, approximately 400-fold induction was observed with E. coli MG1655 culture supernatants.
Figure 3
Figure 3
E. coli MG1655 pgi-EDP luxS produces extracellular AI-2 activity. (A) Conventional V. harveyi BB170 bioassay [17] with E. coli DH5α culture supernatants obtained from cultures growing in LB + 0.5% (w/v) glucose. Turquoise, grey, pink, bright green, dark green, blue, and red lines indicate the bioluminescence observed for E. coli DH5α culture supernatants after 0, 1, 2, 3, 4, 5, and 6 h of growth, respectively. Open circles, negative control (LB medium + 0.5% glucose); closed circles, AI-2 containing positive control (E. coli MG1655 culture supernatant after 3 h of growth). (B) Modified V. harveyi BB170 bioassay with the same E. coli DH5α culture supernatants as analysed in (A). 25 mM HEPES was present in the bioassay medium to prevent acidification. For figure legends, see (A). (C) Growth (lines) and AI-2 activity profiles (bars) for E. coli MG1655 luxS (blue) and E. coli MG1655 pgi-EDP luxS (red). Each strain was grown in LB medium + 0.5% (w/v) glucose and samples removed hourly. For each sample the optical density at 600 nm was recorded and AI-2 activity in culture supernatants recorded using the modified V. harveyi BB170 bioassay containing 25 mM HEPES. Results represent the mean of three independent bioassays. For AI-2 activity, error bars represent the standard deviations. The experiments were repeated five times with similar results.

Similar articles

See all similar articles

Cited by 12 articles

See all "Cited by" articles

References

    1. Bassler BL. How bacteria talk to each other: regulation of gene expression by quorum sensing. Curr Opin Microbiol. 1999;2:582–587. doi: 10.1016/S1369-5274(99)00025-9. - DOI - PubMed
    1. Henke JM, Bassler BL. Three parallel quorum-sensing systems regulate gene expression in Vibrio harveyi. J Bacteriol. 2004;186:6902–6914. doi: 10.1128/JB.186.20.6902-6914.2004. - DOI - PMC - PubMed
    1. Vendeville A, Winzer K, Heurlier K, Tang CM, Hardie KR. Making 'sense' of metabolism:autoinducer-2, LuxS and pathogenic bacteria. Nat Rev Microbiol. 2005;3:383–396. doi: 10.1038/nrmicro1146. - DOI - PubMed
    1. Schauder S, Shokat K, Surette MG, Bassler BL. The LuxS family of bacterial autoinducers:biosynthesis of a novel quorum sensing signal molecule. Mol Microbiol. 2001;41:463–476. doi: 10.1046/j.1365-2958.2001.02532.x. - DOI - PubMed
    1. Xavier KB, Bassler BL. LuxS quorum sensing:more than just a numbers game. Curr Opin Microbiol. 2003;6:191–197. doi: 10.1016/S1369-5274(03)00028-6. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback