Communications
to cyclized DPD that has been observed as a by-product of the
Keywords: autoinducers · bioluminescence ·
molecular recognition · sensors · signal transduction
.
[
9,10]
in vitro degradation of RH.
MHF has been shown to be
roughly 1000-times less active than biosynthetic AI-2.
[
9]
Schauder et al. determined EC50 values of approximately
0 nm for biosynthetic AI-2 versus approximately 100 mm for
[
1] a) J. Engebrecht, K. Nealson, M. Silverman, Cell 1983, 32, 773;
b) W. C. Fuqua, S. C. Winans, E. P. Greenberg, J. Bacteriol. 1994,
8
[
10]
MHF, while Winzer et al. reported higher values (ca. 1 mm
versus ca. 1 mm) but a comparable difference in activities.
Figure 2 shows light-inducing activity in V. harveyi mutants
1
76, 269.
2] J. W. Hastings, K. H. Nealson, Annu. Rev. Microbiol. 1977, 31,
49.
[
5
[
14]
[18]
BB170
synthetic DPD or MHF. In our assays we observed a roughly
00-fold higher activity for synthetic DPD than for MHF
EC = ca. 250 nm for DPD versus ca. 125 mm for MHF), a
and MM30
with different concentrations of
[3] a) B. L. Bassler, Cell 2002, 109, 421; b) S. Schauder, B. L. Bassler,
Genes Dev. 2001, 15, 1468; c) M. E. Taga, B. L. Bassler, Proc.
Natl. Acad. Sci. USA 2003, 100, 14549.
5
(
[
[
[
4] K. Winzer, K. R. Hardie, P. Williams, Curr. Opin. Microbiol.
002, 5, 216.
5] B. L. Bassler, M. Wright, M. R. Silverman, Mol. Microbiol. 1994,
3, 273.
6] B. L. Bassler, E. P. Greenberg, A. M. Stevens, J. Bacteriol. 1997,
179, 4043.
5
0
2
result indicative of the strong relationship between DPD and
AI-2.
1
Addition of boric acid to synthetic DPD did slightly
increase the activity of DPD in both strains BB170 and
MM30. Figure 2, however, clearly shows that addition of boric
acid by itself induces bioluminescence in BB170 and not in
MM30, an expected result since MM30 lacks the synthase
LuxS. Addition of boric acid to MHF did significantly
increase the activity of MHF in both bacterial strains, but its
EC50 value remained roughly 50-times lower than that of
DPD with boric acid. One explanation could be that a small
amount of the MHF is converted into AI-2 upon reaction with
borate. Additional evidence for the necessity of boron for
induction of bioluminescence was demonstrated by using
BB170 grown in a boron-free autoinducer-bioassay medium.
These bacteria showed no bioluminescence until boric acid
was added to the medium. Finally, preliminary studies that we
[7] X. Chen, S. Schauder, N. Potier, A. Van Dorsselaer, I. Pelczer,
B. L. Bassler, F. M. Hughson, Nature 2002, 415, 545.
[
[
8] W. D. Loomis, R. W. Durst, Biofactors 1992, 3, 229.
9] S. Schauder, K. Shokat, M. G. Surette, B. L. Bassler, Mol.
Microbiol. 2001, 41, 463.
[
10] K. Winzer, K. R. Hardie, N. Burgess, N. Doherty, D. Kirke,
M. T. G. Holden, R. Linforth, K. A. Cornell, A. J. Taylor, P. J.
Hill, P. Williams, Microbiology 2002, 148, 909.
[11] J. Zhu, M. B. Miller, R. E. Vance, M. Dziejman, B. L. Bassler,
J. J. Mekalanos, Proc. Natl. Acad. Sci. USA 2002, 99, 3129.
[
12] J. Zhu, X. Hu, E. Dizin, D. Pei, J. Am. Chem. Soc. 2003, 125,
3379.
1
[
13] a) M. G. Surette, M. B. Miller, B. L. Bassler, Proc. Natl. Acad.
Sci. USA 1999, 96, 1639; b) V. Sperandio, J. L. Mellies, W.
Nguyen, S. Shin, J. B. Kaper, Proc. Natl. Acad. Sci. USA 1999, 96,
15196; c) M. H. Forsyth, T. L. Cover, Infect. Immun. 2000, 68,
1
11
have carried out with H and B NMR spectroscopy as well as
mass spectrometry confirm that synthetic DPD is capable of
chelating borate at high (> 8) pH values.
3193; d) W. A. Day, A. T. Maurelli, Infect. Immun. 2001, 69, 15;
e) M. P. DeLisa, C. F. Wu, L. Wang, J. J. Valdes, W. E. Bentley, J.
Bacteriol. 2001, 183, 5239; f) K. Winzer, Y. H. Sun, A. Green, M.
Delory, D. Blackley, K. R. Hardie, T. J. Baldwin, C. M. Tang,
Infect. Immun. 2002, 70, 2245.
The described synthesis of enantiopure DPD and our
biochemical examination of its properties substantiates the
argument that it is the true precursor to AI-2 and adds a
unique analytical tool to the quorum-sensing field. From our
studies it can be deduced that DPD does not require LuxS to
direct it into a chemical state that can be readily utilized by
LuxP and will result in induction of bioluminescence.
Furthermore, chelation of boron by cyclic DPD appears to
be a requirement for full induction of bioluminescence.
Additional studies will be required to determine whether
alternative metals can coordinate to cyclic DPD and generate
bioluminescence. Synthetic DPD is stable at moderate
concentrations in buffer solution. This observation is in
contrast with previous reports from research groups that were
unable to synthesize DPD or claimed that the compound was
too unstable to isolate. Our straightforward synthetic route
could be used to produce DPD as a reliable standard to
readily investigate bacterial coordination of gene expression,
biofilm formation, and other AI-2 quorum-sensing-regulated
processes. Our findings also provide a foundation for a
structure- and mechanism-based approach to develop an
innovative antimicrobial therapy targeting the disruption of
quorum-sensing networks.
[
14] B. L. Bassler, M. Wright, R. E. Showalter, M. R. Silverman, Mol.
Microbiol. 1993, 9, 773.
[15] a) M. W. Bernart, W. H. Gerwick, E. E. Corcoran, A. Y. Lee, J.
Clardy, Phytochemistry 1992, 31, 1273; b) I. A. Kennedy, T.
Hemscheidt, J. F. Britten, I. D. Spenser, Can. J. Chem. 1995, 73,
1
329.
16] B. Witulski, A. Zimmermann, N. D. Gowans, Chem. Commun.
002, 24, 2984.
[
[
2
17] CCDC-221569 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cam-
bridge Crystallographic Data Centre, 12, Union Road, Cam-
bridge CB21EZ, UK; fax: (+ 44)1223-336-033; or deposit@
ccdc.cam.ac.uk). See the Supporting Information for experi-
mental details.
[13a]
À
[
18] V. harveyi strain MM30
is a a LuxS mutant that is not able to
generate AI-2.
Received: October 24, 2003
Revised: January 13, 2004 [Z53150]
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108
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2004, 43, 2106 –2108