25-08-2017, 09:32 PM
Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover
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INTRODUCTION
Many Gram-negative bacterial species employN-acyl-lhomoserine
lactones (AHLs) to control the synthesis of
products that facilitate interactions with the surrounding
environment, including interactions with eukaryotic
host species (reviewed by Eberl, 1999; Kievit&Iglewski,
2000). AHL dependent gene expression has been suggested
to constitute a mechanism by which bacteria can
alter their behaviour in response to cell density and is
thus commonly referred to as quorum sensing (reviewed
by Swift et al., 1999).
METHODS
Strains, plasmids and culture conditions. The LuxR overproduction
strain Escherichia coli XL-1 Blue(pHK724)
(pGroESL) and the control strain E. coli XL-1 Blue(pGroESL)
were cultured and washed during assays in Luria±Bertani
broth (containing 10 g NaCl l−"). Plasmid pHK724 carries a
Ptac±luxR fusion and encodes resistance to 50 lg ampicillin
ml−" (Hanzelka&Greenberg, 1995). Plasmid pGroESL carries
Plac±groES and Plac±groEL fusions and encodes resistance to
25 lg chloramphenicol ml−" (Goloubinoff et al., 1989). The
LuxR-based green ¯uorescent AHL monitor strain E. coli
MT102(pJBA89) was cultured and diluted during assays in
minimal ABT medium [(NH%)#SO%, 15±1 mM; Na#HPO%.
2H#O, 33±7 mM; KH#PO%, 22±0 mM; NaCl, 51±0 mM;
MgCl#.6H#O, 1±0 mM; CaCl#, 0±1 mM; FeCl$, 10lM;
thiamin, 7±4 lM] (Clark & Maalùe, 1967) supplemented with
0±5% glucose and 0±5% Casamino acids. Plasmid pJBA89
carries luxR and PluxI±gfp(ASV) fusion and encodes resistance
to 50 lg ampicillin ml−" (Andersen et al., 2001). The clp
lon double mutant E. coli KY2347 [MG1655 D(clpPX±lon)
1196: : cat] (Zhu & Winans, 2001) and the isogenic parent E.
coli MG1655 (wild-type E. coli K-12 ilvG rph-1) (Guyer et al.,
1981), both harbouring pJBA89, were cultured in LB diluted
20 times in ABT medium. Unless otherwise stated, the relevant
antibiotics at the above-mentioned concentrations were included
in cultures to ensure plasmid maintenance.
Furanone-promoted degradation of LuxR is protease
independent
Comparable results were obtained when the above
experiments were conducted in a clpP strain and in a lon
strain. In both cases activity was tested [LuxR controlled
PluxI±gfp(ASV)] and protein half-life was investigated
(data not shown). This indicated that neither the clpP
protease nor the lon protease affected stability of the
LuxR protein. We therefore tested expression of Gfp
from pJBA89 in a clp lon background as well as in the
isogenic clp+ lon+ parent. Strains KY2347 and MG1655
grew more slowly than our preferred MT102 strain. In
MG1655 the IIX%! value for compound 30 was found to
be 0±23 (compared to 0±16 in MT102). In the KY2347 (clp
lon) the IIX%! value for compound 30 was found to be
0±34.
DISCUSSION
From the few biochemical studies that have been
performed on the function of LuxR-type regulatory
proteins, a model of how AHLs cause transcription of
speci®c genes is beginning to emerge (Zhu & Winans,
2001; Welch et al., 2000; Qin et al., 2000; Zhu &
Winans, 1999). In vitro investigations with the CarR
protein of E. carotovora and the TraR protein of A.
tumefaciens, including DNA bandshift, ¯uorescence
quenching and tryptic digestion experiments, have
revealed that cognate AHLs directly interact with and
induce conformational changes in these regulatory
proteins (Zhu & Winans, 2001; Welch et al., 2000).
What remains uncertain is the effect these conformational
changes have on the behaviour of the protein.