(vzw)
VLAAMS INSTITUUT VOOR DE 2E(
FLANDERS MARINE INSTITUTE
Oostende - Belgium
E n v iro n m e n ta l M ic ro b io lo g y (2 0 0 7 ) 9 (1 0 ), 2 4 8 6 -2 4 9 5
1
d o i: 10.1111/j. 1 4 6 2 -2 9 2 0 .2 0 0 7 .0 1 367 .x
The natural furanone (5Z)-4-bromo-5(bromomethylene)-3-butyl-2(5W)-furanone disrupts
quorum sensing-regulated gene expression in Vibrio
harveyi by decreasing the DNA-binding activity of the
transcriptional regulator protein luxR
Tom D e fo ird t,1’2 C arol M. M iya m o to ,3
T h o m a s K. W o o d ,4 Edw ard A. M e ig h e n ,3
P a trick S o rg e lo o s ,2 W illy V erstraete1* and
Peter B o s s ie r2
le vels o f the L u x R Vh re s p o n s e re g u la to r p ro te in w ere
able to bind to its ta rg e t p ro m o te r seq ue nce s in w ild typ e V. ha rveyi. F in a lly, te sts w ith p u rifie d LuxRvh
p ro te in also sho w e d le s s s h ifts w ith fu ra n o n e -tre a te d
1L a b o ra to ry o f M ic ro b ia l E c o lo g y a n d T e c h n o lo g y
LuxRvh, w hereas the LuxR vh c o n c e n tra tio n w as fo u n d
(L a b M E T ), G h e n t U n iv e rs ity , C o u p u re L in k s 6 5 3 , 9 0 0 0
n o t to be alte red by th e fu ra n o n e (as de te rm in e d by
SDS-PAGE). T here fore, o u r data in d ic a te th a t th e fu ra ­
G h e n t, B e lg iu m .
B e lg iu m .
none b lo cks q u o ru m s e n s in g in V. h a rv e y i by re n d e r­
in g the q u o ru m s e n s in g m aster re g u la to r p ro te in
LuxRvh unable to bind to the p ro m o te r seq ue nce s o f
3D e p a rtm e n t o f B io c h e m is try , M c G ill U n iv e rs ity , M c In ty re
qu o ru m s e n s in g -re g u la te d genes.
2L a b o ra to ry o f A q u a c u ltu re a n d A rte m ia R e fe re n c e
C e n te r, G h e n t U n iv e rs ity , R o z ie r 4 4 , 9 0 0 0 G h e n t,
M e d ic a l S c ie n c e s B u ild in g , R o o m 8 1 8 , 3 6 5 5
P ro m e n a d e S ir W illia m O sier, M o n tre a l, C a n a d a
In tro d u c tio n
H 3 G 1Y6.
4A rtie M c F e rr in D e p a rtm e n t o f C h e m ic a l E n g in e e rin g ,
V ib rio h a rv e y i is a G ra m -n e g a tiv e , lu m in e s c e n t m a rin e
T e x a s A a n d M U n iv e rs ity , 3 1 2 2 T A M U , C o lle g e S ta tio n ,
b a c te riu m
T X 7 7 8 4 3 -3 1 2 2 , U S A .
c o lu m n a s w e ll a s in a s s o c ia tio n w ith m a rin e a n im a ls
th a t c a n
be
fo u n d
fre e -liv in g
in th e w a te r
(T h o m p s o n e ta !., 2 0 0 4 ). W ith th e ra p id d e v e lo p m e n t o f
th e a q u a c u ltu re in d u s try , th e s p e c ie s is b e c o m in g in c re a s ­
S u m m a ry
in g ly re c o g n iz e d a s a n im p o rta n t p a th o g e n o f m a rin e v e r ­
T h is s tu d y aim ed at g e ttin g a deeper in s ig h t in the
m o le c u la r m ech an ism b y w h ic h th e n a tu ra l fu ra n o n e
te b ra te s a n d
(5Z )-4-b rom o-5 -(bro m o m ethylen e)-3-b utyl-2(5 H )fu ra n o n e d is ru p ts q u o ru m se n s in g in V ib rio harveyi.
B io lu m in e s c e n c e e xp e rim e n ts w ith sig n a l m o le cu le
re c e p to r d o u b le m u ta n ts revealed th a t the fu ra n o n e
b lo c k s a ll three ch a n n e ls o f the V. h a rv e y i qu o ru m
re s is ta n c e
s e n s in g system . In fu rth e r e x p e rim e n ts u s in g
m u ta n ts w ith m u ta tio n s in the q u o ru m s e n s in g sig n a l
tra n s d u c tio n pathway, th e co m p o u n d w as fo u n d to
b lo c k q u o ru m s e n sin g -re g u la te d b io lu m in e s c e n c e by
s e n s in g (D e fo ird t e ta !., 2 0 0 4 ).
in te ra c tin g w ith a co m p o n e n t lo ca te d d o w n stre a m o f
th e Hfq p ro te in . F u rth e rm o re , reve rse tra n s c rip ta s e
re a l-tim e p o lym e ra se ch a in re a ctio n w ith s p e c ific
p rim e rs sho w e d th a t th e re w as no e ffe c t o f th e fu ra ­
a te d b y th e H a rv e y i A u to in d u c e r 1 (H A I-1 ), a n a c y la te d
none on luxRvh m RN A levels in w ild -ty p e V. h a rv e y i
ce lls. In co n tra s t, m o b ility s h ift a ssa ys sho w e d th a t in
the p re se n ce o f th e fu ra n o n e , s ig n ific a n tly lo w e r
in v e r te b ra te s
(A u s tin
a n d Z h a n g , 2 0 0 6 ).
B e c a u s e o f th e d e v e lo p m e n t a n d s p re a d o f a n tib io tic
in th e s e
b a c te ria , a n tib io tic tre a tm e n ts a re
b e c o m in g in e ffic ie n t (K a r u n a s a g a r e ta !., 1 9 9 4 ; M o ria rty ,
1 9 9 8 ) a n d th e re fo re , a lte rn a tiv e c o n tro l s tra te g ie s a re
b e in g d e v e lo p e d . O n e o f th e s e s tra te g ie s is th e d is ru p tio n
o f b a c te ria l c e ll-to -c e ll c o m m u n ic a tio n ,
c a lle d
q u o ru m
U n lik e m a n y o th e r G ra m -n e g a tiv e b a c te ria , V. h a rv e y i
h a s b e e n re p o rte d to u se a m u ltic h a n n e l q u o ru m s e n s in g
s y s te m (F ig . 1). T h e firs t c h a n n e l o f th is s y s te m is m e d i­
h o m o s e rin e la c to n e (A H L ) (C a o a n d M e ig h e n , 1 9 8 9 ). T h e
s e c o n d c h a n n e l is m e d ia te d b y th e s o -c a lle d A u to in d u c e r
2 (A I-2 ), w h ic h is a fu ra n o s y l b o ra te d ie s te r (C h e n e t a i ,
2 0 0 2 ). T h e c h e m ic a l s tru c tu re o f th e th ird a u to in d u c e r,
c a lle d C h o le ra e A u to in d u c e r 1 (C A I-1 ) is s till u n k n o w n
(H e n k e a n d B a s s le r, 2 0 0 4 a ). A ll th re e a u to in d u c e rs a re
d e te c te d a t th e c e ll s u r fa c e a n d a c tiv a te o r in a c tiv a te
R eceived
11 May, 2007; accepted
15 May, 2007. ‘ For
correspondence. E-mail W illy.V erstraete@ U G ent.be ; Tel. (+32) 9
264 59 76; Fax (+32) 9 264 62 48.
ta rg e t
gene
e x p re s s io n
by
a
p h o s p h o ry la tio n /
d e p h o s p h o ry la tio n s ig n a l tra n s d u c tio n c a s c a d e . P h e n o ­
ty p e s th a t w e re fo u n d to b e c o n tro lle d b y th is q u o ru m
© 2007 The Authors
Journal com pilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd
M o d e o f a c tio n o f b r o m in a te d fu r a n o n e in V ib rio h a rv e y i
2487
HAI-1
CAI-1
T> LuxP
LuxN
LuxN
LuxM
LuxU
LuxO
54
□
LüxO
G
sRNAs + Hfq
sRNAs + H fq
l
LuxRvh C
LuxR
;>
Target genes
F ig . 1. Quorum sensing in Vibrio harveyi. The LuxM , LuxS and C qsA e nzym es synthesize the a u to in d u c e rs HAI-1, AI-2 and CAI-1
respectively. These autoinducers are detected at the cell su rfa ce by the LuxN, LuxP -LuxQ and CqsS re c e p to r proteins respectively.
A. A t low signal m o lecule concentration, the receptors a uto ph o sp ho rylate and tra n sfe r phosphate to L u x O via LuxU. Phosphorylation activates
LuxO, w hich to g e th er w ith a 54 activates the production o f sm all regulatory R N As (sR NAs). These s R N A s, together with the chaperone Hfq,
d estabilize the m R N A e ncoding the response regulator LuxRvh. Therefore, in th e absence of a utoinducers, the L u x R vh protein is not produced.
B. In the presence of high concentrations of the autoinducers, th e receptor proteins sw itch from kinases to phosphatases, w hich results in
dephosphorylation of LuxO . D ephosphorylated LuxO is inactive and therefore, th e sR N A s are not fo rm e d and the response regulator LuxRvh is
produced (adapted from H enke and Bassler, 2004a).
s e n s in g s y s te m in c lu d e b io iu m in e s c e n c e (B a s s le r e t ai.,
h a lo g e n a te d fu ra n o n e s . T h e a u th o rs c o u ld n o t c o n c lu d e
1 9 9 3 ) a n d th e p r o d u c tio n o f s e v e ra l v iru le n c e fa c to rs s u c h
th a t th e fu ra n o n e s b in d to th e A H L -b in d in g c a v ity a n d
as
s u g g e s te d th a t fu r a n o n e s d o n o t c o m p e te in a c la s s ic w a y
a ty p e
III
s e c r e tio n
s y s te m
(H e n k e
and
B a s s le r,
2 0 0 4 b ), e x tr a c e llu la r to x in (M a n e fie ld e t a l. , 2 0 0 0 ) a n d a
s id e ro p h o re
M e a n w h ile , s e v e ra l re s e a rc h g ro u p s p ro v id e d e v id e n c e
fo u n d th a t v iru le n c e o f th e b a c te riu m to w a rd s th e b rin e
th a t h a lo g e n a te d fu r a n o n e s a ls o d is ru p t q u o ru m s e n s in g -
A r te m ia
and
B a s s le r, 2 0 0 0 ).
fra n c is c a n a
is
a ls o
R e c e n tly ,
w ith th e s ig n a l m o le c u le s .
we
s h rim p
(L ille y
re g u la te d
by
its
q u o ru m s e n s in g s y s te m (D e fo ird t e t a l. , 2 0 0 5 ).
re g u la te d g e n e e x p re s s io n in V. h a rv e y i (M a n e fie ld e ta l.,
2 0 0 0 ; R e n e t a l. , 2 0 0 1 ; M c D o u g a ld e t a l. , 2 0 0 3 ; D e fo ird t
O n e o f th e m e c h a n is m s to d is ru p t b a c te ria l q u o ru m
e t a l. ,
2 0 0 6 ).
H o w e v e r,
because
th e
q u o ru m
s e n s in g
s e n s in g is th e u s e o f h a lo g e n a te d fu ra n o n e s , s u c h a s th e
s y s te m o f V. h a rv e y i is q u ite d iffe re n t fro m th e L u x l/L u x R V(-
n a tu ra l
(5 Z )- 4 -b r o m o -5 - (b ro m o m e th y le n e )- 3 -
ty p e o f q u o ru m s e n s in g s y s te m a n d d o e s n o t c o n ta in a
b u ty l-2 (5 H )-fu ra n o n e . T h e s e fu ra n o n e s (n a tu ra l o c c u rrin g
L u x R v i h o m o lo g u e (M ilto n , 2 0 0 6 ), th e m o le c u la r m e c h a ­
fu r a n o n e
c o m p o u n d s a s w e ll a s s y n th e tic d e riv a tiv e s ) w e re fo u n d
n is m o f q u o ru m s e n s in g d is ru p tio n in th is s p e c ie s m u s t b e
to d is ru p t th e e x p re s s io n o f d iffe re n t A H L -re g u la te d p h e ­
d iffe re n t fro m th a t in th e L u x l/L u x R Vr ty p e o f s y s te m . C o n ­
n o ty p e s in s e v e ra l G ra m -n e g a tiv e s p e c ie s , w ith o u t a ffe c t­
s e q u e n tly , in th is stud y, w e a im e d a t d e fin in g th e m o le c u la r
in g th e ir g ro w th (H e n tz e r a n d G iv s k o v , 2 0 0 3 ; R a s m u s s e n
m e c h a n is m b y w h ic h th e n a tu ra l fu r a n o n e [5 7 ) - 4 -b ro m o -
a n d G iv s k o v , 2 0 0 6 ). B e c a u s e o f th e s tru c tu ra l s im ila rity
5 -(b ro m o m e th y le n e )- 3 -b u ty l-2 (5 H )- fu ra n o n e (F ig . 2 ) d is ­
b e tw e e n A H L m o le c u le s a n d th e fu ra n o n e s , it w a s o r ig i­
ru p ts q u o ru m s e n s in g -re g u la te d g e n e e x p re s s io n in th is
n a lly h y p o th e s iz e d th a t th e s e c o m p o u n d s d is ru p t A H L -
d iffe re n t ty p e o f q u o ru m s e n s in g s y s te m .
m e d ia te d
q u o ru m
s e n s in g
in th e
L u x l/L u x R Vr t y p e
of
q u o ru m s e n s in g s y s te m b y c o m p e titiv e ly b in d in g to th e
A H L re c e p to r s ite o f th e
V ib rio fis c h e ri L u x R vl p ro te in
(G iv s k o v e t a l. , 1 9 9 6 ). L a te r o n , it w a s s h o w n th a t th e
h a lo g e n a te d
fu r a n o n e s p ro m o te
ra p id tu rn o v e r o f th e
L u x R v r ty p e A H L re c e p to r p ro te in , re d u c in g th e a m o u n t o f
L u x R v i a v a ila b le
to
in te ra c t w ith A H L a n d
tra n s c rip tio n a l r e g u la to r (M a n e fie ld
to
a c t as
e ta l., 2 0 0 2 ).
M o re
re c e n tly , K o c h a n d c o lle a g u e s (2 0 0 5 ) u s e d s ite -d ire c te d
m u ta g e n e s is to s tu d y in te ra c tio n s b e tw e e n L u x R v i a n d
F ig . 2. S tructure o f the natural furanone used in this study.
© 2007 The A uthors
Journal com pilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environm ental M icrobiology, 9, 2 4 8 6 -2 4 9 5
2488
T. D e fo ir d t e t al.
th e im p a c t o f th e fu r a n o n e o n th e d iffe re n t c h a n n e ls , th e
10-9
s ig n a l m o le c u le re c e p to r d o u b le m u ta n ts J A F 3 7 5 (s e n s o r
H AI-1 ” , s e n s o r A I-2 ” , s e n s o r C A I-1 +), J M H 5 9 7 (s e n s o r
H A M ",
sensor
A I-2 +,
sensor
C A I-1 ” )
and
JM H 612
(s e n s o r H A I-1 +, s e n s o r A I- 2 ” , s e n s o r C A I-1 ” ) w e re u se d .
B e c a u s e o f th e m u ta te d re c e p to rs , b io lu m in e s c e n c e in
th e s e m u ta n ts is o n ly re s p o n s iv e to o n e o f th e th r e e s ig n a l
m o le c u le s (H e n k e a n d B a s s le r, 2 0 0 4 a ). T h e m u ta n ts w e re
g ro w n to h ig h c e ll d e n s itie s , a n d a fte r fu ra n o n e a d d itio n ,
b io lu m in e s c e n c e w a s fo u n d to b e b lo c k e d in all th re e
d o u b le m u ta n ts in a c o n c e n tr a tio n -d e p e n d e n t w a y s im ila r
to th e o n e o b ta in e d f o r th e w ild ty p e (F ig . 5). T h is in d i­
1e+3
-------------------- 1-----------------------1-----------------------1-----------------------1---------------------- 1----------------------
0.0
05
1.0
1.5
20
2.5
c a te s th a t a ll th r e e c h a n n e ls
o f th e q u o ru m
s e n s in g
s y s te m w e re b lo c k e d .
Time (h)
F ig . 3. B iolum inescence per unit cell d en sity in w ild-type Vibrio
harveyi B B 120 as a function o f tim e, w ithout (open sym bols) and
with (filled sym bols) the n atural fu ranone (5Z)-4-brom o-5(brom om ethylene)-3-butyl-2(5H )-furanone (50 m g I"1). The error
bars represent the standard d eviation of th re e replicates. Note that
RLU is the relative unit o f lum inescence reported by the Lum ac
B iocounter M 2500 lum inom eter.
Im p a c t o f th e fu ra n o n e o n b io lu m in e s c e n c e o f
c o n s titu tiv e ly lu m in e s c e n t V. h a rv e y i m u ta n ts w ith
m u ta tio n s in th e q u o ru m s e n s in g s ig n a l tra n s d u c tio n
cascade
B e c a u s e th e th r e e s ig n a l m o le c u le s h a v e q u ite d iffe re n t
c h e m ic a l s tru c tu re s (H e n k e a n d B a s s le r, 2 0 0 4 a ), w e s u s ­
R esults
p e c te d th a t th e fu r a n o n e d id n o t b lo c k q u o ru m s e n s in g Im p a c t o f th e fu ra n o n e o n q u o ru m s e n s in g -r e g u la te d
re g u la te d
b io lu m in e s c e n c e o f w ild -ty p e V. h a rv e y i
a u to in d u c e rs fo r re c e p to r s ite s b u t ra th e r b y in te rfe rin g w ith
B io lu m in e s c e n c e
is
one
of
th e
p h e n o ty p e s
th a t
is
re g u la te d b y th e V. h a r v e y i q u o ru m s e n s in g s y s te m a n d
th e re fo re ,
n a tu ra l
in
a
fu r a n o n e
firs t
e x p e rim e n t,
th e
im p a c t
of
th e
(5 Z )- 4 -b r o m o -5 - (b ro m o m e th y le n e )- 3 -
b u ty l-2 (5 H )-fu ra n o n e o n th e b io lu m in e s c e n c e o f w ild -ty p e
V. h a r v e y i w a s d e te rm in e d . S tra in B B 1 2 0 w a s g ro w n in
L B 20 m e d iu m
to h ig h c e ll d e n s ity in o r d e r to a c tiv a te
q u o ru m s e n s in g -r e g u la te d b io lu m in e s c e n c e , a fte r w h ic h
b io lu m in e s c e n c e
by
c o m p e tin g
w ith
th e
th e q u o ru m s e n s in g s ig n a l tra n s d u c tio n . In o r d e r to te s t th is
h y p o th e s is , th e e ffe c ts o f th e fu r a n o n e o n b io lu m in e s c e n c e
o f m u ta n ts th a t w e re fix e d in th e h ig h c e ll-d e n s ity c o n fig u ­
ra tio n a t d iffe re n t s ta g e s in th e q u o ru m s e n s in g s ig n a l
tra n s d u c tio n
c a s c a d e w e re
in v e s tig a te d . T h e
m u ta n ts
J A F 5 5 3 a n d J A F 4 8 3 c o n ta in a p o in t m u ta tio n in th e lu x U
a n d lu x O g e n e s , re s p e c tiv e ly , th a t re n d e r th e L u xU a n d
L u x O p ro te in s in c a p a b le o f p h o s p h o re la y (F re e m a n a n d
th e fu ra n o n e w a s a d d e d to th e m e d iu m a t 5 0 m g I"1. T h e
c o m p o u n d b lo c k e d b io lu m in e s c e n c e o f B B 1 2 0 , w ith o v e r 3
10 0 0
lo g u n its d iffe re n c e b e tw e e n th e fu r a n o n e -tre a te d a n d th e
u n tre a te d c u ltu re s a lre a d y 0 .5 h a fte r th e a d d itio n o f th e
c
fu ra n o n e (F ig . 3). C o n s is te n t w ith th is , lu c ife ra s e a c tiv ity in
0)
o
p ro te in ly s a te s o f fu r a n o n e -tre a te d B B 1 2 0 c e lls w a s fo u n d
I
Q.
100
to h a v e d e c re a s e d w ith a p p ro x im a te ly 1 lo g u n it 0 .5 h a fte r
th e a d d itio n o f 5 0 m g I” 1 fu r a n o n e (F ig . 4). T h e b a c te ria l
a lk a lin e p h o s p h a ta s e a c tiv itie s o f th e p ro te in ly s a te s w e re
a ls o m e a s u re d in o r d e r to v e r ify th a t th e fu ra n o n e h a d no
e ffe c t o n p h e n o ty p e s th a t a re n o t re g u la te d b y th e q u o ru m
ill
s e n s in g s y s te m . A s e x p e c te d , th e r e w a s n o s ig n ific a n t
d iffe re n c e
in
b a c te ria l
a lk a lin e
p h o s p h a ta s e
a c tiv itie s
b e tw e e n fu r a n o n e -tre a te d a n d u n tre a te d c e lls (F ig . 4).
0.0
0.5
1.0
1.5
2.0
Time (h)
Im p a c t o f th e fu r a n o n e o n b io lu m in e s c e n c e o f V. h a rv e y i
a u to in d u c e r r e c e p to r d o u b le m u ta n ts
T h e V. h a rv e y i q u o ru m s e n s in g s y s te m c o n s is ts o f th re e
c h a n n e ls , w ith e a c h c h a n n e l b e in g a c tiv a te d b y a d iffe re n t
ty p e o f s ig n a l m o le c u le (F ig . 1). In o r d e r to d e te rm in e
F ig . 4. Luciferase (circles) and bacte ria l alkaline phosphatase
(triangles) activitie s in protein lysates of w ild-type Vibrio harveyi
B B120 as a function o f time, w itho u t (open sym bols) and with (filled
sym bols) the n atural furanone (5 Z)-4-brom o-5-(brom om ethylene)3-butyl-2(5/-/)-furanone (50 mg I 1). The error bars represent the
standard d eviation of fo u r independent experim ents.
© 2 0 0 7 T h e A u th o rs
Jo u rn a l co m p ila tio n © 2 0 0 7 S o c ie ty fo r A p p lie d M ic ro b io lo g y and B la c k w e ll P u b lis h in g Ltd, Environm ental M icro biolog y 9, 2 4 8 6 -2 4 9 5
M o d e o f a c tio n o f b r o m in a te d fu r a n o n e in V ib rio h a rv e y i
2489
re v e rs e tra n s c rip ta s e r e a l- tim e p o ly m e ra s e c h a in re a c tio n
(P C R ) w ith s p e c ific p r im e r s a n d th e R N A p o ly m e ra s e A
120
s u b u n it ( rp o A ) m R N A w a s a n a ly s e d a s a c o n tro l o f a
n o n -q u o ru m s e n s in g -r e g u la te d g e n e . F o r b o th g e n e s , th e
100
m R N A le v e ls fo llo w e d a s im ila r tre n d , w ith n o s ig n ific a n t
8
c
d iffe re n c e b e tw e e n fu r a n o n e -tre a te d a n d u n tre a te d c e lls
<u
o
0)
Ç
£
(F ig . 7). F ro m th e s e re s u lts , w e c o n c lu d e th a t th e fu r a ­
n o n e h a s n o e ffe c t o n lu x R v i, m R N A le v e ls .
.2
o
co
B 8120 (Wildlypo)
JAF375 (CAI-1)
JM H597 (AI-2)
JM H612 (HAI-1)
001
0 1
1
10
[Furanone] (mg I-1)
F ig . 5. B iolum inescence of the Vibrio h a rv e y i w ild type (BB120)
and the double m utants JA F375 (sensor HAI-1 ", sensor AI-2",
se nso r C AI-14), JM H 597 (sensor HAI-1", se nso r A I-2 1', sensor
C AI-1") and JM H 612 (sensor HAI-14, se nso r AI-2", sensor C AI-1")
as a function of the concentration of the n atural furanone
(5Z)-4-brom o-5-(brom om ethylene)-3-butyl-2(5H )-furanone.
L um inescence m easurem ents w ere perform ed 0.5 h after the
addition of the furanone. For each strain, the biolum inescence
w itho u t the addition of furanone w as set at 100% and the other
sa m ple s w ere norm alized accordingly. T h e e rro r b ars re pre se n t the
standard deviation of three replicates.
B a s s le r, 1 9 9 9 a ,b ). S tra in B N L 2 5 8 h a s a T n 5 in s e rtio n in th e
h fq g e n e , re s u ltin g in a n o n -fu n c tio n a l H fq p ro te in (L e n z
e t al., 2 0 0 4 ). H e n c e , b e c a u s e o f th e n a tu re o f th e s e m u ta ­
tio n s , th e th re e m u ta n ts a re c o n s titu tiv e ly lu m in e s c e n t a n d
th e re fo re , b lo c k in g lu m in e s c e n c e in o n e o f th e m w o u ld
in d ic a te th a t th e fu ra n o n e a c ts d o w n s tre a m o f th e m u ta te d
c o m p o n e n t. T h e fu r a n o n e , a t 5 0 m g I-1, b lo c k e d lu m in e s ­
F ig . 6. B iolum inescence of w ild -typ e Vibrio h a rve yi B B120 and the
m utants JA F553 (luxU H 58A ), JA F483 (luxO D47A) and BN L258
(hfq::Jr\5lacZ) w ithout (white bars) and with (striped bars) the
natural furanone (5Z)-4-brom o-5-(brom om ethylene)-3-butyl-2(5/-/)furanone (50 mg h1). M e asu re m e nts w ere perform ed 0.5 h after the
addition of the furanone. The e rro r bars represent the standard
deviation of three replicates. N ote that RLU is th e relative unit of
lum inescence reported by the Lum ac B iocounter M 2500
lum inom eter.
c e n c e in all th re e m u ta n ts (F ig . 6). In w ild -ty p e V. h a rv e y i,
th e H fq p ro te in a c ts to g e th e r w ith s m a ll re g u la to ry R N A s to
d e s ta b iliz e th e m R N A o f th e m a s te r re g u la to r LuxR vh (se e
F ig . 1). In th e m u ta n t B N L 2 5 8 , a tr a n s p o s o n in s e rtio n h a s
re n d e re d th e H fq p ro te in n o n -fu n c tio n a l a n d th e re fo re , in
th is m u ta n t, th e luxR vh m R N A c a n n o t b e d e s ta b iliz e d b y th e
q u o ru m s e n s in g s ig n a l tra n s d u c tio n s y s te m , re s u ltin g in a
c o n s titu tiv e ly
e x p re s s e d
b io lu m in e s c e n c e
(L e n z
e ta l.,
2 0 0 4 ). T h e fa c t th a t th e fu ra n o n e b lo c k e d b io lu m in e s c e n c e
in th is m u ta n t in d ic a te s th a t it a c ts d o w n s tre a m o f H fq , i.e.
a t th e le v e l o f th e luxR vi, m R N A a n d /o r th e LuxR vh p ro te in .
Im p a c t o f th e fu ra n o n e o n luxR vi, m R N A le v e ls
In o r d e r to v e rify w h e th e r th e fu r a n o n e in d e e d a ffe c ts th e
q u o ru m s e n s in g m a s te r re g u la to r, a n e x p e rim e n t w a s s e t
u p in w h ic h th e im p a c t o f th e a d d itio n o f th e c o m p o u n d on
luxR vh m R N A w a s s tu d ie d . W ild -ty p e
V. h a r v e y i B B 1 2 0
w a s g ro w n to h ig h c e ll d e n s ity , a fte r w h ic h th e n a tu ra l
fu r a n o n e w a s a d d e d a t 5 0 m g I-1. T h e a d d itio n o f th e
fu ra n o n e re s u lte d in a ra p id d e c re a s e in lu m in e s c e n c e
(F ig . 3 ). luxR vh m R N A le v e ls w e re q u a n tifie d re la tiv e ly b y
T im e (h )
F ig . 7. D ifference in luxRvh (filled sym bols) and rpoA (open
sym bols) m R NA levels between w ild-type Vibrio h a rve yi B B120
cells tre a te d w ith the natural furanone (5Z)-4-brom o-5(brom om ethylene)-3-butyl-2(5H )-furanone (50 mg b1) and untreated
cells, as determ ined by reverse tra n scrip ta se real-tim e PCR with
specific prim ers. The error bars represent the standard deviation of
two independent experim ents.
© 2007 The Authors
Journal com pilation © 2007 S ociety for Applied M icrobiology and Blackwell Publishing Ltd, E nvironm ental Microbiology, 9, 2 4 86-2 495
2490
T. D e fo ir d t e t al.
A
B
- Fu ran one
Oh
0.5h
1
i
2
%
1h
3
< ■»
+ F u ra n o n e
2h
4
Oh
5
0.5h
6
1h
7
2h
8
B lank
9
<T
^
^
A^
A^
<c°
m %
LuxR
F ig . 8. LuxRvh D NA binding as determ ined by m obility shifts.
A. A utoradiograph after 5% polyacrylam ide gel electro p h ore sis of 5 ' ’•'P-labelled luxRvn prom oter DNA c o n ta in in g the LuxRVh binding sites,
m ixed w ith 0.5 pg of protein lysates from w ild-type Vibrio h a rve yi BB120: lanes 1 -4 , addition of lysates ta k e n at d iffe re nt tim e points from an
untreated culture; lanes 5 -8 , addition of lysates taken at d iffe re nt tim e points from a cu lture treated w ith 5 0 m g I”1 o f the natural furanone
(5Z)-4-brom o-5-(brom om ethylene)-3-butyl-2(5H )-furanone; last la n e . b la n k (addition of lysate from the /uxf?-negative strain MR1130).
B. A utoradiograph after 5% polyacrylam ide gel e lectrophoresis of 5 ' ??P -labelled lu x R .i, p rom oter DNA c o n ta in in g the LuxRvh binding sites,
m ixed w ith purified LuxRvh, which w as incubated in vitro fo r 1 h w ith or w itho u t the furanone (50 mg I*1).
C. S D S -P A G E of the sam e sam ples as in panel B.
Im p a c t o f th e fu ra n o n e o n LuxR vh p r o te in le v e ls a n d
B B 1 2 0 c u ltu re s in o r d e r to s to p tra n s la tio n o f LuxRvh- In
Lu xR vh b in d in g to its ta rg e t p r o m o te r s e q u e n c e s
th e s e c u ltu re s , th e re w a s n o e ffe c t o n LuxR vh m o b ility
M o b ility s h ifts u s in g ra d io la b e lle d lu x R Vh p ro m o te r D N A
c o n ta in in g th e LuxR vh b in d in g s ite s s h o w e d le s s s h ifts
w ith c e ll ly s a te s fro m fu r a n o n e -tre a te d c e lls w h e n c o m ­
p a re d w ith u n tre a te d c e lls (F ig . 8 A ), in d ic a tin g th a t th e re
w e re s ig n ific a n tly lo w e r le v e ls o f th e p ro te in a b le to b in d
th e p r o m o te r D N A . In o r d e r to q u a n tify th e d iffe re n c e in
bound
Lu xR vh
le v e ls
b e tw e e n
fu r a n o n e -tre a te d
and
u n tre a te d c e lls , a titra tio n fo r m o b ility s h ifts o f luxR vt, p r o ­
m o te r D N A u s in g th e 2 -h s a m p le s w a s p e rfo rm e d . T h is
titra tio n re v e a le d th a t 0 .0 2 5 p g ly s a te o f u n tre a te d c e lls
g a v e th e s a m e s h ift a s 0 .5 g g ly s a te o f fu r a n o n e -tre a te d
c e lls (d a ta n o t s h o w n ), w h ic h in d ic a te s th a t th e re w a s a
2 0 -fo ld d iffe re n c e in LuxR vn le v e ls b o u n d to th e p r o m o te r
D N A . T h e lu x C D A B E G H p r o m o te r D N A w a s a ls o u s e d
to c h e c k fo r LuxR vn s h ifts , w ith s im ila r fin d in g s (F ig . 9).
s h ifts in th e a b s e n c e o f fu r a n o n e th r o u g h o u t th e 1 h o f
in c u b a tio n , w h e r e a s in th e p r e s e n c e o f fu ra n o n e , a g a in
le s s s h ifts o c c u rre d (d a ta n o t s h o w n ). T h is s u g g e s ts th a t
LuxRvh h a s q u ite a s lo w tu r n o v e r a n d th a t th e fu ra n o n e
a c ts o n p re -e x is tin g L u x R Vh. F in a lly, th e fu r a n o n e w a s
a d d e d to p u rifie d L u x R v„, a n d a fte r s u b s e q u e n t in c u b a ­
tio n o f th e
m ix tu re , a g a in s ig n ific a n tly lo w e r le v e ls o f
LuxR vh w e re fo u n d to b in d to th e p r o m o te r D N A w h e n
c o m p a re d w ith u n tre a te d LuxR vt, (F ig . 8 B ). In te re s tin g ly ,
S D S -P A G E s h o w e d th a t th e c o n c e n tra tio n o f LuxR vh in
th e m ix tu re s w a s n o t a ffe c te d b y th e fu ra n o n e (F ig . 8 C ).
T o g e th e r, th e s e d a ta lead to th e c o n c lu s io n th a t th e fu r a ­
n o n e c o m p o u n d re n d e rs
ta r g e t
p r o m o te r
L u x R Vh u n a b le to b in d to its
sequences,
w ith o u t
d e g ra d in g
th e
p ro te in .
T h e s e o b s e rv a tio n s c o u ld b e e x p la in e d b y th e fu r a n o n e
e ith e r d e c re a s in g tra n s la tio n o r in c re a s in g tu r n o v e r o f
LuxR vn, o r a lte rin g th e p ro te in in s u c h a w a y th a t it is
re n d e re d
D iscu ssio n
u n a b le to b in d to its ta rg e t p ro m o te r. S o m e
D is e a s e c a u s e d b y a n tib io tic re s is ta n t lu m in e s c e n t v ib rio s
a d d itio n a l te s ts w e re p e rfo r m e d to c le a r th is u p . F irs t,
is a s e r io u s p ro b le m in th e a q u a c u ltu re in d u s try (A u s tin
5 0 m g I 1 c h lo r a m p h e n ic o l
a n d Z h a n g , 2 0 0 6 ). R e ce n t in v e s tig a tio n s h a v e p o in te d o u t
w as
added
to
V. h a r v e y i
© 2007 The A uthors
Journal com pilation © 2007 Society for Applied M icrobiology and Blackwell Publishing Ltd, Environm ental M icrobiology, 9, 2 4 8 6 -2 4 9 5
M o d e o f a c tio n o f b r o m in a te d fu r a n o n e in V ib rio h a rv e y i
- F u ra n o n e
Oh
0 .5 h
b lo c k b io lu m in e s c e n c e in w ild -ty p e V. h a rv e y i B B 1 2 0 in a
+ F u ra n o n e
1h
2h
Oh
0 .5 h
1h
24 91
2h
B la n k
c o n c e n tr a tio n -d e p e n d e n t w a y . M o re o v e r, lu c ife ra s e a c tiv ­
ity
w as
s ig n ific a n tly
d e c re a s e d
in
p ro te in
ly s a te s
of
fu r a n o n e -tre a te d B B 1 2 0 c e lls . In o rd e r to d e te rm in e th e
e ffe c t o f th e fu ra n o n e o n th e th re e d iffe re n t c h a n n e ls o f
th e I/, h a r v e y i q u o ru m s e n s in g s y s te m , its im p a c t o n b io lu ­
m in e s c e n c e
m u ta n ts
of
th e
JAF375,
s ig n a l
m o le c u le
JM H 597
and
re c e p to r
JM H 612
d o u b le
(H e n k e
and
B a s s le r, 2 0 0 4 a ) w a s s tu d ie d . T h e c o m p o u n d w a s s h o w n
to b lo c k b io iu m in e s c e n c e in all th re e d o u b le m u ta n ts in a
p a tte rn s im ila r to th e o n e o b ta in e d fo r th e w ild ty p e .
B e c a u s e o f th e m u ta te d re c e p to rs , b io lu m in e s c e n c e in
th e s e m u ta n ts is o n ly r e s p o n s iv e to o n e o f th e th r e e s ig n a l
m o le c u le s , w h ic h im p lie s th a t a ll th re e c h a n n e ls o f th e
q u o ru m
s e n s in g
s y s te m
w e re
b lo c k e d .
These
d a ta
c o n firm th e re p o rts b y R e n a n d c o lle a g u e s (2 0 0 1 ) a n d
M c D o u g a ld a n d c o lle a g u e s (2 0 0 3 ), w h o d e te rm in e d th e
im p a c t
of
th e
fu r a n o n e
on
th e
A I-2 -
a n d /o r
H A I-1 -
m e d ia te d c h a n n e ls o f th e s y s te m b y u s in g th e A I-2 a n d /o r
H A I-1 re c e p to r m u ta n ts B B 8 8 6 a n d B B 1 7 0 (w h ic h th u s
w e re s till re s p o n s iv e to b o th H A I-1 a n d C A I-1 a n d A I-2
a n d C A I-1
re s p e c tiv e ly ). M o re o v e r, o u r re s u lts in d ic a te
th a t th e fu ra n o n e a ls o b lo c k s th e C A I-1 -m e d ia te d c h a n n e l
o f th e V. h a r v e y i q u o ru m s e n s in g s y s te m .
F ig . 9. LuxRvt, D N A bin d ing as dete rm in e d b y m obility shifts.
Autoradiograph after 5% p olyacrylam ide gel electrophoresis of 5 '
32P-labelled luxC D A B E G H p rom oter D NA co ntaining the LuxRvh
binding sites, m ixed w ith 0.5 p g of protein lysates from w ild-type
Vibrio h a rv e y i BB120: lanes 1 -4 , addition of lysates taken at
different tim e points from an untreated culture; lanes 5 -8 , addition
o f lysates ta ke n a t different tim e points from a culture treated with
50 mg I"1 of the natural furanone (5Z)-4-brom o-5-(brom om ethylene)3-butyl-2(5H )-furanone; last lane, blank (addition o f lysate from the
/ux/?-negative strain MR1130).
P re v io u s ly , th e h y p o th e s is th a t p re v a ile d in lite ra tu re
w as
th a t
th e
fu r a n o n e s
d is ru p t
q u o ru m
s e n s in g
in
V. h a r v e y i b y d is p la c in g th e s ig n a l m o le c u le s fro m th e ir
re c e p to rs
(M a n e fie ld
e t a l. ,
2000;
R en
e t a l. ,
2 0 0 1 ).
H o w e v e r, b e c a u s e th e th r e e s ig n a l m o le c u le s h a v e q u ite
d iffe re n t c h e m ic a l s tru c tu re s (a lth o u g h th e e x a c t s tru c tu re
o f C A I-1 is s till u n k n o w n ; H e n k e a n d B a s s le r, 2 0 0 4 a ), w e
s u s p e c te d
th a t
th e
s e n s in g -r e g u la te d
fu r a n o n e
d id
not
b lo c k
q u o ru m
b io lu m in e s c e n c e b y c o m p e tin g
w ith
th e a u to in d u c e rs fo r re c e p to r s ite s b u t ra th e r b y in te rfe rin g
th a t d is ru p tio n o f th e q u o ru m s e n s in g s y s te m o f th e s e
w ith th e q u o ru m s e n s in g s ig n a l tra n s d u c tio n . In o r d e r to
b a c te ria
te s t th is h y p o th e s is , th e e ffe c ts o f th e fu ra n o n e o n b io lu ­
by
u s in g
h a lo g e n a te d fu r a n o n e s c o u ld
be a
p ro m is in g a lte rn a tiv e b io c o n tro l s tra te g y (M a n e fie ld e t al.,
m in e s c e n c e o f m u ta n ts th a t w e re fix e d in th e h ig h c e ll-
2 0 0 0 ; D e fo ird t e t a l., 2 0 0 6 ). In v ie w o f th e p o te n tia l p ra c ­
d e n s ity c o n fig u ra tio n a t d iffe re n t s ta g e s in th e q u o ru m
tic a l a p p lic a tio n s o f th is ty p e o f c o m p o u n d s , it is o f s ig n ifi­
s e n s in g s ig n a l tra n s d u c tio n c a s c a d e w e re in v e s tig a te d
c a n t in te re s t to d e fin e th e m o d e o f a c tio n o f th e fu ra n o n e s
(i.e . L u x U , L u x O a n d H fq). W e fo u n d th a t th e fu ra n o n e
in th e s e b a c te ria .
in th is s tu d y , w e a im e d a t
b lo c k e d b io lu m in e s c e n c e in th e h fq m u ta n t B N L 2 5 8 . H fq
e lu c id a tin g th e m o le c u la r m e c h a n is m o f q u o ru m s e n s in g
is a c h a p e ro n e p ro te in th a t a c ts to g e th e r w ith s m a ll re g u ­
d is ru p tio n
la to ry R N A s to d e s ta b iliz e th e m R N A o f th e m a s te r re g u ­
by
H ence,
th e
n a tu ra l
fu ra n o n e
(5 Z )-4 -b ro m o -5 -
(b ro m o m e th y le n e )-3 - b u ty l- 2 (5 H ) -fu r a n o n e in V. h a rv e y i.
In a firs t s e rie s o f e x p e rim e n ts , th e im p a c t o f th e n a tu ra l
fu ra n o n e
(5 Z )- 4 -b r o m o -5 - (b ro m o m e th y le n e )- 3 -b u ty l-
la to r LuxRvh- T h e H fq p ro te in is n o n -fu n c tio n a l in s tra in
B N L 2 5 8 , re s u ltin g in c o n s titu tiv e ly e x p re s s e d b io lu m in e s ­
c e n c e (L e n z
e ta l., 2 0 0 4 ). T h e fa c t th a t th e fu ra n o n e
2 (5 F /)-fu ra n o n e o n q u o ru m s e n s in g -r e g u la te d b io lu m in e s ­
b lo c k e d b io lu m in e s c e n c e in th is m u ta n t in d ic a te s th a t it
c e n c e o f V. h a rv e y i w ild ty p e a n d q u o ru m s e n s in g m u ta n ts
a c ts d o w n s tre a m o f H fq, i.e . a t th e le v e l o f th e luxR vi,
w as
m R N A a n d /o r th e LuxRvt, p ro te in a n d n o t b y d is p la c in g th e
d e te rm in e d .
Im p o rta n tly ,
h a lo g e n a te d
fu ra n o n e s
w e re s h o w n b e fo re n o t to b lo c k b io lu m in e s c e n c e w h e n
e x p re s s e d
fro m
an
e x te rn a l p r o m o te r (G iv s k o v
s ig n a l m o le c u le s fro m th e ir re c e p to rs .
e ta l.,
T h e q u o ru m s e n s in g m a s te r re g u la to r p ro te in L u x R Vh
1 9 9 6 ), in d ic a tin g th a t th e b io c h e m ic a l fu n c tio n o f th e L u x
h a s b e e n s h o w n b e fo re to b e a tra n s c rip tio n a l a c tiv a to r
p ro te in s is n o t a ffe c te d . C o n s is te n t w ith th e w o rk o f M a n ­
th a t is re q u ire d fo r e x p re s s io n o f th e lu x o p e ro n (S w a rtz -
e fie ld a n d c o lle a g u e s (2 0 0 0 ), th e c o m p o u n d w a s fo u n d to
m an
e t a l. ,
1992;
S w a rtz m a n
and
M e ig h e n ,
1 9 9 3 ).
© 2007 The Authors
Journal com pilation © 2007 Society for Applied Microbiology and Blackwell P ublishing Ltd, Environm ental Microbiology, 9, 2 4 86-2 495
2492
T. D e fo ir d t e t al.
C o n s e q u e n tly , in a fin a l s e rie s o f e x p e rim e n ts , th e e ffe c t
e t a l. , 2 0 0 0 ).
o f th e fu ra n o n e o n th is re s p o n s e re g u la to r p ro te in w a s
b e e n s h o w n b e fo re to re g u la te v iru le n c e fa c to r p ro d u c ­
in v e s tig a te d . R e v e rs e tra n s c rip ta s e re a l-tim e P C R w ith
tio n in th e s e s p e c ie s a s w e ll (H e n k e a n d B a s s le r, 2 0 0 4 b ;
p rim e rs s p e c ific fo r f/. h a rv e y i luxR vh re v e a le d th a t th e
M ilto n , 2 0 0 6 ) a n d c o n s e q u e n tly , th e fu ra n o n e s m ig h t be
fu r a n o n e h a s n o e ffe c t o n th e c o n c e n tra tio n o f th e lu x R Vh
u s e fu l to
m R N A . In c o n tra s t, m o b ility s h ift a s s a y s s h o w e d th a t th e
p a th o g e n s .
c o n c e n tra tio n o f th e LuxR vh re s p o n s e re g u la to r p ro te in
M o re o v e r,
c o n tro l
th e L u x R Vh h o m o lo g u e s
in fe c tio n s
caused
In a d d itio n to a ff e c tin g b o th
by
th e s e
h a ve
hum an
V. fis c h e ri L u x R v i (M a n ­
a b le to b in d to its ta rg e t p r o m o te r s e q u e n c e s s ig n ific a n tly
e fie ld
d e c re a s e d a fte r fu ra n o n e a d d itio n , b o th in in ta c t c e lls a n d
c o v a le n t b in d in g o f th is n a tu ra l fu r a n o n e to th e E s c h e r i­
e t a l. , 2 0 0 2 ) a n d
V. h a rv e y i LuxR vh (th is s tu d y ),
In p u rifie d LuxR vh e x tra c ts . In te re s tin g ly , th e c o n c e n tra tio n
c h ia c o li L u x S p ro te in
o f th e LuxR vh p ro te in w a s s h o w n n o t to b e a ffe c te d b y th e
2 0 0 4 ). H e n c e , it c a n b e
fu ra n o n e (a s d e te rm in e d b y S D S -P A G E a n a ly s is ). LuxR vi,
nones
is a m e m b e r o f th e T e tR fa m ily o f tra n s c rip tio n a l re g u la to rs
p re s e n t
c o n ta in in g
s e e m s th a t th e m a c r o - a lg a D e lis e a p u lc h ra h a s e v o lv e d
a
h e lix - tu r n - h e lix
DNA
b in d in g
d o m a in
re a c t
in
w ith
has been
c e r ta in
d iffe re n t
re p o rte d
(R e n e t a l. ,
h y p o th e s iz e d th a t th e s e fu r a ­
re a c tiv e
r e g u la to r y
g ro u p s
p ro te in s .
w h ic h
It
a re
th e re fo re
(R a m o s e ta l., 2 0 0 5 ). M e m b e rs o f th e T e tR fa m ily th a t
a
h a v e b e e n s tu d ie d In d e p th , h a v e b e e n s h o w n to b in d
d e fe n c e s y s te m , p r o d u c in g c o m p o u n d s th a t a ffe c t d iffe r ­
s o p h is tic a te d
q u o ru m
s e n s in g
d is ru p tio n -b a s e d
D N A a s d im e rs , b u t th is h a s n o t y e t b e e n p ro v e n fo r
e n t ty p e s o f s ig n a l r e g u la to r s in a n o n -g ro w th in h ib ito ry
LuxRvh- B e c a u s e h a lo g e n a te d fu r a n o n e s a re k n o w n to be
w a y . In v ie w o f th e b r o a d ra n g e o f b a c te ria th a t c a n
v e r y re a c tiv e c o m p o u n d s (H e n tz e r a n d G iv s k o v , 2 0 0 3 ),
p o te n tia lly c o lo n iz e m a r in e o r g a n is m s , th e p ro d u c tio n o f
w e h y p o th e s iz e th a t th e fu r a n o n e re a c ts w ith th e LuxR vi,
b ro a d s p e c tru m q u o ru m
p ro te in , th e re b y a lte rin g It in s u c h a w a y th a t it c a n n o t b in d
by
th e D N A a n y m o re , e ith e r b y c h a n g in g th e s tru c tu re o f th e
a d v a n ta g e .
D N A b in d in g d o m a in o r th e re g io n s in v o lv e d in d im e r
fo rm a tio n (th a t is, If LuxR vi, a ls o n e e d s to d im e riz e in o r d e r
th e
In
show
a lg a
p r o b a b ly
c o n c lu s io n ,
th a t
th e
th e
s e n s in g -d is ru p tin g c o m p o u n d s
c o n fe rs
re s u lts
n a tu ra l
a
s tro n g
p r e s e n te d
fu r a n o n e
e v o lu tio n a ry
in th is
a rtic le
(5 Z )-4 -b ro m o -5 -
to b in d D N A ). H o w e v e r, th e e lu c id a tio n o f th e e x a c t b io ­
(b r o m o m e th y le n e )-3 - b u ty l- 2 (5 H ) -fu r a n o n e
c h e m ic a l re a c tio n m e c h a n is m b e tw e e n h a lo g e n a te d fu r a ­
q u o ru m s e n s in g -r e g u la te d g e n e e x p re s s io n in V. h a rv e y i
d is ru p ts
n o n e s a n d LuxR vh (w ith th e a id o f m a s s s p e c tro m e try ) w ill
b y d e c re a s in g th e D N A - b in d in g a c tiv ity o f th e q u o ru m
b e p a rt o f fu tu re w o rk a t o u r la b o ra to rie s .
s e n s in g m a s te r re g u la to r p ro te in L u x R Vh. T h e s e re s u lts
T h e fa c t th a t th e fu ra n o n e a ffe c ts th e m a s te r re g u la to r
a re o f s ig n ific a n t p r a c tic a l in te re s t b e c a u s e th e y s u g g e s t
ra th e r th a n s e le c tiv e ly b lo c k in g o n e o f th e c h a n n e ls o f
th a t th e fu r a n o n e c o u ld b e u s e d a s a b ro a d s p e c tru m
th e V. h a rv e y i q u o ru m s e n s in g s y s te m is q u ite im p o rta n t
b io c o n tro l a g e n t to p r o te c t a v a rie ty o f h o s ts fro m d iffe re n t
w ith re s p e c t to p o s s ib le p ra c tic a l a p p lic a tio n s b e c a u s e
p a th o g e n ic v ib rio s in w h ic h v iru le n c e fa c to r p ro d u c tio n is
th e re s e e m s to be a d iffe re n c e in th e re la tiv e im p o rta n c e
re g u la te d b y a LuxR vh h o m o lo g u e .
o f th e th r e e c h a n n e ls fo r a s u c c e s s fu l In fe c tio n o f d iffe r­
ent
h o s ts .
In d e e d ,
c h a n n e l o f th e
d is ru p tin g
o n ly
V. h a rv e y i q u o ru m
th e
A I-2 -m e d ia te d
s e n s in g s y s te m
b e e n s h o w n to s ig n ific a n tly in c re a s e s u rv iv a l o f th e b rin e
s h rim p
A.
fra n c is c a n a , w h e r e a s
th e
E xpe rim e ntal p ro c e d u re s
has
F u ra n o n e p r e p a ra tio n
H A I-1 -m e d ia te d
T h e na tura l fu ra n o n e (5 Z )-4 -b ro m o -5 -(b ro m o m e th y le n e )-3 -
c h a n n e l h a d n o e ffe c t o n in fe c tio n o f th e s h r im p (D e f­
o ird t e t a l. , 2 0 0 5 ). In c o n tra s t, b o th th e H A I-1 - a n d A I-2 -
bu tyl-2 (5 /-/)-fu ra n o n e w as s y n th e s iz e d a s d e scrib e d p re v i­
o u s ly (R en e ta l., 20 01). T h e fu ra n o n e w a s disso lve d and
m e d la te d c h a n n e l n e e d e d to b e d is ru p te d in o r d e r to
d ilu te d in p u re e th a n o l and sto re d a t -2 0 ° C .
d e c re a s e m o rta lity o f g n o to b io tic ro tife rs ( B ra c h io n u s p li­
c a tilis ) c a u s e d b y V. h a r v e y i (T in h e t a i , 2 0 0 7 ). B e c a u s e
th e fu ra n o n e b lo c k s a ll th re e c h a n n e ls o f th e s y s te m a t
o n c e b y a c tin g a t th e e n d o f th e q u o ru m s e n s in g s ig n a l
tra n s d u c tio n c a s c a d e , it w ill n o t b e n e c e s s a ry to d e v e lo p
d iffe re n t fu ra n o n e c o m p o u n d s to p r o te c t d iffe re n t h o s ts .
C o n s is te n t w ith th is , th e n a tu ra l fu r a n o n e w a s s h o w n to
p r o te c t b o th b rin e s h rim p a n d ro tife rs fro m lu m in e s c e n t
v ib rio s (D e fo ird t e ta l., 2 0 0 6 ; T in h e t a l. , 2 0 0 7 ). In a d d i­
tio n to th is , s e v e ra l h u m a n p a th o g e n s , In c lu d in g
V ib rio
c h o le ra e , V ib rio p a ra h a e m o ly tic u s a n d V ib rio v u ln ific u s ,
have
been
fo u n d
to
c o n ta in
a
LuxRVh h o m o lo g u e
(J o b lin g a n d H o lm e s , 1 9 9 7 ; M c C a rte r, 1 9 9 8 ; M c D o u g a ld
B a c te r ia l s tra in s a n d g r o w th c o n d itio n s
T h e stra in s th a t w e re used in th is s tu d y a re sh o w n in T a ble 1.
A ll stra in s w e re grow n in L u ria -B e rta n i m e dium con ta in in g
2 0 g I ' 1 N aC I (LB H0) a t 2 8 ’C u n d e r co n s ta n t ag ita tio n . S p e c ­
tro p h o to m e try a t O D got w as u se d to m e a s u re grow th. L u m i­
n e sce n ce w as m e a su re d w ith a L u m a c B io co u n te r M 2 500
lu m in o m e te r (L u m a c b.v., L a n d g ra a f, th e N eth erlan ds).
F o r all e x p e rim e n ts , V. h a rv e y i s tra in s w e re grow n to an
O D 600 o f a p p ro x im a te ly 0.75, a fte r w h ic h th e fu ra n o n e w as
a d d e d to th e c u ltu re s in the a p p ro p ria te c o n c e n tra tio n a n d th e
cu ltu re s w e re fu rth e r incub ated a t 2 8 °C w ith sh a kin g . U nless
o th e rw ise Ind ica te d , sam ples w e re ta ke n in trip lica te 0 .5 h
a fte r fu ra n o n e ad d itio n .
© 2007 The A uthors
Journal com pilation © 2007 Society fo r Applied M icrobiology and Blackwell Publishing Ltd, Environm ental M icrobiology, 9, 2 4 8 6 -2 4 9 5
M o d e o f a c tio n o f b r o m in a te d f u r a n o n e in V ib rio h a rv e y i
2493
Table 1. S tra ins used in this study.
Strain
Phenotype
R eference
Vibrio h a rve yi BB120
W ild type from w hich s tra ins B N L258, JAF375, JAF483,
JAF553, JM H 59 7 and JM H 61 2 are derived
h fq ::T r\5lacZ
luxN ::C m R luxQ ::K anR
luxO D 47A linked to K a n R
luxU H 58A linked to K a n 11
luxN::Tn5 cgsS ::C m R
luxPQ ::T n 5 cqsS::C rr\"
Isogenic L uxR m utant
B assler e ta l. (1997)
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
h a rve yi BNL258
h a rv e y i JAF375
h a rve yi JAF483
h a rv e y i JAF553
h a rve yi JM H597
h a rve yi JM H612
h a rv e y i M R 1130
Lenz e t al. (2004)
Freem an and B assler (1999a)
Freem an and B assler (1999a)
Freem an and B assler (1999b)
H enke and B assler (2004a)
H enke and B assler (2004a)
M artin e ta l. (1989)
tra n scrip tio n u sing th e Q ia g e n O ne S tep R T-P C R
(Q ia gen), acco rd in g to th e m a n u fa c tu re r’s in stru ctio n s.
P ro te in a s s a y s
kit
W ild -ty p e V. h a rv e y i B B 1 2 0 w as use d in th e lu c ife ra s e and
R eal-tim e P C R w a s p e rfo r m e d w ith th e sp e c ific lu x R Vh and
ba cterial a lk a lin e p h o s p h a ta s e a ssa ys. Im m e d ia te ly b e fo re
a n d 0.5, 1 a n d 2 h a fte r th e ad dition o f th e fu ra n o n e , 2 un its
rp oA prim ers. A m p lico n le n g th s a re 8 4 bp a n d 197 bp fo r
luxRvt, a n d rp oA re s p e c tiv e ly . A m p lific a tio n w as p e rfo rm e d in
25 p i re action m ixtu res u s in g th e S Y B R G re en P C R M a ste r
[O D SOo X v o l (m l)] w ere p e lle ted, s o n ic a te d in 0 .5 ml o f 0 .2 5 M
Tris, pH 8 a n d th e c e llu la r de bris re m o v e d (a s d e s c rib e d by
M iy a m o to e t a i, 1990). A m o dified L o w ry a s s a y w a s use d to
d e te rm in e th e p ro te in c o n te n t (M arkw ell e t a i , 1 9 8 1 ). In vitro
lu c ife ra s e a n d b a cte ria l a lk a lin e p h o s p h a ta s e a c tiv itie s w e re
d e te rm in e d a s d e s c rib e d b y G u n s a lu s -M ig u e l a n d c o lle a g u e s
(1 972) a n d G a re n and Le vin th a l (1960).
P r im e r d e s ig n
S p e c ific p rim e rs fo r th e a m p lific a tio n o f lu x R Vh a n d R N A p o ly ­
m e ra s e A s u b u n it ( rp o A ) m R N A w e re d e s ig n e d u sin g th e
P rim e r E x p re s s 2 .0 so ftw a re (A p p lie d B io s y s te m s, F o ste r
City, U S A ). T h e p rim e rs w ere d e s ig n e d b a s e d o n th e c o n ­
s e n s u s o f th e s e q u e n c e s th a t ha ve been d e p o s ite d b e fo re in
G e n B a n k . T h e c o m b in a tio n s of th e tw o p rim e r s e q u e n c e s
w e re b la s te d a g a in s t G e n B a n k . The p rim e r s e q u e n c e s are
M ix kit (A pp lie d B io s y s te m s , N ie u w e rk e rk a /d Ijssel, th e N eth­
e rla n d s), acco rd in g to th e m a n u fa c tu re r's in stru ctio n s, in
o p tica l 96-w ell re action p la te s w ith o p tical ca p s (A p p lie d
B io syste m s). T h e th e rm a l p ro file w a s as fo llo w s : 50 °C fo r
2 m in and 95 °C fo r 10 m in fo llo w e d by 4 0 c y c le s o f 95 °C fo r
3 0 s, 54°C fo r 1 m in, and 6 0 ° C fo r 1 m in. A m p lic o n d is s o c ia ­
tion cu rve s w e re d e te rm in e d b y co n sta n t flu o re s c e n t m e a ­
s u re m e n t du ring a fina l h e a tin g ste p a t 60 °C to 95 °C a t 0.1 °C
s~’ ra m p in g spe ed. T h e m e ltin g te m p e ra tu re s fo r th e a m p lic o n s w ere 7 7 'C a n d 82 °C fo r luxRvh a n d rp o A respectively.
T h e te m p la te D N A in th e re a c tio n m ixtu re s w a s a m p lifie d and
m o n ito re d w ith an A B I P rism S D S 7 0 0 0 in s tru m e n t (A pp lie d
B io syste m s). T h e d iffe re n c e in m R N A leve ls b e tw een
fu ra n o n e -tre a te d and u n tre a te d e xtra cts w a s ca lcu la te d as
fo llo w s:
log (d iffe re n ce in c o n c e n tra tio n ) = A C ,/reg ression c o e ffic ie n t
sh o w n in Ta ble 2.
w ith AC, th e d iffe re n c e in C, v a lu e be tw e e n fu ra n o n e -tre a te d
R N A e x tr a c tio n a n d re v e rs e tra n s c rip ta s e re a l-tim e P C R
T h e e ffe c t o f th e fu ra n o n e on luxR ^h m R N A c o n c e n tra tio n s
w a s s tu d ie d in w ild -ty p e V. h a rv e y i B B 1 20. Im m e d ia te ly
b e fo re a n d 0 .5 , 1 and 2 h a fte r fu ra n o n e a d d itio n , lu m in e s ­
c e n c e a n d cell d e n s ity (O D 600) o f the c u ltu re s w e re m e a su re d
a n d 0 .5 m l o f s a m p le s fo r R N A extra ctio n w e re ta k e n , w h ich
w e re im m e d ia te ly fro z e n in cold e th ano l (-8 0 ° C ). R N A w as
iso la te d u s in g th e Q ia g e n R N e a s y M ini Kit (Q ia g e n , H ilden ,
G e rm a n y ) a c c o rd in g to th e m a n u fa c tu re r’s in s tru c tio n s. R N A
e x tra c ts w e re tre a te d w ith R N A s e -fre e D N A s e I (F e rm e n ta s,
St. L e o n -R o t, G e rm a n y ), a fte r w h ic h th e R N A q u a n tity w as
checked
s p e c tro p h o to m e tric a lly .
RNA
c o n c e n tra tio n s
o b ta in e d w e re all a ro u n d 3 0 0 ng p i“ 1. R N A q u a lity w a s c o n ­
firm e d by e le c tro p h o re s is . c D N A w a s o b ta in e d b y re verse
Table 2. Prim ers used in this study.
a n d u n tre a te d e xtracts. T h e re g re s s io n c o e ffic ie n ts be tw e e n
lo g (co n ce n tra tio n ) and C, v a lu e w ere d e te rm in e d by a n a ly s ­
ing a 10-fold d ilu tio n se rie s o f a V. h a rv e y i B B 1 2 0 D N A extract
a n d w e re -3 .3 8 0 {R 2 = 0 .9 9 8 ) and -3 .5 9 7 (R 2 = 0 .9 9 3 ) fo r
lu x R Vh a n d rp oA respectively.
M o b ility s h ift a s s a y s
T h e e ffe ct o f th e fu ra n o n e o n LuxRvn protein co n c e n tra tio n s
w a s stu d ie d
in w ild -typ e
V. h a rv e y i B B 1 20.
Im m e d ia te ly
b e fo re and 0.5, 1 a n d 2 h a fte r th e a d dition o f th e fu ra n o n e , 2
u n its [O D 600 x vol (m l)] w ere p e lle te d , so n ic a te d in 0 .5 m l o f
0 .2 5 M Tris, pH
8 a n d th e c e llu la r d e b ris re m o ve d (as
d e scrib e d by M iya m o to e ta l., 1990). In th e e x p e rim e n t th a t
a im e d at te stin g w h e th e r the fu ra n o n e a ffe cte d tra n s la tio n o r
a cte d on p re -e x is tin g tra n s la te s , cu ltu re s w e re tre a te d w ith
5 0 m g I“ 1 c h lo ra m p h e n ic o l S ig m a -A ld ric h (B o rn e m , B elgium )
Target
Primer
Sequence
be fo re fu ra n o n e ad d itio n . M o b ility sh ift a s s a y s w e re c o n ­
d u c te d as d e s c rib e d p re vio u sly (S w artzm an a n d M e ighen,
rpoA
rpOAlorv.:ird
rpoA !e.0..a
5 '-C G TA G C TG A A G G C A A A G A TG A -3'
5'-A A G C TG G A A C A T A A C C A C G A -3'
luxRvn
LuxR i0,„„d
LUXRre,srsG
5'-TC A A TTG C A A A G A G A C C T C G -3'
5 '-A G C A A A C A C T T C A A G A G C G A -3 '
1993) using ra d io la b e lle d lu x R Vh p ro m o te r D N A (C h a tte rje e
e ta l., 1996) or lu x C D A B E G H p ro m o te r D N A (M iya m o to
e t al., 1996) co n ta in in g the L u xR Vh b ind ing site s. T h e a m o u n t
o f re ta rd e d D N A w as q u a n tifie d as d e s c rib e d in M iya m o to
and co lle a g u e s (1 9 9 6 ) using a F u ji b ioim age .
© 2007 The A uthors
Journal com pilation © 2007 S ociety for Applied M icrobiology and Blackwell Publishing Ltd, E nvironm ental Microbiology, 9, 2 4 86-2 495
2494
T. D e fo ir d t e t al.
E x p e r im e n ts w ith p u r ifie d LuxR vh
LuxR vh p u rific a tio n w a s c a rrie d o u t as d e s c rib e d pre vio u sly
(S w a rtz m a n a n d M e ig hen , 1993). T h e p u rified LuxR vh w a s
s to re d in 5 0 m M N a P 0 4, 3 0 0 m M NaCI, pH 8.0. T h e protein
c o n c e n tra tio n (as d e te rm in e d by B io -R a d protein a ssa ys)
w a s 0 .2 m g m l 1. To g la ss tu b e s co n ta in in g 40 pi of th e
protein p re p a ra tio n , 0 .2 pi o f a b s o lu te e th a n o l o r 0 .2 p i of
fu ra n o n e (1 0 m g m l 1 in e th a n o l) w e re d ire c tly a d d e d . T h e
tu b e s w e re in c u b a te d in a 3 7 '’C w a te r bath fo r 1 h and then
stored a t 4°C . F o r m o b ility s h ift a n a ly s e s , th e s a m p le s w ere
d ilute d 10 -fold in s to ra g e b u ffe r and 1 p i (0.02 pg ) w as
a s s a y e d . F o r S D S -P A G E , 1 p g of protein w as a p p lie d and
10% S D S -P A G E w a s p e rfo rm e d a cc o rd in g to M a n ia tis and
c o lle a g u e s (1 982).
A c kn o w le d g e m e n ts
W e th a n k B o n n ie B a s s le r fo r g e n e ro u s ly pro vid in g us w ith the
V. h a rv e y i m u ta n ts . T h is w o rk w a s s u p p o rte d b y the ‘ Instituut
v o o r d e a a n m o e d ig in g v a n In n o v a tie d o o r W e te n sch a p en
T e ch n o lo g ie in V la a n d e re n ’ (IW T G ra n t no. 3 3 2 0 5 ), the
‘Fo nds v o o r W e te n s c h a p p e lijk O n d e rz o e k ' (p ro je ct no.
G 0 2 3 0 .0 2 N ), th e N atio n a l In stitu te s o f H ealth (E B 0 0 3 8 7 2 0 1 A 1) a n d the C a n a d ia n In s titu te s o f H ealth R e se a rch
(M T 7 6 7 2 ).
R eferences
A ustin, B., a n d Z h a n g , X .-H . (2 0 0 6 ) V ibrio h a rv e y i: a s ig n ifi­
c a n t p a th o g e n o f m a rin e v e rte b ra te s and in ve rte b ra te s.
Le tt A p p l M ic ro b io l 4 3 : 1 1 9 -1 2 4 .
B assler, B .L., W right, M ., S h o w a lte r, R .E ., and S ilve rm a n,
M .R. (1 993) In te rc e llu la r s ig n a lin g in V ibrio h a rveyis e q u e n c e a n d fu n c tio n o f g e n e s re g u la tin g e x p re ssio n of
lu m in e s c e n c e . M o ! M ic ro b io l 9 : 7 7 3 -7 8 6 .
B assler, B .L., G re e n b e rg , E .P ., a n d S teven s, A .M . (1997)
C ro s s -s p e c ie s in d u c tio n o f lu m in e s c e n c e in th e qu o ru m s e n s in q b a c te riu m V ib rio h a rveyi. J B a c te rio l 1 7 9 : 4 0 4 3 40 45.
C ao, J .G ., and M e ig h e n , E .A. (1 989) P u rificatio n a n d s tru c ­
tu ra l id e n tific a tio n o f an a u to in d u c e r fo r th e lu m in e sce n ce
D e fo ird t, T ., C ra b , R., W o o d , T.K ., S o rg e lo o s, P., V e rstra e te ,
W ., and B ossier, P. (2 0 0 6 ) Q u o ru m s e n sin g -d isru p tin g brom in a te d fu ra n o n e s p ro te c t th e g n o to b io tic brine sh rim p
A rte m ia fra n cisca n a fro m p a th o g e n ic Vibrio harveyi, V ibrio
ca m p b e llii, a n d V ib rio p a ra h a e m o ly tic u s isolate s. A p p l
E n v iro n M ic ro b io l 7 2 : 6 4 1 9 -6 4 2 3 .
F re e m a n , J.A ., a n d B a s s le r, B.L. (1 999a ) A g e netic a n a ly s is
of th e fu n c tio n o f L u x O , a tw o -c o m p o n e n t re sp o n se re g u ­
la to r in vo lve d in q u o ru m se n sin g in V ibrio h a rveyi. M o l
M ic ro b io l 3 1 : 6 6 5 -6 7 7 .
F re e m a n , J.A ., a n d B a s s le r, B.L. (1 999b ) S e q u e n ce and
fu n c tio n o f LuxU : a tw o -c o m p o n e n t p h o sp h o re la y protein
th a t re g u la te s q u o ru m s e n s in g in V ibrio ha rveyi. J B a c te rio l
1 8 1 : 8 9 9 -9 0 6 .
G a re n , A ., a n d Le vin th a l, C . (1 960) A fin e -stru ctu re g e n e tic
an d ch e m ic a l s tu d y of th e e n zym e a lk a lin e p h o s p h a ta s e o f
E. coli. I. P u rifica tio n a n d ch a ra c te riz a tio n of alka lin e
p h o s p h a ta s e s . B io c h im B io p h ys A c ta 3 8 : 4 7 0 -4 8 3 .
G ivsko v, M ., de Nys, R., M a n e fie ld , M ., G ra m , L., M a xim ilie n ,
R., E berl, L., e ta l. (1 9 9 6 ) E u ka ryo tic in te rfe re n ce w ith
h o m o s e rin e
la c to n e -m e d ia te d
p ro ka ryo tic
sig n a llin g .
J B a c te rio l 1 7 8 : 6 6 1 8 -6 6 2 2 .
G u n s a lu s -M ig u e l, A ., M e ig h e n , E .A ., N icoli, M .Z., N e a lso n ,
K .H ., a n d H asting s, J.W . (1 972) P u rifica tio n and pro p e rtie s
of b a cte ria l lucife ra se s. J B io l C h e m 2 4 7 : 3 9 8 -4 0 4 .
H enke ,
J .M .,
and
B a s s le r,
B.L.
(2 004a ) T h re e
p a rallel
q u o ru m se n sin g s y s te m s re g u la te g e n e e xp re ssio n in
V ibrio h a rveyi. J B a c te rio l 1 8 6 : 6 9 0 2 -6 9 1 4 .
H enke , J .M ., a n d B a ssle r, B.L. (2 0 0 4 b ) Q u o ru m se n sin g
re g u la te s typ e III s e c re tio n in V ibrio h a rv e y i a n d V ibrio
p a ra h a e m o ly tic u s . J B a c te rio l 186: 3 7 9 4 -3 8 0 5 .
H e n tze r, M ., a n d G ivskov, M. (2003) P ha rm a co lo g ica l in h ib i­
tio n o f q u o ru m se n sin g fo r the tre a tm e n t of ch ro n ic b a c te ­
rial in fe ctio n s. J C lin In v e s t 1 1 2 : 1 3 0 0 -1 3 0 7 .
Jo b lin g , M .G ., a n d H o lm e s, R .K. (1 997) C h a ra c te riz a tio n
o f ha pR , a p o sitive re g u la to r o f th e V ibrio c h o le ra e
H A /p ro te a s e gene hap, a n d its id e n tifica tio n a s a fu n ctio n a l
h o m o lo g u e o f th e Vibrio h a rv e y i lu x R ge ne. M o l M ic ro b io l
2 6 : 1 0 2 3 -1 0 3 4 .
K a ru n a s a g a r, I., Pai, R., M a la hti, G .R ., a n d K a ru n a sa g a r, I.
(1 994) M a ss m o rta lity o f P e n a e u s m o n o d o n larva e d u e to
a n tib io tic -re s is ta n t
1 2 8 : 2 0 3 -2 0 9 .
Vibrio h a rv e y i infe ction. A q u a c u ltu re
s y s te m o f V ibrio h a rv e y i. J B io l C h e m 2 6 4 : 2 1 6 7 0 -2 1 6 7 6 .
K och, B., Lilje fo rs, T., P ersso n, T., N ielsen , J., K je lle b e rg , S.,
C h a tte rje e , J., M iy a m o to , C .M ., and M e ig hen , E .A. (1 996)
A u to re g u la tio n o f luxR : th e Vibrio h a rv e y i lu x -o p e ro n a c ti­
v a to r fu n c tio n s as a re p re s s o r. M o l M ic ro b io l 2 0 : 4 1 5 -4 2 5 .
a n d G ivsko v, M. (2 005) T h e L u xR re ce p to r: th e s ite s of
C hen, X., S ch a u d e r, S ., P otier, N., Van D orsse laer, A.,
P elczer, I., B assler, B .L., a n d H u g h so n , F.M . (2 002) S tru c ­
tu ra l id e n tific a tio n o f a b a cterial q u o ru m -s e n s in g signa l
Le nz, D .H ., M ok, K .C ., Lilley, B .N ., K ulkarn i, R .V ., W in g re e n ,
c o n ta in in g b o ro n . N a tu re 4 1 5 : 5 4 5 -5 4 9 .
D efo irdt, T., B oon, N., B o s s ie r, P., a n d V e rstra e te , W . (2 004)
D is ru p tio n o f b a c te ria l q u o ru m se n sin g : an u n e xp lo re d
s tra te g y to fig h t in fe c tio n s in a q u a c u ltu re . A q u a c u ltu re 2 4 0 :
6 9 -8 8 .
D efo irdt, T ., B ossier, P., S o rg e lo o s , P., a n d V e rstra e te , W.
(2 0 0 5 ) T h e im p a c t o f m u ta tio n s in th e qu orum se n sin g
s y s te m s o f A e ro m o n a s h y d ro p h ila , V ibrio a n g u illa ru m and
Vibrio h a rv e y i on th e ir v iru le n c e to w a rd s g n o to b io tica lly
c u ltu re d A rte m ia fra n c is c a n a . E n v iro n M ic ro b io l 7: 1 2 3 9 1247.
in te ra c tio n w ith q u o ru m -se n sin g sig n a ls a n d inh ib ito rs.
M ic ro b io lo g y 1 5 1 : 3 5 8 9 -3 6 0 2 .
N .S ., a n d B assler, B.L. (2 004) T h e sm all R N A c h a p e ro n e
H fq a n d m u ltip le sm all R N A s co n tro l q u o ru m se n sin g in
V ibrio h a rv e y i an d Vibrio cho le rae . C e ll 118: 6 9 -8 2 .
Lille y, B .N ., a n d B assler, B .L. (2 000) R e g u la tio n o f q u o ru m
se n sin g in V ibrio h a rve yi b y Lu xO and 854. M o l M ic ro b io l
3 6 : 9 4 0 -9 5 4 .
M cC a rte r, L.L. (1 9 9 8 ) O paR , a h o m o lo g o f Vibrio h a rv e y i
LuxR , co n tro ls o p a city o f V ibrio p a ra h a e m o lyticu s.
J B a c te rio l 1 8 0 : 3 1 6 6 -3 1 7 3 .
M cD o u g a ld , D., R ice, S.A., a n d K jellebe rg, S. (2 000) T h e
m a rin e p a th o g e n Vibrio vu ln ificu s e n codes a p u ta tive h o m o ­
lo g u e o f th e Vibrio h a rve yi re g u la to ry gene luxR : a g e n e tic
a n d p h y lo g e n e tic co m p a riso n . G e n e 2 4 8 : 2 1 3 -2 2 1 .
© 2007 The A uthors
Journal com pilation © 2007 Society for Applied Microbiology and Blackwell P ublishing Ltd, Environm ental M icrobiology, 9, 2 4 86-2 495
M o d e o f a c tio n o f b r o m in a te d fu r a n o n e in V ib rio h a rv e y i
M cD ou gald , D., S rinivasa n, S., R ice, S .A ., a n d K je lle b e rg , S.
(2003) S ig n a l-m e d ia te d c ro s s -ta lk re g u la te s s tre ss a d a p ta ­
tion in V ibrio sp e c ie s . M ic ro b io lo g y 1 4 9 : 1 9 2 3 -1 9 3 3 .
M a n e fie ld , M ., H arris, L., R ice, S .A ., de Nys, R., a n d K je lle ­
berg, S. (2 0 0 0 ) Inhibition o f lu m in e s c e n c e a n d viru le n ce in
th e b la c k tig e r praw n (P e n a e u s m o n o d o n ) p a th o g e n Vibrio
h a rv e y i b y in te rc e llu la r sig n a l a n ta g o n is ts . A p p l E nviron
M ic ro b io l 6 6 : 2 0 7 9 -2 0 8 4 .
2495
F rontie rs. P ro c e e d in g s o f the E ig h th In te rn a tio n a l S y m p o ­
siu m on M ic ro b ia l E c o lo g y . Bell. C .R., B rylinsky, M. and
J o h n s o n -G re e n , P. (e d s ). H alifax, N S , C ana da: A tlantic
C ana da S o c ie ty fo r M ic ro b ia l E cology.
R am o s, J .L ., M a rtin e z -B u e n o ,
M ., M o lin a -H e n a re s, A .J.,
Terán, W ., W a ta n a b e , K ., Z h a n g , X ., e ta l. (2 005) T h e TetR
fa m ily o f tra n s c rip tio n a l re p re s s o rs . M ic ro b io l M o l B io l R e v
6 9 : 3 2 6 -3 5 6 .
M a n e fie ld , M ., R asm u ssen, T.B ., H e n tz e r, M., A n d e rse n ,
J.B ., S te in b e rg , P., K jellebe rg, S., a n d G iv s k o v , S. (2002)
H a lo g e n a te d fu ra n o n e s in h ib it q u o ru m s e n s in g throug h
R asm u ssen, T .B ., a n d G iv s k o v , M. (2 006) Q u o ru m -se n sin g
a c c e le ra te d LuxR tu rn o v e r. M ic ro b io lo g y 148: 1119—
1127.
M a niatis, T., F ritsch, E.F., a n d S a m b ro o k , J . (1 982) M o le c u ­
la r C lo n in g : A L a b o ra to ry M a n u a l. C old S p rin g H arbor, NY,
R en, D., S im s, J., and
USA: C old S p rin g H a rb o r L a b o ra to ry .
M arkw ell, M .A .K ., H aas, S .M., Tolbert, N .E ., and B ie b e r, L.L.
R en, D., B edzyk, L., Ye, R .W ., T h o m a s, S .M., a n d W ood , T.K.
(2 004) D iffe re n tia l g e n e e x p re s s io n s h o w s na tura l b ro m i­
na ted fu ra n o n e s in te rfe re w ith th e a u to in d u ce r-2 b a cterial
(1 981) P rote in d e te rm in a tio n in m e m b ra n e a n d lip o p ro te in
sam ples: m a nual and a u to m a te d p ro c e d u re s . M e th o d s
E n z y m o l 7 2 : 2 9 6 -3 0 3 .
M a rtin, M .O ., S how alte r, R .E ., a n d S ilv e rm a n , M. (1 989)
Ide n tific a tio n o f a lo cu s c o n tro llin g e x p re s s io n o f lu m in e s ­
c e n c e g e n e s in Vibrio h a rveyi. J B a c te rio l 1 7 1 : 2 4 0 6 -2 4 1 4 .
M ilton, D.L. (2 006) Q uo rum s e n s in g in v ib rio s : c o m p le x ity fo r
div e rs ific a tio n . In t J M e d M ic ro b io l 2 9 6 : 6 1 -7 1 .
M iya m o to , C .M ., M e ig hen , E .A ., a n d G ra h a m , A .F . (1990)
T ra n s c rip tio n a l re gulatio n o f lu x g e n e s tra n s fe rre d into
Vibrio h a rveyi. J B a c te rio l 1 7 2 : 2 0 4 6 -2 0 5 4 .
M iya m o to , C .M ., C h a tte rje e , J ., S w a rtz m a n , E., S zittn e r, R.,
a n d M e ig hen , E.A. (1 9 9 6 ) T h e role o f th e lu x a u to in d u c e r in
re g u la tin g lu m in e s c e n c e in V ibrio harveyi', c o n tro l o f lu xR
e xp re ssio n . M o l M ic ro b io l 1 9 : 7 6 7 -7 7 5 .
M o riarty, D .J.W . (1 998) D is e a s e c o n tro l in s h rim p a q u a c u l­
tu re w ith p ro b io tic b a cteria. In M ic ro b ia l B io s y s te m s : N e w
inh ib ito rs a s a n ti-p a th o g e n ic d ru g s. In t J M e d M ic ro b io l
2 9 6 : 1 4 9 -1 6 1 .
W o o d , T.K . (2 001) Inh ib itio n of
b io film fo rm a tio n and s w a rm in g o f E sch e rich ia c o li by
(5 Z )-4 -b ro m o -5 -(b ro m o m e th y le n e )-3 -b u ty l-2 (5H )-furanone.
E n viro n M ic ro b io l 3 : 7 3 1 -7 3 6 .
s ig n a lin g s y ste m o f E s c h e ric h ia coli. B io te c h n o l B io e n g 88:
6 3 0 -6 4 2 .
S w a rtzm a n , E., a n d M e ig h e n , E .A. (1 993) P u rifica tio n and
c h a ra c te riz a tio n o f a p o ly -(d A -d T ) /ux-specific D N A b ind ing
protein fro m V ibrio h a rv e y i a n d id e n tifica tio n as LuxR .
J B io l C h e m 2 6 8 : 1 6 7 0 6 -1 6 7 1 6 .
S w a rtzm a n , E., S ilve rm a n , M ., a n d M e ig hen , E .A. (1 992) T h e
lu x R ge ne p ro d u c t o f V ib rio h a rv e y i is a tra n scrip tio n a l
a ctiva to r o f th e lu x p ro m o te r. J B a c te rio l 174: 7 4 9 0 -7 4 9 3 .
T h o m p s o n , F.L., lida, T ., a n d S w in gs, J. (2 0 0 4 ) B io d ive rsity
o f vib rio s. M ic ro b io l M o l B io l R e v 6 8 : 4 0 3 -4 3 1 .
Tinh, N .T.N ., L in h , N .D ., W ood , T.K ., D ie rcke n s, K.,
S o rg e lo o s, P., a n d B o s s ie r, P. (2 007) In te rfe re n ce w ith the
q u o ru m s e n s in g s y s te m s in a V ibrio h a rv e y i stra in alte rs
th e grow th ra te o f g n o to b io tic a lly cu ltu re d ro tife r B ra c h io ­
n u s p lica tilis. J A p p l M ic ro b io l (in press).
© 2007 The Authors
Journal com pilation © 2007 Society fo r Applied Microbiology and Blackwell Publishing Ltd, Environm ental Microbiology, 9, 2 4 86-2 495