Vol.
176,
No.
8
JOURNAL
OF
BAC ERIOLOGY,
Ap .
1994,
p.
2454-2457
0021-9193/94/$04.00+0
Copy igh
X
1994,
Ame ican
Socie y
o
Mic obiology
Analysis
o
he
Lipid
Moie y
o
Lipopolysaccha ide
om
Rhizobium
opici
CIAT899:
Iden i ica ion
o
29-Hyd oxy iacon anoic
Acid
A.
M.
GIL-SERRANO,'*
I.
GONZALEZ-JIMINEZ,1
P.
TEJERO-MATEO,1
M.
MEGIAS,2
AND
M.
J.
ROMERO-VAZQUEZ2
Depa amen o
de
Quimica
O gdnica,
Facul ad
de
Quimica,
I
and
Depa amen o
de
Mic obiologia
y
Pa asi ologia,
Facul ad
de
Fa mnacia,2
Uni e sidad
de
Se illa,
41071
Se ille,
Spain
Recei ed
1
Oc obe
1993/Accep ed
3
Feb ua y
1994
The
lipid
moie ies
o
wo
lipid
A's
isola ed
om
he
phenolic
and
aqueous
ac ions
o
lipopolysaccha ide
om
Rhizobium
opici
CIAT899
ha e
been
s udied.
Se e al
3-hyd oxy
a y
acids
and
wo
long-chain
hyd oxy
a y
acids,
27-hyd oxyoc acosanoic
acid,
and
29-hyd oxy iacon anoic
acid
we e
iden i ied;
he
a ios
o
hese
acids
a e
he
same
in
bo h
lipid A's.
These
esul s
can
be
used
o
chemo axonomic
pu poses.
Rhizobia
a e
g am-nega i e
bac e ia
and
a e
able
o
o m
ni ogen- ixing
symbio ic
ela ionships
wi h
leguminous
plan s.
As
g am-nega i e
bac e ia,
hey
ha e
he
usual
su ace
polysac-
cha ides,
and
hese
molecules
ha e
been
hypo hesized
o
be
in ol ed
in
he
molecula
mechanism
o
symbio ic
in ec ions.
The
lipopolysaccha ide
(LPS)
is
essen ial
o
la e
symbio ic
s eps
(1,
3,
4,
6,
13,
14).
The
LPS
a e
ancho ed
in
he
ou e
memb ane
by
hei
lipid
A
componen ,
which
exhibi s
a
e y
low
s uc u al
di e si y
and
is
he
main
endo oxic
componen .
Lipid
A
gene ally
con ains
a
(1-6)-linked
glucosamine
disaccha ide
and
di e en
a y
acids.
The
lipid
moie y
o
lipid
A
has
been
analyzed
in
ela ion
o
chemo axonomic
aspec s,
and
3-hyd oxy
a y
acids,
cis-11,
12-me hyleneoc adecanoic
acids,
and
a
se ies
o
ing-opened
p oduc s
ha e
been
iden i ied
(7,
8,
12,
16,
17,
20).
Recen ly,
he
iden i ica ion
o
27-hyd oxyoc acosanoic
acid
om
mem-
be s
o
he
amily
Rhizobiaceae
(9,
10,
15)
and
o he
bac e ia
(11,
18)
was
desc ibed.
In
his
communica ion,
we
epo
he
s uc u es
o
he
a y
acids
ound
in
he
lipid
A
om
wo
LPS
(phenolic
and
aqueous
ac ions)
isola ed
om
Rhizobium
opici
CIAT899
and
he
iden i ica ion
o
29-hyd oxy iacon anoic
acid.
Bac e ia
we e
g own
o
3
days
a
28°C
in
shaken
TY
medium
con aining
(pe
li e )
5
g
o
yp one,
3
g
o
yeas
ex ac ,
and
0.83
g
o
CaCl2.
The
cul u e
was
cen i uged,
and
he
pelle
was
washed
h ee
imes
wi h
0.5
M
NaCl
o
emo e
he
exopolysaccha ide.
The
LPS
was
ex ac ed
om
he
bac-
e ial
cells
by
he
ho
phenol-wa e
me hod
(19),
and
wo
ac ions
(phenolic
and
aqueous
phases)
we e
isola ed.
Bo h
phases
we e
dialyzed
agains
wa e ,
concen a ed,
and
passed
h ough
an
anionic
exchange
esin,
Ambe li e
IRA
400
(ace-
a e
o m)
wi h
wa e
as
eluen .
The
elua es
we e
lyophilized
and
hen
dissol ed
in
10
mM
MgSO4
and
50
mM
T is-HCl
solu ion
(100
ml,
pH
7.0).
DNase
I
(1
mg)
and
RNase
A
(1
mg)
we e
added,
and
he
solu ion
was
s i ed
o e nigh
a
5°C
and
hen
lyophilized
(5).
LPS
ac ions
we e
hyd olyzed
wi h
1%
ace ic
acid
a
100°C
o
90
min,
and
he
lipid
A's
o med
p ecipi a es.
The
supe na an
was
ex ac ed
wi h
dichlo-
ome hane,
and
he
combined
o ganic
laye s
we e
added
o
he
o me
p ecipi a e
and
e apo a ed.
Two
lipid
A's
we e
ob-
*
Co esponding
au ho .
Phone:
54557151.
Fax:
54557134.
ained,
one
om
LPS
in
he
aqueous
phase
(LAW)
and
he
o he
om
LPS
in
he
phenolic
phase
(LAP).
Fa y
acids
we e
de e mined
as
me hyl
es e s
by
gas
ch o-
ma og aphy-mass
spec ome y
a e
elease
by
me hanolysis
wi h
0.625
M
me hanolic
hyd ogen
chlo ide
a
80°C
o
16
h.
The
hyd oxy
a y
acid
me hyl
es e s
we e
silyla ed
[py idine-
bis( ime hylsilyl) i luo oace amide
(1:1),
2
h,
a
oom
em-
pe a u e]
and
also
ace yla ed
(1-me hylimidazole-ace ic
anhy-
d ide
[1:10]).
Unsa u a ed
a y
acids
we e
con e ed
in o
he
dime hyldi hio
de i a i es
(2).
The
me hyl
es e s
we e
ea ed
wi h
iodine,
die hyle he ,
and
dime hyldisul ide
o
24
h
a
58°C.
Na2S203
(5%
aqueous)
was
hen
added,
and
he
de i -
a i es
we e
ex ac ed
ou
imes
wi h
hexane.
Gas
ch oma og-
aphy-mass
spec ome y
was
pe o med
wi h
a
Ca lo
E ba
model
MFC
500
capilla y
gas
ch oma og aph
i ed
wi h
a
CP-Sil
5
CB
WCOT
column
(25
m
by
0.32
mm)
a ached
o
a
K a os
MS-80RFA.
Fo
he
me hyl
es e s,
me hyl
es e
ace-
a es,
and
me hyl
es e
di hio
de i a i es,
he
empe a u e
p og am
was
iso he mal
a
100°C
o
2
min,
ollowed
by
a
10°C/min
g adien
up
o
280°C,
ollowed
by
holding
a
280°C
o
30
min.
Fo
he
ime hylsilyl
de i a i es
o
me hyl
es e s,
he
iso he mal
s ep
was
a
140°C
o
2
min
ollowed
by
an
8°C/min
g adien
up
o
280°C.
The
ioniza ion
po en ial
was
70
eV.
In
he
ch oma og am
o
LAW,
he
ollowing
sa u a ed
a y
acids,
as
hei
me hyl
es e s,
we e
iden i ied:
14-me hylpen a-
decanoic
acid
(i-16:0),
cis-11,12-me hyleneoc adecanoic
acid
(19:0
cyc,
lac obacillic
acid)
and
11-me hoxynonadecanoic
acid
(11-OMe-19:0).
Th ee
3-hyd oxy
a y
acids
and
wo
long-chain
hyd oxy
a y
acids
we e
also
de ec ed.
To
iden i y
he
hyd oxy
a y
acids,
he
co esponding
me hyl
es e s
we e
ime hyl-
silyla ed
and
ace yla ed.
3-Hyd oxy e adecanoic
acid
(3-OH-
14:0),
3-hyd oxypen adecanoic
acid
(3-OH-15:0),
and
3-hy-
d oxyhexadecanoic
acid
(3-OH-16:0)
we e
iden i ied.
The
elec on
impac
mass
spec um
o
he
27-0-ace yl
de i a i e
o
he
me hyl
es e
o
he
27-hyd oxyoc acosanoic
acid
(27-OH-
28:0)
(Fig.
1)
was
iden ical
o
ha
p e iously
epo ed
(10).
The
El
mass
spec um
o
he
29-0-ace yl
de i a i e
o
he
me hyl
es e
o
29-hyd oxy iacon anoic
acid
(29-OH-30:0)
(Fig.
1)
showed
he
expec ed
agmen s:
m/z
464
(M-60,
loss
o
ace ic
acid),
m/z
432
(M-92,
loss
o
ace ic
acid
and
me hanol),
and
m/z
390
(M-134,
loss
o
ace ic
acid,
me hanol,
and
ke ene).
The
spec um
o
he
ime hylsilyl
de i a i e
o
he
me hyl
es e
o
27-OH-28:0
is
shown
in
Fig.
2.
The
main
agmen s
2454
VOL.
176,
1994
NTS
25
1
00
43
190
_
1071
100
4:
60
5
0
302
20
1
0
404'
'II
20)7
120
62
23-:
1
240
360
480
600
432
55
97
281
L1i.
*1.k
I.00
I!
II
ii111III
IIII1IIlII1ihIIiI.iI.I
111
aI.il.1.
11
ORi
oIi
II,.
L.
L
zU
aq
i
iliIiIIi
uIu
iB~lli11.pMUIFpi.
I',
II
9
,Mi b'9l
..
1IIIi.
*!I'
plls
11"
1'
I
I00
200
300
400
FIG.
1.
Mass
spec a
o
he
ace yla ed
me hyl
es e s
o
27-hyd oxyoc acosanoic
acid
( op)
and
29-hyd oxy iacon anoic
acid
(bo om).
we e
m/z
117
(C5H13OS'i
om
he
clea age
be ween
C26
and
C27,
base
peak),
m/z
511
(M-15,
loss
o
me hyl),
m!z
482
(M-44,
loss
o
ca boni
dioxide)
and
m/z
479
(M-47,
loss
o
me hyl
and
me hanol).
The
mass
spec um
(Fig.
2)
o
29-OH-
30:0
showed
he
expec ed
agmen s,
m/z
117,
539,
510,
and
508,
which
co espond
o
he
same
losses
men ioned
abo e.
Fo
he
lipid
A
isola ed
om
aqueous
LPS
(LAW),
he
majo
componen s
we e
hyd oxy
a y
acids,
and
app oxima ely
54%
o
he
o al
a y
acid
con en
co esponds
o
3-hyd oxy
a y
acids,
acco dingly
o
he
da a
epo ed
(12,
16).
A
40%
componen
o
he
o al
a y
acids
co esponds
o
he
long-
chain
hyd oxy
a y
acids.
27-OH-28:0
is
he
majo
componen ,
in
ag eemen
wi h
he
esul s
o
Ramadas
Bha
e
al.
(15).
29-OH-30:0,
which
has
been
iden i ied
in
Ag obac e ium
spp.
and
B ady hizobium
spp.
(15),
was
iden i ied
and
es ima ed.
These
wo
long-chain
hyd oxy
a y
acids
a e
in
a
3:2
a io.
Vaccenic
acid
was
no
ound,
bu
he
ela ed
lac obacillic
acid
and
he
ing-opened
p oduc
11-OMe-19:0
(7,
8)
ha e
been
iden i ied.
The
gas
ch oma og am
o
he
me hyl
es e s
o
he
a y
acids
om
LAP
allowed
he
iden i ica ion
o
he
me hyl
es e s
o
he
ollowing
sa u a ed
a y
acids:
i-16:0,
19:0
cyc,
11-OMe-19:0,
13-me hoxynonadecanoic
acid
(13-OMe-19:0),
and
11-me-
hoxy-12-me hyloc adecanoic
acid
(1II-OMe-12-Me-18:0).
The
.111..
II
i
NOTES
2455
3
-+
-;C:.
LA."I'l
3
.Oql
RI
L.111.
.11111
L.
II
Ill..
li il
II
II.
11
.11
2456
NOTES
I
,0
10e
Ch
100
80
10
_
le
1.
I
i
L
I
l
4j2
s
I11
L
,-
_1
1
500
30040
1
7
510
53
1
L-
.11
I
A.
-
.
-.
d
_
IF
IM
P
l
-.
p
i-
-!
,-
.,
.,I
I
.
.,,
.
-
I
100
200
300
400
500
600
FIG.
2.
Mass
spec a
o
he
silyla ed
me hyl
es e s
o
27-hyd oxyoc acosanoic
acid
( op)
and
29-hyd oxy iacon anoic
acid
(bo om).
ollowing
hyd oxy
a y
acids
we e
iden i ied
as
he
0- ime h-
ylsilyl
and
0-ace yl
de i a i es
o
hei
me hyl
es e s:
3-OH-
14:0,
3-OH-15:0,
3-OH-16:0,
3-hyd oxyhep adecanoic
acid
(3-
OH-17:0),
3-hyd oxyoc adecanoic
acid
(3-OH-18:0),
27-OH-
28:0,
and
29-OH-30:0.
Two
unsa u a ed
a y
acids
we e
also
de ec ed.
To
iden i y
hem
and
o
loca e
he
double
bond
posi ions,
he
me hyl
es e s
o
he
co esponding
a y
acids
we e
ea ed
wi h
dime hyldisul ide.
Oc adec-11-enoic
acid
[ accenic
acid,
18:1(11)]
and
11-me hyl-oc adec-11-enoic
acid
[11-Me-18:1(11)]
we e
hus
iden i ied
as
dime hyldi hio
de i -
a i es
o
hei
me hyl
es e s.
The
analysis
o
LAP
showed
an
inc ease
o
he
ela i e
p opo ion
o
i-16:0
and
a
dec ease
o
he
ela i e
p opo ions
o
3-hyd oy
a y
acids
(38%)
and
long-chain
hyd oxy
a y
acids
(18%)
compa ed
wi h
hose
o
LAW.
In
LAP,
he
27-OH-28:0
is
no
he
majo
componen ,
bu
he
a io
o
27-OH-28:0
o
29-OH-30:0
is
he
same
as
o
he
aqueous
ac ion.
Addi ionally,
wo
3-hyd oxy
a y
acids
(3-OH-17:0
and
3-OH-18:0)
we e
iden i ied.
Vaccenic
acid,
he
biosyn-
he ically
ela ed
lac obacillic
acid,
and
ou
ing-opened
p od-
uc s,
11-OMe-19:0, 13-OMe-19:0,
11-OMe-12-Me-18:0,
and
11-Me-18:1
(11)
ha e
been
also
iden i ied.
Gas
ch oma og aphy-mass
spec ome y
esul s
a e
summa-
ized
in
Table
1.
Ia
.
p
-1
I
.-.
11
l
-q
1-
.
J.
BACTERIOL.
0
-7
3
55
43
I
S.
.-
1.11
NOTES
2457
TABLE
1.
Fa y
acid
composi ion
o
lipid
A's
om
R.
opici
CIAT899
%
Fa y
acid
composi ion
o ':
Fa y
acid
LAW
LAP
i-16:0
4
10
19:0
cyc
1
10
3-OH-14:0
18
13
3-OH-15:0
19
14
3-OH-16:0
17
8
3-OH-17:0
I
3-OH-18:0
2
11-OMe-19:0
1
11
13-OMe-19:0
I
11-OMe-12-Me-18:0
3
18:1(11)
6
11-Me-18:1(11)
5
27-OH-28:0
24
11
29-OH-30:0
16
7
'
Calcula ed
as
ela i e
pe cen ages
o
he
o al
peak
a eas
in
he
me hyl
es e
ch oma o,g ams.
In
conclusion,
he
analysis
o
he
lipid
moie y
o
he
lipid
A
ob ained
om
R.
opici
CIAT899
showed
he
p esence
o
29-hyd oxy iacon anoic
acid,
which
may
be
used
as
chemo-
axonomic
ma ke .
We
hank
he
Comisi6n
In e minis e ial
de
Ciencia
y
Tecnologia
(g an
B10090-0520-C02)
o
inancial
suppo
and
he
Fundaci6n
Cama a
o
he
g an
o
a
ellowship
o
I.G.-J.
We
also
hank
he
Se icios
de
Espec ome ia
de
Masas
y
Reso-
nancia
Magne ica
Nuclea ,
Uni e sidad
de
Se illa,
o
he
spec a.
REFERENCES
1.
B ink,
R.
A.,
J.
Mille ,
R.
W.
Ca lson,
and
K.
D.
Noel.
1990.
Exp ession
o
Rhizobium
leguminosa um
CFN42
genes
o
lipo-
polysaccha ide
in
s ains
de i ed
om
di e en
R.
leguminosa um
soil
isola es.
J.
Bac e iol.
172:548-555.
2.
Buse ,
H.
R.,
H.
A n,
P.
Gue in,
and
S.
Rausche .
1983.
De e mi-
na ion o
double-bond
posi ion
in
mono-unsa u a ed
ace a es
by
mass
spec ome y
o
dime hyldisulphide
adduc s.
Anal.
Chem.
55:818-822.
3.
Ca lson,
R.,
R.
Sha e s,
J.
Duh,
E.
Tu nbull,
B.
Hanley,
B.
Rol e,
and
M.
Djo dje ic.
1987.
The
isola ion
and
pa ial
cha ac e iza ion
o
he
lipopolysaccha ides
om
se e al
Rhizobium
i olii
mu an s
a ec ed
in
oo
hai
in ec ion.
Plan
Physiol.
(Be hesda)
84:421-
427.
4.
Ca lson,
R.
W.,
S.
Kalembasa,
D.
Tu owski,
P.
Pacho i,
and
K.
D.
Noel.
1987.
Cha ac e iza ion
o
he
lipopolysaccha ide
om
a
Rhizobium
phaseoli
mu an
ha
is
de ec i e
in
in ec ion
h ead
de elopmen .
J.
Bac e iol.
169:4923-4928.
5.
Ca lson,
R.
W.,
R.
E.
Saunde s,
C.
Napoli,
and
P.
Albe sheim.
1978.
Hos -symbion
in e ac ions.
III.
Pu i ica ion
and
pa ial
cha ac e iza ion
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