BIOCHEMICAL
Vol. 169, No. 1, 1990 May 31, 1990
LIPID
A MUTANTS
OF VIBRIO
AND BIOPHYSICAL
CHOLERAE:
RESEARCH COMMUNICATIONS Pages 116-122
ISOLATION
AND
PARTIAL
CHARACTERIZATION SAPTARSHI
PAUL’,
ANADI
ASISH
K. SEN*,
NILIMA
BANERJEE’,
and JYOTIRMOY
N. CHATTERJEEq
DAS’
’ Biophysics
Division and *Organic Chemistry Department Indian Institute of Chemical Biology 4 Raja SC. Mullick Road, Calcutta 700032, India ‘Department Jadavpur
Received
April
9,
of Food University,
Tech. & Biochemical Calcutta 700032,
Eng. India
1990
Vibrio cholerae mutants resistant to common antibiotics and neutral and anionic detergents were isolated. Analysis of isolated outer membranes revealed a sionificant deficiency in the acylation of lipid A in the resistant strains. The contknt of amide-linked and ester-bound fatty acids in the lipid A of the mutant strains compared to that of the wild type was about 50-56% and 29-37% respectively. This defect was specific for lipid A as there was no change in the acylation of phospholipids. The reduction in fatty acid content of lipid A was reflected in 81990 Academic Press, Inc. the altered endotoxic properties in the mutant strains. The
amphiphilic
membrane
macromolecule
of
gram
polysaccharide plays lity
is buried
eliciting
nated
matrix
polysaccharide lipid
A
outer
of
endotoxin
of
role moiety
moiety
*Corresponding
and
of
LPS this
membrane of gram that
like
pyrogenicity,
of
the
The
specific structure
biosynthesis
including
lipid
the
steps
LPS
permeabiof LPS
demonstrated (2,3).
etc.
it is capable dissemi-
(3). biosynthesis
is initiated sugar
has of
the
from
the
KDO
author.
Abbreviations KDO : 3-deoxy-D-manno-2-octulosonic acid; TLC : Thin layer chromatography; GLC : Gas liquid chromatography; SDS : Sodium dodecyl sulphate; PAGE : Polyacrylamide gel electrophoresis; RF3 : Boron trifluoride. 0006-291X&W Copyright All rights
$1.50
0 1990 by Academic Press, of reproduction in any form
Inc. reserved.
116
to
Importance
relationship core
The
membrane
hypotension,
function of
outer
been
in LPS
innermost
interest.
amounts
reaction
outer
A moiety
bacteria
in nanogram
the
specificity,
The
even
at
(4).
in the
and has now
blocked
region
(1).
negative
Schwartzmann
in
biomedical
exposed
phages
responses
elucidation
as
of
present
as antigenic
localised
mutants
in
is relatively
fact
biological
(LPS)
considerable
phenomena
and binding
coagulation,
Isolation key
which
by the
of
is of
diverse
of the
is illustrated a host
a
LPS
in such
component
intravascular
played
of
bacteria
compounds
in the active
of endotoxin of
role
to exogenous
be the
negative
moiety
a crucial
Iipopolysaccharide
is
Vol.
169,
No. 1, 1990
essential tive
for
in the
reported would
cell
survival,
synthesis
of
(5).
Obviously
be of help The
tion
of
in the
BIOCHEMICAL
two
KDO
a relatively
which
availability
present
study strains
of
of the
mutants
v.
cholerae
glucosamine
Organisms
and growth
strain
disaccharide
Materials
defective
isolation
lethal
A precursors
biological
the
RESEARCH COMMUNICATIONS
conditional lipid
of the
describes of
few
accumulate
in our understanding
mutant
acylation
only
AND BIOPHYSICAL
role and
569B of lipid
have
in
lipid
of
LPS.
preliminary which
mutants
have
defec-
so far been
A biosynthesis characterizaspecific
blocks
A.
and Methods
media
The hypertoxinogenic strain 5696 of v. cholerae carrying a streptomycin-resistant marker (50 us/ml) was used as the parental strain. Cells were grown in DIFCO nutrient broth (NB) adjusted to pH 8.0 on a gyratory shaker at 37°C as described previously (6). Isolation
of
mutants
For isolation of mutants, cells were mutagenised with N-nitroso-N’nitro-N-methylguanidine (100 ug/ml, final) in 1OmM phosphate buffer (pH 6.0) for 70 min at 25°C. The surviving cells were grown overnight and two classes of mutants were isolated by appropriate plating. The first class, designated Ipa-l was isolated from plates containing novobiocin (10 yg/ml), nalidixic acid (5 ,ug/ml) and rifampicin (0.5 fig/ml) while the second type designated lpa-2 was selected from plates containing SDS (0.05%) and Triton-X-100 (0.1%). Concentration of the inhibitory agents in the plates was at least 10 fold higher than the minimum growth inhibitory concentration of wild type cells. Both Ipa-l and lpa-2 maintained their resistance profile after repeated growth in control medium. Isolation
of
membranes,
LPS and lipid
A
Crude cell membranes and outer membranes were isolated after mild sonication of cells suspended in 1OmM HEPES buffer (pH 7.0) as described previously (6). LPS was extracted from crude membranes by hot phenol at 68°C (7) and purified by repeated centrifugation (105,OOOxg, 4 hrs, 10°C) in presence of 1mM MgCI.. The final pellet was extracted 4 times with chloroform : methanol (2:1, v/v) to remove phospholipids. Contamination of protein in LPS was less than 2%. Lipid A was split off by treating LPS with 1% acetic acid at 100°C for 150 min. Preliminary experiments established that treatment with acetic acid for 150 min was both necessary and sufficient for quantitative release of lipid A. The insoluble lipid A was centrifuged off and the pellet dissolved in chloroform and the yield was assayed gravimetrically. The supernatant containing the liberated polysaccharide (PS) was dialysed extensively and Iyophilised. Phospholipids were extracted from crude membranes by chloroform : methanol (2:1, v/v) (8). Analysis
of fatty
acids
ester and amide- bound fatty acids of LPS were isolated as Total, described by Rietschel methods. Ester bound fatty --et al (9) for assay by chemical acids were assayed by the method of Snyder (10) while total and amide-bound acids by the method of Duncombe (11). For analyses by GLC, derivatisation of total fatty acids with 2M methanolic HCI or 14% BF,/methanol, ester-bound fatty acids with 0.25M sodium methoxide and amide-bound fatty acids by treatment of de-0-acylated preparation with 14% BFs/methanol were done as described by Mayer --et al (12). The column used was a 5% SE-30 at 160°C using nitrogen as a carrier gas. For analysis of fatty acids in phospholipids, the crude membranes (2:1, v/v) and the fatty acids assayed were extracted by chloroform : methanol of 3-OH-tetradecanoic by GLC as described above for total fatty acids. Absence acid in such preparations ruled out contamination by LPS. 117
Vol.
169, No. 1, 1990
Assay
BIOCHEMICAL
of endotoxic
AND BIOPHYSICAL
activity
Standard methods were followed for (13), localised Schwartzmann reaction (14).
activity gelation Other
RESEARCH COMMUNICATIONS
analytical
assay and
of anticomplementary Limulus amoebocyte
methods
Standard methods were followed for estimation of protein (151, total carbohydrate (161, sugars in LPS (12) and phospholipids (6). Glucosamine in lipid A was assayed (17) after removing the solvent under Nz and hydrolysis with 4N HCI for 4 hrs. Phospholipid was quantitated from the content of bound phosphate (18). Outer membrane proteins were analysed on 12% SDS-PAGE according to the method of Laemmli (19). Assay of perosamine was done according to the method of Redmond (20).
General and
properties
lpa-2
of
in NB and
comparable
with
multiple a change
in
difference
profile
of
outer
(data
not
shown).
and
fructose
and
mutant
was
detectable
in the
the
in the
Analysis
of
treatment strains
in
make
lipid of
and
that
proportion
of
wild
type
mutant
The
LPS visual
A from
the
wild
LPS
in Ipa-l
and
lpa-2
Chemical
Table
assay
1.
amount
and
of
the
Composition
Total
be was 50%
total,
of the of wild
strains
less
confirmed strains. and
amide
like
strains
heptose,
glucose
of
26% and
analysed Glucose
A liberated
acetic
from by The
the
LPS
of
the
a quantitative yield
of
major
as compared ester-bound
to fatty
wild acids
lipopolysaccha-
0.4
1.4
0.31
0.26
0.5
1.5
0.31
0.26
0.5
in text.
mutant
A per
(umoles/mg LPS) Fructose
as described
acid
analysis
lipid
lpa-2
118
glucose
during
Ipa-l
were assayed chemically
and
quan-
any
0.26
Components
type
cholerae,
against
0.31
type
wild v.
heptose
1.25
Wild
compo-
perosamine
suggested
pulysaccharide moiety of type and mutant strains
Component carbohydrate
significant
in the
assay
to
from
mutant
sugar
not
lpa-2
and lpa-2. lipid
much
similar
sugars
of Ipa-l
mutant
was about
core
insoluble
to
impression type
ride Strain
of
was
0-antigenic we did
the
O-antigen
appeared
lipid 5696.
and
up of the
A.
this
Although
no
and
were
result
phospholipid
type
Ipa-l
(21)
and
could
carbohydrate,
LPS
an
Ipa-l
However,
wild
total of
Perosamine,
strains.
of
or in the
the
strains
IV phage
selection)
proteins of
mutant
group
membrane.
moiety
1).
their
between
content
polysaccharide
fact
same
change
seen
the
three
outer
the
phenotypes
during
membrane
was
(Table
in all
the
the
of
specific
Resistant (used
of
Moreover,
strains
titatively,
strain.
of outer
membrane
Growth
to vibrio
detergents
composition
in the
sition
strains.
sensitivity
parental and
the
mutant
their
the
antibiotics
were
the
type
of
mg of strain
confirmed
Vol.
169,
No. 1, 1990
Strain
BIOCHEMICAL
Table
2.
Lipid
Yield m/w
of
lipid Lfs
A composition
of
wild
GlcN
type
RESEARCH COMMUNICATIONS
and
mutant
strains
flmoles/mg LPS Total Ester-bound FA FA
A
Wild type
PO4
0.264
0.204
1.18
Amide-hound FA
Ipa-l
240
0.3
0.2
0.58
0.38
0.22
lpa-2
128
0.254
0.21
0.41
0.28
0.18
analysis
carried
Chemical
this
AND BIOPHYSICAL
deficiency
amount
of
well
with
in the total
fatty
that
reported
components, ting
that
city
of
e.g.
whole
experiments behaved
similarly
extraction
of
3.
lipid
in 5696 previously
molecule
cells using
acids
of
glucosamine
,the LPS
Table
acylation
of
the
wild
type,
respect
2.0 ml cell
suspension
Composition
of
fatty
(1.18
umole/mg
(22).
There
was
in the
mutant
Ipa-l
and
non-polar
(23).
NaCl)
in lipid
A of
wild
fatty
acids
acids
Ipa-
Ester-bound yd YW
Ipa-l
0.50
0.45
0.40
0.15
12:1
0.30
0.16
0.15
0.60
0.27
0.13
3-O-Me-12:O
n.d.
n.d.
n.d.
2.77
1.31
0.72
3-OH-12:O
5.18
2.72
1.51
1.94
1.35
0.67
3.74
1.89
1.18
3.44
1.66
1.04
0.51
0.25
0.14
0.10
tr.
tr.
a A2
14:l a1 AZ-14:1 30H
3.59
1.91
0.80
n.d.
n.d.
n.d.
2.43
1.38
0.66
2.35
1.34
0.56
16:0
3.42
2.06
0.76
2.88
1.95
0.92 0.28
18:l
0.95
0.59
0.21
1.06
0.64
0.56 ---21.58
0.47
0.12
0.39
0.40
11.93
5.98
15.93
Values represent a, al : artifacts produced during
-
9.07
per cent fatty acids by wt. of 3-OH dodecanoic and 3-OH sample preparation.
(Table
all
60%
polar
hydrophobiby partition three
strains
in turbidity
after
ml of xylene.
type
and
--
mutant
Amide-bwnd
strains fatty
4.98
2.45
4.98
2.45
0.18 4.62
of LPS. tetradecanoic
n-d.
acids
1.87
1.87
: Not detected. acids respectively
: 3-OH-lO:O, 3-hydroxydecanoic acid; A’-12:1, dodecenoic acid; Abbreviations 3-O-Me-12n0, 3-acyloxyacyl derivative of dodecanoic acid (formed due to @-eliminot detected in assay for total fatty acids); 3-OH-12:0, 3-hydroxydodenation, A’-14:1, tetradecenoic acid; 3-OH-14:0, 14:0, tetradecanoic acid; canoic acid; 3-hydroxytetradecanoic acid; 16:1, hexadecenoic acid: 16:0, hexadecanoic acid; 18:1, octadecenoic acid; 18:0, octadecanoic acid.
119
The
2) indica-
The
compared
However,
the
0.12
16:l
l&O
in
lpa-2
-14:0
TOTAL
were
by 0.2
fatty Ipa-
30t-k10:o
strains
2).
2) corresponded
change
a loss of about
(in 0.85%
(Table
Table
no
in nature.
Total yd Ype 0.90
Fatty acid analysed
strains
LPS,
lpa-2
solvent
registering
acids
in text.
mutant
be less hydrophobic
as the
in this
A in the
or phosphate could
xylene
out as described
Vol.
169, No. 1, 1990
Table
BIOCHEMICAL
4. Endotoxicity
of
Local 50 type
Limulus
10
5
mutant
Amoebccyte LPs(1.19) 1.0
10
strains Gelaticn
0.1
0.01
7mm
4mm
++++
++++
4mm
0
0
++++
+++
++
+
lpa-2
0
0
0
0
+++
++
+
0
of
was
Ipa-l
and
lpa-2
LPS).
The
reported
the
acylation
reduction
more acid
level
of
both
profile
determine
strains
the
lpa-2
lipid
A of
lipid
strains
and
the
+++
A by GLC (Table
3).
amide-bound
strain,
20%,
zone
defect
and
LPS
seen
9 mm at 50 pg of
the
fatty
(Table
in acylation
was
confirmed
There
fatty
acid
was
a
acids.
This
of
5698,
content
11% and 6% respectively 5696
were total
seen
in
defective
from
both
in
diameter
lpa-2
with
1 : Anticomplementary strains Ipa-l is expressed concentrations
( l as of
fatty these
mutant
(as % wt of
3) was
similar
at
Ipa-l
specific
were
parameters
to
that
was restricted
strains
exhibited
Thus,
the
highest
LPS
was
only
15 IO LPSQJg)
20
while
A, GLC.
No
5696
and
the
to lipid
A.
markedly
concentration 4 mm
low
of at
20 pg
equivalent
endo-
lesion
LPS
tested
LPS amounts
25
) and
the
by
no hemorrhagic
activity of LPS of wild type ( l ) and lpa-2 ( o ). Anticomplementary per cent inhibition of hemolysis LPS added to the undiluted serum
120
lipid
quantitatively
analysed between
to 12 mm and 15 mm for
5
for
and analysed
acids
reaction.
even
LPS compared
extracted
acylation
Schwartzmann
2.5 Fig.
ester
membrane
that
localised
was
the
crude were
activity.
(whealing)
isolated
mutant
about
of
if
the
indicating
in the
in the
A being
differences
biological
in the
phospholipids
significant
in the
A
++++
(22).
from
different
acids
lipid
marked
in lipid
previously To
fatty
of
in the
fatty
phospholipid
(50 Ng),
and
12mm
reduction
toxicity
Reaction
type
9mm
reduced
mutant
wild
15mm
general
for
from
RESEARCH COMMUNICATIONS
Ipa-l
Analysis the
isolated
Schwartzmann Lps(#Q) 20
Strain Wild
LFS
AND BIOPHYSICAL
mutant activity at different (100-150 ~1).
and of
Vol.
169, No. 1, 1990
BIOCHEMICAL
LPS
from
5698
(Table
wild
type
LPS
remained
LPS
was
less
active
than
assay around
reduced
halved
the at
was for
the LPS
5698,
ability
increased
the
the
of
and
4).
of
Ipa-l
lpa-2
was
even
In the
LPS,
complement of
respectively was
the
hemolysis
(Fig. seen
1). This
as the
LPS
per
cent
corresponding
were
of the
potency
inhibition
strains
lpa-2
activity
LPS of
cent
lpa-2
the the
(Table
mutant
25 fig
Ipa-l
The
per
and
while
lo0 ng,
Ipa-l the
Ipa-l
at
type,
to range.
from
of
and
wild
same
RESEARCH COMMUNICATIONS
assay,
lo ug
concentration,
0 for
fixing
gelation
from
over
LPS
10 and
complement
inhibitions
limulus
corresponding
2.5 Ng
30,
concentration
In the unchanged
approximately
fixation was
4).
AND BIOPHYSICAL
around
58,
25
and
20
respectively.
Discussion Recent
studies
forms tive
the
both
with
bioactive
bacteria
natural
centre
(2,3,14).
of endotoxic
In addition
as maintaining
the
microviscocity
To
of
the
date
polar
most
substituents
synthesis
of
also
defect
lpa-2
appear acylation
and
divide in
the this
acylation
we led
at
may
be
cannot
to
results
mutant
It would
essential explain
mutants
of wild appear
for how
defective
the
that
even
that
survival
the the
role
acylation
lpa-2
detergents
in acylation
of
lipid
than methods
such
a serious could
grow
of
fatty
complement
to
acids
assay
strain
laboratory
resistance
of
in endotoxi-
less acylated with
in
A (3,4,5).
of fatty
three
full
under
lipid
in the
was
in all type),
defective
by a fall
which
to note
(24).
of
the
nega-
functions in the
precursors blocks
A
defects
mutants
confirmed lpa-2
lipid
membranes
have
paralleled
activity
A (30%
to
lethal
specific
clearly
biological
lipid
37°C.
not
have
was nicely
important
outer
seem
accumulate
It is interesting
of
of the
conditional
to
as the
lowest
study.
normally
moment
compounds
the
present
LPS
I). The
specially
exhibited
in the
acids
to
in such
endotoxicity in the
shown
and
3 and 4, Fig.
Ipa-l
while
Ipa-l
A and decrease
used
(4,25)
been
fluidity
that
by LPS of gram
in such
A synthesis
A
have
mutants
in the
and proper
indicate
exhibited
A is involved
in lipid
KDO
lipid
(Tables
lipid
preparations
properties
lipid
mutants
of
The city
and synthetic
conditions. and
A and
At
hydrophobic
work
to
clarify
is in progress.
Acknowledgments The
investigation
was
supported
BT/TF/O3/026/009/88), of
W. Germany
(S.P.)
Govt. for
is a recipient
Industrial
Research,
Biophysics
Division
his
kind
of
Senior
Govt. for
their
by
the
of India. help
Department
in the
Research
of
India.
help
of
We are grateful identification Fellowship We
wish
to
and encouragement. 121
Biotechnology
to Prof. of from thank
Ernst
fatty
(Grant Th.
acids.
One of us
Council
of Scientifis-
all
members
the
No.
Rietschel and of
the
Vol.
169,
No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
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Westphal, 0. and Jann, K. (1965) Methods Carbohydr. Chem. 5, 83-91. Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917. Rietschel, E.T., Gottert, H., LUderitz, 0. and Westphal, 0. (1972) Eur. J. Biochem. 28, 166-173. Snyder, F. and Stephens, N. (1959) Biochim. Biophys. Acta 34, 244-245. Duncombe, W.G. (1963) Biochem. J. 88, 7-10. Mayer, H., Thranathan, R.N. and Weckesser, J. (1985) Meth. Microbial. Acad. Press 18, 157-207. Galanos, C., Rietschel, E.T., Liideritz, 0. and Westphal, 0. (1971) Eur. J. Biochem. 19, 143-152. Galanos, C., Liideritz, O., Rietschel, E.T., Westphal, O., Brade, H., Brade, L., Freudenberg, M.A., Schade, U., Imoto, M., Yoshimura, H., Kusumoto, S. and Shiba, T. (1985) Eur. J. Biochem. 146, l-5. Markwell, M.A.K., Hass, S.M., Bieber, L.L. and Tolbert, N.E. (1978) Anal. Biochem. 87, 206-210. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956) Anal. Chem. 28, 350-356. Park, J.T. and Thompson, R.E. (1959) J. Biol. Chem. Strominger, J.L.,
463-468.
Rosenberg, M., Gutnick, D. and Rosenberg, E. (1980) FEMS Microbial. Lett. 9, 29-33. Vaara, M. and Nikaido, H. (1984) Handbook of Endotoxin. Vol. 1. Chemistry of Endotoxin. pp.l-45. Elsevier Science Publishers. B.V., Amsterdam. Sukupolvi, S. and Vaara, M. (1989) Biochim. Biophys. Acta 988, 377-387.
122
Vol. 169, No. 1, 1990 May 31, 1990
LIPID
A MUTANTS
OF VIBRIO
AND BIOPHYSICAL
CHOLERAE:
RESEARCH COMMUNICATIONS Pages 116-122
ISOLATION
AND
PARTIAL
CHARACTERIZATION SAPTARSHI
PAUL’,
ANADI
ASISH
K. SEN*,
NILIMA
BANERJEE’,
and JYOTIRMOY
N. CHATTERJEEq
DAS’
’ Biophysics
Division and *Organic Chemistry Department Indian Institute of Chemical Biology 4 Raja SC. Mullick Road, Calcutta 700032, India ‘Department Jadavpur
Received
April
9,
of Food University,
Tech. & Biochemical Calcutta 700032,
Eng. India
1990
Vibrio cholerae mutants resistant to common antibiotics and neutral and anionic detergents were isolated. Analysis of isolated outer membranes revealed a sionificant deficiency in the acylation of lipid A in the resistant strains. The contknt of amide-linked and ester-bound fatty acids in the lipid A of the mutant strains compared to that of the wild type was about 50-56% and 29-37% respectively. This defect was specific for lipid A as there was no change in the acylation of phospholipids. The reduction in fatty acid content of lipid A was reflected in 81990 Academic Press, Inc. the altered endotoxic properties in the mutant strains. The
amphiphilic
membrane
macromolecule
of
gram
polysaccharide plays lity
is buried
eliciting
nated
matrix
polysaccharide lipid
A
outer
of
endotoxin
of
role moiety
moiety
*Corresponding
and
of
LPS this
membrane of gram that
like
pyrogenicity,
of
the
The
specific structure
biosynthesis
including
lipid
the
steps
LPS
permeabiof LPS
demonstrated (2,3).
etc.
it is capable dissemi-
(3). biosynthesis
is initiated sugar
has of
the
from
the
KDO
author.
Abbreviations KDO : 3-deoxy-D-manno-2-octulosonic acid; TLC : Thin layer chromatography; GLC : Gas liquid chromatography; SDS : Sodium dodecyl sulphate; PAGE : Polyacrylamide gel electrophoresis; RF3 : Boron trifluoride. 0006-291X&W Copyright All rights
$1.50
0 1990 by Academic Press, of reproduction in any form
Inc. reserved.
116
to
Importance
relationship core
The
membrane
hypotension,
function of
outer
been
in LPS
innermost
interest.
amounts
reaction
outer
A moiety
bacteria
in nanogram
the
specificity,
The
even
at
(4).
in the
and has now
blocked
region
(1).
negative
Schwartzmann
in
biomedical
exposed
phages
responses
elucidation
as
of
present
as antigenic
localised
mutants
in
is relatively
fact
biological
(LPS)
considerable
phenomena
and binding
coagulation,
Isolation key
which
by the
of
is of
diverse
of the
is illustrated a host
a
LPS
in such
component
intravascular
played
of
bacteria
compounds
in the active
of endotoxin of
role
to exogenous
be the
negative
moiety
a crucial
Iipopolysaccharide
is
Vol.
169,
No. 1, 1990
essential tive
for
in the
reported would
cell
survival,
synthesis
of
(5).
Obviously
be of help The
tion
of
in the
BIOCHEMICAL
two
KDO
a relatively
which
availability
present
study strains
of
of the
mutants
v.
cholerae
glucosamine
Organisms
and growth
strain
disaccharide
Materials
defective
isolation
lethal
A precursors
biological
the
RESEARCH COMMUNICATIONS
conditional lipid
of the
describes of
few
accumulate
in our understanding
mutant
acylation
only
AND BIOPHYSICAL
role and
569B of lipid
have
in
lipid
of
LPS.
preliminary which
mutants
have
defec-
so far been
A biosynthesis characterizaspecific
blocks
A.
and Methods
media
The hypertoxinogenic strain 5696 of v. cholerae carrying a streptomycin-resistant marker (50 us/ml) was used as the parental strain. Cells were grown in DIFCO nutrient broth (NB) adjusted to pH 8.0 on a gyratory shaker at 37°C as described previously (6). Isolation
of
mutants
For isolation of mutants, cells were mutagenised with N-nitroso-N’nitro-N-methylguanidine (100 ug/ml, final) in 1OmM phosphate buffer (pH 6.0) for 70 min at 25°C. The surviving cells were grown overnight and two classes of mutants were isolated by appropriate plating. The first class, designated Ipa-l was isolated from plates containing novobiocin (10 yg/ml), nalidixic acid (5 ,ug/ml) and rifampicin (0.5 fig/ml) while the second type designated lpa-2 was selected from plates containing SDS (0.05%) and Triton-X-100 (0.1%). Concentration of the inhibitory agents in the plates was at least 10 fold higher than the minimum growth inhibitory concentration of wild type cells. Both Ipa-l and lpa-2 maintained their resistance profile after repeated growth in control medium. Isolation
of
membranes,
LPS and lipid
A
Crude cell membranes and outer membranes were isolated after mild sonication of cells suspended in 1OmM HEPES buffer (pH 7.0) as described previously (6). LPS was extracted from crude membranes by hot phenol at 68°C (7) and purified by repeated centrifugation (105,OOOxg, 4 hrs, 10°C) in presence of 1mM MgCI.. The final pellet was extracted 4 times with chloroform : methanol (2:1, v/v) to remove phospholipids. Contamination of protein in LPS was less than 2%. Lipid A was split off by treating LPS with 1% acetic acid at 100°C for 150 min. Preliminary experiments established that treatment with acetic acid for 150 min was both necessary and sufficient for quantitative release of lipid A. The insoluble lipid A was centrifuged off and the pellet dissolved in chloroform and the yield was assayed gravimetrically. The supernatant containing the liberated polysaccharide (PS) was dialysed extensively and Iyophilised. Phospholipids were extracted from crude membranes by chloroform : methanol (2:1, v/v) (8). Analysis
of fatty
acids
ester and amide- bound fatty acids of LPS were isolated as Total, described by Rietschel methods. Ester bound fatty --et al (9) for assay by chemical acids were assayed by the method of Snyder (10) while total and amide-bound acids by the method of Duncombe (11). For analyses by GLC, derivatisation of total fatty acids with 2M methanolic HCI or 14% BF,/methanol, ester-bound fatty acids with 0.25M sodium methoxide and amide-bound fatty acids by treatment of de-0-acylated preparation with 14% BFs/methanol were done as described by Mayer --et al (12). The column used was a 5% SE-30 at 160°C using nitrogen as a carrier gas. For analysis of fatty acids in phospholipids, the crude membranes (2:1, v/v) and the fatty acids assayed were extracted by chloroform : methanol of 3-OH-tetradecanoic by GLC as described above for total fatty acids. Absence acid in such preparations ruled out contamination by LPS. 117
Vol.
169, No. 1, 1990
Assay
BIOCHEMICAL
of endotoxic
AND BIOPHYSICAL
activity
Standard methods were followed for (13), localised Schwartzmann reaction (14).
activity gelation Other
RESEARCH COMMUNICATIONS
analytical
assay and
of anticomplementary Limulus amoebocyte
methods
Standard methods were followed for estimation of protein (151, total carbohydrate (161, sugars in LPS (12) and phospholipids (6). Glucosamine in lipid A was assayed (17) after removing the solvent under Nz and hydrolysis with 4N HCI for 4 hrs. Phospholipid was quantitated from the content of bound phosphate (18). Outer membrane proteins were analysed on 12% SDS-PAGE according to the method of Laemmli (19). Assay of perosamine was done according to the method of Redmond (20).
General and
properties
lpa-2
of
in NB and
comparable
with
multiple a change
in
difference
profile
of
outer
(data
not
shown).
and
fructose
and
mutant
was
detectable
in the
the
in the
Analysis
of
treatment strains
in
make
lipid of
and
that
proportion
of
wild
type
mutant
The
LPS visual
A from
the
wild
LPS
in Ipa-l
and
lpa-2
Chemical
Table
assay
1.
amount
and
of
the
Composition
Total
be was 50%
total,
of the of wild
strains
less
confirmed strains. and
amide
like
strains
heptose,
glucose
of
26% and
analysed Glucose
A liberated
acetic
from by The
the
LPS
of
the
a quantitative yield
of
major
as compared ester-bound
to fatty
wild acids
lipopolysaccha-
0.4
1.4
0.31
0.26
0.5
1.5
0.31
0.26
0.5
in text.
mutant
A per
(umoles/mg LPS) Fructose
as described
acid
analysis
lipid
lpa-2
118
glucose
during
Ipa-l
were assayed chemically
and
quan-
any
0.26
Components
type
cholerae,
against
0.31
type
wild v.
heptose
1.25
Wild
compo-
perosamine
suggested
pulysaccharide moiety of type and mutant strains
Component carbohydrate
significant
in the
assay
to
from
mutant
sugar
not
lpa-2
and lpa-2. lipid
much
similar
sugars
of Ipa-l
mutant
was about
core
insoluble
to
impression type
ride Strain
of
was
0-antigenic we did
the
O-antigen
appeared
lipid 5696.
and
up of the
A.
this
Although
no
and
were
result
phospholipid
type
Ipa-l
(21)
and
could
carbohydrate,
LPS
an
Ipa-l
However,
wild
total of
Perosamine,
strains.
of
or in the
the
strains
IV phage
selection)
proteins of
mutant
group
membrane.
moiety
1).
their
between
content
polysaccharide
fact
same
change
seen
the
three
outer
the
phenotypes
during
membrane
was
(Table
in all
the
the
of
specific
Resistant (used
of
Moreover,
strains
titatively,
strain.
of outer
membrane
Growth
to vibrio
detergents
composition
in the
sition
strains.
sensitivity
parental and
the
mutant
their
the
antibiotics
were
the
type
of
mg of strain
confirmed
Vol.
169,
No. 1, 1990
Strain
BIOCHEMICAL
Table
2.
Lipid
Yield m/w
of
lipid Lfs
A composition
of
wild
GlcN
type
RESEARCH COMMUNICATIONS
and
mutant
strains
flmoles/mg LPS Total Ester-bound FA FA
A
Wild type
PO4
0.264
0.204
1.18
Amide-hound FA
Ipa-l
240
0.3
0.2
0.58
0.38
0.22
lpa-2
128
0.254
0.21
0.41
0.28
0.18
analysis
carried
Chemical
this
AND BIOPHYSICAL
deficiency
amount
of
well
with
in the total
fatty
that
reported
components, ting
that
city
of
e.g.
whole
experiments behaved
similarly
extraction
of
3.
lipid
in 5696 previously
molecule
cells using
acids
of
glucosamine
,the LPS
Table
acylation
of
the
wild
type,
respect
2.0 ml cell
suspension
Composition
of
fatty
(1.18
umole/mg
(22).
There
was
in the
mutant
Ipa-l
and
non-polar
(23).
NaCl)
in lipid
A of
wild
fatty
acids
acids
Ipa-
Ester-bound yd YW
Ipa-l
0.50
0.45
0.40
0.15
12:1
0.30
0.16
0.15
0.60
0.27
0.13
3-O-Me-12:O
n.d.
n.d.
n.d.
2.77
1.31
0.72
3-OH-12:O
5.18
2.72
1.51
1.94
1.35
0.67
3.74
1.89
1.18
3.44
1.66
1.04
0.51
0.25
0.14
0.10
tr.
tr.
a A2
14:l a1 AZ-14:1 30H
3.59
1.91
0.80
n.d.
n.d.
n.d.
2.43
1.38
0.66
2.35
1.34
0.56
16:0
3.42
2.06
0.76
2.88
1.95
0.92 0.28
18:l
0.95
0.59
0.21
1.06
0.64
0.56 ---21.58
0.47
0.12
0.39
0.40
11.93
5.98
15.93
Values represent a, al : artifacts produced during
-
9.07
per cent fatty acids by wt. of 3-OH dodecanoic and 3-OH sample preparation.
(Table
all
60%
polar
hydrophobiby partition three
strains
in turbidity
after
ml of xylene.
type
and
--
mutant
Amide-bwnd
strains fatty
4.98
2.45
4.98
2.45
0.18 4.62
of LPS. tetradecanoic
n-d.
acids
1.87
1.87
: Not detected. acids respectively
: 3-OH-lO:O, 3-hydroxydecanoic acid; A’-12:1, dodecenoic acid; Abbreviations 3-O-Me-12n0, 3-acyloxyacyl derivative of dodecanoic acid (formed due to @-eliminot detected in assay for total fatty acids); 3-OH-12:0, 3-hydroxydodenation, A’-14:1, tetradecenoic acid; 3-OH-14:0, 14:0, tetradecanoic acid; canoic acid; 3-hydroxytetradecanoic acid; 16:1, hexadecenoic acid: 16:0, hexadecanoic acid; 18:1, octadecenoic acid; 18:0, octadecanoic acid.
119
The
2) indica-
The
compared
However,
the
0.12
16:l
l&O
in
lpa-2
-14:0
TOTAL
were
by 0.2
fatty Ipa-
30t-k10:o
strains
2).
2) corresponded
change
a loss of about
(in 0.85%
(Table
Table
no
in nature.
Total yd Ype 0.90
Fatty acid analysed
strains
LPS,
lpa-2
solvent
registering
acids
in text.
mutant
be less hydrophobic
as the
in this
A in the
or phosphate could
xylene
out as described
Vol.
169, No. 1, 1990
Table
BIOCHEMICAL
4. Endotoxicity
of
Local 50 type
Limulus
10
5
mutant
Amoebccyte LPs(1.19) 1.0
10
strains Gelaticn
0.1
0.01
7mm
4mm
++++
++++
4mm
0
0
++++
+++
++
+
lpa-2
0
0
0
0
+++
++
+
0
of
was
Ipa-l
and
lpa-2
LPS).
The
reported
the
acylation
reduction
more acid
level
of
both
profile
determine
strains
the
lpa-2
lipid
A of
lipid
strains
and
the
+++
A by GLC (Table
3).
amide-bound
strain,
20%,
zone
defect
and
LPS
seen
9 mm at 50 pg of
the
fatty
(Table
in acylation
was
confirmed
There
fatty
acid
was
a
acids.
This
of
5698,
content
11% and 6% respectively 5696
were total
seen
in
defective
from
both
in
diameter
lpa-2
with
1 : Anticomplementary strains Ipa-l is expressed concentrations
( l as of
fatty these
mutant
(as % wt of
3) was
similar
at
Ipa-l
specific
were
parameters
to
that
was restricted
strains
exhibited
Thus,
the
highest
LPS
was
only
15 IO LPSQJg)
20
while
A, GLC.
No
5696
and
the
to lipid
A.
markedly
concentration 4 mm
low
of at
20 pg
equivalent
endo-
lesion
LPS
tested
LPS amounts
25
) and
the
by
no hemorrhagic
activity of LPS of wild type ( l ) and lpa-2 ( o ). Anticomplementary per cent inhibition of hemolysis LPS added to the undiluted serum
120
lipid
quantitatively
analysed between
to 12 mm and 15 mm for
5
for
and analysed
acids
reaction.
even
LPS compared
extracted
acylation
Schwartzmann
2.5 Fig.
ester
membrane
that
localised
was
the
crude were
activity.
(whealing)
isolated
mutant
about
of
if
the
indicating
in the
in the
A being
differences
biological
in the
phospholipids
significant
in the
A
++++
(22).
from
different
acids
lipid
marked
in lipid
previously To
fatty
of
in the
fatty
phospholipid
(50 Ng),
and
12mm
reduction
toxicity
Reaction
type
9mm
reduced
mutant
wild
15mm
general
for
from
RESEARCH COMMUNICATIONS
Ipa-l
Analysis the
isolated
Schwartzmann Lps(#Q) 20
Strain Wild
LFS
AND BIOPHYSICAL
mutant activity at different (100-150 ~1).
and of
Vol.
169, No. 1, 1990
BIOCHEMICAL
LPS
from
5698
(Table
wild
type
LPS
remained
LPS
was
less
active
than
assay around
reduced
halved
the at
was for
the LPS
5698,
ability
increased
the
the
of
and
4).
of
Ipa-l
lpa-2
was
even
In the
LPS,
complement of
respectively was
the
hemolysis
(Fig. seen
1). This
as the
LPS
per
cent
corresponding
were
of the
potency
inhibition
strains
lpa-2
activity
LPS of
cent
lpa-2
the the
(Table
mutant
25 fig
Ipa-l
The
per
and
while
lo0 ng,
Ipa-l the
Ipa-l
at
type,
to range.
from
of
and
wild
same
RESEARCH COMMUNICATIONS
assay,
lo ug
concentration,
0 for
fixing
gelation
from
over
LPS
10 and
complement
inhibitions
limulus
corresponding
2.5 Ng
30,
concentration
In the unchanged
approximately
fixation was
4).
AND BIOPHYSICAL
around
58,
25
and
20
respectively.
Discussion Recent
studies
forms tive
the
both
with
bioactive
bacteria
natural
centre
(2,3,14).
of endotoxic
In addition
as maintaining
the
microviscocity
To
of
the
date
polar
most
substituents
synthesis
of
also
defect
lpa-2
appear acylation
and
divide in
the this
acylation
we led
at
may
be
cannot
to
results
mutant
It would
essential explain
mutants
of wild appear
for how
defective
the
that
even
that
survival
the the
role
acylation
lpa-2
detergents
in acylation
of
lipid
than methods
such
a serious could
grow
of
fatty
complement
to
acids
assay
strain
laboratory
resistance
of
in endotoxi-
less acylated with
in
A (3,4,5).
of fatty
three
full
under
lipid
in the
was
in all type),
defective
by a fall
which
to note
(24).
of
the
nega-
functions in the
precursors blocks
A
defects
mutants
confirmed lpa-2
lipid
membranes
have
paralleled
activity
A (30%
to
lethal
specific
clearly
biological
lipid
37°C.
not
have
was nicely
important
outer
seem
accumulate
It is interesting
of
of the
conditional
to
as the
lowest
study.
normally
moment
compounds
the
present
LPS
I). The
specially
exhibited
in the
acids
to
in such
endotoxicity in the
shown
and
3 and 4, Fig.
Ipa-l
while
Ipa-l
A and decrease
used
(4,25)
been
fluidity
that
by LPS of gram
in such
A synthesis
A
have
mutants
in the
and proper
indicate
exhibited
A is involved
in lipid
KDO
lipid
(Tables
lipid
preparations
properties
lipid
mutants
of
The city
and synthetic
conditions. and
A and
At
hydrophobic
work
to
clarify
is in progress.
Acknowledgments The
investigation
was
supported
BT/TF/O3/026/009/88), of
W. Germany
(S.P.)
Govt. for
is a recipient
Industrial
Research,
Biophysics
Division
his
kind
of
Senior
Govt. for
their
by
the
of India. help
Department
in the
Research
of
India.
help
of
We are grateful identification Fellowship We
wish
to
and encouragement. 121
Biotechnology
to Prof. of from thank
Ernst
fatty
(Grant Th.
acids.
One of us
Council
of Scientifis-
all
members
the
No.
Rietschel and of
the
Vol.
169,
No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
References
1.
Lugtenberg,
6.
and
Van
Alphen,
L.
(1983)
Biochim.
Biophys.
Acta
737,
51-115. 2. 3.
4.
5. 6.
Takada, l-l. and Kotani, S. (1989) CRC Critical Rev. Microbial. 16, 477-523. Rietschel, E.T., Brade, H., Brade, L., Brandenburg, K., Schade, U., Seydel, U., Zshringer, U., Galanos, C., Liideritz, O., Westphal, O., Labischinski, H., Kusumoto, S. and Shiba, T. (1987) Progr. Clin. Biol. Res. Vol. 231, 25-53. Alan R. Liss, Inc. Mskelii, P.H. and Stocker, B.A.D. (1984) Hand Book of Endotoxin. Vol. 1: Chemistry of Endotoxin, pp.59-137. Elsevier Science Publishers B.V., Amsterdam. Raetz, C.R.H. (1986) Ann. Rev. Genet. 20, 253-295. Lohia, A., Mazumdar, S., Chatterjee, A.N. and Das, J. (1985) J. Bacterial. 163,
7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17.
234,
18. 19. 20. 21. 22.
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