Sugar nucleotide transferases in Escherichia coli lipopolysaccharide biosynthesis

Vol.

16,

No.

6, 1964

BIOCHEMICAL

SUGAR

NUCLEOTIDE

AND

BIOPHYSICAL

TRANSFERASES

LIPOPOLYSACCHARIDE Ronald Rackham

D.

Arthritis The

IN

RESEARCH

COMMUNICATIONS

ESCHERICHIA

COLI

BIOSYNTHESIS’ 2,3

Edstrom

Research University

and

Edward

Unit and of Michigan,

C.

Heath’

Department of Microbiology Ann Arbor

and

The

Department Johns Hopkins

of Physiological Chemistry School of Medicine, Baltimore

Received June 22, 1964 The which

cell

wall

contains

(Heath

and

fatty

acids,

Ghalambor,

L-xylohexose). the

lipopolysaccharide

enzyme

an incomplete

tains

amounts

reduced for

mine

J-5

.

wild-type

Mclterials noted

enzymatic their

Further,

nucleotide

by and

Heath,

(Ghalambor

chemical

All

KDO-1-C14(3

and

tose-Cl4

(GDP-Col,

previously

(Heath

x lo6

Heath,

19630). with 7.8

and

acceptor

compounds

for

the crude

this

1962),

in the colitose

paper

conevi-

glucosa-

deficient

LPS of

of KDO,

extracts

transferase

absence

presents

transfer

lacks

and

N-acetyl to the

that

Elbein,

obtained

from

was

prepared

was

generated

just

GDP-mannose-C14(7.8

extracts x IO5

were

cprr/prnole)

CMP-KDO-I-C14

mannose-U-C14

derivatives)

dideoxy-

from

f.rom

either

is a fragment

of

degradation.

Methods---

1963b).

the

Elbein,

and This

by

(3,6

is grown

glucose,

indicating

(KDO)

and

golactose

is catalyzed mutant;

polymer

colitose

(Heath

glucosamine.

is presented

or the

and

of golactose,

respective

is a corrplex

organism

lacks

N-acetyl transfer

evidence

organism

below.

from

and

J-5

the

which

monophosphate-KDO(CMP-KDO)

LPS obtained

and

When

of glucose

(from

galactose

--E. coli

LPS is formed

sequential

colitose

cytidine the

the

and

E. coli --

organism,

0711

3-deoxy-octulosonate

glucose,

UDP-galactose-depimerase.

of golactose,

dence

heptose,

of this

of --E. coli

hexosamine,

phosphorus, 1963),

A mutant

(LPS)

of a mutant cpm/)lmole)

before x IO5

prepared

sources

enzymatically

of Salmonella was

commercial

from

(Ghalambar

use from

CTP and

cpdpmole)

typhimurium GDP-mannose

unless

was 4.

KDO

prepared

GDP-colias described

1962).

i

Supported by grants from the National Institutes of Health: AM-08318 and AM-06278. Present address: Department of Physiological Chemistry, The Johns Hopkins School of 3 Medicine. 4 U . S. P. H. S. postdoctoral fellow. We would like to thank Dr. S. Rosen, Department of Molecular Biology, Albert Einstein School of Medicine, New York, N. Y. for providing this organism and details of preparation of GDP-mannose prior to the publication of his results.

576

Vol. 16, No. 6, 1964

BIOCHEMICAL

AND BIOPHYSICAL

UDP-galactose-C14(UDP-Gal, Wiesmeyer

and

(1961) with

Jordan

l x lo6 cpm/pmole) 1961).

and

a gift

of Mr.

was

0.05

borate,

0.06

--et al.,

1959).

pH

5 .O (Bourne

were

as follows:

(7:3);

Ill.

of 3:

I.

by the

Duncombe

(1956),

Weissbach

the

prepared

and

0111

transferred

and

samine,

and

and

contained

polymer

this

sugar that

the

of

No.

Unit.

1) solvents

of the

method

cultures

(upper

determined fatty

--et al .,

acids and

method

by the KDO

of Waravdekar

by

and

Trypticase

by sonic

phase

by the

standard5,

on

used

(Trevelyan

procedure, as the

molybdate,

propane-2-ol:water

nitrate

acid

Paper

in either

acid:water

was

x 105

M sodium

II.

silver

disrupted

Soy

Saslaw

broth6

oscillation.

until

LPS was

1952). 12,000-40,000

the

cell

deficient

fraction

X g particulate LPS and

was the

various

constituents

sugar

as follows:

the

results

with

shown

transferase

various

combina-

in Table

of the

golactose,

fraction

glycosyl

incubated

nucleotides,

sequentially,

isolated

from ion

contained

incubation

hydrolyzed

active

sugars

were

characterized

borate

buffer)

and

as follows:

of the

C

in 2 N H2S04 by paper glucose 1962).

like to thank Dr. W. Biological Laboratories,

by

(Osborn

70 to 90%

were

(Doudoroff,

mixtures

precipitation

precipitates)

would Baltimore

both

or 0.1

(5:3:2);

Boas (1953)

et al., --

8.7 Research

(Whatman

in shake

(Westphal

suggest

4.6,

Phosphorus

Cel Is were

When

pH

alkaline

The washed,

J-5

magnesium

(which

dehydrogenase

grown

method

--et al.,

at 40 to 60 volts/cm

acetate:acetic

lauric

the

1 were

ob-

polysaccharide

are

N-acetyl

gluco-

glucose,

colitose.

LPS was

rations

were

unlabeled

data

to the

paper

modification

reached.

to be described.

These

3 MM

with

B-hydroxy

biosynthesis--of --E. coli

tained.

tation

or J-5

extraction

of labeled

Arthritis

by the

(1959)

by phenol

activities tions

Hurwitz

was

of extracts

Rackham

(1960).

hexosamine

log phase

Polysaccharide

of the

ethyl

by

(Roseman

(UDP-GlcNAc,

1 N HCl

detected

using

prepared

labeled)

acetate,

IV.

prepared

UDP-glucose-C14(UDP-Glc,

chromatographic

of Warren

(1963)

--E. coli

early

were

method

method

(1959).

Paper

(8:2);

Sugars

--et al.,

No.

M pyridinium

propane-2-ol:water

KDO

of Chen

on Whatman

J-5.

(acetyl

Distler

was

chemically

butane-I-ol:pyridine:O.

1:3 mixture).

1950);

the

done

l4

Jack

electrophoresis M sodium

were

glucosamine-C

was

cpm/pmole)

use of E. -- coli

UDP-galactose

UDP-N-acetyl

cpm/pmoIe)

1 x lo6

RESEARCH COMMUNICATIONS

et al., --

14

originally

for

J.

2 hours

digestion,

1962).

oxidase,

conditions

Lennarz Baltimore,

577

The

present at

of hydrolysis

for

this compound. Maryland.

purified

The

paper and

ethanol

in the

100’.

chromatography,

by glucose The

Pronase

precipiLPS prepa-

washed

ethanol

liberated

radio-

electrophoresis

galactose necessary

(in

by galactose to cleave

the

Vol.

16,

No.

6, 1964

hexosaminyl acetyl

BIOCHEMICAL

bond

labeled

also

caused

N-acetyl

AND

BIOPHYSICAL

deacetylation

and

thus

RESEARCH

prevented

characterization

C14SUBSTRATE

I

ADDITION

UDP-Gal*

mpmoles

UDP-Gal

10.2

UDP-Glc

UDP-Glc

15

0.8

UDP-Gal

UDP-GlcNAc

5.5

None UDP-Gal

or UDP-Glc

0.9

UDP-GlcNAc

UDP-Gal

and

3.3

UDP-Glc

None UDP-Gal UDP-Gal,

7.9

0.7

UDP-GlcNAc

GDP-Co1

R

10.0

None

UDP-Glc

M RAyL!$

INCORPORATED

None

GDP-Co1

of the

glucosamine. Table

GDP-Co1

COMMUNICATIONS

4.7

0.1

and

UDP-Glc

UDP-Glc

and

0.1 UDP-GlcNAc

1.0

1.4

*Incubation mixtures contained: Tris, pH 8.5, 25 pmoles; Mg C12, 2.5 pmoles; chlorarrphenicol, 25 pg; sugar nucleotide, 0.1 pm&e; enzyme particulate, 0.4 mg protein. Final volume was 0.25 ml. The mixtures were incubated at 370 for 2 hours. The reactions were stopped by the addition of 0.5 ml of ethanol and the precipitates were washed 4 times with I ml of 70% ethanol. The precipitates were then dissolved in 2 ml of 1 .O M Hyamine and counted in a toluene based scintillation solvent. **The amount of incorporated sugar relative to 100 moles of endogenous glucose in the LPS acceptor of the active particle.

Evidence added

that

golactose

tures

which

residue

contained

hydrolyzed mixture

(0.1

exhaustively galactose.

tose

type

obtained

various

and

combinations 1 hour) first

buffer.

The

of disaccharide

the

results

were labeled

An additional the

products of these

was

being

transferred LPS,

as follows:

100°,

disaccharides

hydrolyzed;

and

indeed

chromatography The

molybdate

was the

in 2 N H2S04,

sodium

was

N H2S04,

by paper

(RGlc=0.48). hours

glucose

and

a disaccharide

was

III

(RGl

with

products

characterized

portion

formed.

518

from indicated

reduced

experiments

isolated

to the

of substrates

in solvent

were

directly

shown

=0.61),

NaBL4,

of the

in

indicated

was

each

by solvent at

100°

1

for

2

electrophoresis disaccharide

quantities that

II,

from

followed

labeled

mix-

Table

isolated

by paper

to be equimolar clearly

incubation

hydrolyzed

doubly

previously

in was

of glucose

a &cosyl-galac-

Vol.

16, No.

6, 1964

BIOCHEMICAL

AND

BIOPHYSICAL

Table UNLABELED

C’SUBSTRATE

RESEARCH

COMMUNICATIONS

II C’4PRODUCT

SUBSTRATE

REDUCTION

AFTER

AND

HYDROLYSIS

UDP-galactose

UDP-glucose

Galactitol

UDP-glucose

UDP-galactose

Glucose

UDP-golactose UDP-glucose *Equimolar

Incorporation

quantities

of KDO---

matically

catalyzed

of CMP-KDO scribed KDO

with

to the

prepared (0.2

with

Addition

chemical

and

crude

extract

was

electrophoretically

fied

by a combination

S Blue

Ribbon,

of this

material

gave

was

acid

0.12;

hydrolysis

shown Heath, by paper

The

from

authentic

KDO

material

obtained

by only

electrophoresis).

product

on the

drolysis,

and

basis that

the

The

KDO

group

III)

0.07

N HCI,

containing

of this assumed

to alkaline could

hydrolysis.

579

not

of the

hydrolysis, be reduced

was and

puri-

paper from

the

100;

or-

was present).

After

in a compound

which

and

electro-

paper

product

was

and

(determined

glycosidically susceptibility

chemically

further

(Ghalambor

was pyruvate

to be linked

acid.

which

elution

KDO-aldolase reaction

fatty

hexosamine,

chromatography

specific

and

buffer)

heptose

alkali 30 min.),

a product

After

was

by hydrolysis

lODo,

product

ratios:

of

CMP-KDO-l-Cl4

into

1).

transfer

of LPS with

acetate

(no

(de-

to an acceptor

radioactive

molar

incubation

in the

phosphorus

radioactivity

with

was

(0.5

RKDO=l.

by paper

product

of its resistance carbonyl

of the

its reactivity

radioactive

II,

KDO,

all

to be KDO-l-Cl4 the

0.99;

treatment

The

following

product,

radioactive

1963b);

acid,

the

After

(pyridinium

solvent

result

CMP-KDO

of radioactivity

substrate.

of the

indistinguishable

phoresis.

fatty

the

not

organic

Thus,

enzy-

preparations

from

(Table

electrophoresis

(S and

ganicphosphorus,

mixtures

incorporation

did

hydrolysis

hexosamine,

from

of paper

LPS,

by acid

contained

enzyme

LPS.

the

unsuccessful.

transferred of the

followed

to demonstrate

particulate added

was

degradation

distinct

chromatography analysis

KDO

in the

LPS were

or the

to incubation

resulted

designed

into

LPS or with

which

material

KDO

extracts

30 min.),

isolated

of this

paper,

crude

endogenous

60°,

was

experiments of

However,

by partial N NaOH,

mild

either

and

product.

Preliminary

polymer.

a fragment

of each

incorporation

above),

Galactitol* Glucose*

None

and

in the to acid

prior

to acid

hy-

Vol.

16, No.

6, 1964

BIOCHEMICAL

AND

BIOPHYSICAL

Table INCUBATION

RESEARCH

III mpmoles

MIXTURE

INCORPORATED

Complete*

0.90

-acceptor

0.10

-acceptor

+ purified

-crude

COMMUNICATIONS

0.13

LPS

extract

0.12

-CMP-KDO

synthetase

1 .Ol 0.45

-CTP

*Incubation mixtures were as foil?? (pmoles): Tris, pH 8, 15; MgCl , 1; 4.08 pg; final cytidine triphosphate, 1.25; KDO-1-C , 0.03; CMP-KDO synthetase, volume, 0.075 ml. After incubation at 370 for 30 min., 0.05 ml (250 pg) of acceptor and 0.2 ml of crude extract were added. After incubation for 20 min. at 370, an aliquot was applied to a paper strip and subjected to electrophoresis in pyridinium acetate buffer. The radioactivity remaining at the origin was determined in a scintillation spectrometer in a toluene solvent.

Discussion--J-5

In contrast

is deficient

presented

are

to the added

the

acetyl

been

apparent

in the

GlcNAc.

The

of

of?.

7 stein

ruled

mutants 1964;

GI c,

out.

each

3964;

same

incorporation

typhimurium

Zeleznick

has also

--et al.,

Personal commun ication College of Medicine,

Col)

1964;

and

phosphate in the

transthat

the

have

rhamnose

mono-

demonstra-

glucose

and

N7

typhimurium

1964).

These

sugars

of the

deficient

Glc,

Gal,

-i.e.

sugars

intermedi-

of Salmonella D’Ari,

data

nucleotide

co-workers

(Nikaido,

E. coli --

The

oligosaccharide

studies;

of galactose,

from

suggest

“backbone”

present

reported

colitose.

galactose,

of mutants

Osborn

and

respective

their

of glucose,

polymer

results

their

and

the

be enzymatically

of nucleotide

transfer

been

can the

from

Horecker

polyheptose

as observed

sugars While

LPS of a variety

to the

enzymatic

system.

and

Rosen --et al.,

order

glucosamine

involvement

enzymatic

to deficient

organism,

of these

GlcNAc,

0 s b orn

sequential

attached

acceptor

that

the possible

--et al.,

apparently

aido,

(Gal,

wild-type

N-acetyl

LPS in an in vitro --

glucosamine

(Rothfield are

deficient

the

glucose,

indicate

derivatives,

has not

ted

report

sequentially

saccharide ates

in galactose,

in this

ferred

to LPS from

and

mannose

1962;

Nikaido

LPS Glc,

into

LPS

and

Nik-

1964).

from New

Drs. York,

Hilberman, N. Y.

580

Osborn

and

Horecker,

Albert

Ein-

Vol.

16, No.

Osborn phosphate position

BIOCHEMICAL

6, 1964

(1963)

has observed

“backbone”

for the

of the

KDO

of the

acceptor

The

enzymatic

in this and

that

is presumably

7 or 8 of KDO.

acceptor

AND

polymer the

in LPS from bound

apparent

transfer

of KDO

to KDO

from

the

of the

degraded

CMP-KDO

of KDO

to the are

under

COMMUNICATIONS

the

through

attached

of attachment

RESEARCH

S. typhimurium

requirement

is glycosidically site

BIOPHYSICAL

hydroxyl

group

lipid

suggests lipid

polyheptose

that moiety.

at either

fragment

as

at least

some

The

structure

investigation.

References Boas, N. F., J. Biol. Chem., 204, 553 (1953). Bourne, E. F., Hutson, D. H. andweigel, H., Chem. and Ind. (London) 1047 (1959). Chen, P. S., Toribara, T. Y., and Warner, H., Anal. Chem., 28, 1756 (1956). Doudoroff, M., In Methods in Enzymology, edited by S. P. Colozck and N. 0. Kaplan, 5, 339 (1962). Duncombe, W. G., Biochem. J., 88, 7 (1963). Ghalambor, M. A., and Heath, E.., Biochem. Biophys. Res. Communs., 210 346 (1963a). Ghalambor, M. A., and Heath, E. C., Biochem. Biophys. Res. Communs., -11, 288 (1963b). Heath, E. C. and Elbein, A. D., Proc. Nat. Acad. Sci., 48, 1209 (1962). Heath, E. C. and Ghalambor, M. A., Biochem. Biophys. Res. Communs., E, 340 (1963). Nikaido, H., Proc. Nat. Acad. Sci., 48, 1542 (1962). Nikaido, H. and Nikaido, K., Abstracx6th Intern. Congr. Biochem., Section VI, No. 78 (1964). Osborn, M J., Rosen, S. M., Rothfield, L. and Horecker, B. L., Proc. Nat. Acad. Sci., 48, 1831 (1962). Osborn, M.1, Proc. Nat. Acad. Sci., 50, 499 (1963). Osborn, M. J., and D’Ari, L., Biochem.xiophys. Res. Communs. , 16, 568 (1964). Roseman, S., Distler, J. J ., Moffatt, J. G. and Khorana, H. G., Jxm. Chem. Sot., -83, 659 (1961). Rosen, S. M., Osborn, M. J., and Horecker, B. L., J. Biol. Chem., In press. Rothfield, L., Osborn, M. J., and Horecker, B. L., J. Biol. Chem., In press. D. P. and Harrison, J. S., Nature, 166, 444 (1950). Trevelyan, W. E., Procter, Waravdekar, V. S., and Saslaw, L. D., J. Biol. Chem., 234, 19x(1959). Warren, L., Nature, 186, 237 (1960). Weissbach, A., and Hxitz, J., J. Biol. Chem., 234, 705 (1959). Westphal, O., Luderitz, 0 and Bister, F., Z. Naturforsch., 7b, 148 (1952). Wiesmeyer, H. and Jordan, E., Anal. Biochem., 2, 281 (196r Zeleznick, L. D., Rosen, S. M., Saltmarsh-Andrew, M., Osborn, M. J. and Horecker, B. L., Abstracts 6th Intern. Congr. Biochem., Section VI, No. 125 (1964).

581