Ter mutation and susceptibility to φx174 phage in E. coli K12

Vol. 91, No. 3, 1979 December

BKICHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

14, 1979

Pages

Ter Mutation

and Susceptibility

to @X174 Phage

Tsutomu Department

Received

of Biochemistry, Kanazawa,

October

23,

in E. coli

1051-l

056

K12

Ohkawa

School of Medicine, Ishikawa, Japan

Kanazawa 920

University,

1979

SUMMARY :

The Ter-15 mutant derived from E. coli K12 W2252-lluRCStr (wild type I) is found to be sensitive to $x174 phage infection. Lipopolysaccharide extracted from this mutant inactivates the phage, and has core oligosaccharides identical in amounts to those in the lipopolysaccharide from wild type cells. In contrast, the Ter-21 mutant derived from E. coli K12 W2252-lluRCrel (wild type II) is not sensitive to this phage infection, and its lipopolysaccharide does not inactivate the phage. Its lipopolysaccharide sugars are found to be D-glucose and D-ribose, thus differing from the lipopolysaccharide sugars of the wild type cells. @x174 phage

contains

a single

nucleotides

(1)

in an icosa

From the

reports

of Lindberg

able

to

infect

strains.

the

is

primarily

specific

receptor

binding,

it

releases

the

ved from

sensitive

(7,8).

identified Lindberg

is

strain,

DNA (6).

the

and Kornberg is

of E. coti

be represented

in the

structure

(4),

the

the

on the cell

cells.

wall

purified

of

found

inactivates

the

cell

phage

or semirough

to

4x174

phage

regard

phage

can bind to

to

41x174 phage

(LPS)

deri-

to

for

the

be

$x174

outer

core

Abbreviations used : LPS, lipopolysaccharide ; circular deoxyribonucleic acid ; dTDPG, thymidine KW, 2-keto-3-deoxymannulosoctonic acid.

phage

receptors chain

activity the

the

is

lipopolysaccharides

and showed in the

$x174

(2).

and

receptor that

5375

(5)

the polysaccharide

for

LPS of

With

of

12 particles

smooth

or not

and S. typhinmriwn,

by the

infect

are demonstrated

important

et al

upon whether

the

structure

at the

strain

(4) demonstrated

found

spikes

coli

sites

Further,

cannot

DNA (SS-DNA)

Escherichia

isolated

strains

Also,

but

of the

the

with

(3) and Jazwinski

attachment found

circular

coat

dependent

as crucially

S13 receptor strains

hedral

The susceptibility

infection

phage

rough

strand

in the

LPS is

(3).

Jazwinski,

and the

related

membrane this

this

of

phage

sensitive

binding

and backbone SS-DNA, single 5'-diphosphate

phage

of

site the

to

LPS.

strand glucose

;

0006-291X/79/231051-06$01.00/0 1051

Copyright All rights

@ 1979 by Academic Press, Inc. of reproduction in anyform reserved.

Vol. 91, No. 3, 1979

We are

interested

in resistant dine

coli

in the

and sensitive

5'-diphosphate

precursor

for

synthesis

(wild

mutant type

their

E. cozi

In this

paper,

by the

LPS from

is

cell's

in 9x174

sensitive

type the

nor

its

were

but

its

K12 W2252-llu-

Ter-21

mutant

in the the

as a

phage,

cozi

from

thymi-

Escherichia

9x174

discrepancy

contents

of cells

membrane.

Escherichia I)

infection

in growing

to

extracted

sugar

phage

concentration

formed outer

Neither to

RESEARCH COMMUNICATIONS

decreased

is not

and their

strains

Ter-mutation

wild

types

analyzed

and

in detail

chromatography.

we describe these

the

LPS were

and gas-liquid

occurs

which

To resolve

the

strains,

that

sensitive.

phage.

K12 strains,

Ter mutant

I)

strain

to $x174

by thin-layer

tion

(9) becomes

II)(non-isogenic

sensitive

from

(dTDPG)

type

BIOPHYSICAL

through

of LPS of (wild

AND

change

strains

glucose

K12 W2252-llU-

Ter-15

is

BIOCHEMICAL

the

E. coli

phaqe cells,

sensitivity, and the

the sugar

phaqe

inactiva-

components

of

the

LPS. Materials and Methods Bacteria : Escherichia coli K12 W2252-llU(thy-,ura-,met-) RCStr (wild type I) and its dTDPG pyrophosphorylase deficient mutant is named Ter-15 (9). Escherichia cozi K12 W2252-llu(thy-,ura-,met-) RCrel (wild type II) and its dTDPG pyrophosphorylase deficient mutant is named Ter-21 (9). Escherichia cozi C strain is used as an indicator for 4x174 phage infection. Phaqe

:

@X174 phaqe,

wild

type.

Chemicals : Uracil (Ura) and thymine (Thy) were purchased mica1 Company, LTD. Casamino acid was from Difco laboratories, U.S.A. Methionine, hexose and pentose and other chemicals Pure Chemical Company, LTD, Japan. Adsorption of $X174 by E. cozi The kinetics of attachment starvation buffer, as detailed

from

PL-BiocheDetroit, were from Wako

K12 and to its Ter-mutant strains to whole bacteria were determined by newbold and Sinsheimer (10).

Inactivation of 41x174 phaqe by LPS from E. coli strains : Bacteria were grown in the synthetic 37OC. The LPS in the outer membrane of E. COli E. coli C (as a control) strains were extracted al (11) and of Westphal et al (12). The 50% inactivation doses (ID50 ; see 13) the residual infectivity after incubation (1 hr amounts of homogenized LPS with 5 X lo4 PFU per of Simmon's medium containing magnesium ion.

: in a

K12 and from its Ter-mutant medium (9) with aeration K12, its Ter-mutant and by the methods of Galanos were estimated at 37OC) of ml starvation

at et

by titrating increasing buffer, or

Isolation and analysis of LPS core oliqosaccharides : The LPS samples were degraded by heating (2 hr at 1OO'C) in 1% aqueous acetic acid, the insoluble and the core oliqosaccharides were lipid A was removed by centrifuqation, chromatoqraphed through Bioqel P2 (14).

1052

Vol. 91, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

The aldose analysis of the core oligosaccharides was carried out on the methyl-sugar derivatives formed after methanolysis with 1 N HCl-methanol for 6 - 8 hr at 1OO'C by gas-liquid chromatography (Shimadsu Gas Chromatograph, model GC 6 AM, column 2 m, 2.6 nunID, OV-17, 2%, chromosorb W, 80 - 100 mesh, chromacarrier gas N2, 60 ml/min, 2 Kg/cm2, Temp. 150°C) and by thin-layer E. Merck AG) with solvent ; chlotography (Silica Gel 60 F254, TLC plates, roform : methanol ( 3 : 1, v/v ). 2-keto-3-deoxymannulosoctonic acid (KDO) was determined by the thiobarbituric acid method of Weissbach and Hurwitz (15), as modified by Osborn

(16)

.

Heptose was determined by the (16). The phosphorous determinations (17).

cysteine-H2SOq

reaction

as modified

by

Osborn al

were

Results The susceptibility 4x174 are

phage

type

coli

to

I)

becomes

K12 (wild

that

the

type

cells.

cell

type

the

the

type

Therefore,

structures tions

have

of cozi

membrane

extracted

the

the

LPS from

and measured

the

inactivity

Table

the

LPS from

LPS from

the

inactivate the

the

mutant

phage.

It

but is

could type)

the

et

the

from

Ter-21

surmised from

a rough

should

wild

derived

cells

the

core

strains

E.

from

from

these

that

of

results

the

wild

or semirough

or "deep-rough"

under

K12

coli

mutant

show altered

type

to

types)

are a smooth

to

cells

Ter mutants (wild

differ

cells

may mutate

of

to Lowry

cell

chenosurface

identical

condi-

growth. reported

that

receptor E.

coli

of

$x174

the

for

and its

K12 (wild phage

Ter-15

E.

type)

related and its

by these

mutant

and parent

The LPS from

$x174

component

LPS.

inactivates

cells

(wild

cozi

C cells

phages.

We 1

Ter-mutant

cells,

The results the

type

of LPS of the

show in

phage, but the

I and II)

as a control

cannot inactivates

phage. Further,

oligosaccharide cells.

Ter-21

insensitive. Ter-mutant

(4) have is

mutant

phage,

those

and its

K12 W2252-llU-

The Ter--15

Ter-mutant

cell

et al

coli

K12 (wild

according

Discussion

to the

cells

with

of exponential

1 that

the

the

compared

cell

is

Ter-mutant

Jazwinski outer

II)

E.

chenotype,

phage.

out

K12 W2252-llU-

1. E.

sensitive

surface If

(18).

4x174

and

coli

shown in Table

non-sensitive

(wild E.

is

of E.

carried

Table

we determined of the

the

LPS from

2 shows that

sugar

nature E.

cozi

residues

1053

of the

sugars

K12 (wild of

the

present

types) core

in the

and the

oligosaccharide

core

Ter-mutant in the

Vol. 91, No. 3, 1979

Table

1.

BIOCHEMICAL

Susceptibility

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

to @X174 phage and inactivation

of 9X174 by LPS.

Bacterial

Lipopoly*Whole cells

Strains E. coli

IU2 (wild

type I)

Ter-15 E. cozi

**saccharides

K12 (wild

+

+

+

+

type II)

Ter-21 E. coli 'i

from

;

the

(+)

Ter-15

E. coli

in the

The LPS from core

( R (rfb)

were

2.

type

same amounts sugar

mutant

termed

residues are different

Ter-15 E. coli K12 (wild type II)

heptose sugars

from

oligo-

those

in

of

lipid

of the

E. coli

in the

and KDO from

the

core,

A

K12

common occurrence

of these

oligosaccharides cells

show that

in the

LPS from

galactose,

glucose,

fractions

from the LPS

Glucose

Ribose

Heptose

1.0

2.2

-

2.2

1.4

1.9

1.0

2.0

-

1.9

1.5

1.9

1.0

2.1

-

2.2

1.3

2.0

2.2

1.0

0.004

0.5

2.6

2.3

-

1.03

1.8

1.7

1.0

LPS

sugar

(21 - 24).

of core

Ter-mutant

Ter-21 C

core

They consist

may be complete

Molar ratios of the components of core of E. coti K12 and its Ter-mutants.

E. cozi K12 (wild type I)

in the

of the

R antigens.

Because

(19,20).

Galactose

as those

II).

which

basal

and its

the

Ter-21

K12 are

of analysis

Bacterial Strains

E. coli

but

galactose,

termed

types)

Table

(wild

) cells

glucose,

The results K12 (wild

the

Kl2

in the

I),

oligosaccharide,

glucosamine, components

type

6. cozi

mutants

( - ) : non-sensitive ( - ) is not inactivated

occur

LPS from

E. coti

LPS from

and the

mutant

K12 (wild

saccharide the

: sensitive, : inactivated,

(+I

l *

LPS from

c

1054

RDO Phosphorus

E. COzi

Vol. 91, No. 3, 1979

heptose II)

and KW are present

and the

ribose

Ter-15

core

from

the

Ter-21

However,

cells,

from

core

of E. of,

tial

complement

which

core

To study

presence

30% of the the

heptose-deficient

mutant

membrane

"heptose-less"

amounts thesis

of the

major

apparently

membrane altered the

of these

cells

filament

From these

membrane

formation

(31,21),

galactosamine,

in other

residue

is

and the galactose

also

the

the

linkage

The structure

phage shown to

may be

in strain

E.

contains

proteins,

of

Ter-21

reduction

in the

of

The 020 antigen

10%

and a substancozi

E.

GR 467,

of the

cell

envelope,

coli

(28).

The outer

the

(29).

reduced

mutation into cells protein

in LPS biosynthe

outer

should

have

fraction,

in contrast

W-irradiation O-antigens

(27).

is not

mutant

is made up of

( 25 -

CR 34,

W-irradiation,

after

upon the

LPS core

of proteins

the

susceptibi-

galactose

drastically

as the

incorporation

after

core

to C21, whereas

from

the

to be important

in its

isolated

of the

C21, depend

of

were

and ribose.

cells,

is

cells.

membrane

05 antigen

mutant

type

outer

found

(14).

mutant

in the

strains

the

Ter-15

was found

present

form

because

Ter-15

in why

the

amount

susceptible

reports,

because

explains

adsorption

type)

specific

galactose

mutants

the

the

glucose,

investigation.

relative

membrane

show an elliptical

D-ribose strains

outer

inhibits

(28,18). outer

cells

wild

heptose

heptose

alterations

from

core

the

sugars

the phage of

I and

can be found

infection,

K12 (wild

the

little is

core

type

cells

heptose

phage

C21 has been

of

has

of heptose,

has only

to

in,

which

from

other

mutants

nor

for

core

now under

with

or a reduction

CR34 (28),

in the

is

the

B. coti

mutant

of these

4x174

of the

from

in the

rough

in addition, coti

core

experiment

COli

to

K12 (wild

Ter-21

is necessary

and glucose that

in the

RESEARCH COMMUNICATIONS

coli

E.

The absence

content

oligosaccharide

From the

27);

core

from

galactose

non-sensitive

as in the

different

but

mutant.

galactose

galactose

core

Neither

in the

as the

between

absence

is

residue

same amount

lity

Ter-21

mutant

galactose

E.

mutant

the

AND BIOPHYSICAL

in the

and KDC are present.

the

the

BIOCHEMICAL

as

to the

(30). 05 and 020 of

E.

3-amino-3,6-dideoxyglucose, also

is

made up of

an

cozi

Vol. 91, No. 3, 1979

galactose

and

glucose,

ribose

mutant poration

differs

ribose,

BIOCHEMICAL

but

the

R-antigen

and KDO. The content from

of D-ribose

that into

AND BIOPHYSICAL

of

Ter-21

mutant

in the

LPS of the

of ribose

in O-antigens the

the

of E.

LPS of E.

COzi

RESEARCH COMMUNICATIONS

coli

and the

are now under

contains Ter-21

mechanism

of

incor-

investigation.

References 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

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