BIOCHEMICAL

Vol. 71, No. 2, 1976

OUTER

MEMBRANE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF

ESCHERICHIA

TSX

MUTANTS

(RESISTANT

AND

COLICIN

K)

Paul

A.

AN

and

Department

of

The

TO

LACK

Manning

COLI BACTERIOPHAGE

OUTER

Peter

Adelaide.

T6

MEMBRANE

PROTEIN.

Reeves,

Microbiology

University

K-12:

and

of

Adelaide,

S.A.

500cl.

Immunology,

Australia. Received

11,1976

May

TSX mutants of Escherichia coli are fully resistant to a set of TG-like bacteriophage and are resistant to colicin K. We demonstrate that these mutants are missing an outer membrane protein (the tsx-protein) of molecular weight 32,000 as measured by SDS-polyacrylamide gel electrophoresis. TSX mutants are receptor mutants which are unable to absorb either the bacteriophages or the colicin and the loss of receptor function can be demonstrated using outer membrane preparations. We suggest colicin.

that

aa

the

tsx-protein

is

the

receptor

for

both

the

bacteriophage

Introduction Cross that

resistance these

two

bacteriophage which

resistant

only

membrane

and

between

agents and

mutants,

Sabet

studies

map

share

colicin at

bacteriophage

a common

on

it the

to

a group

of

8 T6-like

Schnaitman

(5)

have

shown

and

Weltzien

receptor

resistance, 11 min.

and

Jesaitis

T6

was

and

(1).

In

found

that

chromosome

of

(6)

466

colicin have

shown

K have

a recent the

E.coli

bacteriophages

that

colicin

it

on

classical

to

lies is

-tsx

(2),

and

K receptor that

study

K-12 (3)

suggested

protein

were colicin

in

K

the in

outer nature

(4).

BIOCHEMICAL

Vol. 71, No. 2,1976

and that tsx

receptor

activity

AND BIOPHYSICAL

is unable

to be detected

RESEARCH COMMUNICATIONS

in membrane

extracts

of

mutants.

In this

communication

protein

which

Materials

we show that

we can readily

tsx

detect

mutants

are missing

on SDS-polyacrylamide

an outer

membrane

gels.

and Methods

The bacterial in nutrient

strains used are broth at 37OC.

listed

in table

1 and all

cultures

were

grown

TABLE 1.

Bacterial

(all

strains

derivatives

Strain

Characteristics

P400

F-/thr argE proA &acY -str e

of E.coli

K-12)

Reference

thi ---

ku mtl

IK&. ara

12.

h

P407

-tsx mutant

of P400

3.

P460

con mutant

of P400

12.

P1731

tsx

of P460

mutant

This

paper

Outer membranes were prepared as previously described (7) using the methods of Schnaitman (8) to obtain the Triton X-100 insoluble component of the cell envelope. Sample preparation and acrylamide gel electrophoresis methods have been described before (8, 9) and are essentially the pH7.2 system of Maize1 (10) and the pH 11.4 system of Braggand HOU (11). Bacteriophage K was prepared

T6 was from by induction

Phage neutralization volumes of dilutions

stocks maintained in this as described elsewhere

laboratory (7).

(2)

and colicin

experiments were carried out by incubating 0.1 ml of the outer membrane preparations (in phosphate

467

Vol. 71, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

buffer O.lM, pH7.2) with 0.1 ml of nutrient broth containing lo3 pfu of bacteriophage T6 for 3 hours, after which 0.1 ml of a culture of indicator bacteria (strain MOO, 2 x lOa cells/ml in nutrient broth) was added and incubation continued for a further 15 minutes. 4 ml of molten 0.7% nutrient agar was added and the whole poured as an overlay on a nutrientiqar plate, incubated overnight and scored for plaque forming units. The amount of colicin added was such that 20% survival of indicator bacteria was obtained under the assay conditions with 0.1 ml buffer substituting for the membrane preparations.

FIGURE 1.

Densitometer tracings tsxmutant, P1731, run system of Maize1 (10) the pH 11.4 system of labelled according to

of outer membranes of strains P460 (con) and its on SDS-polyacrylamide gels using theH7.2 buffer with both unheated and heated samples, and using Bragg and Hou (11) with heated samples. Peaks are Schnaitman (8).

468

BIOCHEMICAL

Vol. 71, No. 2,1976

Results

analysis

of the outer

con and -tsx

con double

results

obtained

membrane

by using

a con mutant

of the acrylamide

Thus

in Figure

1 it

using

unheated

mutant,

or heated

run on Bragg-Hou mutant

samples

with

gels,

when compared

the

then with

tsx

of molecular

weight

32080.

transferrin,

bovine-serum-albumin, type

(P400)

on Maize1

if

unheated

We

have

called

accounts

the

polyacrylamide

gels).

than

the

which

are

outer

membrane.

defect

and colicin

mutant

is

in tsx outer

con

to a protein phosphorylase

the protein

for

However,

of the

A,

mutants,

using

peak

can

heated

3A masks the protein

under

been detected

mutants,

the

membrane

tsx-protein:

proteins

is

thus

bacteriophages

(as measured

trace

present T5 (131,

h(14) gels

our

growth

in tsx

The function

laboratory

mutants of this

other

of the

in much greater

on SDS-polyacrylamide normal

it

than

relatively

and BF23 (15) of the whole conditions,

tsx-protein

corresponds

469

with

the

major

loss

no

the bacteriophage protein

not known. The absence

are

the protein

then

protein

con

anhydrase.)

on the densitometer

The tsx-protein

resistance.

cases

and carbonic

However,

gels.

in the --tsx

used were

in tsx -

a --tsx samples

corresponding

compared,

when

the heated

three

proteins

easier

3A (7, 8).

P1731,

a peak missing

In all

an

C detectable

If

used

We present

of protein

of strain

P460. is

P400. enables

no peak

gels

ovalbumin

of the peaks

undetectable

has so far

is

in a position

8% of the

receptors

normally

there

missing

about

areas

there

we have

used.

the protein

in P400 for

by comparing

amounts

are

absence

P400 and absent

in

or Bragg-Hou

samples

in the

con mutant,

is

strain

12) as this

(The standard

and a tsx

be shown to be present samples

mutants

mutants

(7,

a con mutant.

in the

of tsx

parent

on Maize1

again

peak missing

wild

gels

can be seen that

when compared

proteins of the

mutants

interpretation

If

RESEARCH COMMUNICATIONS

and Discussion

In our z,

AND BIOPHYSICAL

of neutralizing

is

BIOCHEMICAL

Vol. 71, No. 2, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 2.

Neutralization

of bacteriophage

Strain

Amount

required

T6 and colicin

under

the test

Bacteriophage

by outer

K

conditions

for

T6

lll9

P407

>lOO

(a)

50% of the 103pfu of bacteriophage were described in the materials and methods.

neutralized

(b)

Only 50% of the indicator in the test as described.

killed

for

bacteriophage

and Jesaitis

(6)

We are presently receptor

for

both

K

5 lJ9 ,100

bacteria

were

T6 and colicin

and confirmed purifying

50% neutralization Colicin

P400

activity

membrane

for

our

by the

K as previously mutants

the tsx-protein

the bacteriophage.T6

in the test

residual

and colicin

colicin

shown by Weltzien

as shown in table

which

as

we believe

2.

to be the

K.

References

1.

Fredericq,

2.

Taylor,

3.

Hancock,

4.

Hancock,

P. A.L.

(1949)

- C.R.

& Trotter,

R.E.W.

& Reeves,

R.E.W., Davies, in press.

Sot.

Biol.

143,

pp.l014-1017.

C.D.

(1972)

- Bact.

P.

(1975)

- J. Bacterial

J.K.

& Reeves,

470

P.

Rev. 36, pp.504-524.

(1976)

121,

pp.983-993.

- J. Bacterial

BIOCHEMICAL

Vol. 71, No. 2,1976

S.F.

& Schnaitman,

AND BIOPHYSICAL

5.

Sabet,

6.

Weltzien,

7.

Manning,

8.

Schnaitman,

C.A.

(1974)

- J. Bacterial

9.

Schnaitman,

C.A.

(1973)

- Arch.

H.V. P.A.

J.V.

& Jesaitis,

Maizel,

Jnr.

11.

Bragg,

12.

Skurray,

13.

Braun,

14.

Randall-Hazelbauer, ~~-1436-1446.

15.

Sabet,

S.F.

- J. Bacterial

- Science

118,

151,

L. & Schwartz,

P.

M. (1973)

471

133,

pp.534-553.

~~-442-453.

Biophys.

(1973)

(1973)

Med.

~~-422-430.

157,

pp.541-552.

pp.988-990.

& Reeves, H.

105,

in press.

Biophys.

- Biochem.

R.E.W.

C.A.

- J. Exp.

Biochem.

K. & Wolff,

& Schnaitman,

- J. Bacterial

(1971)

(1976)

(1972)

R.A., Hancock, pp.726-735. V., Schaller, pp.87-97.

P.

(1966)

& HOU, C.

(1971)

M.A.

& Reeves,

10.

P.D.

C.A.

RESEARCH COMMUNICATIONS

(1974)

Acta

2,

- J. Bacterial

- Biochem.

Biophys.

- J. Bacterial

- J. Biol.

pp.478-488.

Chem. 248,

119, Acta

323,

116, pp.1797-1806.