Recovery of Bacillus amyloliquefaciens protein synthesis from inhibition by pactamycin

BIOCHEMICAL

Vol. 43, No. 5, 1971

RECOVERY

OF BACILLUS

G.W.

Both,

Department

Received

of

April

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AMYLOLIQUEFACIENS PROTEIN INHIBITION BY PACTAMYCIN

J.L.

McInnes,

Biochemistry,

B.K.

May

and

University S.A., 5001.

of

SYNTHESIS

W.H.

FROM

Elliott

Adelaide,

Adelaide,

20, 1971

--SUMMARY Pactamycin inhibits protein synthesis in g. am.yloliquefa,ciens as expected but the cells quickly recover from this inhibition. This recovery is not due to acquisition of resistance; the results are compatible with the metabolic removal of the antibiotic. -INTRODUCTION Pactamycin initiation on

of

this

organism been

believed 1

synthesis

enzyme

that

not

an antibiotic

protein

extracellular

observed

has

is

the

effect

was

rapidly

,

In

synthesis of

the

by II.

the

specifically

course

of

inhibit other

studies

amyloliquefaciens

antibiotic

it

on protein So far

overcome.

previously

to

as we

was

synthesis are

aware

by this

observed.

EXPERIMENTAL Cultures

of

as previously

medium Samples 30°C

cell

harvested, 2

and

centrifuged previously

amyloliquefaciens

were

grown

at

30°C

for

'25 hr.

described2.

Washed were

g.

experiments twice

and

resuspended

(25

ml.)

at

of

appropriate and

the

were

washed in the

in this

carried

as follows:

a salts-maltose-0,5$ medium

cell

suspension

times,

samples

supernatants

out

assayed

described3.

1095

to

casamino

the

were (1.0 for

Cells

original shaken ml.)

were

a-amylase

cell aerobically

acids density. at

withdrawn, activity

as

Vol. 43, No. 5, 1971

BIOCHEMICAL

To determine protein,

the

a 2 ml.

0.1 ml.

incorporation

was incubated

except

that

liquid

scintillation.

was again

into

used;

2 ml.

total

containing

At various

as previously filters

total

except

describedL

into

1 1°C times

described2

was counted

cellular

RNA was

liquid

scintillation

that

of washed cells

by

were incubated

with

PC of 2-[C14]-uracil. Pactamycin

Dr.

shaking.

on the Oxoid

incorporation

measured as previously

suspension

and treated

the radioactivity

2-[C14]-Uracil

0.6

with

samples were withdrawn

counting

of L-[C14]-valine

sample of washed cell

-valine

of L-[c14l

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

H.F.

was a generous

gift

from the Upjohn

Co. and from

Lodish, RESULTS AND DISCUSSION

Protein

synthesis,

L-[C14]-valine of 20 min.

into under

lag is possibly pool

of cold

protein the

escape from

only

inhibition with

exerting

does not time

pg/ml. l),

protein

but with

greatly

inhibits

The time needed for

increasing

antibiotic,

concentrations escape occurs

5.0 pg/ml.,

[cl41

on

protein

synthesis,

general

toxic

2 where it

inhibit

of the

This

of an intracellular

soon recover. with

1).

full

recovery

is

3 hr.

for

a transient

used (Fig.

initially

increases

of

shows a lag phase

of pactamycin

cells

of pactamycin

specific

shown in Fig.

0.1

(Fig.

after

The effect is

conditions

The addition but the

40-60 min.

observed

this

the experimental

valine.

incorporation

material,

due to the presence

of pactamycin;

here

TCA precipitable

synthesis,

within

as measured by the

-va 1'ine incorporation rather

effect

can be seen that

2-[C14]-uracil synthesis

1096

that

pactamycin

totally

into

(0.5 RNA.

inhibited.

it

is This

on the cells.

incorporation is almost

than

observed

is

iJ-g/ml.) During These

BIOCHEMICAL

Vol. 43, No. 5, 1971

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Time

(mins)

Effect of pactam.ycin on general protein synthesis by Fig. 1. Pactamycin and 2-LCl4]-valine were both added at washed cells. zero time. Curve 1 - no addition; curve 2 - 0.1 rig/ml. pactamycin; curve 3 - 0.5 pg/ml. pactamycin; curve 4 - 1.0 pg/ml. pactamycin; curve 5 - 5.0 rig/ml. pactamycin.

results

are consistent

appreciable

reported

The possibility

-tion

inhibition process

tion,

is artefactual, in the presence

synthesis

the results

was fully That

restored the

that

This

the

is not the

were identical

may allow

case.

instead

However,

no

as has been

escape from pacta-

sense that

of pactamycin

3 was followed,

after

was observed

the apparent in

475 .

observations

of RNA synthesis 4 in g. subtilis . existed

of nonsense protein. a-amylase

previous

stimulation

previously

mycin

with

a faulty the

initiasynthesis

When extracellular of [C 14 I-valine

and the rate

of a-amylase

incorporaproduction

escape.

escape from pactamycin

1097

inhibition

does not

depend on

Vol. 43, No. 5, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(mins)

Time

Fig. 2. Effect of pactamycin on total RNA synthesis by washed Pactamycin and 2-LC14]-uracil were both added at zero cells * -time. Curve 1 - no addition; curve 2 - 0.5 rig/ml. pactamycin.

the acquisition cells

of resistance

were pre-incubated

(during

which

then

transferred

(Fig.

3, curve

identical

inhibited

the

rate

(0.5

indicates

control that

both cells

cells,

for

cultures

once incubated with

explanation inhibition

pactamycin which

1098

70 min.

(0.5

for

and

kLg/ml.) was

70 min.

without

This

pactamycin. with the

pactamycin, antibiotic.

for

was that

removed from the medium.

medium from cells

When

were initially

without

the most likely

without

for

of [C14]-valine

pre-incubated

from pactamycin

was progressively

pactamycin

to a second incubation

that

synthesis

pre-incubated

cells

yg/ml.)

3.

escape from inhibition),

of incorporation

As expected,

2).

susceptible

in supernatant

pactamycin completely

of control

was thought

of protein

cells

cells

as compared with

are equally

biotic

3),

(curve

clearly

It

the

with

is shown in Fig.

to new medium containing

to that

pactamycin

result

time

to the drug

To test

the

escape

the antithis

idea,

70 min. were resuspended

had escaped from pactamycin

BIOCHEMICAL

Vol. 43, No. 5, 1971

AND BtOPHYSlCAL

Time

RESEARCH COMMUNICATIONS

(mins)

Effect on protein synthesis of treating washed cells Fig. 3. Washed cell cultures were pre-incubated twice with pactamycin. in suspending medium without pactamycin (culture 1) and with pactaAfter 70 min., cells were mycin 0.5 pg/ml, (culture 2). (0.5 pg/ml. and resuspended in new sus ending medium. Pactamycin where required, and L-[C 141j_valine (0.5 PC/ml.) were added togethe; Curve 1 - cells from culture 1; immediately after resuspension. curve 2 - cells from culture 1 + pactamycin (0.5 pg/ml.); curve 3 - cells from culture 2 + pactamycin (0.5 iJ-g/ml.). inhibition

(Fig,

identical

4, curve

Control

cells

medium which had not at any stage

(curve

2).

valine

incorporation

originally

70 min.

can be seen that

pared with

there

contained

of [c14]-

suspended in medium which

pactamycin.

This

shows that

2 and 3 (Fig.

(The absence

in the medium after

1) is attributed 70 min.

1099

the

antibiotic

during

the initial

of a significant

4) and the greater (curve

cells

overall

lag phase rate

to the

pre-incubation.)

in

pactamycin

was no inhibition

removed from the medium by the

the control

were resuspended

in the cells

pre-incubation.

curves

of valine

It

contained

was totally

for

3).

as com-

lower It

level appears

Vol. 43, No. 5, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4

30

60

Time

90

(mins)

Effect on protein synthesis of resuspending new cells Fig. 4. in medium in which cells have escaped from pactam.ycin inhibition. Washed cell cultures were pre-incubated in suspending medium without pactamycin (culture 1) and with pactamycin (0.5 ug/ml.) 1 cells were centrifuged and (culture 2). At 70 min., culture resuspended with L-[C14]-valine (0.5 PC/ml.) in the following medium. Curve 1 - fresh suspending medium; curve 2 - supernatant from culture 1; curve 3 - supernatant from culture 2.

that

the mechanism responsible

the medium is operating for

protein

synthesis

for

the

at a constant to escape is

removal rate,

since

the same during

second treatments

with

the antibiotic.

Furthermore,

this

mechanism is intracellular,

addition

of pactamycin

pre-incubated degraded

during there

Therefore, which

destroys

consistent several

with

with Bacillus

an acquired

(0.5

pg/ml.)

pactamycin

a further

to the

(0.5

pg/ml.) incubation

70 min.

is no extracellular the pactamycin findings species

inducible

in the

concerning 6

.

taken

the first

and

because a second of cells

for

is not

70 min.

of the supernatant. produced

external

medium.

the

from

supernatant

inactivation

by the This of nisin

cells is in

et al. 7,S have characterised

to tetracyclines

1100

the time

product

Sompolinsky

resistance

of pactamycin

in S. aureus and

Vol. 43, No. 5, 1971

in this

system,

drug accounts resistance

a decrease for

in the

However,

seem unlikely

It

in the

acetylation

cells

in S.

is induced

by sub-inhibitory

does not

of the fact

that

for

concentration.

a given

treatment

of the

a longer

recovery

process

faciens

1.

2.

2:

7. 8. 9.

inactivates

with for

within

Cohen, L.B., Goldberg, Biochemistrv, 8, May, B.K. and Elliott,

synthesis, short

inducible

that

same time

a second

would result

unless

or

in view

removed in the

at 70 min.

has

Our data

more likely

be expected

pactamycin

of the pactamycin

is an

enzyme which

system is

appears

might

a very

there

is

the pactamycin.

the

is always

protein

that

of chloramphenicol.

latter

It

permeability

the pactamycin

is an inducible

whether

the

this

by an acetyltransferase

this

the

to a tetra-

situation

because all

levels

has not been further

157,

3. 4.

time

is complete

The nature

which

the pactamycin

cells

Furthermore,

seems more probable

establish

However,

non-inducible.

in the present

epidermidis9;

equivocally

cells.

accumulate

were transferred

of chloramphenicol

been found

RESEARCH COMMUNICATIONS

to actively

of these

to be involved

removed from the medium.

Indeed,

ability

when the cells

medium.

enzyme system

AND BIOPHYSICAL

the resistance

declined

cycline-free effects

BIOCHEMICAL

in

the induction

time.

removing

system

in g.

am.vlolique-

examined.

I.H. and Herner, A.E. 1312. W.H. (1968). Biochim.

607.

(1969). biophys.

Acta,

W.H. (1962). Biochem. J., 83, 256. Coleman, G. and Elliott, Kersten, H., Kersten, B., Emmerich, B. and Chandra, P. (1967). Hoppe-Sevler's Z. Physiol. Chem., B, 1424. Biochem. Pharmacol., l6, 1411. Bhuyan, B.K. (1967). J. Gen. Microbial., 3, 33. Jarvis, B. (1967). Sompolinsk , D., Krawitz, T., Zaidenzaig, Y. and Abramova, N. 62, 341. (1970 ;r . J. Gen. Microbial., Sompolinsky, D., Zaidenzaig, Y., Zeiglerschlomowitz, E. and J. Gen. Microbial. 62, 351. Abramova, N. (1970). D.W. and Sands, L. (197Oj. J, Bacterial., Shaw, W.V., Bentley, 104, 1095.

1101