Control by phosphate of candicidin production

BIOCHEMICAL

Vol. 71, No. 4, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

CONTROL BY PHOSPHATE OF CANDICIDIN Juan

F. Martin*

and Arnold

PRODUCTION L. Demain

Department of Nutrition and Food Science Massachusetts Institute of Technology Cambridge, Massachusetts 02139 Received

May 3,1976

SUMMARY: synthesis

Phosphate concentration was found to control the bioof the antibiotic candicidin by resting cells of Streptomyces griseus. Phosphate concentrations above 1 mM decreased the rate of incorporation of [14C]propionate and [14C]paminobenzoic acid into candicidin in relation to the concentration of phosphate. The inhibitory effect of phosphate on incorporation of labeled precursors into candicidin was not caused by inhibition of cellular uptake of precursors. Protein synthesis, sensitive to chloramphenicol, was not affected by phosphate levels that inhibit antibiotic synthesis. Similarly, phosphate concentrations inhibitory to antibiotic synthesis did not affect rifampinsensitive RNA synthesis. INTRODUCTION Inhibition (idiolites)

of

the

by inorganic

mentations

(1,2).

concentrations

of

Streptomyces

griseus

phosphate

tion

accumulation

polyene

of reserve

such

an antibiotic-synthesizing

from

antibiotic

mary

metabolism).

glucose

has been idiolites

Liu

utilization,

are produced

candicidin

sensitive

system

is

produced

when net

mass

continues

(3).

(secondary

and dry weight

results

1103 0 1976 by Academic Press, of reproduction in any form

Inc. reserved.

in

studying

accumuladue to to

a reversal to growth

increases

following

for

of phosphate

metabolism)

(4) found

by

to increase

Addition

at

growth.

a very

*Present address: Departamento de Microbiologia, Universidad de Salamanca, Spain. de Ciencias,

Copyright All rights

only

antibiotic

culture

al.

in many fer-

macrolide

materials

et

observed

for

cell

production

metabolites

suboptimal

Candicidin

although

secondary

phosphate

provides

effect.

of DNA stops

these

inorganic

of the

of

phosphate

Some of

Biosynthesis

the

biosynthesis

in

(pri-

respiration,

phosphate

addition

CSIC,

Facultad

BIOCHEMICAL

Vol. 71, No. 4, 1976

to a candicidin-synthesizing cells

system.

to a phosphate-free,

antibiotic

synthesis

mitted

for

molecular

and halted

mechanisms This

incorporation synthesis

by which report

protein

metabolic

in

the

initiated

study

sub-

of

influence

influence

into

growing

and McDaniel,

signals

the

precursors

medium

(Martin

involved

describes

of young

defined

growth

We are

of labeled of

Transfer

chemically

publication).

expression.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the gene

of phosphate

candicidin

on

and on the

and RNA. MATERIALS

AND METHODS

Resting ceZZ system. S. griseus IMRU 3570 was grown in a which supports high antibiotic proglucose-soy peptone medium, duction, as described previously (3). At 18 hr, when the cells started to produce antibiotic, they were collected, washed, and resuspended in a phosphate-limited defined medium, as described Candicidin by Martin and McDaniel (submitted for publication). synthesis proceeded linearly in this system without increase in dry cell weight. Incorporation of 1abeZed precursors into candicidin. Candicidin production and incorporation of labeled precursors, [14Clpropionate and [ 14C]p-aminobenzoic acid (PABA), were estimated as Labeled candicidin was puridescribed by Martin and McDaniel (5). fied by thin-layer chromatography, identified by spectrodensitounder UV light, scraped and counted. Cellumetry (6), visualized and [ 14C]PABA was determined as lar uptake of [ 14C]propionate previously described (5). Protein and RNA biosynthesis. Incorporation of a 14C-amino acid mixture into macromolecules insoluble in trichloroacetic acid was measured as described by Martin and McDaniel (5). Incorporation of [14C]uracil was determined in loo-u1 aliquots of the Cells were incubated during 6-min phosphate-limited culture. in 0.02 mM unlabeled uracil. pulses with 0.5 pCi/ml of [ 14C]uracil Incorporation was stopped by addition to the incubation mixture of and 2.5 ml ice-cold 20 mM uracil in 5 percent TCA. Counting quenching correction were performed as described before (5). [7-14C]PABA (4.26 Ci/mol) was obtained from Mallinckrodt Sodium [U-14C]propionate (48.7 Ci/mol) and [U-14C]LNuclear. amino acid mixture (226 Ci/mol) were products of New England NUclear. [2-14C]Uracil (62 Ci/mol) was purchased from Amersham/ Searle.

RESULTS Phosphate

of candicidin fashion

for

phosphate (Fig.

1).

inhibition

of candicidin

by phosphate-limited at least

The decrease

cells

36 hr without

at 12 hr decreased in

synthesis.

the rate

cell rate

proceeded

in

a linear

Addition

growth. of antibiotic

of antibiotic 1104

Biosynthesis

formation

of

formation was

5 mM

BIOCHEMICAL

Vol. 71, No. 4, 1976

0

AND BIOPHYSICAL

1

12

13

TIME

14

15

RESEARCH COMMUNICATIONS

16

17

ihr)

Phosphate inhibition of candicidin synthesis. 1. hr, phosphate (5 mM) was added to candicidin-synthesizing Controls (A) and (0); with 5 mM phosphate (A) and (0).

At 12 cells.

Fig.

oLOO OIii4 rc3 Phosphate

lb2 (Molar1

IO’

of different phosphate concentrations on candiFig. 2. Effect cidin synthesis and dry cell weight increase. Phosphate was added to the resting cell system at the final concentrations Dry cell weight (0) and percentage of inhibition of indicated. total candicidin synthesis (A) were determined after 23 hr of incubation.

proportional phosphate bition

to the was not

was observed

concentration inhibitory at

of phosphate. up to 1 m&l whereas

5 to 10 mM.

1105

Inhibition

Fig.

2 shows that

considerable

inhi-

of antibiotic

syn-

Vol. 71, No. 4, 1976

BIOCHEMICAL

thesis

was accompanied

by phosphate

cose

utilization

weight. cell

(not

A high weight

tures

prior

the

preformed

into

inhibition

candicidin

drastically onate

and

whether

in dry

cell

phase

dry

of batch

cul-

incorporation

into

addition

by degrading

or by inhibiting the

into

TIME

of

Addition decreased

candicidin

acts

candi-

the biosynthesis

incorporation

and immediately

‘3

of glu-

and increasing growth

phosphate

molecule.

[14C]PABA

the

of precursor

we studied

the

of

rate

formation.

antibiotic

antibiotic,

increase

utilization

characteristic

To determine

by an increased

and a large

of glucose

to antibiotic

Phosphate cidin.

shown)

rate

are

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

labeled

precursors

5 or 10 mM phosphate

incorporation (Fig.

(mln)

of the

of

[14Clpropi-

3).

TIME

(mln)

Fig. 3. Effect of phosphate on incorporation of labeled precursors into candicidin. Phosphate was added to the resting cell system at the time indicated by the arrow.

Lack of

Inhibition been

the

Our results

effect

of

the

result (Fig.

of phosphate

incorporation

of decreased 4) indicate

on ceZZuZar

of labeled uptake that

1106

uptake

of precursors.

precursors

of precursors a phosphate

could

by the concentration

have

cell. which

BIOCHEMICAL

Vol. 71, No. 4, 1976

AND BIOPHYSICAL

TIME

RESEARCH COMMUNICATIONS

(mid

effect on cellular Fi 2. 4. Lack of phosphate I1 CIPABA was added at time zero. Phosphate at the time indicated by the arrow. Control added (A).

inhibits take

antibiotic of

effect

biotic

production.

protein

turnover

and streptomycin presence

antibiotic

of

the

studies

the

cellular

is

(not

shown).

sensitive

inhibitors

up-

Antibiotic

phosphate

addition

of phosphate

a small

of

leucine turnover

carry

indicating

In

concentrations

of

1107

5a).

5a) in that

in

unpublished).

synthesis.

(Fig.

out

decreases

synthesis

on protein

anti-

(Fig.

replenished

and Demain,

had no effect

by incorporation detected

being

on antibiotic

of protein

synthesis

synthesis

(Martin

effect

cells

synthesis

continuously

cultures

during

to chloramphenicol

of protein

are

biosynthesis

resting

by an inhibition

experiments,

mined

affect

on macromoZecuZar

that

synthases

However,

antibiotic

not

Phosphate-limited

phosphate-limited

caused

does

PABA.

Phosphate

the

formation

uptake of PAPA. (10 mM) was added (0); phosphate

is

short-term which

synthesis Pulse

rifampin-sensitive

not

inhibit as deter-

labeling RNA syn-

Vol. 71, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

18

I

T; ; 80 -E 2n 9 fj 60

B 5z 40 0” 2 2 2o f & s

0

a

0

40 TIME

80

120

b

(min)

TIME

(min)

of phosphate on total protein synthesis. Fig. 5. a. Effect The 14C-amino acid mixture was added at time zero and phosphate (10 mM) or chloramphenicol (100 pg/ml) was added to separate flasks at the time indicated by the arrow. Control (0); phosphate added (A). With chloramphenicol (0). b. Effect of phosphate on RNA synthesis by candicidin-producing cells. [14CIUracil was incorporated in 6-min pulses. Phosphate (10 mM) and rifampin (10 ug/ml) were added at the time indicated by the arrow. Control (A); with rifampin (Cl). (0) i with phosphate

thesis

in

thesis

is not

which

phosphate-limited

cells

affected

inhibits

(Fig.

by adding

antibiotic

5b).

phosphate

synthesis

Overall,

RNA syn-

at a concentration

(Fig.

5b).

DISCUSSION Our data

show that

of candicidin. occurs

Biosynthesis

only

molecular

at

been observed

posed

regulates this

polyene

for

in many antibiotic including

and control

the

this

at the

of

transcription

antibiotic

of phosphate.

phenomenon,

processes, regulation

biosynthesis

macrolide

concentrations

responsible

mechanisms

synthases

of

growth-limiting

mechanism

several

phosphate

is

not

activity level

which yet

The

has

known;

of antibiotic have been pro-

(7,8,9). Our results

indicate

that

the

1108

inhibition

of

antibiotic

accu-

BIOCHEMICAL

Vol. 71, No. 4,1976

mulation

is

into

antibiotic

the

Since

caused

cursors

into

by a decreased rather

phosphate

does the

On the that

primary term

(Fig.

phosphate

for

at

least

inhibited

10 mM phosphate.

mitted

to antibiotic

We conclude din

rapidly

is

that

not mediated

thesis)

but

effector)

by the

noted

on formation

that

(Martin

control

of

by an effect

or action

shortProtein

stimulated

by 10 mu

antibiotic

synthesis that

remained

by addition

of

content

addition

to cells

and Demain,

of antibiotic

to

theory.

intracellular

(protein

of com-

unpublished).

biosynthesis

of phosphate

commit-

a return

stimulated

on growth

action

cell

RNA synthesis

the

a$.

et

of our

this

phosphate

bio-

Liu

the in

time

the

pre-

of precur-

data,

was not

not

following

specific

support

1 and 3).

of labeled

of antibiotic

The results

by this

antibiotic.

inhibition

results

cells

synthesis

uptake

by reversing

do not

was likewise

phosphate

the

which

100 min;

We have

that

growth.

(Figs.

to rifampin

of

fermentation

acts

5a,b)

by phosphate-limited

rate

incorporation

of the

inhibition

of batch

synthesis

ATP increases

a true

and cell

experiments

sensitive

the

biosynthesis

metabolism

was greatly

alter

phosphate

of precursor

an inactivation

we conclude

basis

to antibiotic

rate

than

indicates

(4) postulated ment

not

cell,

sor incorporation synthesis.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

candici-

or RNA syn(or a related

synthases.

REFERENCES 1. 2. 3.

Weinberg, E.D. (1974) Develop. Ind. Microbial. 15, 70-81. Martin, J.F. (1976) In MicrobioZogy-1976. (D. Schlessinger, ed.) American Society for Microbiology, Washington, D.C. In press. Martin, J.F., and McDaniel, L.E. (1975) Biotechnol. Bioeng. 17,

4. 5. 6. 7.

925-938.

Liu, C.M., McDaniel, L.E., and Schaffner, C.P. (1975) Antimicrob. Agents Chemother. 7, 196-202. Martin, J-F., and McDaniel, L.E. (1975) Biochim. Biophys. Acta 411, 186-194. Martin, J-F., and McDaniel, L.E. (1975) J. Chromatog. 104, 151-160. Mertz, F.P., and Doolin, L.E. (1973) Can. J. Microbial. 19, 263-270.

8. 9.

Miller, Pass, Polon.

A.L., and Walker, J.B. (1970) J. Bacterial. L., and Raczynska-Bojanowska, K. (1968) Acta 15, 355-367. 1109

104, 8-12. Biochim.