Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Vol. 62, No. 3,1975

BIOCHEMICAL

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS

UNCOUPLER ANDANAEROBICRESISTANTTRANSPORT OF PHOSPHATE IN ESCHERICHIACOLI A. S. Rae and K. P. Strickland Department of Biochemistry University of Western Ontario London, Ontario, Canada N6A 3K7 Received

November

11,1974 SUMMARY

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from Escherichia & K12 was found to be partially resistant to 50 JM carbonyl cyanide Echlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCPcompletely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mMKCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additsqnal s$$di es of transport in a mutant (CBT302) deficient in membraneboundCa -s Mg -ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions. INTRODUCTION Inorganic phosphate (Pi) transport

in Escherichia coli has been defined

as an active process since the substrate is concentrated many hundred fold within the cells under conditions requiring metabolic energy (1). However, detailed

study of this transport

system is made difficult

by the inherent

metabolic drag in which a significant percentage of the phosphate transported is rapidly modified chemically. Presumably this is one major reason why those studying transport have, in general, addressed themselves to the phenomenonof biological transport with other systems (e.g. amino acids, sugars, etc.). However, it was considered that, if a suitable means could be devised for observing the size of the Pi intracellular pool generated under a variety of metabolically inhibited conditions, this inherent problem would be largely overcome. To determine this pool size, we have refined a technique of whole cell high voltage electrophoresis combined with radioautography (1) and applied this procedure to two strains of Escherichia coli. It was observed Abbreviations used: DTT: dithiothreitol.

cccp:

Copyright o 19 75 by Academic Press, Inc. AN rights of reproduction in any form reserved.

Carbonylcyanide g-chlorophenylhydrazone;

568

BIOCHEMICAL

Vol. 62, No. 3,1975

that

a significant

of the It

transport

uncoupler,

was also

ported

Pi under These

membrane Thus,

is

the mutant

some alteration would

anomalous

active

and uncoupler

accepted

in

gradient

existing

Curtin eristics media

of Medical

Cell

counts

(1).

were

at timed

intervals.

(140 mM) solution. using

Warburg

and the

flasks

sealed

were inserted.

provide cell

a vent

was added

was also

for

solution

a serum

maintained

under

to give

which

filtration

twice

cell

Pi Co.,

with

three

x 10'

were

taken

2 ml of KH2P04

conditions the

(2-5

Ann Arbor, were

suspension in

in the main

side

arm.

hypodermic

The

needles

with N2 (99.99%), one to sampling. Preflushing of the anaerobic assembly

Chemical

5 min before

569

by Medveczky

0.5 ml samples

to be added

CCCP (Sigma

a 50 PM concentration

and incubated

labelled

anaerobic the

a completely the

at 37°C in

by centri-

by centrifugation

Instrument

was washed

cap through

over

studies

(1)

as described

p, Gelman

containing

5 min produced

In the uncoupler

collected

[ 32 PI-

of

Uptakes

32 Pi-stock with

then

One was used to flush the media for pressure release and one for

suspension

nitrogen assay.

concentration

flasks

and 2%

harvested (1)

solid

studies. essentially

Each sample

(7 mM)-NaCl compartment

were

of this

on rich

extract

TSG media

was 100 ~JM. Normally (0.22

B. D. Sanwal

maintained

were

John

(charact-

in 60 ml TSYG media

The cells out

studies.

H. Rosenberg,

0.3% yeast

phase

or transport

carried

nanomole)

by Dr.

in phosphate-free

uptake

these

University

were

0.5% NaCl, overnight

carried

were

hitherto,

used in

National

strains

stationary

on Gelman membranes

out

2).

to bacterial

this,

by Dr.

supplied

Both

grown

The final

per min per

Mich.)

supplied

in the

at 37°C.

uptakes

and filtered

4).

resuspended

shaking

and Rosenberg

of a trans-

see Harold,

coupling

for

were

Australian

extract,

were Cells

to phosphate

Phosphate

that

AND METHODS

kindly

Research,

1% beef

and then

prior

met-)

in ref.

cultures

2 h with

just

trans-

activity

generation

discussion

of energy

from K12,

3) and CBT 302 kindly

shaker.

fugation

indicate

of respiratory for

to account

derived

(Reeves

(1% tryptone,

agar).

coli,

(described

a gyrotory for

of E.

in ref.

Department

actively

transport.

AB3311

School

studies

(for

models

MATERIALS were:

or by KCN treatment. mutant

the absence

as necessary

seem to be necessary

Two strains These

in the presence

by anaerobiosis

in

or electrochemical

transport

persisted

an ATP'ase-negative

concentrated

traditionally

proton

phosphate

unaffected that

RESEARCH COMMUNICATIONS

conditions.

from both

apparently

and conditions

is

to note

anaerobic

data

phosphate

of inorganic

CCCP which

surprising

AND BIOPHYSICAL

Co.,

system.

A flow

during

the uptake

St.

the initial

Louis, uptake,

of

MO.) A

Vol. 62, No. 3,1975

preincubation Chemical 32

BIOCHEMICAL

of 5 min was also Co.,

St.

P-labelled

Louis,

intermediates

to 3 MM Whatman

filtration

and washing

Before

application

onto

by gentle

suction.

was glued

(book

three

drops

formic

acid

par

wetted

with

buffer

surface

of buffer,

action).

and a current Model

Automatic

were

collected

were

right

glue)

then to the (43.5

applied wetting

side

voltage

marked

this

acetic

Counting

filters

acid

(High

were

Voltage Wise.).

Two or

and 12.4

ml 90%

to occur

was by

out at 3000 volts Electrophorator, After

located

by exposure

by cutting

out

each labelled

using

a gas-flow

System,

Nuclear

counter Chicago,

dried

membrane

The 3 MM paper

was allowed

Middleton,

present

cakes of dry ice.

3 MM paper.

was carried

45 min

and directly after

Each folded

on the

filters

were

flat

procedure.

electrophoresis

was achieved

radioactivity

up onto

(57 x 46 cm) the

ml glacial

areas

by high-voltage

The membranes

origin

Electronics,

phosphate

separated

and glued.

around

indicated.

ester

folded

during

DTT (Sigma

times

on Gelman membranes

as follows:

applied

radioactive

Low Background

etc.)

Whatman 3 MM paper

(final

Medical

Quantitation the

were

at the

and water-soluble

paper

pH 2.0

1) were High

electropherogram, counting

were

of ZOO-250 mamps for

D, Gilson

film.

that filter

These binder

KCN (1 or 10 mM).

nucleotides,

of cells

applied

for

10 mM was added

phosphate

(glycolytic,

electrophoresis

used

MO.),

inorganic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

drying to Kodak

of the X-ray

area.and (Model

1043

Des Plaines,

111.).

“p

x IO’

UPTAKE E.coJ

(AB3311)

l bEWBlC(coNTRou 4-

o -E; l

CN-.

N, l-5’) I-lOrnM(-5’1

TIME ( min I

Fig.

1

AB3311 under aerobic and anaerobic Uptake of Pi by E. coli strain with N2 or conditions and in the prescence of KCN. Preflushing the addition of KCN was carried out 5 min before the uptake commenced. Uptakes were done as described in the methods.

570

BIOCHEMICAL

Vol. 62, No. 3, 1975

x IO’ I

‘s

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

UPTAKE IN ATPose hWlM4-r

ApTpoSe CET302

TIME (mm)

Fig.

2

Uptake mutant)

of Pi by 2. coli strains, AB3311 and CBT302 under aerobic and anaerobic conditions.

09

6-

(ATP'ase

negative

=P, “‘TAKE IN ECCCI lAB33,l) / . /

/

. AERCBIC,CONTROL 0 m-.I0pmMl-5’)

.

A CCCP,5opM i-S) A CCB.CN- 1-5’1 0 CCCP.Np C-5’)

/

TIME ( m m 1

Fig.

3

Uptake of 10 mM, (b) (d) CCCP, bitors was

Pi by E. coli strain AB3311 in the presence of (a) KCN, CCCP, 50x (c) CCCP, 50 PM plus KCN, 10 mM, and 50 PM and N . Preflushing with N or addition of inhicarried ou 2 5 min before the uptike commenced.

571

Vol. 62, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTSANDDISCUSSION Phosphate uptakes were performed on cells grown to the stationary phase and starved for 2 h in phosphate-free TSGmedia. These yielded a biphasic curve (fig. 1) under aerobic conditions similar to that reported by Medveczky and Rosenberg (1). Pig. 1 depicts also that the anaerobic technique used is valid in that the uptake curves for both anaerobiosis and for 10 mu KCN (a concentration that completely abolishes respiration) were practically superimposable. Analysis of the electropherogram (not shown) from a similar experiment showed the pool of intracellular phosphate in the aerobic to be approximately

twice that of the KCN-treated cells.

Fig. 2 shows the sameuptake (aerobically

for both the wild-type

strain

(AB3311) and the ATPase-negative mutant (CBT302). Anaerobiosis lowered the uptake of the mutant by about one-half as observed for A83311 (Fig. 1). Thus, it appears that a functional

ATPase is not obligatory

under both aerobic and anaerobic conditions. surprising

for phosphate uptake

The latter

in view of the many studies which implicate

finding was somewhat an ATPase in anaerobic

or glycolytic supported transport (5-9). Since a number of workers (5, 7, 10) have found that the presence of uncouplers (e.g. DNPor CCCP)completely inhibits the transport of S-galactosides (lactose or thiomethyl-g-D-galactoside) or amino acids (proline, leucine),

it was decided to test the effect

transport

of uncouplers on the phosphate Results from this study appear in

system that we had under study. Fig. 3-5 for the uncoupler, CCCP. Two observations of interest appear in Fig. 3. First, there is still a significant phosphate uptake (which as discussed later

is mainly active

transport)

Second, this residual uptake apparently

in the presence of CCCP(50 PM). is independent of any respiratory

activity since it is identical to the uptake obtained in the presence of This uncoupler-resistant transport would 10 mMKCNor under anaerobiosis. appear to occur independently of any "energized-state"

or"proton motive"

force (both dissipated by uncouplers) generated (see 2 for ref.) in the membraneby either respiration or glycolysis. Kaback et al (11) have been concerned that certain of the uncouplers might simply be acting as sulfhydryl reagents. They have demonstrated not only protection against but also release of inhibition by CCCPwith the DTT, to membrane vesicles carrying out amino addition of the thiol agent, A similar effect has been observed here in respect to acid transport. phosphate uptake (Fig. 4). DTT (10 mM) added before the CCCP(not shown) prevented any inhibition by the uncoupler. DTT, added 5 min after the transport assay started, returned the uptake rate back to that of the control. The intracellular Pi pools included in Fig. 4 come from radioactive

572

BlOCHEMlCAL

Y

I 5

OO

Fig.

4

1 10 TIME ( m m )

of the areas

Cells

used

three

columns

in

cells

regions

this

depicted

in the

electrophoresis

on the

left

incubated

with

of interest

run were

may be noted:

(1)

is

correspond

intermediates

the

result

(1,

also

to glycolytic of incorporation unpublished

a direct

function with

50 PM CCCP.

Fig.

4) is

is

definitely

active

25

a concentrating by one-half.

reduced.

Under

This incorporation

columns

by CCCP.

into

three

In the presence

spot

areas

is

that

two thirds this

acids

from

control.

pool This

on the

at

reduced

intermediates

glycolytic

of

uptake

control

and CCCP has only formation

as

cells

intracellular

the glycolytic

is

to increase

arise

of the

the

area

and nucleic

performed

of 400 times

con-

at the origin

observed the

The for Three

above

the activity

conditions

would seem to indicate of 32 P label into

to the right

circular

columns

as calculations effect

4.

4, 10 and 20 min.

dark

5.

profile

phospholipids

one-half

in Fig.

as in Fig.

metabolite

region

Interestingly

deduction is further supported the phospholipids and nucleic The three

treated

and (3)

these

shown

radioactive

The central

transport

20 min give

the

the

latter

approximately

this

ATP for

This

of time.

(see

greatly

(2)

primarily

treated

effect

Pi,

data).

Pi

tion

20

autoradiograph

show the radioactive 32 P-labelled Pi for

of the way up the column

led

L-A I5

The effect of DTT on CCCP inhibition of Pi uptake and intracellular Pi of &. coli strain AB3311. CCCP ( 50 PM added 5 min before and Intracellular Pi DTT (10 mxdded 5 min after uptake commenced. measured by high voltage electrophoresis technique d5scribed in the methods. One nanomole of Pi corresponds to 1.8 x 10 c.p.m.

analysis

trol

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of little

intermediates.

are labelThis

by the almost total loss of incorporati.on into acids as judged by the activity at the origin. demonstrate of DTT,

573

the

increased

relief

by DTT of the

activity

occurred

inhibiin all

Vol. 62, No. 3, 1975

Fig.

the

5

BIOCHEMICAL

Radioautograph electrophoresis as in Fig. 4

areas

phosphate

of electropherogram obtained followed of cells as described in the methods.

labelled,

that

compounds

at the origin.

Independent while

this

uncoupling,

the

inhibition

that

independent

proton

or electromotive

glycolysis. suggest

in respect

seem to occur

transport

via most in 2. directly

shown

gradient

(see

a mechanism closely coli

where

the membrane the ATPase

12).

Thus,

than

that

energy

574

that

either

state" mutant,

with or or

or anaerobitransport

of glycolysis

"glutamine

of

CBT302

some of phosphate

by Berger

formed

dissipation

by respiration

negative

involving

observed

affect

complete

Our findings

aerobically

to a step

he concludes

by phosphate-bond

either

coupling those

and the

an "energized

transport. other

parallel

systems

of either

ATP'ase

direct

of CCCP cause

of Pi in E.

role

for

50 vrn CCCP does not

1, 7, 10,

with

high voltage Conditions

the Pi and the

transport

obtained

to phosphate via

in

that

concentrations

of the active

a non-obligatory

cally

intermediates,

of many transport

The results

utilization

12) have

of the generation

would

driven

(7,

gradients

part

lished

the glycolytic

and much lower

or electrochemical

CCCP suggest

findings

is

studies

respiration proton

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

or to ATP

ATP'ase. (13) uptake

These for

glutamine is

by way of oxidative

apparently or sub-

BIOCHEMICAL

Vol. 62, No. 3, 1975

Fig.

level

to both

phosphorylations".

phosphate

and implicated substances exhibit

whose further

scheme shown the

It

and glutamine (14,

15, 16).

transport

Boos is

and additional

in Fig.

active

The support acknowledged.

interesting binding

(16)

mediated data

of the Medical

in

binding

lead

isolated that

protein

may

us to propose

the

mechanisms

that

coupling

in _E_. coli.

ACKNOWLEDGEMENTS Research Council of Canada

is

gratefully

REFERENCES 1. Medveczky, 2. 3. 4. 5. 6. 7. 8. 9.

N., and Rosenberg, H. (1971) Biochim. Biophys. Acta 241, 494-506 Harold, F. M. (1974) Ann. N. Y. Acad. Sci. 227, 297-311. Young, I. G., McCann, L. M., Stroobant, P., and Gibson, F. (1971) J. Bacterial. 105, 769-778. Lo, T. C. Y., Rayman, M. K., and Sanwal B.D. (1974) Canad. J. Biochem. In press. Klein, W. L., and Boyer, P. D. (1972). J. Biol. Chem.3, 7257-7265. Simoni, R. D., and Shallenberger, M. K. (1972) Proc. Nat. Acad. Sci. USA 2, 2663-2667. Schairen, H. U., and Haddock, B. A. (1972) Biochem. Biophys. Res. Commun. 48, 544-551. Or, A., Kanner, B. I., and Gutnick, D. L. (1973) FEBS Letters 35, 217-219. Yamamoto, T. H., Mevel-Ninio, Mr. and Valentine, R. C. (1973) Biochim. Biophys. Acta. 2, 267-275.

575

drive

respect

have been

to transport.

the energy

of phosphate

that

that

on the possibility

by a periplasmic

to be reported for

to note proteins

has commented

in respect

6 to account

transport

is

transport

common features

The above drive

RESEARCH COMMUNICATIONS

Scheme to account for possible energy coupling mechanisms the active transport of inorganic phosphate in g. coli.

6

strate

AND BIOPHYSICAL

Vol. 62, No. 3, 1975

BlOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

98, 198-204. 10. Pavlasova, E., and Harold, F. M. (1969) J. Bacterial. 11. Kaback, H. R., Reeves, J. P., Short, S.A. and Lombardi, F.J. (1974) Arch. Biochem. Biophys. 160, 215-222. 12. Hertzberg, E. L., and Hinkle, P. C.974) Biochem. Biophys. Res. Commun. 58, 178-184. 13. Berger, E. A. (1973) Proc. Nat. Acad. Sci. USA70, 1514-1518. 14. Medveczky, N. and Rosenb erg, H. (1969) Biochim. Biophys. Acta. 192, 369-371. 15. Medveczky, M. and Rosenberg, H. (1970) Biochim. Biophys. Acta 2ll, 158-168 16. Boos, W. (1974) Ann. Rev. Biochem. 43, 123-147. l

576