Repression of oxidative phosphorylation in Escherichia coli B by growth in glucose and other carbohydrates

Vol. 41,No.

1, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REPRESSION OF OXIDATIVE

PHOSPHORYLATION

IN ESCHERICHIA COLI B BY GROWTH IN GLUCOSE AND OTHER CARBOHYDRATES

Walter Department

P. Hempfling

of Biology,

River

University

Campus Station,

of Rochester,

Rochester,

N.Y.

14627

Received August 5, 1970 Sumnary A simplified method for the assay of oxidative phosphorylation in intact bacteria is described. Using this method it is shown that the efficiency of oxidative phosphorylation in Escherichia coli B grown in glucose-containing media is less than 10% of that of organismsgrown in media containing noncarbohydrate substrates. Growth in media containing galactose, lactose or mannitol yields cells capable of only half the efficiency of oxidative phosphorylation of fully competent cells. The incorporation of casamino acids into the growth medium has no effect on glucose repression of oxidative phosphorylation. Stimulation of respiration by the uncoupler 2,4-dibromophenol occurs only in organisms in which all three sites of oxidative phosphorylation are functional. Introduction Escherichia tionary

coli

period

B, grown

of growth,

is

in tryptic capable

soy broth

of oxidative

and harvested phosphorylation

in the stawith

an

. apparent

efficiency

Bacteria

harvested

efficiency

during

f 0.3 moles the

of phosphorylation,

cose.

Using

based

on that

cally

in a variety

which

are

suggest phorylation Materials

of 3.5

a method previously

part is

logarithmic

growth

however,

apparently

of oxidative

described

of media

a function that

of assay

carry

it

of the enzymatic

period

due to the

repression.

--and Methods

Escherichia

cqJ

B was maintained

as described'. 9

cells

growing

lower of glucells aerobi-

at efficiencies

medium.

apparatus

presence in intact

phosphorylation

of the growth

of NADH oxidized'.

show considerably

can be shown that

or structural

to catabolite

per mole

phosphorylation

out oxidative

of the composition

subject

Pi esterified

The results

of oxidative

phos-

VoL41,No.

BIOCHEMICAL

1, 1970

The media of growth

employed

in the

composition:

presence

- 10 ml/l;

5 g/l;

and substrate E. coli

times within removed after

when

the cold in cold buffer

grown

at 12,000

the effects

of the

NaCl - 0.3

- 30 mfi, pH 7.0;

Difco

to be used for

700 ml of medium

in tryptic were

the optical

Tris-Cl

into

When the effect

inoculation)

g/l;

at 300 rpm on a New Brunswick

the contents

10 min.

to test

a solution

NH4Cl - 0.5

in the medium

(5%, v/v)

cells

stated,

and,

COMMUNICATIONS

following

g/l;

trace

Casamino

Acids

- 15 mM_.

shaken

When examining

Soy Broth

substrates,

phosphate

B was cultivated

were

Tryptic

- 0.8 g/l;

potassium

inoculation

Cultures

Fisher

of various

MgS04'7H20

metals2

before

were

AND BIOPHYSICALRESEARCH

soy broth

chilled

of various

substrates

imnediately

x g for

Cells

buffer,

10 min.

0.1 1,

at a concentration

pH 7.5,

of about

in 2.8

1 Fernbach

flasks.

platform

shaker

were in ice,

were

washed

and were

15 mg/ml

finally

(dry

at the

and then

1.0 * 0.1

harvested

at 37'.

removed

was tested

at 650 nni reached

and the contents

of 12 hr

flasks

immediately

density

a period

harvested

flasks

(2.5

were

to 6 hr

by centrifuging

twice

by resuspending

suspended

weight)

in

in the same

and kept

in ice

until

used. The respiratory with

a Clark

piration

oxygen

3 fl

the anthrone

were

of bacterial

electrode

glucose

in 0.1 MTris-Cl Glucose

activity

in the

in a vessel

was added

buffer,

oxidative

to a washed

portion

incubated

at 30"

32

phosphorylation for

7 min under

, pH 7.0,

cell

suspension

of the culture

to each of 4 samples

added

5 ~1 of 0.1!2,4-dibromophenol The 4 samples

The P/2e'

value

(Set

To initiate (0.6

res-

- 0.8 mg/ml)

fluid

was assayed

a stream

1).

1 ml suspensions

of bacteria

of He'.

time

activity

(Set

expressed

3) received is

10

At zero 500-1000

To each of 4 other in methanol

remaining finally

twelve

0.5 I+, specific

added

7.0.

of 3.5 ml volume,

at 23-25'

pH 7.5

supernatant

To assay

Pi32.

was measured

by

procedure3.

of Tris-phosphate

Of

suspensions

samples

(DBP) 30 set

20 ~1

cpm/mumole, (Set before

2) was addition

20 ~1 of 0.5 MTris-Pi,

the amount

of pi32

esterified

was

pH after

-

Vol. 41, No. 1, 1970

5 set

of exposure

presence (Set

BIOCHEMICAL

of the uncoupler

3).

the

Pi32

injected

intra-

addition into

later

(Set (Set

under

two samples

of

of each set

the addition

of aerobic

in the cold

method

of Hagihara

for

pH 7.5,

Tris-Cl

buffer

5 set

ethanol'.

The remaining 5 - 10 set

were

of total

and organic

portion

phosphate

Mk II scintillation

An Ansitron

before

centrifuged

and 1 ml of the supernatant

the measurement

and Lardy'.

followed

samples

5 set

To each of two

reagent

The acid-treated material

was rapidly

of Set 2 followed

1 ml of the terminating

buffer.

of

at 15 min

1.8 fi HC104 and 1.2 5 Na2S044.

to remove denatured

of each was removed

0.15,

of 1.4 L KOH in 85% (v/v)

received

the

equilibration

Accordingly,

buffer,

1 ml of aerobic

in

of NADH oxidized

isotopic

spaces. Tris-Cl

incorporated

amount

of Set 1 and two samples

by 1 ml of a solution

samples

by the

He assured

1 ml of aerobic

of Set 3 was added

later

the amount

and extracellular

by 1 ml of a solution

samples

1) minus 2) divided

15 min of incubation

P 32 between alter

to oxygen

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by the

counter

was

used. NADH was determined Set 3 as previously

in heated,

neutralized

portions

of the samples

in which

the time

of

1.

described

Results Table growth, coli

1 shows the results

glucose 6 growing

lation

within

out full

oxidative

prevents

both

yield

dependent

compounds phosphorylation

were

tested of cells

at the

at the time

for

11

in

the

glucose

phosphoryto

acquisition

of

by the

is exhausted,

phosphorylation. of glucose

r.

The

disappearance,

utilized.

the ability grown

of

the ability

is demonstrated

of oxidative

of glucose

oxidative

That

time

course

was followed.

of glucose

upon growth

added

of such a culture, per mole

of full

is gained.

and the development

60 - 70 g dry weight Other

incapable

phosphorylation is

efficiency

the exhaustion

chloramphenicol,

growth

is

90 min after

oxidative

that

growth

oxidative

soy broth

phosphorylation

observation

molar

and phosphorylation

in tryptic

although

carry

is

utilization

of an experiment

their

to affect presence,

the efficiency and the results

of

VoL41,No.

Table

BlOCHEMlCALANDBlOPHYSlCALRESEARCH

1,197O

Efficiency of oxidative phosphorylation growth in tryptic soy broth.

1.

Time

0

Cella density

hr

in r.

nPib

Residual glucose

coli

B during

ANADHb

Apparent P/2e'

0.07

14.2

2.0

0.62

9.2

0.47

1.43

0.33

3.5

1.92

0.6

0.30

0.82

0.36

5.2

2.48

0

3.69

1.13

3.3

5.8

2.70

0

3.48

0.81

3.9

6.3'

1.84

0.12

0.53

0.23

aOptical

density dry

'30

ug/ml

chloramphenicol

are

given

in Table

weight.

in media

oxidation previously

titious

Pi. carry

contains

Class

out

reported

that

are

of cells

capable grown

but only

during

the

to the class

the rate

of substrate

little

by DBP.

rate

15 min anaerobic employed

for

addition

glucose

grown

12

is

(Class

uptake

less

than

the medium also glucose

containing

intermediate A compounds

consumption of DBP.

C) do

during

in media

Class

preincubation growth

during

even when

containing

by the

found

phosphorylation

of oxygen

oxidative

(some 5 - 10%) by adven-

of oxygen

Cells

of oxidative

of full

value

of Pi32

rate

Organisms

consumption

phosphorylation,

and their

separable.

capable

of oxygen

of dilution

in media

their

are

were

The mean P/2e'

oxidative

A compound,

changed

esterified

of compounds

in a medium containing

not much increased

glucose, is

grown

hr.

A compounds

because

significant

B compounds

containing dation

Class

Organisms

is

between

classes

some 1.7 - fold.

a Class

oxidation

Three

2.

at 3.6

and DBP increases

that

not

added

containing

phosphorylation glucose

-

at 650 nm.

bvmoles/g

grown

mII

COMMUNICATIONS

during

and those glucose

The amounts period

in a manner

differ perhaps

oxi-

of Pi according related

to

BIOCHEMICALANDBIOPHYSICALRESEARCH

Vol.41,No.1,1970

Table

2.

Oxidative of various

Substrate

AP.a1

--Class

phosphorylation substrates.

A

in g. coli

NADHa

Apparent P/2e'

4.63 3.52 6.24 6.07 3.60 4.80 5.36 4.15 4.60

1.52 1.06 1.61 1.83 1.14 1.70 1.92 1.08 1.18

APi

presence

GlucoseC oxidation

4.05 7.05 6.24 9.14 8.83 8.21 8.73 7.14 10.63

Mean (12)e S.D.

3.3 0.4

RCRd

2

1.80 1.45

s:

1.47 1.81

:;

1.88 1.87

7.9 1.0

iI::

B

Lactose Mannitol Galactose

3.16 1.98 2.25

1.91 1.00 1.15

:?I 2:o

9.00 5.76 7.14

ii:

7.1 1.1

Mean (6) S.D. --Class

Anaerobicb

in the

A

None Formate Acetate Lactate Glycerol Succinate Malate Glutamate Ribose

--Class

B grown

COMMUNICATIONS

0.93 1.00 1.14

zi 52

A::

C

Glucose Glucose t glycerol Glucose + lactate

0.17 0.31

1.83 2.08

0.10 0.15

2.36 1.74

88 74

1.15 0.92

0.58

3.03

0.20

2.10

65

1.19

Mean (6) S.D.

apmoles/g

dry weight.

bPi esterified 'dj d

during

15 min anaerobic

preincubation

(pmoles/g

dry weight).

02/min/mg.

Respiratory the presence

eNumber

glucose oxidation of DBP.

rate

in

of samples.

the ability capable

ratio of steady state Control Ratio: of 140 IJM DBP to that in the absence

to carry of glucose

out oxidative oxidation

as well

phosphorylation. being

tested. 13

able

All to oxidize

preparations the compound

are

BIOCHEMICALAND

Vol.41,No.1,1970

Variation no effect

of the concentration

on the

although

it

is

ability

by the presence

of casamino

of glucose

possible

that

BIOPHYSICALRESEARCH

to repress

repression

or absence

acids

of added

carbon

(zero

oxidative

by Class

COMMUNICATIONS

to 10 g/l)

had

phosphorylation,

B compounds

could

be affected

sources.

Discussion Growth to all

in glucose

three

is

growth

is

not

sites

membranes,

derepression

of new cytochromes

have not

but

totally

unable

fully

uniformly

throughout

or

B compounds it

if

site

in a mannitol-containing

medium,

they

not did

permit note

following

the

glucose-grown that

oxidative

that

the

aerobic

is

effect

anaerobic

and Johnson"

found

that

molar

growth

yield

I.

e,

the are repressed

of the absence

grown

of a has

aerobically.

oxidation

However,

on glucose

but

in respiration DBP, fi-Cl-CCP

by r.

coli

growth

predicted Hadjipetrou,

of Aerobacter

methods

efficiency,

Like

molar

aerobic

Their

m-Cl-CCP

conditions.

than

anaerobically

uncoupler-sensitive

uncoupler

lower

14

with

of phosphorylation

the aerobic

operated.

of the popu-

595 , and glucose

mitochondria

grown

glucose

was considerably

phosphorylation

have appeared

of less

to aerobic during

a fraction

of

mitochondria'.

capable

of the

present

of phosphorylation

that

of changes

consumption

E.

of yeast

in

repression

phosphorylation

in yeast

same organism

calculation

from

oxygen

Hernandez

the

an increased

a shift

stimulates

is

partial

whether

synthesis

Hempfling,

bring

Reports

evidence

Massive

of quinones

about

it

of new proteins,

component. (W.P.

but

and amounts

not clear

I)

of synthesis,

synthesis

involved

out oxidative

(Site

have provided

did

is

the synthesis

than

by the

one or two sites

--et al8

Pi esterification

kinds

the population.

to repress

Cavari,

is not

to carry

competent

phosphorylation

been shown

about

common

to chloramphenicol,

or some other

of the

phosphorylation,

remainder

single

changes Class

is

brought

of the two,

been excluded.

oxidative lation

is

of components

Derepression

by the response

and flavoproteins

although

the synthesis

phosphorylation.

as indicated

a combination

preparation)

to repress

of oxidative

dependent

known if

appears

if

8'. yield

it

of

was assumed 11 reported --et al

aerogenes

was

Vol.41,No.1,1970

that

BIOCHEMICAL

expected

from

Such inconsistencies repressive

the

operation

might

effects

reported

related

compounds.

affects

repression

of other

some effects

of glucose

that

be due to a general terminal

electron

of three

be resolved

on oxidative

The results

AND BIOPHYSICALRESEARCH

of oxidative

by an investigation

phosphorylation

here

To what

sites

extend

extent systems

repression as yet

in the ability

of the

properties

of oxidative undetermined,

on enzyme synthesis

deficiency

phosphorylation. possible

of the substrates

the repressive

is

COMMUNICATIONS

in growing

used. of glucose

and

phosphorylation but

it

is

possible

microorganisms

to conserve

energy

technical

assistance.

may

through

transport.

Acknowledgments Thanks work

are

was supported

due Diane by Grant

K. Beeman for No. 68-8217

expert from

the

National

Science

This Foundation.

References Hempfling, W.P. Biochim. Biophys. Acta, 205, 169 (1970). Vishniac, W. and M. Santer. Bacterial. Rev., 21, 195 (1957). Ashwell, G. in S.P. Colowick and N.O. Kaplan (ed), Meth. Enzymol., Vol. III, P. 84 (1957). Hagihara, B. and H.A. Lardy. J. Biol. Chem., 235, 889 (1960). 54: Ohnishi, R., K. Kawaguchi and B. Hagihara. J. Blol. Chem., 241, 1797 (1966). 6. Kovac, L., H. Bednarova and II. Greksak. Biochim. Biophys. Acta, 153, 32 (1968). and C.W. Sharp. Arch. Biochem. 7. Jayaraman, J., C. Cotman H.R. Mahler 116, 224 (19661. Biophys., Cavari, B.r Y. Avi-Dar and Ii. Grossowicz. J. Bacterial., 96, 751 (1968). and N. Grossowicz. Biochem. J., 103; 601 (1967). :: Cavari, B.Z., Y. Avi-Dor Hernandez, E. and M.J. Johnson. J. Bacterial., 94, 99G (lm). E: Hadjipetrou, L., J. Gerrits, F.A.G. Teulings andT.H. Stouthamer. J. Gen. Microbial., 36, 139 (1964).

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