The uptake system of free thiamine in mutants of Escherichiacoli

Vol. 47, No. 2, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE UPTAKE SYSTEM OF FREE THIAMINE OF ESCHERICHIA COLI

Takashi

Hiroshima

Received April

Kawasaki

IN MUTANTS

and Kazuo Yamada

Department of Biochemistry University School of Medicine Hiroshima, Japan

3, 1972

SUMMARY Mutants of Escherichia coli auxotrophic for thiamine monophosphate took up I%-thiamineas its free form from the external medium against a concentration gradient regardless of the presence of glucose. The radioisotope was then transported outward in the presence of glucose, but not in its absence. 2,4-Dinitrophenol and sodium azide eliminated the effect of added glucose. The other mutant auxotrophic for thiamine pyrophosphate showed the same profile of uptake as the parent strain except for the accumulation of the radioisotope as 14C-thiamine monophosphate in the cytoplasm. The uptake a manner

similar

(2) that

the

first,

then

which

uptake

in

a membrane It

second

step,

not

conclusively

crosses

the

diffusion

or of

active

whether

the

of at

the

membrane

is

membrane

Press, Inc.

free

Thiamine

is

in

the

catalyzed

cyto-

by

(3).

It

reaction

two steps:

as its

(TPP)

probably

in

was proposed

least

carrier.

however,

by a process

transport.

accumulation

0 1972, by Academic

cell

established,

465 Copyright

2) and it

pyrophosphate which

proceeds

composed

pyrophosphokinase

thiamine

coli

(1,

by a specific

as thiamine

thiamine

is

is

through

is mediated

the

Escherichia

transport

process

passes

accumulated

plasm

in

to active

thiamine

form,

of thiamine

is

of also

as TPP is

whether

free

facilitated not

involved

clear in

(2, the

4)

Vol. 47, No. 2, 1972

thiamine

BIOCHEMICAL

uptake.

to separate been

tried

form

either

In order

these with

obtained

monophosphokinase

thiamine

in

(5,

cytoplasm

thiamine

taken

depending

--E. coli

K12.

(KG1673) 1.3)

have been

been

isolated

strains

the

after

which

or TPP alone

Cells

were grown

stationary been

described

14C-thiamine in

the

obtained

the

not

method

into

defi-

membrane

the

cell.

all

derivatives parent

from

KG1673 have (8) (KG1674

as -

of

14C-thiamine

(1).

of Sigma.

mCi/mmole)

at the uptake

analysis

has been

previously

Centre,

with

and harvested

cell

466

strain

(EC 2.5.

(9) supplemented

Chromatographic the

14.0

of the

of

on 1 x 10q5M thiamine.

products

Radiochemical

is

the

TMP or TPP

indicated

(1).

are the

to hold

nitrosoguanidine

medium

as described

thiazole-2-14C; from

but

The assay

same manner

and that

enzyme

derived

1 x lo-8M

otherwise

transported

presence

pyrophosphorylase

with

on a minimal

of TMP and TPP used (thiamine

are

Mutants

(7).

previously

the

a role

through in

is

source

and properties

(KG1679),

phase.

to

itself in

When this

outward

mutagenesis

1 x 10B8M TPP unless

ability

AND METHODS

grow on either

1678)

has

or TPP.

energy

thiaminephosphate described

the

membrane

state

an attempt

uptake

thiamine

experiments

The isolation

lacking

lack

6) may play

up passes

in

that

cell

on energized

used

thiamine

that

as TMP.

MATERIALS Strains

the

or exogenous

thiamine

the

the

problems,

(TMP)

suggest

across

an endogenous

probably

of

of --E. coli monophosphate

transported

cient,

these

mutants

thiamine

of either

to solve

two processes

The results actively

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

carried

of out

Preparations Thiamine-14C

hydrochloride England.

has

was

BIOCHEMICAL

Vol. 47, No. 2, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION The parent

strain

(KG1673) and its

(KG1679) took

up 14C-thiamine

tially

same fashion

in the

of glucose, glucose

although

shown in Fig.

the rate

strain

of

a rapid

the uptake

l4 C-thiamine profile

was essentially

or the

of

the

uptake

higher

than

amount of

14C-thiamine

at 0".

by KG1675 was lost

found This

cell

suggests

of KG1673, free

tively.

These results

of

cells

1675,

with

incubated

formed

with

the to

the

taken the

up

cell

by chromatographic

of the

uptake

14C-thiamine

in the

cell

weight

as 2.55 ml per g dry

14C-thiamine transport

analysis

the

concentra-

of KG1675 was calculated space of -E. coli

which

external

thiamine

467

to be

2 min at 37'.

(2 min),

(lo),

KG1675 cell.

for

water

in the of

in the presence

and 14C-TMP, respec-

14C-thiamine

taken

this

37',

and 1679 was found

level

an active

of uptake

at

14C-thiamine

14C-thiamine,

cellular

suggests

that

were obtained

incubated

of

rate

medium through

to be 2.4 x 10e5M when the

concentration

the

incubation

in the cell

form of thiamine

14C-TMP and 14C-TPP,

At the maximal

and then

absence

was present.

The intracellular by cells

glucose

of KG1675 was reduced

to the external

membrane when glucose

of

shown in its

10 - 15 min of in the

of the parent

presence

that

After

as that

time;

In the

slowly.

of free

as

in the absence of

decreased

tion

presence,

with

a peak at 2 min,

these

in its

absence of

a TMP auxotroph,

reaching

of glucose

the presence

by KG1675,

occurred,

radioisotope

of

in the

same as that

TPP auxotroph.

initial

the same level

medium essen-

regardless

50 % of that

showed a characteristic it

time

external

mutant

1.

The uptake

glucose

from the

with

was approximately

TPP-requiring

as its

is

24 times

is the

medium.

This

free

form in

Vol. 47, No. 2, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

h 0

A

t

a

//

/

5

10 blin

Min

uptake by three Fig. 1 (left). Time course of 14C-thiamine mutants of E. coli. Cells of three mutants grown and harvested as given in the text were washed and resuspended in minimal medium (9) containing 100 pg/ml chloramphenicol with or without The uptake of 14C-thiamine was measured in the 0.4% glucose. same manner as described previously (1). The concentration of 14C-thiamine (specific activity, 14.0 mCi/mmole) added to the Open symbols represent the uptake uptake medium was 1 x lo-6M. in the presence of 0.4% glucose and closed symbols, in the A, A: KG1673; o, l : KG1675; 0, l : KG1679. absence of glucose. x: uptake of 14C-thiamine at O", which was practically identical in these three strains regardless of the presence of glucose. Fig. 2 (right). Uptake of 14C-thiamine by 4 auxotrophs for TMP. Uptake of thiamine was carried out under the same conditions as shown in Fig. 1. These 4 strains could be divided into 2 groups (KG1674 and 1678; KG1676 and 1677), which show slightly different patterns of uptake. Open symbols represent the uptake in the presence of glucose and closed symbols, in the absence of glucose. o, l : KG1674 and 1678; A, A: KG1676 and 1677.

468

15

Vol. 47, No. 2, 1972

In order up is

to show that

specific

thiamine

for

(KG1674,

although

identical

mutants

glucose

trated

for

containing

3.

decrease

KG1675 and a rapid conversely

demonstrated

This in which

assumption

was present thiamine

in the

uptake

DNP to the of

uptake

absence of

this

is

of

not

showed the

illustrated. same effect of

free

phosphorylating

with

cells

uptake

of

was

4).

glucose,

When DNP

the rate

by 40%.

of

Addition

resulted

same as that

The addition

medium

of

in a time

obtained

in the

of the uncoupler

brought

about

a rapid

at

rise

when glucose

was present,

Sodium azide

at 0.02M concentration

although

as DNP. thiamine systems

isolating

mutants

of -E. coli

dependent

loss

14C-thiamine

of

illus-

at 37O, a rapid

(Fig.

glucose

incubation

the uptake

Separation coupled

the

is

by the experiment

approximately

the

and DNP.

This

presence

KG1673 and 1679.

supported

medium without

essentially

glucose

rate

of both

medium containing

2 and 10 min after in the

uptake

completely

in these

of the

(DNP) was added

was reduced

uptake

occurs

incubation

rate

cells

was further

2,4-dinitrophenol

was not

was observed

in the

with

showed

was added to the uptake

uptake

increase

tested

as found with

of energy.

at 10 min after

in amount of the

TMP auxo-

assumed to be due to the

supply

of

2). which

When glucose

no glucose

strains

uptake

(Fig.

TMP is

TMP, the uptake

uptake

of the

strains

as an exogenous

in Fig.

course

of thiamine

once taken

4 additional

All

the radioactivity

auxotrophic

for

of

1678).

the pattern

of

of 14C-thiamine

cells

1617,

among these

The loss

loss

auxotrophic

with

same characteristics

KG1675,

of

1676,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

mutants

was determined

trophs the

BIOCHEMICAL

transport

system

from the

has been established

auxotrophic taken

469

for

TMP.

up by these

by The energy-

mutants

is

Vol. 47, No. 2,1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5

0

10

Min

Min

Fig. 3 (left). Effect of adding glucose on the uptake of thiamine by three mutants. At the time indicated by arrow, glucose was added at 0.4% concentration to the uptake medium without glucose and incubation was continued for another 10 min. Closed symbols show the uptake after addition of glucose. A, A: KG1673; o, l : KG1675; t3, l : KG1679. The other conditions for the uptake were same as shown in Fig. 1.

Fig. 4 (right). Effect of 2,4-dinitrophenol (DNP) on the uptake by KG1675. DNP was added to the uptake medium at 2 x 10-3M. The medium was preincubated for 5 min at 37' prior to addition of 14C-thiamine. The other conditions for the uptake were same as given in Fig. 1. o: glucose added; l : both glucose and DNP added; 4: no glucose added; A: no glucose, but DNP added. assumed

to be due to

1) the

and 2) energetic

state

specific

protein

carrier It

has been

lack

of thiamine

monophosphokinase,

of the

cell

membrane

for

thiamine

is

shown recently

that 470

active

in which

the

functioning. transport

of

amino

Vol. 47, No. 2, 1972

acids

(ll),

sugars

a D-lactate it

was not

of free the

compound

probably is

supported

involved by the

to trap ly

either

removed

through the

is

property

of these

proteins

(enzymes)

(14)

an electron

transport

whether coupled

described

generated in

experiments that

suggest

that

to the

due to the or the

therefore,

concentrative

energy transport This

in Fig.

is

4.

are present, system

cytoplasm formed negatively

accumulate

as above

a high

thiamine.

binding

to

chain.

the

thiamine

TMP or TPP thus

either

coupled

same system

phosphorylation thiamine

is

to electron

of free

phosphorylate

compounds and,

to the

demonstrated

oxidative

cell

or not

by coupling

transport

transported

the

and manganese

determined

as TMP or TPP. from

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

via

results

When enzymes generated

13),

thiamine

mentioned,

system

(12,

dehydrogenase

Although uptake

BIOCHEMICAL

with

ATP

is utilized concentrativemight

not

be

charged cellular

in

the

cytoplasm.

REFERENCES T., Miyata, I., Esaki, K., and Nose, Y., Arch. 1. Kawasaki, Biochem. Biophys., 131, 223 (1969). 2. Kawasaki, T., Miyata,T, and Nose, Y., Arch. Biochem. Biophys., 131, 231 (1969). 3. Miyata, I., Kawasaki, T., and Nose, Y., Biochem. Biophys. Research Commun., 27, 601 (1967). J., and Stadtman, E. R., J. Biol. Chem., 4. Hochstadt-Ozer, 246, 5304 (1971). H., and Hayashi, R., J. Bacterial., 109, 936 5. Nakayama, (1972). A., Nishino, H., and Nose, Y., Biochim. Biophys. 6. Iwashima, Acta, 258, 333 (1972). 7. Kawasaki,., Nakata, T., and Nose, Y., J. Bacterial., 95, 1483 (1968). 8. Adelberg, E. A., Mandel, M., and Chen, G. C. C., Biochem. Biophys. Research Conunun., 2, 788 (1965). B. D., and Mingioli, E. S., J. Bacterial., 9. Davis, -60, 17 (1950). S. G., and Solomon, A. K., J. Gen. Physiol., 10. Schultz, -45, 355 (1961). 11. Kaback, H. R., and Milner, L. S., Proc. Nat. Acad. Sci., 66, 1008 (1970). E. M., Jr., and Kaback, H. R., Proc. Nat. Acad. 12. Barnes, Sci., 66, 1190 (1970). x M., Jr., and Kaback, H. R., J. Biol. Chem., 13. Barnes, 246, 5518 (1971). P., J. Bacterial., 104, 1307 (1970). 14. Bhaacharyya, 471