Selection of fructose 6-phosphate kinase mutants in escherichia coli

Vol. 32, No. 3, 1968

BIOCHEMICAL

SE:LECTION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF FRUCTOSE

6-PHOSPHATE

IN ESCHERICHIA A. T. E. Morrissey Department Harvard

Received

enzyme

6-phosphate

which

Escheric:hia Blangy,

coli But,

purified

(Atkinson

a critical

affected

role

Massachusetts

1968).

Because

with in this

1964;

this

1966).

Griffin,

Houck,

In this

from

include

and Brand,

ease

it would

of

enzyme

sources

of the relative

activity.

control

The

these

organism,

enzyme

is a complex

in the metabolic

in Stadtman,

and Walton,

and Monod,

mutants

i967;

of genetic

be useful report

and

to have

we describe

selection. immediate

suggested

fructose

that

metabolism

functioning, fructose

in vitro --

reason fructose in E. --

for

searching

6-phosphate coli.

of the pathway:

i,6 -diphosphate.

and Roseman

the involvement

anticipated, to grow

since

Tanaka

on fructose

et al. --

such mutants

was

not a primary

fructose

system of this

sugars)

467

was

in for

the

i-phosphate4 by the

by Kundig,

in fructose a pleiotropic

lacking

recent

elsewhere

discovered

that

that

intermediate

is catalyzed

system

showed

was

be presented

phosphorylation

(1967)

(or other

will

Fructose+

(Th e f irst phosphotransferase

(1964);

for

Evidence

phosphoenolpyruvate

failed

Boston,

and characterized;

experimentation

The work

and Immunology

(“phosphofructokinase”)

(reviewed

has been

physiological

their

to have

metabolism

sources

coli

School,

kinase

is thought

carbohydrate

g.

and D. G. Fraenkel

June 24, 1968

Fructose

many

MUTANTS

COLI

of Bacteriology

Medical

KINASE

enzyme

utilization mutant I of this

Ghosh, was which system,

Vol. 32, No. 3, 1968

while

a fructose-positive

has recently Anderson, tose

(Fig.

been

1968).

The

second

kinase,

like

) If this

were

(1966).

1) that

in growth

a mutant

used

teriol.

lacking

a similar

the main

this

in A. -

aerogenes

one might

expect

kinase

might

as one criterion

in the

screening

learned

to select

that

then

lacking

Dr.

such mutants

not be affected of pleiotropic

fructose

Anderson in -A.

frucand

6-phosphate

mutants

and

by Hanson

in vivo,

three

reaction

by a kinase,

pathway

found

recently

This

aerogenes((Hanson is catalyzed

discovered

fructose

rationale

enzyme.

in Aerobacter

that

we have

We have

this

phosphorylation

Using

mutants,

activity.

had regained

demonstrated

on fructose.

carbohydrate kinase

revertant

also

i-phosphate

Anderson

have

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6-phosphate

and his

colleagues

aerogenes

(J. Bac-

, in press).

Fig. 1. Pathways of sugar metabolism (a gene for) phosphoglucose isomerase, and 9, for fructose diphosphatase.

in _E. coli. Abbreviations are p@, pfk, for fructose 6-phosphate kinase,

RESULTS According phate

kinase

neogenic glucose metabolism plates

to Fig. would

i,

the phenotype

be normal

growth

substances,

but very

6 -phosphate,

galactose,

is via fructose

to reveal

mutants

of a mutant

on fructose,

slow

growth,

defective

in glucose

fructose

primary or mannitol

6-phos-

and other

on substances

mannitol, Our

468

glycerol,

if any,

mannose,

6-phosphate.

lacking

or pentose, selection utilization.

like

glucoglucose,

whose used

indicator These

Vol. 32, No. 3, 1968

isolates allow

were

8lOCHEMlCAL

screened

us to recognize

by replica pleiotropic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

plating mutants Table

Growth

Carbon

D-Glucose

AMI butant)

2.0

0.3

>2

(0.2

sources

potential

to

fructose

1 plates

Colony KIO $vild type)

of carbon

and to distinguish

on minimal

source

D-Galactose

on a variety

AMlR3 (revertant)

size

AM2 butant)

>2

(0.2

>2

co.2

a,b

(mm) AM2Ri (revertant) 1.5 >2

AM3 butant)

AM3Rl (revertant)

0.3 (0.2

2.0 >2

2.1

0.7

-

0.3

1.0

0.7

>2

D -Mannitol

2.0

0.0

>2

0.0

1.6

0.0

>2

D -Xylose

1.0

0.3

.

-

L-Arabinose

1.5

0.3

-

-

D-Fructose

1.5

1.3

1.5

0.2-1.0

D -Gluconate

1.5

1.5

-

-

Glycerol

1.3

1.1

1.3

0.9

D-Glucose

6-P

1.0

0.6

co.2

1.2

1.3

1.2

a Mutant selection: Strain KiO, a prototrophic Hfr, was grown to stationary phase in medium 63 (Sistrom, 1958) supplemented with 1% Bacto-tryptone (Difco). 0. 1 ml of this culture was added to 0. 9 ml 63 containing 4% ethyl methanesulfonate (Loveless and Howarth, 1959) at 37O. After 20 min (survival was ca. 1OOoJo) the cells were recovered by centrifugation, allowed to reach full turbidity in 63-Bacto-tryptone, and then subcultured to minimal medium containing 0.4% glycerol. The glycerol-grown cells were resuspended in minimal medium with 0.4% mannitol and after 2 doublings (Gorini and Kaufmann, 1960) treated with penicillin, 2000 u/ml. After 5 hr incubation at 37O dilutions of the lysed culture (survival was 0. 02%) were spread on plates AMi was one of the containing MacConkey agar base (Difco) and 1% glucose. apparent glucose-negative isolates. In another experiment mutagenesis of KiO was like the above but for 84 min, with 0.2% survival. No penicillin step was used; instead, after outgrowth in broth a portion of the mutagenized culture and AM2 was one of the glucosewas spread on MacConkey-glucose plates, negative clones appearing. Another portion wasbcultured 1: 100 in fructose minimal medium and then spread on MacConkey plates containing 1% mannitol; AM3 was an apparent mannitol-negative mutant. Spontaneous revertants were8 selected on minimal mannitol plates, and appeared with a frequency of ca. 10 . b Growth: Stationary phase broth cultures were diluted, spread on minimal plates containing 0.4% of the indicated carbon source so as to give ca. 100 colonies/plate, and average colony size estimated after 48 hr incubation at 37O.

469

Vol. 32, No. 3, 1968

6=phosphate

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

kinase

mutants

lacking

phosphoglucose

lacking

enzyme

(Tanaka

--et

al.,

from

known

isomerase

I of 1967;

the

pleiotropic

(Fraenkel

et al.,

1968;

Enzyme

Fox

those

1967),

and

and

Wilson,

those

system 1968

(_E.

c&);

2 a

activities

Enzyme

Strain KiO

6phoskinase

Phosphoglucose isomerase Fructose phosphatase

Levisohn,

- e. g.

phosphotransferase

Table

Fructose phate

and

phosphoenolpyruvate Bourd

mutants

AM1

83b

710

AMlR3

9c

710

AM2

65

AM2Rl

gd

-

384

AM3

62e

-

AM3R

jf

1

758

466

-

di21

16

-

21

-

23

-

a Extracts were prepared from cells harvested from logarithmic growth in a broth containing medium 63, 1% Bacto-tryptone, and 0.4% glycerol. The cells were washed once in cold 0.9% NaCl, resuspended in (3 ml/g wet wt) 0. i M tris-HCl, 0. 02 _M MgC12, p H 7.6, and treated 1 min/ml in an MSE ultrasonicator; the crude extract was centrifuged 17, 000 x g 30 min and the pellets discarded. (In a few cases, instead of sonication the cells were disrupted in a French pressure cell at 15, 000 lb/in2 and the lysate treated with DNAase, 10 pg/ml, before centrifuging. ) The fructose 6-phosphate kinase assay mixture (1 ml) contained 0. 05 g tris-HCl, pH 8. 5, 0. 01 _M MgC12, I. 3 -mM fructose 6-phosphate (calcium salt), i mM ATP, 50 pg aldolase and 23 pg glycerol phosphate dehydrogenase-trz phosphate isomerase (Boehringer, rabbit muscle), 0.1 mM DPNH, 0. Of M NaCN (to eliminate DPNH oxidase activity), and 10-100 pg Fructose 6-phosphate kinase activity was calculated from the extract-protein. difference in the rate of DPNH oxidation between the complete assay and one lacking ATP (the minus ATP-control gives a substantial rate of DPNH oxidation, which is fructose 6-phosphate reductase activity not depending on the auxilliary enzymes). Assays for phosphoglucose isomerase, fructose diphosphatase, and protein were as described elsewhere (Fraenkel and Levisohn, 1967; Fraenkel et al. , 1966). Reactions were followed with a Gilford 2000 spectrophotometerwith the cell chamber at 25O, and are expressed as m~moles/min/mg protein; for fructose 6-phosphate kinase the values are given as 112 the DPNH equivalents oxidized. b average 6; d av.

of separate experiments of 4, 9, 4, 5, 3; e av. of

49,

(97, 76;

470

64, 94, 63, 86, f av. of 4, 4,

120, 56); 2; g av.

’ av. of 79,

of 10, 71.

11,

Vol. 32, No. 3, 1968

Tanaka

BIOCHEMICAL

a,nd Lin,

1968

A phosphoglucose as it grows

nizing

fructose

more

type

or mannitol sixty

screened, like

that

Spontaneous

of apparent

and three

of them,

AMI,

fructose

6-phosphate

expected

for

caused

by a single

gene

2 shows

the results

phate

kinase,

phosphoglucose

three

mutants

are

revertants

they

have

have

regained

(Table

be among

to grow

on

or mannitol

kinase

had a growth mutants

a wild-type Thus

defectives

(Table

growth

the mutant

phenoi).

response phenotype

on is

lesion. of -in vitro

enzyme

assay

and fructose

activities

wild

might

6-phosphate.

in phosphofructokinase,

normal

two

Recog-

somewhat

the inability

and AM3

1).

the other

I mutants differ

glucose

gave

isomerase,

deficient

share

AM2,

revertants sources

enzyme

on glucose

isolates

of carbon

level;

all

from (unpublished).

phenotypes

primary

series

Table

type

the growth

normally

(2. typhimurium)).

distinguished

among

as is known

but grow

1967

and on mannitol

mutants

because

mannitol-positive

the whole probably

kinase

, as far

--et al.,

is readily

on pentoses

however,

About were

normally

nonetheless

the latter;

Simoni

mutant

6-phosphate

difficult,

fructose

(A. aerogenes);

isomerase

types,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

type

6-phos-

diphosphatase. with

of the other levels

of fructose

two

of fructose

The

5-100/o of the wild enzymes.

The

6-phosphate

kinase.

DISCUSSION

ments

Three

fructose

6-phosphate

are

in progress

on their

ization.

It should

unexpected. fructose, be necessary the apparent

One and thus for

be noted

that

such finding

kinase further

abnormality

genetic

some

of their

is that

not all

the expectation fructose

mutants

metabolism in fructose

that

fructose

have

471

selected.

and physiological properties three

mutants 6-phosphate

is not confirmed. growth

been

of two

Experi-

character-

reported grow

here normally

kinase

on

should

It is possible

of the isolates

are

(AM2

not

that and

Vol. 32, No. 3, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AM3)

is a secondary

ways

apparently minimal

and AM3

may

some mutants

plates be less

may

role

that

phosphate

mutants such enzyme

several

metabolism

fail

AMi

that

(Table

their

metabolism,

by the pentose

seems

complete

while

does

have

of the present

growth

is not equally

according

to Fig.

small

AM2

responses

kinase

cycle;

of

hand,

characteristic

phosphate

in path-

colonies

so the different

Another

is that

to form

6-phosphate

metabolism.

mutants

On the other 2),

fructose

other

impaired

1, is restricted it is unexpected

colonies

appear

on

6-phosphate. of these

be particularly

in the

than

whose

formation

since

in preparation).

clarification

negativity

In view will

fructose

substances

mannitol

glucose

(Fraenkel,

be an indication

needs

on the various

to fructose

“leaky”

in normal

which

to triose

of the lesion,

unrelated

fructose

to fructose

effect

structural

would

(between

interesting gene

, or their strains

differences

for

to learn

fructose

revertants, be a useful

mutants

whether

6-phosphate

might

contain

probe

for

and between

these

strains

kinase. a kinetically

the further

substrates) carry

If they abnormal

understanding

do,

it

mutations then

these

enzyme; of this

function.

ACKNOWLEDGEMENTS This (GB-7207)

work was supported and the U.S. Public

by grants from Health Service

the National (GM-00177-09

Science Foundation and 5K03 GM07344).

REFERENCES Atkinson, D. E. and Walton, G. M., J. Biol. Chem. , 240, 757 (1965). J. , J. Mol. Biol. , 31,fi 3 (1968). Blangy, D., But, H. and Monod, V. P. and Gershanovitch, V. N. , Bourd, G. I., Andreeva, I. V., Shabolenko, Molekuliarnaya Biologiya 21 89 (1968). Fox, C. F. and Wilson, G. , Proc. Natl. Acad. Sci. U. S. , 2, 988 (1968). Fraenkel,D. G. and Levisohn, S. R., J. Bacterial., 2, 1571 (1967). S. and Horecker, B. L., Arch. Biochem. Biophys., Fraenkel, D. G. , Pontremoli, 114, 4 (1966). Gorini, L. and Kaufmann, H., Science, 131, 604 (1960).

472

Vol. 32, No. 3, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Griffin,

C. C., Houck, B. N., and Brand, L., Biochem. Biophys. Res. Comm., 27, 287 (1967). HansonTT. E. and Anderson, R. L., J. Biol. Chem., 2&i, 1644 (1966). Hanson, T. E. and Anderson, R. L., Fed. Proc., 27, 644 (1968). Kundig, ‘W. , Ghosh, S. and Roseman, S., Proc. Natl. Acad. Sci. U.S., 52, 1067 (1964). Lovele s s , A. and Howarth, S. , Nature, 184, 1780 (1959). Simoni, :R. D., Levinthal, M. , Kundig, F.. , Kundig, W. , Anderson, B. , Hartman, P. E. and Roseman, S., Proc. Natl. Acad. Sci. U.S., 58 -’ 1963 (1967). Sistrom, W. R., Biochim. Biophys. Acta, 2, 579 (1958). Stadtman, E. R., Advances in Enzymology, 28, 41 (1966). Tanaka, S. and Lin, E. C. C. , Proc. Natl. Acad. Sci. U. S. , 57, 913 (1967). Tanaka, S. , Fraenkel, D. G. and Lin, E. C. C., Biochem. Biophys. Res. Comm., 2J, 63 (1967).

473