Requirement of cyclic AMP for induction of GMP reductase in Escherichia coli

Vol.

43,

No.

5, 1971

BIOCHEMICAL

REQUIREMENT OF

Department Philadelphia,

Received

April

AND

OF

GMP

BIOPHYSICAL

CYCLIC

AMP

REDUCTASE

Charles E. Benson, of Microbiology, Pennsylvania

IN

RESEARCH

FOR

INDUCTION

ESCHERICHIA

Bette Ann Brehmeyer School of Medicine, 19104

COMMUNICATIONS

COLI

and Joseph University

S. Gots of Pennsylvania

20, 1971

Summary: Guanosine-5’-monophosphate reductase is induced in Escherichia coli by growth -bacteria in the presence of guanine or guanosine. In mutants that lackadenyl cyclase GMP reductase is not this induction does not occur unless cyclic 3’,5’-AMP is present. subject to catabolite repression. Guanosine-%monophosphate catalyzes

the

irreversible,

and

role

in the

by

Mager

and

by ATP AMP

and

NADPH-dependent

interconversion Magasanik

was

or GMP.

not

(1).

influenced

In this

Nijkamp --E. coli

and

reductase

IMP

dehydrogenase

increased

enzyme

increased (guaB)

reported

paper

we

cyclic

the

and

In this

to be sensitive

They

also

mapped

present

AMP

to catabolite

evidence

is required

Stmin

GP-1

-coli

(HfrH)

by

K-12

is a -cya Yokota

GMP

They

that

(3).

GMP

the

induction.

enzyme

found

of guanine

were

that

the

coordinately

reductase The

inhibition

biosynthesis

controlling

map

described

was

of

inducible.

reductase the

in the

level

medium

repressed

in of while

by the

in E. coli --

is an inducible

reductase

does

not

appear

repression. MATERIALS

Bacteria:

the --de novo

gene

(guac)

first

to feedback

that

[guaA)

for

Its occurrence

was

sensitive

for

concentration

aminase

bacteria

it was

EC 1.6.6.9)

to IMP.

no indication

Taylor

increasing

XMP

that

the

on the

oxidoreductase

of GMP

essential

was

GMP

in enteric

enzymes

there

(2)

with

of guanine.

that

nucleotides

88 minutes

levels

and

of purine

report,

DeHaan

(NADPH: conversion

by other

early

at approximately

GMP

reductase

mutant

and Gots

AND

METHODS

deficient (4).

Both

in adenyl strains

1089

cyclase

require

derived

thiamine

from for

growth

Escherichia and

Vol.

43,

GP-1

No.

5, 1971

has additional

Media:

The

(0.2%)

unless

(0.5 and

BIOCHEMICAL

minimal

requirements

for

salts

(E) of Vogel

otherwise

&ml)

and

medium indicated.

GP-1

methionine,

AND

isoleucine

methionine,

All

cultures

were and

BIOPHYSICAL

valine

isoleucine and

cultures

further

RESEARCH

and

Bonner

were

valine.

(5) was

routinely

supplemented

supplemented

with

supplemented

with

at 20 to 40 pg/ml

COMMUNICATIONS

casein

with

thiamine

hydrolysate

as indicated

in the

glucose

(0.1%) individual

experiments. Preparation

of Extracts:

at 37’C

on a New

prototrophy, sodium

buffer

cultures

Brunswick

harvested phosphate

fresh

All

were

rotatory

shaker.

by centrifugation

buffer,

containing

pH 7.5. 2 mM

The

of 20 second

cell

was

by centrifugation

by the

Lowry

Enzyme

assay:

Magasanik

reductase

Dithiothreitol

in a Gilford was

patterned

assayed after

GMP sine

was

at 29,000

measured

was

used

Table guanosine. levels. that

The Strain

induction

x g for

to 100 ml medium) for

(30 ml

resuspended

gross

in 0.5 were

by brief

cooling

to

with

0.03

to 2.0

ml of

prepared

by sonic

periods.

Protein

1 hour.

reversion

per wash)

extracts

used 1 shows

was

The determined

amount GP-1, of GMP

of cysteine at 293

(7) and

AND

guanine

guanine

at

assay

1 .O mM

nm.

Purine

uptake

and

of Mager

and

the

was

and

assay

nucleoside

phospho-

incorporation

was

the

inducing

levels

agent

of GMP

of induction

DISCUSSION

or guanosine

to circumvent as the

of the

(8).

of either

the

by a modification

in place

to Kalckar Gots

In order

routinely

times

interrupted

spectrophotometer

according

presence s

were

(50

checked

Cell-free

was

recording

Kal le and

reductase

flasks

were three

pellets

duration,

RESULTS The

liter

(6).

GMP

(1).

measured rylase

method

cell

washed

2-mercaptoethanol.

3 to 4 bursts removed

in one Cultures

and

disruption, debris

grown

cyclase

reduciase

might

in these reductase

ranged

an adenyl

problem

from

deficient be analogous

1090

in the

medium

of poor

solubility

an increase

of guanine,

in

guano-

studies. in --E. coli

2.5

caused

to 7.8

mutant, to the

fold was cyclic

HfrH

grown

increase examined

with over for

AMP-facilitated

and the

the

without noninduced

possibility induction

M

Vol.

43,

No.

BIOCHEMICAL

5, 1971

AND

BIOPHYSICAL

RESEARCH

in

Escherichia

-coli

GMP

reductase

Table GMP

reductase

1

induction

lAdditions

type HfrH)

s mutant (strain GP-1)

0.17

0.40

guanosine

1.69

0.56

guanosine plus cyclic AMP

1.27

3.28

cyclic

0.31

0.41

AMP

were grown (200 &ml) with amino

of other

inducible

of cyclic

AMP

overnight in Medium E, 0.2% glucose, and cyclic AMP (1 .O mM) as indicated. acids as indicated in the text.

enzymes

The

1).

type

strain.

in the

wild

high

levels

of the

GMP

(9.

(Table

tase

cyclic The

required

reductase AMP

optimal

amino

is not

alone

does

conditions

acids

induced not

for

(40 pg/mI)

thiamine

in ihis

act

(0.5

Strain

mutant

was

in the absence

as an inducer

induction

&ml)

GP-1

of

GMP

in the -cya

mutant plus

o r amino

acids

at 20 rg/ml

in a system

which

is induced

reducwere 0.1%

hydrolysate. One

of the

lizable

compound

partially

alleviate

night

induction.

AMP

to the

trated

protein)

none

Bacteria guanosine supplied

strain,

(nmoles/min/mg

to media Wild (strain

casein

COMMUNICATIONS

the

this

problem,

inducing the -cya

problems

is that

Table

however, in the

inherent

involved

inducer

is constantly

the

2 shows

the

cells induction

medium

had

definite

requirement

mutant.

Analogues

were

no effect

being induced

for

of HfrH on the for

of guanine

1091

removed

and

levels

cyclic such

AMP

two

from hours

GP-1. of GMP in the

OS B-azaguanine

the

by a metabosystem.

instead

The

of an over-

addition

reductase induction

To

of cyclic

in the

parent

is clearly and

mercaptoguan-

illus-

Vol.

43,

No.

5, 1971

BIOCHEMICAL

AND

BIOPHYSICAL

Table induction

of GMP

RESEARCH

COMMUNICATIONS

2

reductase

in log

phase

cultures

Strain

Fold increase no addition

HfrH

5.3

4.5

GP-1

1.2

4.4

in GMP with

reductase cyclic AMP

GMP reductase was measured as described in the Materials and Methods. Samples were taken before guanosine was added to the cultures and after 2 hours induction. Cyclic AMP (1 mM) was added along with the guanosine where indicated. Activity at time zero was 0.46 and 0.79 nmoles/ min/mg protein for HfrH and GP-1 respectively.

osine

could

been

found.

not

The sensitive

repression

at least

reductase

cultures utilization

and

The GP-1

of guanosine

indicates

that

uptake

the

and

as was These

to cyclic

possible

effect

examined.

nucleoside

similar

are

GMP

reductase

usually

those

repression

cultures in Table

3,

has

which on the

were

conditions,

phosphorylase

of this

enzyme

comparable. limiting

of purine

induced

are inin

no catabolite

glucose-&phosphate

This

factor

unique

has been

in both

nucleoside

the

eliminates

in the

of guanine-8-C-14

are

for

of catabolite Log phase

Under

hypoxanthine-guanine findings

AMP

As shown

observed.

levels

is the

induction

inducer

of B-galactosidase.

were

incorporation the

was

of purine

11).

respond

therefore

reduction

induction

of HfrH

The

no gratuitous

of glucose-&phosphate.

reductase

a 50%

as yet

which

was

absence

(10,

and

repression.

of GMP

investigators

strains

enzymes

and

The

The

inducible

of GMP

presence

caused

as inducers

to catabolite

duction the

serve

of GMP

phosphorylase

is not

and

an inducible

enzyme

by several and

possibility

induction

in HfrH

1092

noninduced the

phosphoribosyltransferase in that

reported

GP-1

induced

that

Feductase cyclic was

AMP

similar

the

initial

and

also

dependent. in the

activity. which

is neither

catabolic

two

Vol.

43, No.

5, 1971

BIOCHEMICAL

AND

Table Effect

BIOPHYSICAL

on the

inductiot

reductase

GMP time

COMMUNICATIONS

3

of glucose-&phosphate of GMP

Induction (min)

RESEARCH

reductase

Guanosine

(nmoles/min/mg

Guanosine

t

protein)

glucose-6-PO4

0

0.503

30

0.870

1.39

60

1.76

2.26

An overnight culture of HfrH grown in medium E plus glycerol (0.5%) was collected by centrifugation, and resuspended in 5 times the original volume with medium E plus glycerol (0.5%). The cells were incubated with shaking at 37oC for 3 l/2 hours, a sample taken and the culture divided equally between 2 flasks. Both flasks contained guanosine (0.5 mM); glucose-bphosphate (1 x 10m2 M) was added to one flask and samples were taken at 30 and 60 minutes. GMP reductase was assayed as described in the Materiols and Methods.

nor

subject

GMP

to catabolite

nsductose

perties

of mutants

of purine

reports

Under have

the K-12

with

medium strain

of endogenous completely

these

in

the

and

Salmonella the

was

K-12

necessary

except

that

inducers abolished

enzyme

would

thot

requires

(2,

control composition

(l),

Aerobacter

for

and

the

there

(12)

are

B stroin

strains Our

endogenous

1093

used. GMP

not

the

(1) ondfj.

findings

confirm attribute

constitutive with

interconversions and

influence

addition

level

pro-

a deficiency

be desirable.

of --E. coli

The

induction.

prototrophic

of GMP

of E. coli --

We

its

in the

would

does

detectable.

strains

by the

is an excess

medio the

AMPfor

primarily

induction

activity.

levels

the

It serves

of the (I),

of cyclic

as indicated

substrate

H orvord

prototrophic

by decreasing

13). when

by

demonstrable

constitutive in the

participation enzyme

12,

be required

the

(2)

the

biosynthetic

conditions

indicated

activity

However,

an obligate

lacking

nucleotides

of AMP.

tase

is not

repression

GMP

reduc-

subtilis

of guanine this this

GMP

(13). to

with

the

to the

activity the

Previous

synthesis can

inhibitor,

be

Vol.

43,

No.

5, 1971

psicofuronine the

BIOCHEMICAL

(unpublished

level

of GMP

addition

of guanosine

induction

of GMP

AND

observation).

reductase

In contrast

in Salmonella

to the reductase

medium. does

BIOPHYSICAL

not

to the

typhimurium However,

require

RESEARCH

above

is also

addition

report

(1) we

markedly

in an adenyl the

COMMUNICATIONS

find

that

influenced

cyclase

mutant

of cyclic

AMP.

Service

grant

by the

of Salmonella,

ACKNOWLEDGEMENT This the

National

investigation Cancer

was

Institute

supported and

by

by Public

research

Health

grant

GB-7453

from

the

CA-02790 National

from Science

Foundation. REFERENCES 1.

2. 3. 4. 5. 6.

Mager, J. and Magasanik, B. J. Biol. Chem. 235, 1474 (1960). Nijakamp, H.J.J. and DeHaan, P.G. BiochimTBiophys. Acta, Taylor, A. L. Bacterial. Rev. 34, 155 (1970). Yokota, T. and Gots, J.S. J.Bacteriol. 103, 513 (1970). Vogel, H.J. and Bonner, D.M. J. Biol. Chem. 218, 97 (1956). Rosebrough, N.J ., Farr, A. L. andRandall, R.J. Lowry, O.H.,

- 145,

J.

31 (1967).

Biol.

Chem.

-’193

265 (1951). 7. 8. 9. 10. 11.

12. 13.

Kalckar, Kalle, Pastan, Ahmad,

H.M.

J.

Biol.

Chem.

167,

429

(1947).

G.P. and Gots, J .S. Bioxm. Biophys. I. and Wrlman, R. Science, 169, 339 S.I., Barth, D.T. and Pritchard, R.H.

Robertson, B.C., Jargiello, P. Blank, J. (1970). Magasanik, B. and Karibian, D. J. Biol. Nishikcrwa, H. and Katsuya, Momose , H.,

and

Acta, 51, 130 (1961). (1970). Biochim. Biophys. Acta, Hoffee,

Chem. 235, N. Jxen.

(1965).

1094

P.A.

J.

61,

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Bacteriol.-TTII2, -

2672 (1960). Appl. Microbial.

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211