The effect of nalidixic acid on the expression of some genes in Escherichia coli K-12

Vol. 64, No. 1, 1975

BIOCHEMICAL

THE EFFECT OF NALIDIXIC

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACID ON THE EXPRESSION OF SOME GENES

IN ESCHERICHIA COLI K-12 -H.Shuman and M.Schwartz Unite de GBnetique Moleculaire Departement de Biologie Moleculaire Institut Pasteur,Paris. Received

March

14,1975

SUMMARY The synthesis of maltodextrin phosphorylase and the phage X receptor of Escherichia coli K-12 is substantially inhibited by the presence of 50 pg nalidixic acid/ml in the culture medium. B-galactosidase synthesis is inhibited to a lesser extent and no inhibition of L-tryptophanase synthesis is observed. The inhibition of enzyme synthesis is apparently not due to the effect of nalidixic acid on deoxyribonucleic acid synthesis.

INTRODUCTION Nalidixic

acid

(1-ethyl-1,4-dihydro-7-methyl-4-0x0-1,

8-naphthydrine-3-carboxylic deoxyribonucleic understood which

for

(2,8).

that

This

in the

they

Escherichia

significantly

(50pg/ml)

is

poorly

literature

DNA synthesis

synthesis

(13,14,

is based on the observation bacteria

do not

incorporate

amino acids from that

into

that thymine

protein

observed

(2).

experiments

K-12 cells,

in the

to stop

on protein

different

drug.

to describe coli

acid

do incorporate

absence of the

We wish

acid

of the drug,

DNA while not

have appeared

is no effect assumption

of bacterial

whose mode of action

Many reports

there

presence

at a rate

synthesis

is an inhibitor

the use of nalidixic

example).

in the into

acid

describe

assuming

acid)

which

a concentration

commonly used to

interrupt

show that

in

of nalidixic DNA synthesis

also

BIOCHEMICAL

Vol. 64, No. 1,1975

inhibits

the

appearance

for

the bacteriophage

in part, the

of maltodextrin

the product

(~~2,4,1,1),

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the

lambda,

gene,

which

malA region

of

the Escherichia

1amB gene is

located

of the malA

and malB regions

ma1T product

and is

(1,3,4,15,Hofnung of

glucose

subject

the products

coli

is

source

in

The expression by the

repression results).

The

and malB regions

the malA

coli

located

controlled

unpublished

K-12 cells

by adding

monophosphate

(3,4).

positively

of both

at least

chromosome and the

to catabolite

in Escherichia

as carbon

adenosine

is determined,

in the malB region

and Schwartz,

can be induced

and the receptor

The malP gene is

by the

synthesis

phosphorylase

maltose

which

are using

and 3':5'

cyclic-

(CAMP) to the medium (Fig.Ia

and Ib).

RESULTS AND DISCUSSION The induction both

in the

in Figure

of the

absence and presence

la.

the receptor

An 80% reduction was observed

same experiment, maltodextrin the

the into

acid

acid

appearance

nalidixic inhibition

not

acid

in the

shown).

This

on the maltose

of DNA synthesis.

effect

suggests

of

with of

phosphorylase of

1Opq

or

14C-thymine

was completely that

system is not

205

of

was observed

material

In order

In the

of synthesis

were observed

incorporation

insoluble

of

in the presence

maltodextrin

acid

shown

of the drug.

per ml. At a concentration

although

is

of synthesis

rate

was observed

of either

trichloroacetic

acid

rate

per ml no significant

lambda receptor,

inhibitcd,(data

in the

The above effects

50 pg of nalidixic of nalidixic

of nalidixic

a 70% reduction

(Fig.lb).

by CAMP and maltose,

in the presence

phosphorylase

drug.

on the

X receptor

to test

the

effect

a consequence this

of of

hypothesis

BIOCHEMICAL

Vol. 64, No. 1,1975

AND BIOPHYSICAL

20 t

RESEARCH COMMUNICATIONS

bJ

I 0.35

0.45 Absorbance

0.55 i600nml

0.65

0.35

0 45 Absorbance

0.55 1600nm)

0.65

04 1600nm,

05

dJ 400

L 02

FIGURE 1 : The effect inducible proteins. The activity plotted as a function

0.3 A bsorboncc

0.4 l6OOnm)

05

t

03 Absorbonce

of nalidixic . -

acid -_

on the synthesis

of each protein per ml of culture of the absorbance of the culture

of some is at 600 nm.

a) Lambda receptor. A culture of exponentially growing Escherichia coli K-12 cells, using glucose (2xl0'2M) as carbon source was divided in half. Nalidixic acid was added to one half of the culture at a final concentration of 50 vg/ml. After shaking at 37 C for 15 minutes, CAMP and maltose were added to both halves of the culture, at final concentrations of 4x10-3M and 10m2M, respectively. One ml samples were taken from each culture into chloramphenicol (100 pg/ml) at 0 C, at 0, 10, 20, 30, 45, and 60 min after addition of CAMP and maltose. After the A600 was determined for each sample, the cells were centrifuged, resuspended in O.lml of tris-cholate-EDTA buffer, (10) and extracted for 30 min at 37 C. Lambda receptor activity was determined in the extracts as described by RandallHazelbauer and Schwartz (10). (O-O without nalidixic acid, PJ -m with nalidixic acid). b) Maltodextrin phosphorylase. The procedure is the same as that used in a) except 2ml samples were taken and maltodextrin phosphorylase activity was determined in sonicated extracts of the bacteria. The sensitive method of Schwartz and Worcel (12) which measures incorporation of 32P inorganic phosphate into glucose-lphosphate was used to determine the amount of maltodextrin phosphorylase activity in the extracts. (O-O without nalidixic acid, a-a with nalidixic acid). c) L-tryptophanase. The procedure used was the same as in a) except that L-tryptophan (lmg/ml final concentration) was added to the culture instead of maltose, to induce synthesis of L-tryptophanase. L-tryptophanase activity was determined in toluenized cells using

206

BIOCHEAKAL

Vol. 64, No. 1, 1975

the procedure p--n with

AND BIOPHYSICAL

of Newton et s.(7). nalidixic azd).

RESEARCH COMMUNKATIONS

(O-O without

nalidixic

acid).

d) @-galactosidase. The procedure was as in a) except that the culture was divided into 4 parts before treatment with nalidixic acid. Nalidixic acid was added to two of the aliquots and after shaking for 15 min at 37 C, CAMP and isopropyl-B-D-thiogalactoside (IPTG) (5x10-3M final concentration) were added to one treated and one untreated aliquot. IPTG (5x10-3M, final concentration) alone B-galactosidase activity was added to the two remaining aliquots. was determined in toluenized cells by the method of Horiuchi et al -(5). IPTG + CAMP O-O IPTG + CAMP + nalidixic acid a-0 I?TG 0-e IPTG + nalidixic acidm-m

we examined

the

synthesis

lambda receptor

in a strain

In such a strain, transfer

of the

cells

synthesis

receptor.

In the

maltodextrin

inhibited view

of these

nalidixic

acid

a consequence

that

it

that

on the expression the effect

these

experiments interfere

induction

the effect

with

that

maltose

of the maltose

to

is not

the possibility of the bacteria the

system. acid

both

system and on the

nalidixic

207

of

system

induce

maltose.

transport

was

the effect

of nalidixic

transport

of

at 30 C or 42 C. In

ability

transport

to transport suggest

synthesis

on DNA synthesis.

the

maltose

of the maltose

the bacteria

the

we have tested with

on the

or lambda

of the maltose

of the drug

enough internal

effect

and lambda receptor

seems likely

was done by examining

of

acid,

either

interferes

directly the

results

induction

after

phosphorylase

incubation

nalidixic

ability of

during

experiments

on the

immediately

of nalidixic

In preliminary

to accumulate This

presence

acid

a dnaBtS mutation.

at 42 C had little

phosphorylase

of

and

to 42 C. In the absence of nalidixic

of maltodextrin

severely

phosphorylase

carries

stops

of the bacteria

of

both

which

DNA synthesis

acid,incubation rate

of maltodextrin

The results acid

but

does not

does prevent

system in addition

of

BIOCHEMICAL

Vol. 64, No. 1, 1975

to lambda receptor shown below of

for

and maltodextrin

the

i3-galactosidase

acid

does not Figure

the

synthesis

without

with

of synthesis

acid

has no effect

in the

of

acid

of L-tryptophanase. synthesis

any involvement

suggest of nalidixic

acid

of L-tryptophanase

sensitive

the effect

that

nalidixic

intercalate

supercoiled inhibit

operons

(11).

that

the

have

not

dehydrogenase

shown).

These repression

is

synthesis. nalidixic

product

acid by

may act

orange

of some catabolite

lines

nalidixic

acid,

or a metabolic agent

of

it,

reasoning

it

product

may preferentially

of some genes such as those

208

as

drugs which

DNA such as acridine

expression

of

DNA (9).

shown that

these

transcription

nalidixic

nor does CAMP reverse

that

in supercoiled

Following

as an intercalating

on the

amount of RNA produced

or a metabolic

and Pogell

bromide

effect

the drug does not affect

repression)

in the

agent

Senkaran

or

of the phage S-13 genome. They postulate

acid,

an intercalating

with

of catabolite

of the drug on %-galactosidase

transcription

synthesis

of homoserine

since

Puga and Tessman have observed

--in vivo

There

In addition,

(data

on

(an enzyme whose synthesis

to catabolite

causes an 80% reduction

nalidixic

acid

either

has no apparent

results

very

of nalidixic

B-galactosidase

dehydrogenase

do not

that

and B-galactosidase.

rate

on the

on the

indicate

effect

or glucose-6-phosphate

synthesis

induction

of the drug when induced

rate

effect

acid

CAMP transport.

of L-tryptophanase

CAMP. Nalidixic

in the

The results

of nalidixic

and Id show the

presence

phosphorylase.

and L-tryptophanase

is a 40% reduction in the

effect

interfere

lc

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sensitive

could of

and ethidium

be proposed it,

inhibit

acting the

of the maltose

BIOCHEMICAL

Vol. 64, No. 1,1975

system.

Whatever

suggest

that

can have

their

nalidixic

interpretation acid,

an effect

AND BIOPHYSICAL

on the

RESEARCH COMMUNICATIONS

, our observations

besides

blocking

expression

of

DNA synthesis

some genes.

ACKNOWLEDGEMENTS This National

investigation

was supported

Institutes

of Health,

the"Centre

Recherche

Scientifique'j

the"Delegation

Recherche

Scientifique

et Technique"

a 1'Energie for

granting

Atomique". him

H.S.thanks

a fellowship

LITERATURE

by grants

Gendrale

the

de Ia a la

and the"Commissariat the

during

National

from

"Fondation the

course

Philippe" of this

work.

CITED

1. Chao,J.,and Weatherbee,C.J. (1974) J.Bacteriol,x,l81-188. 2. Goss,W.A., Deitz,W.H.,and Cook,T.M.(1965) J.Bacteriol.e,1068-1074. 3. Hatfield,D.,Hofnung,M. and Schwartz,M. (1969) J.Bacteriol. !@,559-567. 4. Hofnung,M. Hatfield,D.,and Schwartz,M. (1974) J.Bacteriol. 117,40-47 (1962) Bioch.Biophys.Acta, 5. Horium,T.,Tomizawa,J.,and Novick,A. 55 1152-163. 6. Kohiyama,M.,Cousin,D.,Ryter,A. and Jacob,F. (1966). Annales Inst.Pasteurr110,465-486. (1965) J.Biol.Chem.240,1211 7. Newton,W.A.,Morino,Y.,Snell,E.E. 8. Pedrini,A.M.,Geroldi,D.,Siccardi,A. and Falaschi,A. (1972) Eur.J.Biochem.25#259-365. 9. Puga,A. and Tessman>. (1974) J.Mol.Bio1.75,99-108. 10. Randall-Hazelbauer,L.rand Schwartz,M. (1973) J.Bacteriol.z, 1436-1446. 11. Sankaran,L. and Poge1,B.M. (1973). Differential inhibition of catabolite sensitive enzyme induction by intercalating dyes Nature New Biol. 245,251-260. and Worcelx (1971) J.Mol.Bio1.61,329-342. 12. Schwartz,M. and Schaber,I. (1973). 245,144-145. 13. Traub,W.H.,Kleber,I. 14. Weiner,R.M. and Blackman,M.A. (1973). J.Bacteriol.l16,1398-1404. 15. Yokota,T.tand Kasuga,T. (1972). J.Bacterio1.109,13~1308.

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