Vol. 70, No. 3,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MOLYBDENUM AND CHLORATE RESISTANT MUTANTS IN ESCHERICHIA COLI K12 M. Dubourdie?, Grupo

Received

de Biologia Universidad.de

March

and J. Puig

E. Andrade"* Experimental, 10s Andes,

Facultad de Ciencias Merida, Venezuela

8,L976

SUMMARY Radioactive molybdenum is used to detect the existence of molybdo compounds in E. coli K12. Three membrane bound MO-proteins are found, using sodium dodecyT smte. One of them is the nitrate reductase. The nature of the other two is discussed. The soluble fraction of the cellular extract contains a small MO binding molecule which could be peptidic in nature (MW is about 1,500). Different chlorate resistant mutants are analyzed on the basis of these molybdo-compounds. None of the mutants is found to contain radioactivity bound to nitrate reductase protein. Defects in the biosynthesis of a molybdenum coentyme is deduced for chlorate resistant pleiotropic mutants. INTRODUCTION Most of the trate lack

reductase(') formic

mutants effect

were

dehydrogenase(') was first

cytoplasmic

activity.

xanthine

dehydrogenase

tion

molybdoproteins

are

corporation reductase

is

that

type

coli

reductase

Several

Copyright All rights

which

have lost

since

they

ni-

also

of such pleiotropic In order

produces

of explanation

to explain

an alteration

activity and it

to explain activity

(8,

9).

Both

is supposed into

12, 13). chl

has been proposed.

are also

this on the

pleiotropic

proteins that

In Asper-

since

affected

the mutation

they

lack

by the mutaaltered

the

in-

these proteins (10). --E. coli nitrate The molybdenum hypothesis has already

D mutations

of some mutants

(14,

15) and the deficiency

(16).

The problem is now to decide how far can the molybdenum hypothesis the pleiotropic chlorate mutants. Very little is known about molybde-

address 2 resent o whom reprint d

classes

the mutation

some NR- mutants

of a molybdo-cofactor a MO-protein (11,

been used in i. of nitrate

coli,

to be pleiotropic

(7).

another

and Neurospora

of Escherichia

found

(1, 2, 3, 4, 5, 6, 17).

proposed

membrane Recently,

explain

mutants

have been described it

gillus

chlorate activity,

: LCB, CNRS, 13274 Marseille request must be sent Nitrate reductase and for-mate dehydrogenase FDH respectively.

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

766

Cedex 2, France will

also

be written

NR and

BIOCHEMICAL

Vol. 70, No. 3, 1976

num processing type

strain

nitrate it

in r.

A first

set

of questions

: does the molybdenum

enter

the cell

reductase

? What is

biosynthetized

step

it

is

necessary

MO-processing.

rectly

labeling

The present and examining

the

? Are there

normal

MATERIALS

coli.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

report

tries

whether

presence

cellular

in various

proteins

in r.

the pleiotropic

to answer

binding

concern binding

of the molybdenum

many molybdo

to determine

molybdenum their

nature

would before

chlorate

and how is ? In a second

mutants

some of these compounds

cofactor coli

with

the wild to membrane

present

questions radioactive

a

by diMO

mutants.

AND METHODS

The following strains were used : r. coli K12, PA601 (356 in Puig's collection) and its chlorate resistant mutants : chl A, B (1, 2, 3), chl D and F (unpublished results), chl E (17, 18). The chl C mutant was obtained from Puig's collection (18). None of the pleiotropic mutants contained nitrate reductase activity. chl C had about 2 % of the wild type activity. Cells were grown anaerobically MO isotope (as molybdate). The medium in order to minimize the concentration mented with selenite (10-6 M) (19) and 50 uCi of radio-active molybdate were 10-6 M). Induction by nitrate was done

on minimal medium in the presence of (prepared with double distilled water of dissolved molybdenum) was supplethe requirements of strain 356 (1, 2). added to each 200 ml of medium (about 3 to 6 hours before harvesting.

Cells were washed once and disrupted by sonication (3' in ice bath). Cells were sedimented from the extract by low speed centrifugation. Then the extract was separated into pellet (P) and supernatant (S) by ultracentrifugation (120 000 g during 2 hours). P (10 mg/ml) was resuspended in buffer Tris 0.02 M pH 8, containing sodium dodecyl sulfate (SDS) 1 %, B-mercaptoethanol 0.05 M and ethylene diamine tetraacetic acid (EDTA) 0.05 M. After one hour of incubation the extract was centrifuged at a low speed to eliminate undissolved material and an aliquot (50 ul) was then applied to the top of polyacrylamide gels. SDS acrylamide gel electrophoresis was performed according to Shapiro et al. (20), (phosphate buffer, pH 7, 0.1 M, SDS 0.1 %, 5 mA/tube) for 8 to 12-h=rs. Gels were run in duplicate. Both gels were treated with methyl viologen reduced by dithionite (gels are deep blue after l/2 hours) and then with nitrate which produced the appearence of a clear band at the level of the nitrate reductase, which was marked by slightly cutting the gel. One of these gels was frozen, then cut in pieces 1.5 mm long. The radioactivity of these gel slices was measured. The other gel was stained for proteins using Amido Schwartz. S was passed through a G-50 3Gphadex column (450 x 20 mm). 2.5 ml fractions were collected and counted. MO radioactivity was measured in a y counter. The life of 99Mo is about 3 days, and each determination had to be corrected for decay. Radioactive molybdate was bought from CEA (Paris, France). RESULTS A - The wild num incorporate (1.2

x lo6

type

cells,

approximately

counts/min/mg

grown in presence of radioactive molybde-9 atomgrams of MO ion/mg of protein 10

of protein).

767

Vol.70,No.3,

BIOCHEMICAL

1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

E u” 5000~

.J(. .L... :_ Ol lo

Fig.

1 -

brane

L”qel

pieces

MO radioactivity profile in polyacrylamide gel electrophoresis of SDS solubilized particulate fraction. (a) represents y radioactivity throughout the gel. Gels are cut and counted as described in "Materials and Methods". (b) represents a gel after amido black staining. Top of the gel is at the left. Bromophenol blue went out in about 5 hours of electrophoresis.

MO binding

molecules

by SDS and one in the

aliquote.

- _._ _.._. 2 a

30

99

Four ty diagram

20

obtained Three

are

soluble

identified,

fraction

by polyacrylamide

proteins

S. Fig.

gel

associated

three

solubilized

1 shows

electrophoresis

radioactive

from mem-

the radioactivi-

performed

peaks

are well

on a P

separated

in

12 hours. 1) The first black.

It corresponds

little

and must

partially,

band

proteic.

peak and protein

protein idea

very

high

narrow

weight

MO/protein

quantity

and poorly

radioactive

molecular

The ratio

stained the

is

to a very

be a high

of each port

("a")

peak.

of some sort binding

(MO is evaluated

is evaluated

of molybdenum of molybdenum

"a"

migrates from

from color

storage

by amido

Its nature

molecule.

band) is very high too (10 arbitrary

of a nonspecific

stained

at least

radioactivity

intensity

units

of the

) and would sup-

molecule.

to a membrane

very

is,

The possibility

particule

cannot

be

discounted. reductase, thods). ratio

2) The second

radioactive

band

characterized

by its

activity

The activity MO/protein

is is about

3) The last migrates

a little

exhibits

the

associated 0.2

faster

than

migrates

on SDS gel

to a stained

arbitrary

radioactive

same ratio

("n")

"n",

level

protein

band

of nitrate and Me-

(Fig.

1).

The

units.

peak corresponds

MO/protein

at the

(see Materials

indicating than

later.

768

"n".

to the protein a lower

molecular

Its function

will

band weight,

"f".

It and

be discussed

Vol.70,No.3,

Fig.

BIOCHEMICAL

1976

electrophoresis

is found

is

performed.

by SDS denaturation list

through

"a"

process

is

and "n"

(75 % of

culate

fraction

separated column

ted to a large

radioactive two successive

in presence to this

(MW < 450). molecule could

Although is

are

1,2,

does not

account

activity

is

sibly

for

associated

molybdate

partially

the present

blue

to already

solubilized

des-

by sonication

100 % of "f"

are

it

weight

in the

parti-

after

treatment

to decide

of trypsin). of molybdenum "n", present

low molecular

and technetate.

769

if

is estimated

"f",

with after

co-

to the four cells

molecules

as-

a mixture FMN of this

apparent

Mo-peptide

in the weight

Sephadex

the nature (its

bound

associa-

The radioactivity

peak a little

Mo-"peptide"

for

molecule weight

as standards.

as a sharp

the molybdenum to very

by passing

on G-25 and G-15

of MW 1500 is lost

call

peaks

Its molecular

2).

10 respectively all

that

been studied.

(fig.

and elutes we shall

5,

radioactive

chromatographies

be due to esterase activity The relative quantities

molecules

can be produced

Peak I corresponds

2).

more has to be done

peptidic,

It follows

and almost

of FMN and dextran enzymes

dye when shorter

molecules

to a low molecular

peak

molecule

of proteolytic

molybdo

four

are

has not

corresponds

at 1,500

of the tracking

proteins.

(fig.

which

Peak II

P).

after

small

into

80 % of "n"

"a",

level

may not be exhaustive.

proteins

4) Peak III

sociated

These

of some molybdo

a G-50 Sephadex

cribed

at the

of molybdomolecules S fraction

lumns

RESEARCH COMMUNICATIONS

2 - " MO radioactivity elution diagram of soluble fraction in Sephadex G-50 column. 0.6 ml of S fraction (about 1 mg/ml of protein) was passed through the column. 2.5 ml fractions were recollected and counted. DB (dextran blue) and FMN (flavin mononucleotide) were used as standards. Dextran blue elution volume indicates column dead volume.

Radioactivity

short

AND BIOPHYSICAL

hydrolysis described and "a".

It

as much radio(2 200),

pos-

Vol.70,No.3,

BIOCHEMICAL

1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I

WT "MO radioactivitv bound to,or at the leljej of : a "n"

chl

t

+

t

-

"f"

t

-

Mo-"peptide"

t

-

The proteins

eluted

through

radioactiv

ity

about

400) .

treated extract

does not

that

of -nit-l Mo"peptide"

that

the

the

molecule

with

"peptide"

pounds served

is

the cofactor.

the peptide.

We cannot

times

chl

described

important

fact

and "f"

peaks associated the

the

particulate finds

(15, the

here.

presence

C

t

and "n"

t

proteins

resulting In this

weight

case

proteins

200 and the that

which

solution the but other

any MO-protein

reductase

activity

of an

would

suggest

But we cannot

discard

the

possibility

of the yet

of Nason --et al. (about 8.0) low molecular weight molybdo

decide

if

MO-cofactor molybdenum,

only

in the on the

The results

while

all

This

is

the

the Mo-

chlorate

less

are

mutants

is

the chl

I.

have con-

radioactive common to

(and may be "f")

in contradiction

with

by immunological techniques, Forget of the NR protein in chl E (personnal communication).

770

B

Mo-com-

in Table

mutants exhibit

characteristic protein

than

fraction.

of the four

summarized

pleiotropic

re-

(x 0.7),

particulate basis

the only of "n"

D and E mutants

Nevertheless,

is

in the same amount

A, E and F accumulate

The presence

of chl

-

chl

which

D less

such bands.

21).

ttt

0

(lo),

bands , none of these

mutants. fraction

studies

crassa

been analyzed

is that,

protein with

pleiotropic

to recall

radioactivity

C) : chl

more and chl

tt

molecular

of radioactive

do not incorporate for

t

of them is about

Neurospora

reassociation

we have just "n"

to high

pH used in the experiments

has then

all

also

from

Each mutant The most

2 ("a"

NAOPH-nitrate

mutant

(except

chl

B

G-50 column.

important

spontaneous

mutants type

two to three

vious

is

to restore

not

chl

at pH 2 and the

described bound

it

F

t

treated

or part of it. B - When grown in presence

sistant wild

remain

point

restoration

may allow

were

able

chl

t

of MW < 450 (one

At that

at pH 2 is

E

t

the previously

as two peaks

appears

chl

in peak I of Fig.

have not been SOS treated) was passed

A

in pre-

BIOCHEMICAL

Vol.70,No.3,1976

These

contradictions

ding

to the

classes

could

is

procedure

of pleiotropic

to "a",

be due to proteases

solubilization

mutants

lack the Mo-"peptide" from the first three which

the only

used

chl

pleiotropic All

which

Table

accorthree

same phenotype.

They

complex. chl D seems to differ of twice more radioactivity bound

Mo-"a"

which

may vary

I distinguishes

A, E and F have the

that

mutant

activity

(22).

but still have Mo-"a" mutants by the presence

seems to indicate

"peptide" complex. mes more Mo-"peptide"

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

complex

can produce

is

not an artefact.

an apparently

the chl B mutants tested than the wild type.

synthetize

chl

well-made

B

Mo-

two to three

ti-

CONCLUSION The non exhaustive ductase

is

lybdenum

not

analysis

the only

bound

molybdo

to both

"n"

of molybdocompounds

protein

and "f"

FDH activities,

and the disappearance

"n"

NR specific

band in the

formate

dehydrogenase

rification then

of a molybdenum

reasonnable

pleiotropic into

mutants

that

cofactor

could

biosynthesis Some authors

rized

as being mutant

(24,

25,

the cofactor. treatment not get

in the

nitrate

wild

presence

NR and FDH in their

form.

of membrane to bind

as substrate. authors

active

chl

bound

B would

lack

here

described

such enzyme.

Mo-"peptide"

only

after

is

being

acidic

et al.

(16)

do

is

not sufficient

to

soluble

Mo-"peptide"

is

an enzyme

proteins, It

mu-

(1 000 < MW < 1 300)

by McGregor

and that

of the

of Neurospora

peptide

to particulate

pre-

has been characte-

activity

that

is

of a cofactor

which

weight

We suggest

It

of pleiotropic

of Mo-"peptide"

MO-cofactor,

MO-cofactor

possibility

obtained

pu-

the

The complexity

the

fractions

(23).

in all

be due to the

number

yields

soluble

recent

of molybdenum

large

reductase

restoration type

would

enzymes.

out

of the

complex

incorporation

Such a cofactor,

confirm this hypothesis. As shown by chl B, the

necessary

the

band may be the

activities

effect

pointed

be in favor

But the poor

bI-FDH

of both

be a low molecular

would

coli

"f"

the very

for

(10).

to restore could

This

of 5.

a precursor is

able (10)

26).

have already

molybdoenzymes

nit-l

that with

lack

NR and to only

suggest

comnon to both account

associated

re-

of mo-

lacking

in agreement

The pleiotropic cofactor

nitrate

The disappearance

the mutants

cytochrome

the

is due to a defect

NR and FDH proteins.

tants.

C),

is

containing

sence of a molybdenum

common to all

(chl

This

to suppose

coli.

in all

of radioactivity

mutant

protein.

in r.

bands

show that

using

in agreement

(transferase Mo-"peptide" with

other

(16). chl

synthesis map in the

A, E and F mutants

would

of Mo-"peptide".

It

same region

of E. coli

is

lack

some enzyme

interesting chromosome

(6). 771

to note (chl

necessary that

F being

to the bio-

these

three

still

uncertain)

genes

?)

BIOCHEMICAL

Vol. 70,No.3,1976

chl tant

which

and the 15).

only

chl

complex in the

D mutant presents

seems to be more complex alteration

one which

D gene product but

it

process.

is

and transfer The alteration

still

can be restored would

impossible

results

support

of a MO-cofactor of the cytoplasmic

of radioactivity

by high

have to deal

molybdate

with

to decide

whether

a regulation

the

idea

membrane

that

the (27)

bound

"a" role

pleiotropic

is

implied

(15). in the biosynthesis chlorate

then

(14,

of Mo-"peptide"

molecule

defects would

muto "a"

concentration

the formation

for

can explain

It is the only

to define.

in the quantity

We have no evidence

The present

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mutants.

be a secondary

phenomenon. ACKNOWLEDGEMENTS We wish to thank the personnel of the "Instituto de Medicina Nuclear, U.L.A., Merida" and particularly its Director, Dr. M. Oropeza for giving us access to their equipment. We also wish to thank the mechanical assistance of J. Ceballos in the building of devices used in the experiments with radioactivity. Financial assistance was from CONICIT (N" 31.26.S1-0637) and from CDCH (U.L.A.) (N" CllR). REFERENCES

1. Puig,

J., Azoulay, E., Pichinoty, F. (1967) C.R. Acad. Sci., Paris, -’264 1507-1509. 2. Puig, J., Azoulay, E. (1967) C.R. Acad. Sci. Paris, 264, 1916-1918. 3. Casse, F. (1970) Biochem. Biophys. Res. Commun., 39,a29-436. A. (1968) Proc. ?I%. Acad. Sci. U.S.A., 4. Adaya, S., Cleary, P., Campbell, 61, 956-962. 5. Glaser, J-H., DeMoss, J.A. (1972) Mol. Gen. Genet., 116, l-10. 6. Taylor, A.L., Trotter, C.D. (1972) Bacterial. Rev., 36; 504-524. 7. Azoulay, E., Puig, J., Pichinoty, F. (1967) Biochem.-Biophys. Res. Cornnun., 2, 270-274. 8. Ketchum, P.A., Cambier, H.Y., Frazier III, W.A., Madansky, C.H. and Nason, A. (1970) Proc. Nat. Acad. Sci. USA, 66, 1016-1023. 9. Pateman, J.A., Cove, D.J., Rever, B.M., Roberts, EB. (1964) Nature (London) 201, 58-60. 10. Nason, A.,Xe, K., Pan, S., Ketchum, P.A., Lamberti, A. and DeVries, J. (1971) Proc. Nat. Acad. Sci. USA, 68, 3242-3246. 11. Tanigushi, S., ftagaki, E. (1960) Biochfi. Biophys. Acta, -44, 263-279. 12. Forget, P. (1974) Eur. J. Biochem., 42, 325-333. 13. MacGregor, C.H., Schnaitman, C.A., Ntiansell, D.E., Hodgins, M.G. (1974) J. Biol. Chem., 249, 5321-5327. 14. Glaser, J. and DeMossJ. (1971) J. Bacterial., 108, 854-860. 15. Sperl, G.T. and DeMoss, J.A. (1975) J. Bacterial., 122, 1230-1238. 16. MacGregor, C.H., Schnaitman, C.A. (1972) J. Bacteri'oTT, 112, 388-392. 17. Puig, J., Azoulay, E., Gendre, J., Richard, E. (1969) C.R.Acad. Sci. Paris, 268, 183-184. 18. Puig, J., AzTay, E, Pichinoty, F. and Gendre, J. (1969) Biochem. Biophys. Res. Cornnun., 35, 659-662. 19. Lester, R.L., DeMoss, J.A. (m71) J. Bacterial., 105, 1006-1014. 20. Shapiro, A.L., Vinuela, E., Maizel, J.V. (1967) Biochem. Biophys. Res. Cornnun., 2, 815-820. 21. MacGregor, C.H. (1974) J. Bacterial., 121, 1117-1121.

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Vol.70,No.3,1976

f:: 24. 25. 26. 27.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MacGregor, C.H. (1974) J. Bacterial., 121, 1X2-1110. Enoch, H.G. and Lester, R.L. (1975) J.-BTol. Chem., 250, 6693-6705. Ganelin, V.L., L'VOV, N.P., Sergeev, I.S., Shanoshnikov, G.L. and Kretovich, V.L. (1972) Ookl. Akad. Nauk, SSSR Bioximiya, -206, 1236-1238. L'vov, N.P., Ganelin, V.L., Alikulov, Z., Kretovich, V.L. (1975) Izv. Akad. Nauk SSSR, 3, 371-376. Zumft, W.G. (1974) Ber. Otsch. Bot. Ges., 87, 135-143. Mutaftschiev, S. and Azoulay, E. (1973) Bizhim. Biophys. Acta, -'307 525-540.

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