The

Journal

of Biochemistry,

Vol.

48, No.

2, 1960

MECHANISM

OF IN

.

CYANIDE

CYANIDE

RESISTANCE

ACHROMOBACTER‡W

RESISTANT

RESPIRATION

OF

CULTIVATED

BY

SHOJI

MIZUSHIMA*,

(From

the

Department

(Received

In

the

previous

cultivated

of

chrome

a2

tion of

of the

that

cells

cells

a2

the

of

in

in

12,

reported

of

1960)

that higher

aerobic

Achrotnobacter

strain

concentration

of

condition

was

reported

or

the

and

this

cells

less

and

sensitive

concerning

iron (2)

in

(3)

the

they

paper,

relation the

the

that

to

(4).

the

medium

the

7 cyto

respira

cyanide

than

doubt

that

and

function

the

concentration

authors

content

the

between

the

these

a2 on

relation

and

However,

cytochrome

threw

studies

between

presence

oxygen

were

the or

the

it

may

mechanism

of

of

of

failed

to

respiratory of

cyanide

the

as

find

activity

cytochrome

the

purpose the

cells

a2

it

of

as

a

bacteria

different

considered

to of

confirm

out activity

under

significance may

finding

respiratory grown

are

physiological as

in

the

relations

well

resistance

the and

of

these

clear

chain,

for

content

cyanide,

on

make

respiratory

out a2

absence Studies

because

in

carried

cytochrome

the

concentrations.

portant, a2

Faculty

oxidase.

In the

been

oxygen

between

bacteria,

ARIMA

Tokyo)

contained under

KEI

aerobically.

parallelism

cytochrome

was

anaerobically

have of

cytochrome out

cultivated

studies

concentration

Tokyo,

February

condition

grown

grown

Many

it

AND

Chemistry,

of

publication,

(1)

anaerobic

than

the

for

OKA

Agricultural

University

paper

under

CELLS

TETSUO

Agriculture,

ANAEROBICALLY

the

be

im

cytochrome view

on

the

(5).

EXPERIMENTALS

The

methods

of

of

DPNH

measurement sorption

spectrum

Cultivation study. contained Air

apparatus

under

very

necessary cylinder

medium the

could

to

Present

a

from

aerobic

which *

of

be

with

easily

the

had

stood ,

in a

"porous

condition

measure

address

shown

few

1.

degree

about

aeration,

Institute

and of

in The of

a

simple

silicon

had

205

is

filled

Microbiology,

intact

oil

as

an

the

air with

for

led

agent.

could When into

water.

a

Using of

this liters)

fish-breeding.

medium

University

for 6

antiforming

minute. was

ab

(5).

(capacity

used

per

cells, of

designed

vessel

apparatus,

out-letting been

apparatus

usually

air

the

measurement

previously

cultivation

7liters the

Applied

this

of and

described

which In

passing of

those

drops

stone"

autoclaved.

upside-down The

to

Fig.

activity

preparation

cultivated

air

by

oxidation

cell-free

similar

were

was

succinic of

were

Bacteria•\Bacteria

bubbled

This

of activity

cytochromes

apparatus 4liters

was

oxidase

of of

The

measurement

Tokyo,

be it

kept was

graduated this

ap-

Tokyo.

206

S.

paratus,

bacteria

meat

were

extract,

The

rate

cultivated

1per of

culture

MIZUSHIMA,

cent

bacterial

in

of

growth

was

AND

nutrient

K

broth

polypeptone

FIG.

air

and

.

ARIMA

(pH

0.5per

measured

Preparations harvested

7.0)

containing

cent

of

1per

sodium

nepherometrically

The

D;

cent

chloride,

by

amount

of

M/15

tant

for fluid

of

at

sampling

32•‹.

10ml.

of

of

a

was

of

for

a

and

intact

cells

and

at

M/15

phosphate

the

was

exposed

turbid

oxidase for

small

to

sonic at

superna

activity.

60minutes

and

cell a

centrifuged

The

particulate

buffer

washed in

was

DPNH

The

solution.

suspended

debris.

145,000•~g

fraction.

of

were

were

saline

suspending

obtained

cell of

conditions

cent

suspension

thus

measurement

centrifuged

volume

by

thick

various

0.5per

cells

this

tube.

under

with

washed

sonicate

particulate

Porous

thermostat.

measured the

The

the

further

and

small

amount

the

used

fluid in

the

7.4)

C;

a

grown

was

(pH

remove

and fluid

suspended

mation

buffer

in

once

Then

filter. F ; Sampling

bacteria

7.4).

cultivation.

air

soaked

washed bacteria

(pH

to

supernatant

again

was

for 15minutes.

diluted

turbid

apparatus

intact

phosphate

20minutes

non-diluted clear

of

KC)

was

E ; Medium.

Cell•\The

buffer

(9-10

5,000•~g

the

bacterial

Gotten

let.

centrifugation,

activity

for B;

Out

Bacterial

phosphate

oscillation

use,

continuous

respiratory M/15

Apparatus

compressor.

stone.

from

by

1.

Air

For

a

a

OKA

medium.

A;

in

T.

The to

obtain

preparation was

used

for

was the

esti

cytochromes.

RESULTS

The

bacteria

7liters

per

these

the kinds

were

number of

was

no

of The significant

cells

were

Using

insensitivity

under

about in

these

difference

to were

to "the

two

respiration results

Fig.

reached called

two

200ml.

shown

of

cells

respectively.

measured.

cultivated and

cultivations

when two

were minute

kinds

2.

shown

In

both

the

cases,

cells" cells, and

in

flow The

the

Table respiratory

"the

Fig. activity

cells"

activity, content 3.

in

these

anaerobic

respiratory

and

about

harvested

Hereafter

cytochromes I

i.e. curves

were

ml. and

the

rates, growth

cells

7•~103per

aerobic of

air

minute.

about

cyanide

between

different

per

the were

Though

there

of

kinds

both

FIG

CYANIDE

of

cells,

the

sensitive

to

succinic

oxidation

cyanide

than

activity that

. Bacteria

were

apparatus

RESISTANCE. ‡W

of

2.

in

Succinic meter.

of

aeration;

7liters

•\•œ•\

aeration;

0.2liters

oxidizing

Each (pH

ment,

0.2ml.

7.4) of

and

center

well.

Total

by

oxidase

oxidase than

buffer

and

of

0.4ƒÊM

addition

of that

the of

of

contained

0.2ml.

per

2.2ml.

activity

of

Each

photo-cell

of

anaerobic

the

case

of

In

lesss DPNH

at

32•‹

using

the

of

in

oxidase cells,

or side

measured

the arm

using

buffer

of

the

activity

of there

of

of

sonicate,

the

volume

aerobic a

remarkable

respiro

M/15

phosphate

main

compart

20%

KOH

in

Photoelectric 2.3ml.

Reaction

Total

was

the

Hitachi

buffer.

water.

Warburg

0.2ml.

using

Cells

of

in

and

0.2ml. or

Anaerobic

1.6ml.

measured

KCN

and

and

4•~109cells,

the

0.2ml.

minute.

Aerobic

was

KCN

was

minute. per

contained

of 10-2M

the

cell

in

sonicate

DPNH

much

I

about

10-2M

volume

sonicate, the

of

intact

succinate

0.3ml.

was

curve. broth

Activities

vessel

2•~10-1M

spectrophotometer. phosphate

Respiratory

activity

Warburg

buffer

DPNH

on

.

cells

1.

•\•›•\

Cyanide

cells

nutrient

T ABLE Effect

anaerobic

growth in

Fig.

the

aerobic

Bacterial

cultivated

shown

of the

207

of was

M/15 started

3.0ml.

cells

was

lower

difference

208

S. MIZUSHIMA, T. OKA AND K. ARIMA

between the the presence

oxidative activity of aerobic cells and of cyanide. As shown in Fig. 3, the

FIG . 3. reduced

Absorption

with ---

„Ÿ„Ÿ

a2

cells,

from

aerobic

Preparation

obtained

from

anaerobic

in

to

those

of

cyanide

the

Nitrogen

of

preparation

cells

was

with

two

kinds

the

cells

cytochrome of

glass

Model

method.

than

that

in

between

These

aerobically

were

Cary

observed

cells.

extracts

preparation

higher

was

cor

crude

using

opal

much

of

of

measured the

.

cells.

anaerobic

on

difference

these

type

was

based

cells

contents

and

Spectrum

significant

of

the

aerobic

100:89.

anaerobic

obtained

results

were

in

presence

grown

the

the

aerobic

amount quite

of similar

or

absence

(5). the

observed.

cells

of

which

of

cytochromes

medium,

2liters

medium,

and

a

shown of the

the

the

culture

studied

Fig.

state, 4,

medium condition

of

from were

these

changed

of by

the to

was

activity

bacteria

4liters

condition growth

conditions. of

replaced was

rapid

respiratory

cultivation the

anaerobic

the

the

under a

under

condition,

properties

steady in

cultivated

aerobic

were

in As

been

under

changes

experiment follows.

had

cultivated The

amount

as

show

Spectrophotometer,

further

this

figure peak.

14

bl

When were

the the

the

no

cytochrome

in to

separation ratio

cells in cytoch

dithionite.

in

whereas,

particulate

obtained

Letters

rome

of

Preparation

responding before

spectrum

that of anaerobic concentration of

To was

the

anaerobic

equal

volume

aerobic

and

one.

the

perform

carried

out culture of

When

fresh the

FIG

CYANIDE

amount

of

again The

bacteria

replaced same

in by

the

the

treatment

medium fresh

was

min.), at

the

A

was

vation

replaced was

at at

A,

II

and

Medium;

C B,

B,

and C

Fig.

the

and

D.

of fresh The

D

were

of

Cyanide

equal

removed

centrifugation and and their sensitivity

from

broth.

on

the

carried

two

(aeration;

volume

liters

on of

the

the

fresh

medium

When

culture

medium

medium.

The

bacteria the

the were

same

in for

0.4Liter/

of

Temperature

Succinic

;

the

culti again

treatment

culture

media

experiments

shown

vessel

32•‹.

II Oxidizing

Activity

A, B, C and D in Column I show harvested at the point A, B, C and D in tively. The conditions of measurement of ing activity were the same those given in medium

each

was

5.

nutrient

TABLE Effect

medium

curve. culture

used

the was

From

(12liter/min.).

2liters of

of

cultivation .

growth

by

volume

the

anaerobic

aeration

to

equal

2liters

more

Bacterial the

rapid

reached

repeated

Table

4.

replaced

by

by

removed in

was

followed

and twice

of

209

doubled,

medium

4liters

2liters

was

repeated

. From

RESISTANCE. ‡W

the

of

In

Cell

the intact cells Fig. 4, respec succinic oxidiz Table I.

bacterial

used for experiments. to cyanide were shown.

Intact

Table Though

cells

were

harvested

II, the oxidase the respiratory

by

activity activity

.

FIG

210

S. MIZUSHIMA, T. OKA AND K. ARIMA

of the the

intact

succinic

cells

remained

oxidizing

reduced

constant

activity

in the

. 5. Absorption with dithionite.

A, B, C and

D show

throughout presence

spectrum

the

the course

spectrum

of

of

of this experiment,

cyanide

particulate

of particulate

was

decreased

preparation preparation

obtained

from the cells harvested at the points A, B, C and D in Fig. 4, respectively. Nitrogen contents of crude extracts before separation of the particulate preparation of A, B, C and D were in the ratio 100:83:105:100. were measured using Cary Model 14 Spectrophotometer based glass

method.

FIG . 6. respiratory

Succinic

rement

of

oxidizing Table

of

Relation activity

•\•œ•\

value

Spectra, on the opal

cytochrorne activity

II.

Cytochrome

cytochrome

content;

between in

presence

cytochrome of

b1. •\•›•\ in

see

cytochrorne

presence

content text.

content

of was

and

cyanide.

the

a2.

cyanide value

(10-3M of

Fig.

was 5.

Measu

the

in

CYANIDE

reverse

propotion

the

to

properties

The

of

absorption

Fig.

5.

The

As

it

from

amount

lines

to

on

estimated

from

at

and

630mƒÊ

amount

a2

of

and

and

was

and

the

and

b1

be

straight

600mƒÊ

line) the

not

First,

a2

and

660

b1

was

and

in

optical

relation

activity

between

could

the

Therefore,

between

shows

respiratory

observed

cyanide. in

follows.

straight

6.

between of

cytochrome

minus Fig.

relation

oxidized).

as

of

b1 bacterial

cytochromes a2

580mƒÊ,

reverse

the

presence

minus

in

cytochrome to

close

oxidize

.

shown in

of

a

cells

were

decreased

the

estimated

(spectrum

a2

parallelism

is

cytochrome

amount

respectively.

be

in to

was

the

difference

561mƒÊ,

A

b1

cells

experiment aerobic

propotion

(reduced

545mƒÊ

cytochrome

cyanide.

and

Then

the

of

these

this the

amount

there

difficult

spectrum

between

the

of of

to

activity

amount

end

those

reverse

that

very

difference

spectrum.

seemed

in

suggest

be

cytochrome

drawn the

also

respiratory

the

the

of

were

mƒÊ

found

the to of

decreased

the

at

Though

not

and

preparation,

estimated

a2

strongly

content

was

particulate

was

results

a2

and

cytochromes

growth.

it

These

cytochrome

the

211

reached

cytochrome

bacterial

changeable,

growth.

the

of

the

growth, system

of

amount to

also

bacterial

respiratory

spectrum

proportion was

the

the

RESISTANCE. ‡W

density

between

in

the

the

presence

of

them.

DISCUSSION

Many

studies

cytochrome

a2

Escherichia tion

was

kinds

of

no

a2

marked

and as

cytochrome

deficiency, iron

rich

activity

of

between In

of

a2

Achromobacter and the

strain the

of

10-3M caused

the at

step

content

In

the

of

(7)

authors and

the

was

no

previous

cyanide

the cytochrome

also

content

failed

to

and

electron

a

increase

Therefore,

that

of cyto

there

was

respiratory

out

the

the

relation

it

but

in

was not

the

reported

in the

the

completely amount

capacity

cytochrome was

activity

in

iron that

of

the

parallelism

respiratory

transfer a2.

find

between

(1),

by

activity.

relation

strongly

though

to role

out

a2

that

the

and

respiratory

a

oxygen

cytochrome

equal

pointed

a2

cyto

on

decreased

on

two

reported

out

almost

doubt

paper

was

the

found was

However, the

the of

also

aerogenes that

was

cytochrome

and

a2 of

of

threw

there

cytochrome

increase the

also

activity.

a2

concentration

he

these

respiratory

(10-3M).

cells

content 7,

view

aerogenes the

Smith the

(3)

(4)

of

of

importance

Aerobacter

the

Aerobacter

and

then,

of

whereas

activity

Moss

Tissieres

result,

a2

cytochrome

cyanide

cyanide

So

cytochrome

oxido-reduction by

of

(6)

that

supported

activity this

physiological

content

hand,

between

bacteria.

content

between

other

content

Keillin

the

and

the

From

parallelism

a2

the

that

concentra

condition,

respiratory

After

the

reported

higher

aerobic

the

between

(2)

contained

under

on

cytochrome

medium,

a2

a2. of

doubt oxidase.

respiratory

cells.

chrome

threw

On

the

lack

the

oxidase.

grown between

relation

Moss

condition

that

terminal of

in

the

a2

he

the

concentration the

than

the

condition.

anaerobic

difference

dependence

was

a

a2

concerning

cultivation

under

significant

cells,

chrome

reported

and

grown

cytochrome

there

been

content

coli

of

have

results

presence that

the

inhibited of

the

observed

cytochrome presence presented

of in

212

S. MIZUSHIMA, T. OKAAND K. ARIMA

this paper could be explained as follows. As the respiration of these bacteria may not be limitted by the reaction catalized by cytochrome a2, the change in the amount of cytochrome a2 seemed to have no influence on the respira tory activity. However, the fact that the respiratory activity was strongly inhibited by 10-3M concentration of cyanide shows that the reaction catalized by cytochrome a2 limits the over-all reaction in the presence of cyanide. Therefore, if cytochrome a2 have an important role in the respiratory chain, there must be a parallelism between the cytochrome a2 content and the respiratory activity in the presence of cyanide. The results given in Fig. 6 show the parallelism between these two. In addition, the fact that there was a parallelism between the cytochrome a2 content and the respiratory activity in the presence of cyanide confirmed the view on the mechanism of cyanide resistance in the bacteria (5). As shown in Table II, when the anaerobic cells were cultivated under aerobic condition, the cyanide resistance in the respiration decreased in reverse proportion to the bacterial growth. This result was quite different from the case that the cells which had been made ressistant to cyanide in the presence of cyanide, was aerobically cultivated in cyanide-free medium (5). In the later case, the insensitivity to cyanide of the respiratory activity could be observed even after 3 or 4 times cell-divisions in the cyanide-free medium. Possible explanation is that in the former case, though the cyto c hrome a2 content was indeed high in anaerobic cells, this does not mean the increase of the cytochrome a2 forming activity, namely cytochrome a2 seemed to be merely accumulated in the cells owing to the slow growth; whereas in the later case, the cytochrome a2 forming activity was really increased, probably owing to the some fundamental changes in the cell constituent such as in the level of nucleic acid. This may be the reason why the cyanide insensitivity obtained in the presence of cyanide was maintained during 3 or 4 times cell-divisions in the cyanide-free medium. SUMMARY 1. Achromobacter strain 7, grown under anaerobic condition was found to possess greater amount of cytochrome a2 than that grown under aerobic condition. 2. Though the respiratory activities of both kinds of cells were quite equal, the anaerobically cultivated cells showed a higher respiratory activity than the aerobically cultivated cells, in the presence of cyanide (10-3M); namely the respiration became cyanide resistant during an anaerobic cul tivation. 3. A parallelism was observed between the cytochrome a2 content and the respiratory activity in the presence of cyanide. 4. A physiological significance of cytochrome a2 as respiratory carrier and the mechanism of cyanide resistance in anaerobically cultivated cells were discussed.

CYANIDE

The encouragement

authors

wish and

to

express

their

RESISTANCE. ‡W

sincere

appreciation

213

to

Dr.

K.

Sakaguchi

interest.

REFERENCES

(1) (2) (3) (4) (5) (6) (7)

Mizushima, S., and Arima, K., J. Biochem., 47, 837 (1960) Moss, F., Aust, J. Exptl. Biol. Med. Sci., 30, 531 (1952) Moss, F., Aust. J. Exptl. Biol. Med. Sci., 34, 395 (1956) Tissieres, A., BiochemJ., 50, 279 (1952) Mizushima, S., and Arima, K., J. Biochem., 47, 351 (1960) Keilin, D., and Hartley, C., H., Biochem.J., 35, 688 (1941) Smith, L., Archiv. Biochem. Biophys., 50, 299 (1954)

for

his