Thermosensitive mutant of Escherichia coli requiring new protein synthesis to recover cellular division ability

Vol. 42, No. 4, 1971

BIOCHEMICAL

THERMOSENSITIVE NEW PROTEIN

Nagai,

RESEARCH COMMUNICATIONS

MUTANT OF ESCHERICHIA

SYNTHESIS

Kazuo

AND BIOPHYSICAL

TO RECOVER CELLULAR DIVISION

Harumi

Department

Kaneko

and Gakuzo

of Agricultural

University

COLI REQUIRING ABILITY

Tamura

Chemistry,

of Tokyo,

Tokyo,

Japan

Received December 30, 1970

SUMMARY: Several thermosensitive mutants deficient in cell division were derived from E. coli by means of membrane filters. Two of the mutants showed normal growth at 30", filament formation (at 42' and rapid increase in cell number when shifted back from 42' to 30". After the temperature shift, the one could not divide in the presence of 150 pg/ml of chloramphenicol while the other could irrespective of the antibiotic. :For the purpose of analysing the biochemical pathways involved

in

mutants

cellular

division

have been

Hirota

et al - -0 occurrence

that

the

several solution formation

isolated

(3)

sis,

finely

(l,Z,3),

of the

genetical the

cellular

materials

directly division

we have

attempted

mutants

by a simplified

to method

has not isolate

at 30" but

cells

at 4Z",

recover min

of

colony Incubation

to retain forming

not

viability ability

attempt

at

on DNA syntheand showed

is

controlled

related

been

by re-

septum In

this

of thermosensitive et

al.

by this

at 4Z",

to form

when shifted

to

successful.

selected

42"

and

of biochemical

of Van de Putte

are

normally

cycle

new kinds

mutants

(2)

etc.

or closely

of the

to divide

et al.

morphology

the

common characteristics

thermosensitive

mutants

division

However,

respect

those

cell

loci.

or cellular

various

Kohiyama

classified

of septation,

process

of

of bacteria

at least back

for

(1).

The

method filamentous 120 min,

to 30"

after

to 120

at 4Z".

* Abbreviations: NBT, nutrient methyl-N,-nitro-N-nitrosoguanidine;

broth 669

containing thymine; CAP, chloramphenicol.

NTG,

N-

Vol. 42, No. 4, 1971

Bacterial

strain

his-

thy-

ara-

ent

strain.

thl-

BIOCHEMICAL

AND WPHYSICAL

MATEHIALS

AND METHODS

and media: lac-

gal-

The strain

obtained

from

Dr.

JElOll

xyl-

H. Matsuzawa

of thymine

of distilled

was solidified tained, acid

with

per 5.804

liter

1.5

g, MgS04.7Ha0

Chemicals:

Sankyo

of mutants:

of cells

with

cubated

agar,

6.057

NBT con-

g, malelc

1 g, sodium

from

Aldrich

citrate

gifted

Chemical

by Dr.

0.5

Co.,

H. Okazaki,

Preparation

of NTG solution

et al.

(4).

0.1

the

ml of the

modified

overnight

10 ml of fresh

at 30" with

reciprocal

shaking.

When the

phase filter

of growth,

(Sartrlus pore

size

a final

concentration

treated

cells

0.45

p pore

size,

p.

buffer.

0.1

in 10 ml of NBT and incubated

at 42" 670

reached on a 25

in

suspension

at

30"

on a membrane saline

suspension for

filter

to

concentr.atlon

was kept

10 ml of cold the

on the

to the

filtered

ml of

culture

were suspended

The final

The mixture

washed with

2 ml of saline.

cells

NTG was added

sample.was

NBT and in-

filtered

washing

the

of 100 &ml.

the

were

culture

GmbH, 34 Goettlngen,

After

buffer,

was log/ml.

incubation,

cells

Membranfllter

10 ml of Tris-maleic

2 ml of Tris-maleic

the

and treat-

little

into

West Germany),

ed in

Tris-HCl

was introduced

mm membrane

0.45

and 10 mg

buffer

JElOll

logarithmic

After

contained

For nutrient

NTG were made following

of Adelberg

of E. &

with

NBT*

Co. Ltd.

method

the

K12 and kindly

n-NaOH.

CAP was generously

Isolation ment

trp-

as a par-

3 g of NaCl

fNH,)zS04

NTG was purchased

Wis.

leu-

was used

Tris-maleic

water,

with

thr-

department.

agar.

g,

(P'

E.

extract,

cent

0.1

to 6.0

from

water.

of distilled

g, pH was adjusted

Milwaukee,

per

strr)

of our

7 g of meat

liter

mtl-

was derived

10 g of polypeptone, per

RESEARCH COMMUNICATIONS

for

of 30 min.

filter

and resuspendwas inoculated

120 mln with

reciprocal

of

Vol. 42, No. 4, 1971

shaking. ter,

Then

8 p pore

the

filter

with

the

culture

size,

AND BIOPHYSICAL

was poured

and filtered

were washed with

10 ml of

of

BIOCHEMICAL

saline.

saline. solidified

melted

0.75

at 30”

the

RESEARCH COMMUNICATIONS

onto

a 25 mm membrane

gently.

10 ml of

The suspension

The cells

saline

fil-

retained

on

and resuspended

was diluted

to 5 x lo3

cells/ml

0.1

ml of the

sample

was pipetted

agar

in petri

dishes

and overspread

with

After

of incubation

per

cent

agar

colonies

at 45”.

were replica

One of the

plates

24 hours.

The colonies

24 hours

plated

was incubated

on a couple

at 30”

formed

on the

in

and the

at 30”

but

surface ml of

3.0

of plates.

other

at 42'

for

not

at 42O were

obtain-

JR1011

was treated

with

ed.

RESULTS Isolation 100 pg/rnl cent

of the

cells

5 per

on a filter tous

(4)

After

NTG treatment,

120 min the

cell

of

the

cent

to the

by this

procedure

and the

any temperature. filter

of 8 u pore

filamentous

size

cells

at

and normal rest

did

not

Repeating size to the

frequency

reveal

frequency 671

at 42”

the

obtained

three

cells

into

a mix-

or at both

any morphological for

in

filamen-

filamentous

developed

only

filtration

resulted

of

of thermosensitive

1) formed

21 strains cells

microscopically.

Among 59 strains

was 0.1.

by

The filtration

The percentage

30”,

reported

in NJ3T following

filaments. the

50 per

by NTG in g.

was studied

(group

min,

30

mutagenesis

increased

cells

for

has been

at 42'

formed

23 strains

of filamentous

for

incubation

cells

cells

at 30”

condition

morphology

filtered

at 42O and normal

and 30”,

of

by 5 to 10 times.

mutants

ture

This

to be optimal

of 8 p pore

cells

buffer

survived.

et al.

:K12.

3 to

When E. -coli

of NTG in Tris-maleic

Adelberg coli

of mutants:

change

times

microscopic

of 70 to 80 per

42O

on the

appearance cent.

at

of

However,

Vol. 42, No. 4, 1971

the

number

of viable

mosensitive -. Strains after

cells

mutants

Most

staining.

increase after

that,

formed

than

filaments

them

density

was more

2

0

1,

shift

for

grown

at 42'

ts 1

to 42O

60 mln,

as normal

4

at 30"

in group

rapid

or

at 42"

temperature

as long

directly

density

strains

Cells

was at 30".

2 to 4 times

of ther-

ts 1 and ts 20

incubation

the

after

it

including

in optical

1 shows that

of optical

yield

by a microscope

after

on two of

final

10m5.

The increases

were compared Fig.

of

filaments

Ts 1 and ts 20:

and ts 20.

than

RESEARCH COMMUNICATIONS

and the

1 were examined

multinucleated

and 42'

AND BIOPHYSICAL

decreased

was less

in group

Giemsa

formed

BIOCHEMICAL

but

for

slower

60 min

cells.

4 time

Othr12

Fig. 1. Increase in optical density of ts 1 and ts 20 grown at different temperature conditions. The culture was grown at 30". When optical density at 550 rnp reached 0.20 the culture was divided into two parts: one (e) was shifted to 42" and the other (0) was kept at 30".

As shown in Fig. at 30". sion dually ble

However,

stopped

when the

immediately.

decreased count

2 both

remained

and then constant

strains cells

showed exponential

were

The cell increased. as long 672

shifted

number In

to 42"

growth cell

of ts 1 at 42' the

as 6 hours.

case of ts When the

divigra20,

via-

cells

Vol.42,No.4,

1971

BIOCHEMICAL

AND .BlOPHYSlCAL RESEARCH COMMUNICATIONS

t

0

60

120

160

.

0

time

I

.

1

60

,

120

I

.

160

(min)

count of ts 1 and ts 20 grown under different Fig. 2. Viable temperature conditions. into two parts: The culture grown at 30" in NBT was divided one (0) was kept at 30" and the other was shifted to 42". (0) was shifted After 60 min of incubation at 42' the culture back to 30" while a part (A) was kept at 42". Viable cells were counted after 24 hr of incubation at 30" on NBT agar plates.

were

shifted

cell

number

and later It

back

to 30"

increased almost

after

more

as slow

was examined

60 min

rapidly

for

as the

whether

this

Ts 1 could

well

absence

of 150 pg/ml

divide

of

ts 20 was completely

inhibited

the

of

the

a little

shift.

was observed

when the

15 min

the

after

However, cells

shift.

were exposed

The results

increase

the

did

at 30"

it

are

in

in the

of CAP.

division time

than

at 42'

growth.

rapid

by CAP.

incubation

60 min

normal

was influenced as in the

of

cell

number

presence

On the

as

other

hand,

when CAP was added increase to the

in

cell

number

antibiotic

shown in Fig.

3.

DISCUSSION An experiment mutants

deficient

stated in

cell

above

shows that

division 673

which

the form

thermosensitive filaments

at

at l+2O

BIOCHEMICAL

Vol. 42, No. 4, 1971

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

. /. /O-o O’O

J

0’

/

0

Pf A-A---A -O-A-1*I . 1. L 30

60

1 30

90

time

.

I 60

.

I . SO

t 120

(min)

increase of colony formers grown with or withFig. 3. Relative out CAP. A) Ts 1 grown at 30" to a density of 7.0 x lo7 cells/ml was At the time of the shift (A) or 60 min after shifted to 42". the culture was shifted back to 30". the shift (O,e), To (A) and (0) CAP was added to a final concentration of 150 pg/ml at the time of temperature shift to 30". B) Ts 20 grown at 30" to a density of 5.5 x lo7 cells/ml was After 60 min the culture was shifted back to shifted to 42". CAP was added to a final concentration of 150 pg/ml 30" (0). at the time of (0) or 15 min after (m) the temperature shift to 30". and can divide

normally

The method

using

lected

filamentous

ing

the

8 p pore

a gentle

the

experiment

siological caused

cell

division

during

is very

filtration

on cells,

successfully the

simple

especially

incubation

sefollow-

and easy except

The low might

yield.

frequency

shown at

be ascribed

to phy-

on filamentous

cells,

repetition.

The nature

in

formed

at a good

filter

is required.

of repeated damages

isolated

membrane

The process

filtration

by the

formed

size

cells

NTG treatment.

that

at 30" were

of two of the

multinucleated number process

for

filaments a while after

the

mutants

were

Both

at 42" and showed rapid

when shifted shift

studied.

was not 674

back

to 30".

influenced

of

them

increase In

ts 1 the

by CAR.

Vol.42, No.4, 1971

Therefore,

the

blocked the

BlOcHEMCAL

but

thermosensltive

accumulated

temperature

However,

In

blocked

shift ts 20,

to divide

because

Inhibited

the

amount

cell

the

the

at least

related

of

thermosensltlve

type

this

analysis Of

synthesis

protein formation of

this

was not

In

time

15 mln

activity.

for shift

is not

clear

the

after

the

shift

or cellular protein

will

in 15 min cells it

cell

completely

that

of the but

the

fact

was synthesized a part

after

was Irreversibly

of the the

reversibly

recovering

was Indispensable

Moreover,

for

only synthesis

protein

at the

new protein

sufficient

to septum

chemical

necessary

division.

RESEARCH COMMUNICATIONS

and new protein

when CAP was added

that

The nature

this

was not

CAP added

Increased

Indicates

mechanism

at 42"

and new protein

number

AND BIOPHYSICAL

cell

to the

to divide.

may be closely

division be possible

process. by means

BIOof

mUtantS.

REFERENCES 1. Van de Putte, P., van Dlllewjn, J. and Rorsch, A., Mutation Res. 1, 121 (1964) 2. Kohlyama, M. D., Cousin, D., Ryter, A. and Jacob, F., Ann. Inst. Pasteur. &&I, 465 (1966) 3. Hlrota, Y., Ryter, A. and Jacob, F., Cold Spring Harbor Symp. '&ant. Biol. 22, 677 (1968) 4. AdeIberg, E. A., Mandel, M. and Chen, G. C. C., Biochem. Blophys. Res. Comm. I8, 788 (1965)

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