Detachment of lambda prophage upon induction

Vol. 29, No. 1, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DETACHMENT

OF LAMBDA

PRUPHAGE

UPON

INDUCTION

Amos B. Uppenheim Department Hebrew

bacteriology

of

University

-

Hadassah

Jerusalem.

Received

August

The

marker

at (Jacob

the

host

DNA

ing

over

between

insertion

of

lambda

prophage

and

Prell

is

of

& Wollman,

1961).

not

lambda into

that

report is

and this

The

experimental

based

on

at

the

experimental

induction

intact

chromosomes.

(1962)

had

Hfr

3000.(

conjugation

Lysogenic

derivatives

A c1657), in

the

phase

ware order

the

for

the

an

induced

was possible

to

induction

the

phage

prophage cell to

to show that

particles.

allow the

phage

F-.

protein

that

(1965)

detachment

to

chromosome

the

Ptashne

mature of

and

to cross-

leads

into

Hfr

detachment

prophage

DNA.

essay

host

a reciprocal

After

way

its

and

of

chromosome

its

the

a chromosomal

that

host

to

as

replicating

transfer

Materials and

behaves

suggested

finds

prophage

attached

attachment

the

of

to

it for

the

is

chmmosome,

ability

approach

Bacterial

and

DNA

time

is necessary

during

of

system

the

same

nature

vegetatively

prophage

an

it

bacterial into

cell

conjugation

DNA

the

is converted

showed

It

In

circular

DNA

bacterial

Campbell

known.

prophage

the

described.

a lysogenic

location.

the

this

of

a specific

is

multiplication use

prophage

(1965) In

School,

30, 1967

lambda

chromosome

Medical

Israel

By

the

synthesis

regenerates

excision

Methods

strains: of used

Hfr as

3000(thy-,

donors.

El;), These

0-leu-gal-tryp.

F-

120

strains strains

Hfr

3000(

A++)

donate DlllEi

their (leu-,

and genes tryp-,

during arg - ,

Vol. 29, No. 1, 1967

met-,

lx-,

which

were

gal-,

Margulies,

and

mal-,

made

/

19651,

were

as

obtained his+)

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A*,

the

were

Matino Hfr

and

(1%

Difco

F-

cultures

were

the

while

at

42’

at

is

46

When

malwas

Hfr

and

(Clark

8.

originally

(tryp-,

AI31 311 1966).

and

parental

h h,

and

(19571,

x x

h

CIB57

which

(Appleyard,

is ?lcGregor

cultures.

least on

x

an

in

To

had

either

test

lost

the

then

15

and

5 ml

in

carried

at

ml to

of

-et

was

recom-

the

broth

for

15

and

min,

1956)

al -09

plates

plates.

tryptone 4.2’

ml

33'

minimal

selective

0,5

100

Mating

content

appropriate

in

pre-

a

out

flask.

lambda

kept

with

selective

( Uppleyard to

min,

the

were

cells/ml

on

into

CR63

cells

Erlenmeyer

transferring

at

they 42’.

A+‘.

iliscussion

was

x cIa57) h c1857

to

the

F-

was

spread

on

resistant

or

ml

was

mating,

l-2xlC* for

picked

or

ability

for

purified

JCl557

A++

of

tryptone

culture

Hfr

initiate

The

a 100 were

After

Hfr(

uninduced

To

Conjugation

hour.

h and

The

El e

into

cultures

34'.

incubated

samples

were

at

a density

and

were

one either

to

30’.

overnight

1 pg/ml

shaking.

and at

fresh

centrifugation.

broth

colonies

to

harbored that

or

strain

between

Kaiser

grown

and

broth without

hr

and

diluted

colonies

at.

tested

NaCl)

Results

nants

h vir

91118

19621,

from

by

shaking

sensitive

binants

of

the

diluted

tryptone

for

for

CR63

f+

x

& Howard-Flanders,

8 Jacob,

lysogenize

F-

a cross

lambda

thymine

was

33’

recombinant

spot

type”

either to

The

Boyce

0.5

Hfr

recombinant

ware

recipients.

concentrated

vigorous

incubated

for

resistance

Chamberlin,

tryptone or

incubated

Single

for

from

to

pg/ml

supplemented

binanta,

) lysogenic

recombinant

were

10

the

flask,

and

used

first

supplemented

by

H

( Sussman

tryptone,

warmed

stopped

as

“wild

cells

with

5 ml

Sm

experiments:

supplemented

in

used

mutant 1956)

, selection

his+

a thermoinducible

cold

by

( Clark,

Phage

broth

x

a tryp-,

JC1557

& Baird,

/A

produce

121

mated (Table lambda

to

an

1,

A). phages.

F-t

A+‘)/ There

x ware

However

the

recom-

no recombi( when

the

Hfr

Vol. 29, No. 1, 1967

ceils

were

nants

did

ok

BIOCHEMICAL

induced not

CIi357)

*

by

heat

contain as

treatment

prophage

would

be

Table

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

before

expected

from

The

I.

1,B).

(Table

They

a cross

Effect

af

Recombinants lambda

their A-

were

obtained content as

= non lysogenic for not easily classified,

contain

a mixture

of

in the described

A++

were

and

X

of

X cIB57)

x++)/x

Control,

3

Heat

treated;

C

Heat

treated; -15 to

from

no

Hfr(

A++,

CAP

X F-t

Heat

lysogenic

treated;

F-

temperature some.

A similat

experimental

result Hfr

3000(

Lambda

spot

tested

from

the

Table

were

II xc

=

tested

xc1857;

recombinants table.

They

appeared

to

Content

of

tiecombinants

the

5ma

leu+

xxc+x-

x

tryp+

5mil

xc A- L--- xc+

++

0

0.00

23

60

16

11

64

0.85

14

11

present

5

74

1

0.01

27

65

0

added

7

10

16

0.61

--

--

--

--

hh

A++

j)-

x-

CAP

Xh)/‘h

A++ x- K A”, x-70 0 0.00

Ah

CAP

18

& Vollman,

induction

a

93

without

(Jacob

in I’ethods.

and

--

E

of

to

7

without

Heat treated; at 0 min

nor

dfr

L’etachment

xc A-

A++

treatment

CAP @ min

recombi-

( A++)

lysoganic

presented

gal+

A

II

the

x

X F-t

neither

the

CIE57.

1eu+

Hfr(

the

of

Treatment

omitted

Treatment

Exp.

on

Heat

majority

a non

experiments in klaterials l-2;:

lambda. and

were

of

CAP

upon

for

the

mating,

of

h

conclusion

1961

We can

) .

c1857

the

was

reached

therefore

prophage by

is Jacob

26

(1956)

0.82 0.00

A+++X-

that

& Wollman

51

64

conclude

from

0.00

--

-

detached

0

0

0.00

after

the

host

chromo-

with

a different

system. To

show

that

of

some

indirect

A++)

and

detachment effect F-

DlllE(

following on Xh)/X

heat

the

mated .

both

treatment cells. these

122

of a mating prophagas

the

Hfr was possess

is

not

the

performed the

between wild

type

Vol. 29, No. 1, 1967

repressor

and

when

Hfr(

that

the

of

are

A++)

was

vacant

the

chosen

to

lambda

site

( A’)

that

can

conclude

also

I,

8)

the

E).

was

know

if

its

II(A the

level,

described

prophage

was

Therefore,

the

in

fl.82-0.85 1.0

result

one of

observed can

thermal

that

is

Hfr

cells

the

conclude

induction

had

I

(A

the

8. II)

been

shows

(Table

be

prophage

This

problem

This

prophage.

it

is

interesting

chromosome.

proximal

to

mating.

the

lambda,

albeit

These

detachment

deserves

Jne

transferred

chromosome when assayed

that

I)

of the Hfr cells.

were transferred, before

a

Hfr

induction.

bacterial is

with

experimants

cannot

which

induced

the

complete

the

There-

recombinants

chromosome,

lambda,

bacterial

chromosomes.

transferred

host

linkage

site.

the resident

of

the

the

from

these

replace the

reflects

the

the majority

marker,

to

of

upon

continuity

distal

attachment

chromosome and

gal

lambda

in

in

to

the

I)

received

expected

place

attached

in

Table

is

prophage

both

which

sites

observed

which

affects

the

(Table

as the ratio

tube

is

a discontinuity

the

of

detached

DNA

the

and

lambda

conjugation

detachment

here.

total

takes

the

lambda

when

suggest

the

of

the

marker,

tryp

to

detachment

B) shows

&

markers

the results

ratio

that

the

selective

limit

through Since

place

upper lambda

effectively

not

I,

among the recombinants

present

The

the

suggests

lower

of

(Table

(Table

A”

have

approaches

and

detachment

mating

xCIBs7

of

C1857 + '-I*

Table

No

before

of

between I

to

induced.

heated

frequency

relationships

x

heat

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

prophage. The

(

not

detachment

this

fore,

BIOCHEMICAL

results

a do

by the system

indeed

further

at

took

investiga-

tion. It

treatment

was of

shown E.coli

It was therefore tion.

Indeed,

prevented colony-forming

by

Lieb

(1966)

that

for

XC,,,,

lysogenic expected

inhibition

that of

no

ability

(Lieb,l966),

will

synthesis

I,

(CAP)

prevents

detachment

protein

lambda detachment(Table

chloramphenicol

3 B C). was

also

123

induction take

by

CAP

of

place during

Induction, prevented(Table

added

during the

under heat

measured

heat

prophage. this

condi-

treatment

by the loss

II,'J&C).

These results

Vol. 29, No. 1, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

The

The

II.

mating

Effect

mixtures

of

CAP

contained

on

Induction

6.0~13~

hfr(

and

x C1857)

Kating

per

ml

and

l.CxlflB

Hfr( A++) per ml and l.~xl$ F-i hh)/h per ml, or 1.5x1f17 The heat treated Hfr cultures were incubated at 42' from -15 to Ll min. treated Hfr cultures were incubated at 33" at the same time. At 0 tine CAP level, when present, was initiated by mixing the Hfr and F- cells. 5urvivors were measured by plating on glycerol minimal plates 25 pg/ml. F-i

per

ml.

The

non

X++)/A

taining

10

@g/ml

thymine

and

1 llg/ml

Recombinants

leu+

;:

Control,

no

Heat

treated;

Heat from

-15

Heat

treated;

aithout

CAP present 0 min

A++,

Heat

CAP

CAP

at

SmH

60

min

tryo+

leu+

5mR

Iifr

Viable cells

at J min*

l.lxlc?

3.5x103

2.7~10~

1.Ex103

4.3x104

2.5x103

2.7x104

2.5xlr12

4$*+

5.2x105

2.7~10~

--

added

X F-t

treated;

gal+

x++1/x

treatment

treated; to

Hfr(

F-t

con-

B,,

Exp.

hfr(&57)

mating was

145;; 1.6:; 55jL

x h)/X

without

CAP

* The

number

XI

Hfr

Viable

suggest

that It

15

min

ments

of it

can

of

heat seems

now

60

-15

interest

to

to did

as

lOO$.

allow not

needed

protein

detachment. prevent

for

a necessary

determine

more

124

for

precisely

detachment.

during

the

added

immediately

detachment

(Table

I,

"early

protein(s)".

detachment step

prophage

synthesis CAP

prophage

needed is

to

is

whether

protein(s)

detachment conducted

taken

induction

determine

induction the

is

during

sufficient

that

min

min.

synthesis

that being

at

at

is

speculate are

cells

cells

period

Therefore Jne

Hfr

protein was

induction the

of

for

are DNA

replication.

the

time

after

D).

Experiof

synthesis

of

Vol. 29, No. 1, 1967

these

BIOCHEMICAL

postulated

protein(s)

and

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

time

of

their

action

following

temperature

induction. These

postulated

m-RNA.

It

lambda

m-RNA

cribed

been is

m-RNA

the

that

fmm

shown

protein(s)”

by

Naono

synthesized

during

present

early

has

“early

in

heat was

& Gros

heat

DNA

by which

& Gros

still

attached

from

Green

by

lysogenic

Naono

is

and

results

The

translated

be

(1967)

induced

treatment.

observed

prophage

may

presented (1967)

here and

to

(1966)

even

cells

by

the

early

lambda that

when

CAP

suggest Green

host

early is

that

(1966)

the is

trans-

chromosome,

Acknowledgement

I

wish and

strains, is

also

to Il.

thank Godinger

R.

acknowledged.

Research

Fund

Ben-Gurion, for her

work

This

of

the

J.

Hebrew

Clark,

and

assistance. was

The

supported

part

in

University-Hadassah

for

A. Honen technical by

Medical

help a grant

the of

gift of Mr. A. Srouji

from

the

Joint

School.

References Appleyard, Clark,

McGregor,

R-K.,

Campbell,

A. A.J.,

(1962). Chamberlin,M.,

J. Mol. Biol., and Margulias,

J.F.,

Advan.

19, A.D.

Clark,

A.J.,

Green,

M. (1966).

Jacob,

F.‘,

and

Wollman,

E.L.

Jacob,

F.,

and

Wollman, Academic

E.L. Press,

Lieb,

J.

(1966).

M.

Naono,

S.,

and

Prell,

H.H.

J. Gros,

Mol.

Mol.

(1965).

J. J.

Ptashne,

M.

(1965).

Sussman,

R.,

and

Jacob,

(1961). New York.

Mol. F.

Biol.,

Virology, P.

Nat.

Acad.

Pasteur, and

Sci., 2.

the

2, 565. (1966).

in 13, 3 C.

press. 329. 829.

R.

125

Acad.

Sci.,

254,

Wash.,

53,

of

Bacteria".

486.

Genetics

149.

16,

(1962).

inst.

“Sexuality

BjOl.

Blol.,

(1956).

Howard-Flanders,

Proc.

134. Ann.

(1956).

Mol.

K.M.

101. and

(1965). 16,

Biol., Mol.

fi, R.P.,

442.

Dial.,

F.J.

Baird,

and

Genet., Boyce,

1517.

451.