Genetic recombination of phage S13 in a recombination-deficient mutant of Escherichiacoli K12

Vol. 22, No. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

GENETIC RECOMBINATION OF PHAGE S13 IN A RECOMBINATION-DEFICIENT

MUTANT OF ESCHERICHIA COLI K12

Irwin

Tessman

Department of Biological Sciences Purdue University, Lafayette, Indiana

Received

that

recombination

in Escherichia

reduces

the frequency

of recombinants

and Margulies,

1965).

the recombination

mechanism bination

used

recombination 2. phage

genetic

material;

To determine

whether

the

under

protein

likely

the

species;

close

S13 presumably stranded

of the

contains

DNA (Tessman,

reduction

implies

must be the major

on phage

for

S13 recom-

I,

simple

II,

IIIa,

genetically

mutants

in group

1.7 x lo6

1958 and 1959;

daltons Sinsheimer,

169

genetic

IIIb,

II,

E.S.

in

of

or of the host

Tessman

(Sinsheimer, 1959).

communication) were

more than

linearity were

cell.

constitution.

and IV)

specify

frequencies 1958;

involved

1965 and personal

does not

to p)X174 (Zahler,

is

in recombination

phage

Tessman,

recombination

only

involved

of the

(labelled

the virus

and except

steps

relatively

(E.S.

groups

relation

here

same mechanism

control

of its

investigation

map and additivity of its

genetic

because

that

of 1000

objectives:

of the phage

5 complementation is

reported

this

are

large

by a mutation

a factor

by the mutation

whether

In a comprehensive

so it

of this

affected

The study

can be blocked

by more than

To determine

S13 was studied

only

cell.

has two specific

coli

The finding

mechanism

by the

1.

this

14, 1965

Genetic

(Clark that

December

of the

demonstrated. and Shleser, 1959)

found, about

5

genetic Because 1963),

of single-

Vol. 22, No. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

METHODS A recombination-deficient isolated Both

by Clark

F- strains

resistant

and Margulies were

kindly

were

S13 were

labelled

-Ret+ and -Ret Because of these

of the mutagenic

respects.

medium with acids.

confirming also strains; (Clark

and Margulies,

phage.

Therefore,

strains

will

are the

sensitive

to

strains

used

be referred

was necessary

to as

still

and also

parent

grew

to ultraviolet

in (leu-)

as the only (try-)

Hfr

Trisadded

strain

than

of -Ret-. light

to be related

strain

on a synthetic

recombinants

property

each

leucine-requiring

and methionine

more u+trp+

to show that

to its

a tryptophan-requiring

is known

(KlO),

did &-,

The derived

strains

(IJV) as the original

to their

abilities

to recombine

1965).

recombination

was compared &)

Q')

recombinants

Cl22

or -Rec- and incubating

type

can form plaques.

-Ret

in tryptone

broth

mutants

were

in TB plus

+

in -Ret

of S13 that by plating

at 43',

a temperature

Crosses

were

(13 g of Bacto

performed tryptone of 4-8

1 x 10 -2 M CaC12.

at a multiplicity

of 5.

170

and -Retcannot

selected

of H20; TB) to a concentration

each mutant

it

at 43'

histidine

same sensitivities

type

added,

these

were

with

W sensitivity

to 2 x 108/m1

K12 strains

and colonies

Both derivatives

1000 times

temperature-sensitive

liter

These

JC-411.

N-methyl-N'-nitro-N-

was similar

the recombination-deficient

Phage

with

derivatives

leucine,

at least

had the

was strain

The two S13-sensitive

treatment

grew

In crosses

showed

the

1960),

and JC-1553.1;

and streptomycin-resistant,

&+

JC-1553,

respectively.

the following

amino

Clark.

to adsorb

plating.

two S13-sensitive

glucose

K12,

the non-deficient

by Dr.

and Greenberg,

by replica

JC-411.5

from

by treatment

(Mandell

isolated

(1965)

of a failure

mutagenized

nitrosoguanidine

of E. coli

supplied

to S13 because

two strains

were

mutant

by crossing grow

progeny

phage

at which by growing plus

Wild-

on _. E &

only the wild + Ret and

7 g of NaCl per

x lo7 cells/ml A pair

above 41'.

and concentrating

of phage mutants

The phage-cell

mixtures

was were

Vol. 22, No. 2, 1966

shaken

for

mixtures

5 min,

were

incubated

diluted

of the

X ~266,

that

at 42'

mixedly

infected;

infection

which

for

took

additional

day to day.

In part,

state

indicator,

sizes

were

this

plates,

the effect

of these

measured

is known

to stimulate

and Doermann,

1958;

Tomizawa

and Anraku,

Tessman,

at 42',

cannot

and Tessman,

by mixed For

of about

in -Ret

errors, +

2 from

such as the

and the exact

1964),

1959;

were

phage/cell.

temperature

for

of

each pair

of

and -Rec- at the same time.

recombination

be compared

cells

only

conditions,

which

E.S.

the

may be due to the assay

was always

(Tessman

that

a cross

of complementation.

30-50

age of the

one cross,

of performing

lethal

fluctuate

for

a factor

To minimize

experiments

except

as a result

between

The

Adsorption

within

the

frequencies

at 37'

assurance

Cyanide,

bination

to burst.

The purpose

growth

frequencies

recombination

the cells

are

adsorbed.

2 x 10 -3 M CaC12 and

TB plus

the t mutations be phage

of the phage

place

was to provide

The recombination

mutants

time

at 42'.

there

the incubator.

into

was performed

the burst

of the

85-95%

loo-fold

always

since

could

crosses,

phage

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

time

40 min to allow

entirely

all

during

then

for

and growth t76

BIOCHEMICAL

in phage

was not

with

those

E.S.

Tessman

used,

found

T4 (Chase

so the

recom-

in previous

and Shleser,

1963;

1965).

RESULTS Table pairwise the L"

1 shows the in

-RecR&c-,

6 different

recombinant

frequencies

of crosses. recombination

the

frequencies

for

5 mutants

crossed

ways in Ret+ and -Ret-.

Since

apparently

only

was observed,

in order

In each cross + than in -Ret . but

recombination

the frequencies

to account

for

the reciprocal

the recombination Not only

reduction

factors

For one class, frequencies

are

frequency

suggest

reduced

that only

was significantly

there group

by a factor

171

shown as twice

the t

+

recombinants.

the recombination

involving are

are

lower

frequencies are at I and IIIb

reduced

least

in

two classes

mutants,

of approximately

in

the 20 to 40

Vol. 22, No. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I RECOMBINATION OF S13 MUTANTS IN -REC+ AND -REC CrOSSeSa Mutants

groupsk

Frequency

units -I-Ret

of 10W5)

ratio

-Rec-

--Rec+/Rec-

2.5

0.13

19

IXI

t43

x

frequency

(in

Complementation

crossed t39

Recombination

t39

X ~266

I X IIIb

15

0.61

25

t39

x y11

I X IIIb

42

42

t39

x t11

I X IIIb

92

1.0 2.2

x I

30

3.9

~76 X t39

II

42 7.7

t76

X tll

II

X IIIb

30

4.7

6.4

t76

X ~266

II

X IIIb

50

12.4

4.0

~76 X t2661

II

X IIIb

32

8.4

3.8

a All crosses were performed at 37' which was performed at 42O.

except

for

the last

t76 X ~266

cross,

b The complementation groups are functional units determined by complementation tests. The mutants were classified by E.S. Tessman (1965) with the exception of tll, which I classified by its failure to complement ~266.

in -Ret

.

For the other

by a factor ratio

each class

Bacterial

one of these

involved

in

step

are

is

under

genetically In g-,

recombination

appears

steps,

cellular

phage is.

of phage

by the phage

or by the cell.

that

reduced

be because activity there

is

172

far

the phage very

also S13.

one cannot and whether

might

many steps.

must

recombination

recombinational

much more likely

in &-,

control,

is

is

of the --Rec+/Rec-

K12 may involve

mechanism

genetic

recombination

This

the reduction

significant.)

one blocked

recombination

in phage controlled

be considered

the

t76,

(The variation

in E. coli

bacterial

are involved

residual

cannot

the primary

mutant

4 to 8.

recombination

At loast

steps

involving

of approximately

within

this

class,

than

utilizes

effectively.

an important

Although

say what

these

less

be

secondary

other

other steps

bacterial the slight But it mechanism

only

Vol. 22, No. 2, 1966

BIOCHEMICAL

of recombination

that

conceivable

one mechanism

other

that

operates

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

only

for

could

the

phage.

involve

For example,

breakage

it

is

and rejoining,

the

copy choice. The secondary

effective mutants

crosses

in groups

group

II

from

all

crosses group

for

mutants the

be crossed

involving

behave

A fact

anomalously

roughly

Tessman,

characteristic

of mutants

In a cross

of 8.5% and 8.0% were data).

Thus phage

primary

recombination

found

mutant

than

set

whether

more

for

the

be related

is

experiments;

that

S13 mutants ordered

map, except

to determine

for

by two-factor

mutants

from

of 513 mutants

the behavior

must

of ~76 is

II.

mutants

of phage +

T4 is different mechanism

~76

may possibly

A more complete

in -Ret

relatively

can be consistently

in group

of two -rI1

that

additive

1965).

in -Ret+ and -Ret-

II

apparently

in mapping

groups

and

is

the group

complementation

(E.S.

mechanism

I and IIIb.

on a linear II

recombination

T4,

recombination

and -Rec- respectively from both 5. coli

does not utilize

frequencies (Tessman,

unpublished

and S13 in that

the step

blocked

its

in -Ret-.

SUMMARY The primary

recombination

recombination-deficient is eliminated, into

the primary the

mutant

a secondary

at least

is

on the basis

and secondary

mechanisms

depends

for

of E. coli

mechanism

two classes

classification

mechanism

phage

K12.

When the primary

revealed. of the

to the

on the mutants

S13 is.blocked

Crosses

relative

in a mechanism

can be grouped

contributions

recombination

frequencies;

crossed.

ACKNOWLEDGMENTS This

research

was supported,

in part,

by NSF research

173

grant

GB-2748.

of

Vol. 22, No. 2, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES Chase, M., and Doermann, A. H. Genetics 43, 332 (1958). A. D. Proc. Natl. --Acad. Sci. u. 2. 53, 451 (1965). Clark, A. J., and Margulies, 2. BiochKBizs. Res. Corm. 3, 575 (1960). Mandell, J., and Greenberg, Sinsheimer, R. L. 2. Mol. Biol. I., 43 (1959). Tessman, E. S. ViroloFgx3 (1965). Tessman, E. S., and Shleser, R. Virology l-9, 239 (1963). Tessman, E. S., and Tessman, I. Virow1, 465 (1959). Tessman, I. Program and Abstracts of the Biophysical Society p. 42 (1958). Tessman, I. Virology7 263 (1959). Tomizawa, J., and Anraku, N. 2. Mol. Biol. 2, 508 (1964). Zahler, S. A. J. Bacterial. 75,310 (1958).

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