Regulation of the synthesis of penicillinase in diploids of Staphylococcus aureus

Vol. 30, No. 3, 1968

REGULATION

OF

THE

SYNTHESIS

DIPLOIDS

OF

Elizabeth

H. Asheshov

OF

PJZNICILLINASE

STAPHYLOCOCCUS

IN

AUREUS

& K.G.H.

Dyke*

Cross-Infection Laboratory Colindale Avenue LONDON, N.W.9

Received

December

Study the

of merodiploids

genetic

cates

the

region

that

mutation

21, 1967

concerned

there

on a plasmid

in

the

aureus,

in penicillinase

is a positional

present

presence

of Staphvlococcus

effect is not

same cell

involving

synthesis, in

that

indi-

a constitutive

completely

repressed

of a chromosomal

by

gene for

inducibility. Pardee, bility is

(i+> dominant

Jacob

gene of the to the

was obtained coccal

& Monod

constitutive

penicillinase

particles PS80

responsible

for

(Asheshov,

1966).

ance

to arsenate

*Present

address:

(1965)

system,

on extrachromosomal

demonstrated

Escherichia

by Richmond

S. aureus

(1959)

coli gene.

synthesis This

ions

with

the

i gene

1'

result

of the

staphylo-

were present

and i-

(plasmids). to

of penicillinase

strain

(AsaRI,

inducisystem

A similar

(NCTC 9789) is believed

the

the

!3-galactosidase

(i-1

when both

that

carries cadmium

genes salts

Department of Biochemistry, Oxford, Oxford, England. 213

carry

the

on its conferring (CdR)

genes

chromosome resist-

and mercury

University

of

Vol. 30, No. 3, 1968

salts

(HgR)

of phage

on a plasmid

(Asheshov,

three

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

but,

20 propagated

ance and the

for

BIOCHEMICAL

despite

certain

on PS 80 co-transduce

metal-ion

resistances

to be published).

penicillin

this,

In

resistance

preparations

penicillin with

a high

the

transductants

and metal-ion

resistance

resistfrequency the are

genes extra-

chromosomal. PS 80 is

inducible

for

A semi-constitutive after

mutant

treatment

strain

with

and the

and mutants from

transduction

was carried

cadmium

sulphate

resistant

transductants

by developing

In

the

and the

& Parker, first

donor

(w/v)

strains

respectively

plates

for

containing

soluble

starch, for

were PS 80 and the

Selection

cadmium

40 W

and cadmium-

penicillinase

production

a benzylpenicillin-iodine

mixture

1966).

transductants

90% appeared

absence

on agar

as recipients

the donor was PS 80 i-p+AsaRCd%gR PS 80 i.+p+Asa (del)Cd(del)Hg(del) . A total

in

the

(i)

p= gene determining

(*) (del)

the

(*)

experiments

recipient

than

were used

resistance.

with

of 409 cadmium-resistant more

(dell

were screened

the plates

Jevons

(Dyket

and 0.2%

at 43.50

metal-ions were isolated PS 80 i+p+Asa (del)Cd(del)Hg(del)

where

out

strain

three

(dell Cd (dellHg

was cadmium

this

The inducible

were grown

and PS 80 ifp+AsaRCd%gR

marker

resistance

mutants,

(i+p+)(+),

from

sulphonate. mutant

experiments

i-pfAsaRCd%gR selected

methane

to the

These

production

was isolated

semi-constitutive

and ps 80 i-p+Asa

in

(i-p+)

ethyl

sensitive

them.

penicillinase

to produce

of inducer.

= deletion

amino

was obtained, large

Several

acid

mutant 214

of

amounts these

sequence

of which

of penicillinase were

isolated,

of penicillinase

and

Vol. 30, No. 3, 1968

quantitative

estimations

produced

Parker,

Table

Table

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the

average

by each of them was measured

(Richmond, in

BIOCHEMICAL

Jevons

& John,

amount under

1964).

of penicillinase

standard

conditions

The results

are

shown

1.

1.

Quantity of penicillinase PS 80, a semi-constitutive resistant transductants.

Culture

Genotype

produced mutant

by S. aureus and cadmium

Units penicillinase mg. dry wt. bacteria

Phenotype

No inducer

Induced methicillin

1. Donor

i-p+CdR

Constit.

90

185

2. Recipient

i+p+Cdcdel)

Inducible

6

140

Constit.

63

625

8

136

3.

Cd-resistant transductants

4.

Cd-(del) mutants derived from Cd-resistant transductants

dell i+pfCd(del)

Inducible

One unit of penicillinase hydrolyses in one hour at 35” and pH 5.9. Cadmium-sensitive

mutants,

after

growth

their

penicillinase-producing

recipient

at 43.50,

(Table diploid

therefore,

Nevertheless,

was produced

in

of the

synthesis the

absence

the

absence

chromosomal

of penicillinase

quantity

from

of penicillinase

of inducer

suggests

benzylpenicillin

three

transductants

to an inducible ability

a considerable of inducer,

gene was only

in

this

gene of the

amount

of penicillinase

the 215

i-

that

slightly

the

repressing

The reduction

diploid.

produced

were,

the i+

indicating

i+

and

same as the

The traqsductants

to carry

that

phenotype

was the

2 and 4).

and continued

recipient.

product

isolated

reverted

1, lines

1 fimole

with

by the

diploid

mutant

is

not

in

in the

of the

0'

Vol. 30, No. 3, 1968

type i+

since

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

there

is

gene product In

the

and the

and the

total

in

selected

three

from

43.5O and these genes

to carry masked

Table

donor

the

Table

i-

i+

chromosome,

Genotype

were

growth

at

to a constitutive were

therefore

production where

its

diploid

and continued presence

Phenotype

produced by S. aureus transductants Units Denicillinas mg. dry wt. bacter:a No inducer

Induced methicillin

1. Donor

i+p+C 8

Inducible

2. Recipient

i-pfCd(del)

Constit.

9:

156

Inducible

10

126

Constit.

96

124

3. Cd-resistant

138

transductants

4.

Cd-sensitive mutants derived from Cd-resistant transductants

i-p+Cd(del)

was

gene.

Quantity of penicillinase PS 80, and cadmium-resistant

Culture

transductants

after

reverted

penicillinase

gene on the

by the plasmid

2.

to have

measure-

mutants

transductants

The transductants

controlling

these

Cadmium-sensitive

of these

2).

of

.

92% were

of quantitative

on several 2.

was PS 80 ifpf (deZ'Cd(del)Hg(del)

transductants,

The results

were found

(Table

the

the

chromosomal

region.

PS 80 i-pfAsa

production

are presented

for

recipient

the

penicillinase

experiment

inducible.

of enzyme

phenotype

between

of 1940 cadmium-resistant

phenotvpically ments

plasmid

reciprocal

AsaRCdRHgR Of the

some interaction

One unit of penicillinase hydrolyses in one hour at 35" and pH 5.9.

216

1 pmole

benzylpenicillin

with

Vol. 30, No. 3, 1968

Revel reported the

BICXHEMICAL

results

where,

is

on the

assumption

are

the

chromosomal in

From the

if

is probably

chromosomal initiation

then only

such an explanation somal

account with

the

synthesized

for

the

diploid

presence

synthesis

chromosomal

are not,

per

for

from

than

the

partial

the

and if the

observed

than

however,

one regulatory ruled

failure

out

region

of plasmid

Although of the over it

of enzyme

positional

the

of the

gene.

is

However,

site

of inducer,

mutation

(1.9631,

initiated

control

amount

i-

gene products.

we conclude

is

same rate,

chromosomal

either

chromosome.

and plasmid

induced

in which

of more

(l965)

far

of

gene products

& Cuzin

?c Richmond

t'ne absence

high

of the

Brenner

can have two copies

more in

very

Explanations the

than

may account

i + gene to exert

of enzyme

amount

one copy of the

amount

of the plasmid

plasmid

i gene is

gene in

based

or that

at the

many cells

basal

genes

chromosome

penicillinase

the

copies

one plasmid

and proceeds

of an i+

explanations

of Jacob,

only

of the

simultaneously

than

are more

(1967 and Novick

replication

gene but

greater

observations

& Jacob there

the presence greater

there

(1965) have

& Rosenbaum

They propose

that

synthesized

that

gene,

synthesized.

than

Cuzin

despite

as an i-

enzyme

genes

(1963) and Markovitz

& Luria

same cell

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

chromothe

does not obtained

on the

plasmid.

effect

based

for

and are under

level

on

penicillinase investigation.

References Asheshov, E.H. (1966) Nature, (Land.) 210, 804. Asheshov, E.H. (Unpublished observatio????. Cuzin, F. and Jacob, F. (1967) Ann. Inst. Pasteur M T (19663 s:e?97i !!$%b,K6G*HB~e$%ns6 ",% %%flarEer{l$jj Cold Spring H&&r Sympkia on Q&an;. Biol. 28: 3i9. 217

835.

Vol. 30, No. 3, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Markovitz, A. and Rosenbaum, N. ('965') Proc. Nat. Acad. Sci. 54, 1084. NovicF R.P. and Richmond, M.H. (1965) J. Bact. 90, 467. Pardee, A.B., Jacob, F. and Monod, J. (1959) J. xl. Biol. Revel, Revel,

1,

165.

H.R. (1965) J. Mol. Biol. 11, 23. H.R. and Luria, S.E. (1963TCold Spring Harbor Symposia Quant. Biol. 28 403. Richmond, M.H. <19T53 J. Bact. 0, 370. &evons, M.P. and John, M. 11964) Richmond, M.H., Parker, M.T., Lancet, ,i, 293.

218