Non-penicillin-like cyclic peptides as inducers of and substrates for Bacillus cereus 569 penicillinase

Vol. 19, No. 1, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NON-PENICILLIN-LIKE CYCLIC PEPTIDES AS INDUCERSOF AND SUBSTRATESFOR BACILLUS CEREUS569 PENICILLINASE

Arthur

K. Saz and Dolores L. Lowery

Georgetown University Dept. of Microbiology, Medical & Dental Schools, Washington, D.C.

Received February 2, 1965 The metabolic linase,

function

which hydrolyzes

and significance

the 6-lactam ring

of various

sensitive

penicilloic

acids,

penicillins

to the antibiotically

not clear.

Possession of such an enzyme seemingly does not afford

competitive

advantage to the producing

numerous efforts, ecological strains

inactive

of the enzyme penicil-

the production

conditions

of inducible

Bacillus

the hypothesis

peptides

enzyme.

inhibited

it

has been reported

capable of acting lococcal

of,

both as inducers

cyclic

a relatively

specialized penicillinase,

1961).

Recently

peptides

of atisubstrates

for

were staphy-

(Saz and Lowery, 1964).

communication,

these compounds as well

for,

certain

(Saz et al,

two synthetic

accounts for

that penicillin

of staphylococcal

hypothesis

that

that

in certain

produced by the cell.

and substrate

The observation

penicillinase

In this

protein

has been entertained

inducer

this

Yet,

cereus , penicillinase

the activity

was in accord with

under natural

has not been demonstrated.

serves as a fortuitous non-specific

since despite

of penicillin

as much as 2% or more of the total Accordingly,

cells

we shall

as other

report

naturally 102

is

on the effects occurring

cyclic

of

Vol. 19, No. 1, 1965

BIOCHEMICAL

peptidesl

on induction

addition,

preliminary

these peptides 569, will

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of penicillinase

in g. cereus 569.

data indicating

by purified

hydrolysis

penicillinase

of certain

extracted

was measured by inoculating

suspension of g. cereus 569 into The cultures the pellet

60 ml of Difco

were shaken at 35 C for

5 hours,

Nutrient

density

of 200-300 Klett

of the appropriate

centrifuged

units.

to yield

0.8 ml of the cells

concentration

of inducer

The mixture period,

City

by Citri

1.

A unit

L-leucyl

Duarte,

1.0 PM of the Na salt

California

L-prolyl

Dept.

L-prolyl

phenylalanyl

and a cyclic

lysyl

valyl

L-arginyl

hexapeptide

phenylalanyl

of amino acids unknown); Dr. J. G Neilands, Univ.

of California,

Berkeley,

two naturally

occurring

sphaerogena,

Ferrichrome

cyclic

and Ferrichrome

hydroxyornithyl;

Dr. H. Koffler,

oxytocin

peptides

for

of benzyl

of the synL-arginyl L-leucyl

(CHP), = cyc

lysyl

(configuration

Dept. of Biochemistry, crystalline

isolated

samples of

from Ustilago

A (FCA) = cyc diseryl-glycyl-triq-N-

hydroxyornithyl

for circulin;

Calif.,

method

of Biochemistry,

for gifts

L-valyl

After

is defined

Homogramicidin S (HGS) = cyc L-valyl

D-phenylalanyl

D-phenylalanyl

l-2 hours.

of penicillinase

We wish to thank Dr. M. Winitz,

peptides

and 0.1 ml

was measured by the iodometric

(1958).

of Hope Hospital,

thetic

valyl

penicillinase

a cell

of distilled

was then shaken at 35 C for

amount which hydrolyzes

Footnote

and

were mixed together

and the volume was made up to 1.0 ml by addition

described

Broth.

was then resuspended in an amount of casein hydrolysate-

medium (Kogut et al 1956) sufficient

as that

from g. cereus

0.05 ml of a spore

citrate

this

of

be presented.

Induction

water.

In

Dr. C. deFiebre,

(FC) = cyc triglycyl-tri-&-NPurdue Univ., Wilson Lab.,

and vasopressin. 103

Lafayette,

Chicago,

Ill.

Ind. for

Vol. 19, No. 1, 1965

BIOCHEMICAL

penieillin/hr.

Hydrolysis

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of inducers was determined by treating

the compounds for 5 minutes at 35 C with penicillinase al,

1960).

preparation

derived

from g. cereus 56914 (Citri

The penicillinase-treated

as described

above for capacity

0.05 ml of a purified

inducers were then tested to induce. I

0

IO

1 -

Induction various

I

20

MICROGRAMS

Figure

30 INDUCER

of penicillinase cyclic

peptides.

have been subtracted. circles

Ferrichrome linase

A at higher

formation.

40

/ML.

in 2. cereus 569 by The endogenous values

Open circles

= FCA; closed triangles

Figure 1 indicates

that

Indeed,

= FC

are both potent

in other

experiments

former compound has been shown to be as potent various

penicillins

tested,

as well as benzyl

penicillin.

differing

= HGS; closed

HGS at low concentrations

levels

et

including

from FCA in the substitution 104

inducers

of penicil-

(See Table l), in this

methicillin

Ferrichrome,

and

respect

as

and oxacillin

a cyclic

of a diglycyl

the

hexapeptide residue

in

Vol. 19, No. 1, 1965

lieu

BIOCHEMICAL

of a diseryl

other two cyclic induction plotted

residue, peptides.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is much less active

However, the probability

mediated by FC is real in Fig.

FC is noted. centrations

is strengthened

2, where a good concentration This figure

also indicates

both the antibiotic

circulin

that

of the the

by the data

response curve for

that

at higher

con-

and the hormone

600

1

MICROGRAMS

Figure 2 -

than either

INDUCER

/ML

Induction of penicillinase in g. cereus 569 by various cyclic peptides. The endogenous values have been substracted Open circles = FC; closed circles = circulin; open triangles = vasopressin; closed triangles = oxytocin.

vasopressin has no effect.

induce activity It

circulin

were

capacity

of various

of the

enzyme.

should be noted that preparations.

crystalline

cyclic

peptides

activity. 105

The hormone oxytocin neither

the hormones nor

Table 1 compares the

to induce penicillinase

Vol. 19, No. 1, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1 INDUCTION OF PENICILLINASE BY CYCLIC PEPTIDES

Peptide

Concentration

HGS

CHP

FCA

Penicillinase

(pg/ml.)

2.0

352.0

4.0

305.0

2.0

402.0

4.0

450.0

119.0

20.0

BP

(u/ml.)

40.0

77.6

1.0

376.0 22.5

HGS = Homogramicidin S;

CHP = cyclic

FCA = Ferrichrome

BP = Benzyl penicillin

It

is evident

A;

that

are extremely

at very

active,

of FCA shown in Figure this

table.

It

hexapeptide

low concentration

indeed as potent 1 is confirmed

is interesting

that

both HGS and CHP

as Pen G.

The activity

in the data presented Gramicidin

in

S and Gramicidin

D

do not induce activity. experiments

Preliminary

indicate

that

prior

treatment

HGS, CHP, FCA, and FC with

a purified

penicillinase

g. cereus 56914 completely

abolishes

the capacity

peptides

to induce penicillinase. penicillinase

sensitise

It

is thus evident

specific

penicillins other.

apparent

the enzyme.

that

so-called

of penicillinase

of these cyclic

penicillinase

inducibility

in producing

wider parameters of inducibility

cells

106

peptides

on the

and substrates

of the metabolic

must take into

and activity

communication.

There

between the various

cyclic

are both inducers

an understanding

is a non-

and activity.

in structure

similarity

these peptides Obviously,

from

HGS and CHP do not

on the one hand and the active

Yet,

derived

to iodine.

enzyme both in its

is no readily

Further

of

for

role

account the

reported

in this

Vol. 19, No. 1, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES Citri,

N.

Biochim.

Citri,

N.,

Garber, N. and Sela, M.

Kogut, M., Pollock, (1956) Saz, A.K.,

Biophys.

Acta 2,

M.R. and Tridgell,

Lowery, D.L.

and Jackson,

Saz, A.K. and Lowery, D.L. (1964)

277, (1958) J. Biol.

Chem. 235, 3454, (1960)

E.J. L.J.

Biochem, J. 62, 391, J. Bact.

Biochem. Biophys.

107

82, 298, (1961)

Res. Comm.15, 525,