The effect of nalidixic acid, rifampicin and chloramphenicol on the mitomycin C — induced synthesis of a bacteriocin of serratia marcescens

Vol. 59, No. 1, 1974

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE EFFECT

OF NALIDIXIC

CHLORAMPHENICOL SYNTHESIS

ACID,

RIFAMPICIN

AND

ON THE M I T O M Y C I N C - INDUCED

OF A BACTERIOCIN R. Eichenlaub

OF SERRATIA MARCESCENS.

and U. Winkler

Lehrstuhl Biologie der M i k r o o r g a n i s m e n Ruhr Universit~t, 4630 Bochum, Cermany Received May 14,1974 SUMMARY Nalidixic acid inhibits the mitomycin C-induced production of the baeteriocin marcescin B in S. marcescens HY indicating that DNA replication is an essential step for its production. Using nalidixic acid, rifampicin and chloramphenicol the temporal sequence of m a c r o m o l e c u l a r synthesis following mitomycin C induction was determined. Marcescin B is effectively produced if (i) DNA synthesis is allowed during the first 30 min, (ii) RNA synthesis can proceed up to about 60 min and (iii) p r o t e i n synthesis continues for about 3 hours. INTRODUCTION Bacteriocins plasmids

(1).

spontaneous mitomycin genic

Recent

studies

colicinogenic

of plasmid

there

is little

which can be induced by

investigations

of the colicinothat induction

copies per cell and thereby

production

cells was determined

of resistance

number

of R-factor in mutants

by a 'gene-dosage-effect'

after

induction

of

did not

coli it was found that the

to antibiotics

copies per cell

was correlated with the (7). A similar

of Serratia marcescens

Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any ]orrn reserved.

in-

On the other hand in an R-factor

strain of Escherichia

degree

was made

culture

are coded by

(5, 6) in which the plasmid DNA content

this hypothesis.

containing

of bacteria

E1 and E2 (3, 4) it was suggested

the bacteriocin

support

production

C (2). In earlier

the number

creases

species

In a bacteriocinogenic

bacterioein

factors

raises

of various

133

observation

HY with increased

Vol. 59, No. 1, 1974

BIOCHI~MICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

spontaneous bacteriocin production.

Such mutants contained

higher amounts of plasmid DNA than the wild type strain (8). The present communication describes the molecular events in S. marcescens HY following mitomycin C induction of marcescin B. MATERIALS AND METHODS Bacterial strain: Serratia marcescens HY, W 831/3. Growth conditions, media, mitomycin C induction, and bacteriocin tests were as described elsewhere (9). Measurement of the synthesis of DNA~ RNA and protein: Cultures of W 831/3 in a shaking water bath (30oC) were grown to a cell titre of 2x108Lml and then diluted I : 4 in fresh YPM. (DH)thymidine, (DH)uridine or (DH)leucine was added as appropriate to a final concentration of 0.1 ~Ci/ml, and the incubation was continued. Samples of 0.5 ml were withdrawn, added to 0.5 ml of ice-cold trichloroacetic acid (TCA)*, mixed rapidly, and cooled with ice for 30 min. The precipitates were collected on glass filters (Whatman GF/C), and washed succesively with 5 ml cold 5 % TCA and 5 ml cold 1 % acetic acid. Filters were dried in glass tubes for 15 min at 100oc. The radioactivity content of the filters was determined after addition of 5 ml scintillant (0.5 % PPO and 0.022 % POPOP in toluene) in a liquid scintillation counter. More information is given in the legends to the figures. RESULTS Mitomycin C induction of logarithmically growing S. marcescens HY stopped growth but did not kill the cells (Fig. 1). This shows that the concentration of mitomycin C used (1 ~g/ml)

is bacteriostatic but not lethal for S. mar-

cescens. Marcescin B production under these conditions yields about 400 AU/ml after 5 h (9). Since the viable cell count is constant after induction,

this might indicate that

marcescin B production is not a lethal event if one does not assume that only a minor fraction of cells was induced. When

*Footnote: Abbreviations used are NA (Nalidixic acid), CMP (Chloramphenicol)~ RIF (Rifampicin), TCA (Trichloroacetic acid).

134

Vol. 59, No. 1,1974

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

16

//./"

501

12

/ ~. 70 ~

/~ o o

s

d

8

/

o

N

i

@ Figure

10 I:i.

o--o

1

i

2 Time (h)

i

i

3

/.

@

3'0

60 Time {rain)

~ 90

' 120

I

Growth of S. marcescens after induction° Two 10ml cultures in YPM were grown at 30°C in a shaking water bath. After 90min one culture was induced with mitomycin C (1~g/ml). The viable cell titre was determined by plating on NB agar. Control (0). Induced culture (O). Figure

2

Synthesis of macromolecules after induction. The incorporation of (3H)uridine (&,A), (3H)thymidine (R,D), and (3H)leucine (0,0) was measured as described in the Methods. M i t o m y c i n C (1~g/ml) was added at 60min (open symbols). Control (solid symbols).

the synthesis

of macromolecules

(Fig.

2) it was observed

normal

rate and finally

that DNA synthesis

We wanted

after mitomycin

whereas

to know whether

C induction

tion of marcesein

was measured

continued

stopped after about 30 min.

of protein was u n i m p a i r e d reduced.

after induction

RNA synthesis

early and late after

for the produc-

3 NA immediately induction.

blocks DNA synthesis

The p r o d u c t i o n

of marcescin

was inhibited when NA was added during a period induction

unimpaired C (Fig.

(Table

1) which coincides

DNA synthesis

observed

135

of

of mitomycin

for an essential

for the induced p r o d u c t i o n

B

of 30 min

with the period

after addition

2). This is taken as evidence

of DNA synthesis

observed

B.

As shown in Fig.

after

Synthesis

was slightly

the DNA synthesis

is of importance

at a

role

of marcescin

B.

Vol. 59, No. 1, 1974

Table

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I Marcescin B production (AU/ml) of mitomycin C-induced cultures after addition of NA RIF CMP

t (min after induction)

Influence

0

5O

5O

10

10

5O

-

-

20

75

-

-

30

150

75

I0

60

45O

35O

I0

9O

-

45O

5O

120

-

500

100

180

-

-

450

of NA~ RI B and CMP on the production

of marcescin

B.

Cultures of S. marcescens (10ml) were grown in YPM at 30°C. At 5x1OScells/ml (t=O) the cultures were induced with mitomycin C (lpg/ml) and NA (10pg/ml), RIF (lOOpg/ml), and CMP (100~g/ml), respectively, were added at the times indicated. Cultures were further incubated for 4h. Cultures were then sonicated with a Branson Sonifier (2min at 9ON) and centrifuged for 10 min at 43300 xg. For the titration of marcescin B in the supernatant, aliquots (0.01ml) of serial dilutions were placed on agar plates, seeded with E.coli W4110. The reciprocal of the greatest dilution which inhibited growth of the indicator lawn was defined as the number of arbitrary units per ml (AU/ml). Addition of NA, RIF, and CMP alone did not raise marcescin B production above the background level of about 50AU/ml, whereas mitomycin C-induction yielded about 500AU/ml.

Since marcescin

B is a protein molecule,

protein are necessary

for its production

synthesis

(9)- The sequence

and the length of time required for these processes studied by means

of specific

RNA synthesis

synthesis

was

inhibitors.

was inhibited by RIF (11).

cens a concentration

of RNA and

of 100 p g / m l

is sufficient

immediately but allows the synthesis

protein for at least I h at a reduced rate

136

In S. marcesto stop RNA Of DNA and

(unpubl.

results).

Vol. 59, No. 1, 1974

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

_ / o . _ . . . . - ~o

Control

Control

eJ

12

Nit.C

"7 o x

%

NA

Addition of Mit C RIF rnin rain

e

o/ojo

2O

~0

-

30

30

x

E8 V

6. ,j

120

150

90

gO

10

o

/.

--

~

~

b i

I

60

i

i

120

i

i

180

240

I

@

Figure

i

I

30

Time (min)

i

90

i 150

Time (rain)

3

Effect of NA on DNA synthesis during induction. DNA synthesis was measured by incorporation of (3H)thymidine into acid insoluble material as described in the Methods. Concentrations of mitomycin C were IDg/ml and of NA 10~g/ml, resp.. Figure

4

Effect of RIF on RNA synthesis during induction. RNA synthesis was measured by incorporation of (3H)uridine into acid insoluble material as described in the Methods. Mitomycin C concentrations used were Ipg/ml and of RIF 100~g/ml, resp..

Even after mitomycin

C induction

(Fig. 4) but reduced the amount

RIF inhibited of marcesein

RNA synthesis

B produced

only

when it was added to the cells during the first 60 min after induction CMP

(Table

(12) at a concentration

synthesis (Fig.

I).

in S. marcescens

5). However,

completely,

marcescin

up to 2 h after induction

of 100 ~ g / m l

even after induction

B production

(Table

blocked protein

was only inhibited

I).

DISCUSSION After thesis

induction

of marcescin

in S. marcescens

HY continued

has been shown to be an essential marcescin

B. This synthesis

or continued

replication

B by mitomycin

C, DNA syn-

for about 30 min and

step for the production

of

might be a result of repair and/

of chromosomal

137

DNA or might be

Vol. 59, No. 1,1974

BIOCHI~MICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

/Control 8 % Addition of M~.C CMP rain min

~: ~ 0 ~ 0

M)

~'o

'

;o

'

60

gO

,;o

T i m e (rain)

Figure

5

Effect of CMP on protein synthesis during induction. Protein synthesis was measured by incorporation of (3H)leucine into acid insoluble material as described in the Methods. Concentrations of mitomycin C were Ipg/ml and of CMP lOOpg/ml, resp..

caused by the start of plasmid DNA replication. the latter

interpretation

because preliminary

indicated that the content

the

duction.

should result

This,

Recently

in fact,

was increased

control.

'gene-dosage-hypothesis',

replication

experiments

of supercoiled plasmid DNA in

induced cells of S. marcescens compared to the uninduced

We favour

2-3 fold as

Therefore,

inhibition

in a decrease

according

to

of p!asmid DNA of marcescin

B pro-

was found.

it has been reported that NA also reduces

rate of $13 m-RNA synthesis

(13).

Inhibition

the

of RNA syn-

thesis by NA does not seem to be the reason for the reduction of marcescin B production S. marcescens

since RNA synthesis

in

is only slightly effected by NA. Furthermore,

NA and RIF should then inhibit marcesein during the same period after induction,

B production but this is not the

case. Addition

of RIF reduced drastically

marcescin

duction within about 60 min after mitomycin has been shown that RIF interferes

138

B pro-

C induction.

It

with the DNA replication

Vol. 59, No. 1, 1974

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Col-factor El by inhibition of RNA-primer synthesis (14). The extended RIF sensitive phase of marcescin B production as compared to the shorter NA sensitive phase is consistent with the interpretation that RIF inhibits m-RNA synthesis under our conditions (Table 1). However, our experiments do not exclude the possibility that RIF also inhibits DNA synthesis at an early stage after induction. In the experiments presented above the approximate temporal sequence of DNA, RNA and protein synthesis essential for mitomycin C-induced production of marcescin B has been determined and is summarized in the following scheme: Ind. I

30 r

60 I

90 I

120 I

150 I

I~0

~min)

DNA replication m-RNA synthesis protein synthesis Similar results have been obtained with E. coli W3110 containing Col-factors EI-K30 or D-CA23 (unpubl. results). REFERENCES ( ~I Clowes,R.C.(1972)Bact.Rev.36,361-h05 lijima,T.(1962)Biken's J . 5 7 - 8

i¼1 Amati,F. 964)J.Mol.Biol.m239-246 DeWitt,W. ,and Helinski,D.~. (1965)J.Mol.Biol.13,692-703 5) Hausmann,C.,and Clowes,R.C.(11971)J.Bacteriol.-~r07,16(33-635

8) I 9) (10) (11) 12 ) (13) (14)

NordstrOm, K., Ingram, L. C o, a~d Lundb~ck, A. (1 972 ) J. Bacteriol. 11 0,562-569 Timmis,K. ,an~-~inkler,U. (1973)Molec.gen.Genet.124,207-217 Eichenlaub,R. ,and Wink!er,U. (1974)J.Gen.Microbio-~. (in press) Goss,W.A., Deitz,W.H.,and Coek,T.M.(1965)J.Baeteriol.89, 1 068-I 074 Hartmann,G., Honikel,K.O., KnHse!,F.,and N~esch,J.(1967) Biochim. Biophys. Acta145,843-844 Pestka, S. (1 971 )Ann. Rev .Microbiol. 25,487-562 Puga,A.,and Tessman,i.(1973)J.Mol.Biol.75,99-108 Clewell,D.B., Evanchik,B.G.,amd Cransto~,J.W.(1972) Nature New Biol. 237,29-31

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