BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

December 29,1978

RESISTANCE

TO (L)-AZETIDIN-2-CARBOXYLIC IN BACILLUS

M.Bazzicalupo,

E.Gallori, Istituto

di

1518-1525

ACID

SUBTILIS

B.Parisi*,

G.Pedaggi

and M.Polsinelli

Anatomia Comparata, Biologia Generale Universita di Firenze, Italy

e Genetica,

and *Istituto

di

Microbiologia

e Fisiologia Pavia, Italy

di Received

September

22,

Vegetale,

Universita

1978

summary: A spontaneous mutant of Bacillus subtilis resistant to (L)-azetidin-2-carboxylic acid, a structural analogue of (L)proline, has been isolated and characterized. Da$$ have been obtained which indicate that in vitro binding of [ C]-proline to tRNAs from the resistant strain is reduced only in part by while a complete inhibition of (L)-azetidin-2-carboxylic acid, binding occurs using tRNAs from parental strain. The mutation conferring resistance to the analogue and a mutation for proline auxotrophy have been mapped. (L)-Azetidin-2-carboxylic and it

is

found

in members

structural

analogue

analogues,

AZC has been

sting

Previous

in

(2).

the

place

eluding cell

line

residues

d-helix gions in

the

of

the

place

have in

been

of

the

shown that proteins

in

a toxic

chain.

a proline

aminoacid intere-

will

of

cells,inof AZC

(3,4,5,6,7,8). in

The insertion

1518

most

a disruption

AZC, (L)-azetidin-2-carboxylic Abbreviations: stance to (L)-azetidin-2-carboxylic acid: z, requirement of (D)-alanine, aromatic methionine, tryptophan, respectively. 0006-291X/78/0854-1518$01.00/0 Copyright 0 I978 by Academic Press, Inc. All rights of reproduction in any form reserved.

natural

a

incorporation

effect

bending

residue,

AZC is

AZC is incorporated,

the

shown to cause sharp

product

of a variety

organisms;

or less

polypeptide

(1).

as one of the

results

have

Liliaceae

described

and higher

proteins

the

a natural

Among the

studies

and a more of

of

(AZC) is

(L)-proline.

of.proline,

bacteria

into

acid

turn

the of the

Pro-

of the non-helical an AZC residue helix

acid; dal, aro, aminoacids,

through azcr,resiE, met, proline,

re-

BIOCHEMICAL

Vol. 85, No. 4, 1978

a smaller

angle

and the

function

of the

has been

also

It Escherichia

coli

of proline stance

(about

so altering

15O),

protein

(4).

shown (9)

that

cells,

by acting

biosynthesis. ability

the

tertiary

studies

the

the

growth

end-product have

resi-

of proline

analogue

of

inhibitor

shown that

to overproduction

to concentrate

structure

AZC inhibits

as false

Previous

to AZC may be due either

or to impaired cell

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

into

(9)

the

(10).

Plant

species

recognize In

the the

analogue

present

terization

of

been obtained mutation

in

producing

a B.subti1i.s

the

tRNA synthases

indicate

transfer

we describe mutant that system

the

resistant the

isolation

do not

have

due to a to tRNAs pro .

H

is

H

-0OH (L)-azetidin-2-carboxylic

and charac-

to AZC. Data

resistance

of proline

.I

MATHERIALS

which

(5).

report

which

AZC have

acid

CT

COOH

(L)-proline

AND METHODS

Strains and growth conditions. The B.subtilis strains used were: PB lg,prototroph, FB6 a+ resistant to AZC,derived from PB 19; PB 1715/l a derivative of QB 820 of J.Le--da1 aroI( pesant); BS 146 pro1 (obtained from N.Harford); PB 1676 metC=C The strains were grown in PY (Antibiotic Medium 3, DifcoEr in Minimal Medium (MM) as described by Sala et al. (11). PY cultu res of PB 1715/l were supplemented with 25pg/ml of (D)-alanine. Transduction and Transformation. PBS1 phage transduction was carried out as described by Yamagishi and Takahashi (12) and transformation as described by Spizizen (13). Recombinants were selected on MM plates containing the appropriare requiDal+ recombinants were selected on PY plates. All recomrements; binants w= cloned once and scored for resistance on minimal plates containing 5OO;ug/ml of AZC ( Sigma ). Preparation of crude aminoacyl-tRNA-synthases Crude enzymes containing from B.subtilis cells. and tRNAs were prepared from the supernatant of

1519

and tRNAs tRNA synthases the first ultra-

.

BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

centrifugation at 200.000 x g of ribosome preparation, as described by Sala et al.(ll). For enzyme preparation, the supernatant was precipitated with protamine-sulphate, as described by Lazzarini and Mehler (14); tRNAs were extracted from the same supernasaturated with 1 mM EDTA; aquetant with two volumes of phenol, ous phase precipitated with ethanol in presence of IM KCl. The pellet was collected again in ethanol at -20%; the pellet containing tRNAs was dissolved in IO mM acetate buffer (pH 5.0). 14 Binding of 14C -proline to tRNAs. Binding of C -proline (Amersham, U.K.) to tRNAs was performed at 37OC for 20' in an incubation mixture (200~11) containing 100 mM tris;-HCl (pH 7.3). 2 mM reduced-glutathione, 2 mM ATP and 350 pmoles of 5,qM MgCl, C -proline, specific activity 285 mCi/rtunole. The reaction was stopped by adding 1.5 ml of a mixture containing 67% ethanol, 0.5 M NaCl and unlabelled (L)-proline (1 mg/ml); the precipitate, collected on GF/C Whatman filters, was washed with 10 ml of stop mixture and dried. Radioactivity was measured in a Packard liquid scintillation counter.

Cl

I: 1

11

RESULTS Isolation

and characterization

Spontaneous cells

of

mutants B.subtilis

500 ug/ml

PB 19 strain

value

numerous

than

has been further in

The effect rental

strain

AZC reduces

presence

and of the almost

affecting

lOpg/ml,

lated

Inhibition understand

medium mutants,

is

of in

growth

growth

of

a few gene-

are much more indicated

MM medium,

(15),

using

shown that

as tester

liquid

medium Fig.

of the the

the

resistance

as cells

of

AZC.

shown in

the

after

of the

parental

mutant.

strain

pa-

lOOug/ml

I;

of

strain,

At a concentra-

completely removes the inhibition carried out as described experiments,

have

to proline

growth

frequency

proline

and Chapman

ne auxotroph,

the

only

On solid

1.5 mg/ml

mutant

completely

Cross-feeding claw

of

of AZC on the

slightly of

characterized,

was about

true

selective

One of these

by plating

mutants

cells

to AZC.

containing

of the

on the

titred.

selected

of resistant

of plated

bacteria

those

FB6 grow also

tion

growth

resistant

on MM medium

an overestimation

the

therefore

while

is

AZC blocks

rations, FB6,

to AZC were

of AZC. The frequency

2x10 -5 . This

since

resistant

of mutants

BS146,

of mutant

by AZC by Holtza proli-

FB6 is

not

re-

overproduction. of synthesis

the resistance

of prolyl-tRNAs. mechanism 1520

to AZC in

In the

attempt

FB6 mutant,

to we ha-

BIOCHEMICAL

Vol. 85, No. 4, 1978

I 0

1 90

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 270

I 180 TIME

Figure FB6 in in the cultures -----o----A----

1 380

(MINUTES)

1. Growth curves of parental strain and of mutant the presence or absence of AZC. Bacteria were grown MM liquid medium. AZC (lOOug/ml) was added to the at zero time. Symbols: -parental strain; mutant strain; --- parental strain plus AZC; L mutant strain plus AZC.

ve tested

the

analogue

The data

indicate

action

that

on the

AZC inhibits

formation only

partially

14

of

to tRNAs of the resistant [ C I- proline inhibition is observed when tRNAs of wild

in

Fig.

resistance

which

the

mutant, type

while

are used,

binding a strong as shown

2.

Mapping the

of prolyl-tRNAs.

code for

ascertain

if

of

azcy

and pro,

to analogues the this

related was also

ne requiring

mutant

phy by using

as donor

(BS146)

markers. are

linked

growth the

Several to the

factors

case for

was transducted

FB6 -0 azcr

loci

(16,

structural

AZC resistance, to proline

in

genes

In order

17).

None of 250 proline

1521

involved

to

a proliprototro-

independent

BIOCHEMICAL

Vol. 85, No. 4, 1978

0

1 20

0

I 10

AND BIOPHYSICAL

,

I 80

(L)-a&din-2-crrboxylic

RESEARCH COMMUNICATIONS

1 200

acid

(amoles/ml)

of AZC on the binding of L- 14C I -proline Figure 2. Effect to tRNAs in extracts of parental (+) and mutant strain FB6 (-+-). The values reported in the figure are the mean of three experiments. Assay conditions were as described in the text. tRNAs and enzymes, prepared as described, were used at the concentration of 250 and 45Opg/ml, respectively. In the trol experiments without analogue, 75 and 100pnoles of -proline were bound per mg of tRNA of parental and mupC] tant strain, respectively.

Figure 3. Location of AZC resistance mutation on B.subtilis chromosome. Map distances are expressed as 100 minus the percent of cotransfer index as described by Nester et al. are computed from data of Table 7. The arrows (16); values point from the selected to the unselected marker. (-transduction;-----*transformation).

transductants between

the

PBS1 phage ~2;

marker

resulted E,

and pro,

transduction is

resistant

linked

to AZC, indicating

markers.

Further

and transformation, to ~1

(Table

1522

no linkage

experiments, have

1 and Fig.

by using

shown that 3).

the

BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BlOPHYSlCAl

Table Mapping

of AZC resistance

Donor strain

Recipient strain

1

by transduction

Selected marker

Dal+

RESEARCH COMMUNlCATlONS

and transformation

Recombinant classes

~N~colonies scored

Dal+Aro+Azcr

19

Dal+Aro+AzcS

12

Dal +ArO-Azcr 2 Vi w

1 84

Dal+Aro-AzcS FB6 azc;

3 5 &

PB 1715/l --aro1

azc-aroI-da1 ---

da1 Aro+

Aro+Dal+Azcr

5

Aro+Dal+AzcS

2

Aro+Dal-Azcr

108

Aro+Dal-AzcS z V-l 44 2

Aro+Azcr FB6 azc:

b cy

PB 1715/l aro1 da1 --

Implied order

1

152

Aro+ ArO+AzCS

10

3 b

Table Mapping Donor strain

Recipient strain

of E, Selected marker

2

marker

Recombinant classes Met+Pro+

BS 146 pr+

PB 1676 ectrpc

by transduction No colonies scored

% cotransfer index (16)

12

Met+

80 Met+Pro-

1523

95

Vol. 85, No. 4, 1978

In

the

maps near

BIOCHEMICAL

course

&C;

of this

the

AND BIOPHYSICAL

work,

cotransfer

it

RESEARCH COMMUNICATIONS

was found

of pr+

with

that

the pro , gene metC was 80% (Table2).

DISCUSSION Bacterial belonging

to

regulation the

mutants two different

of proline

permeability

isolates

the Data

mutation

did

not

have

been obtained

tRNA synthase

here

described,

and tRNAPro,

in

the

alterated

in

was chosen

of the

to the

which

the

alterated

suggested

belonging

modifies

as

(2).

of cross-feeding

mutants

described

and b) mutants

show cross-feeding

from which

a) mutants

analogue

FB6 mutant,

absence

different

classes:

to the

which

to AZC have been

biosynthesis,

The B.subtilis BS146;

resistant

that

first

that

recognition

and allow

this

it

auxotro& could

be

FB6 carries

a

class.

indicate

proline

proline

among

system

mutant

by prolyl-

to be ascribed

to a new class. Further stance

of

studies FB6 is

are

needed

associated

with

to establish

whether

prolyl-tRNA

synthase

the

AX

resi-

or with

tRNAPro. Detailed and in

informations

related

cificity

ones

is

a useful

of aminoacyl-tRNA The mapping

of

on charging

B.subtilis

protein

data

biosynthesis

synthases

show that

chromosome

tool

near

mechanism for

genes

with

this

understanding

or of

azcrmarker

in

mutantthe

spe-

tRNAs. maps at the

are

also

origin

involved

in

(18).

REFERENCES 1. 2. 3. 4.

Fowden, L. (1955) Nature, 176, 347. Fowden, L., Lewis, D., andxistram, zimology, 3, 89-163. Baum, B.J., Johnson, L.S., Franzblau, (1975) J.Bio',L,"~Ghm~, 1464-1471. Fowden, L., , M.H. (1963) 71,

5. 6.

H. (1967) C.,

Advan.

and Troxler,

in

R.F.

Biochim.Biophys.Acta,

459-461.

Peterson, P.J., and Fowden, L. (1963) Nature, 3, 148-151. Trasko, c.s., Franzblau, C., and Troxler, R.F. (1976) Biochim. Biophys.Acta, 447, 425-435.

1.524

En-

BIOCHEMICAL

Vol. 85, No. 4, 1978

8.

Uitto,

R.F., and Brown, A.S. (1974) Biochim.Biophys.Acta, 341-349. J., and Prockop, D.J. (1974) Biochim.Biophys.Acta,

336,

234-25-l.

9. IO.

Baich, Neale, viii. Sala,

7.

Troxler,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

336,

11.

119,

A. (1968) Experientia, 24, 1107. S., and Tristram, H. (1965) J.Gen.Microbiol., F.,

Bazzicalupo,

M.,

and Parisi,

B.

(1974)

-'39

vii-

J.Bacteriol.,

821-829.

12. 13. 14.

Gagishi, Spizizen, Lazzarini,

15.

Holtzclaw,

H., J.

and Takahashi, I. (1968) (1958) Proc.Nat.Acad.Sci.U.S.A. R.A., and Mehler, A.H. (1964)

Virology, 36, 639-645. 44, 1072-1078. Bioct;;T=mistry, 3,

1445-1449. 88, 16. 17. 18.

W.D.,

and Chapman,

L.F.

(1975)

J.Gen.Microbiol.,

289-294.

Nester, E.W., Shafer, M., and Lederberg, J. (1963) Genetics, 48, 529-551. Polsinelli, M. (1965) Giorn.Microbiol., 13, 99-110. Young, F.E., and Wilson, G.A. (1976) Handbook of Biochemistry and Molecular Biology, vol. II, pp. 686-703, CRC Press, Cleveland.

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