DNA gyrase (Topoisomerase II) from Pseudomonasaeruginosa

BIOCHEMICAL

Vol. 110, No. 2, 1983 January 27, 1983

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

DNAGYRASE(T~PoIsOMBRASEII)

694-700

PROM

PSBUDOWONAS AERUGIBOSA Robert V. Miller

and Ted R. Scurlock

Department of Biochemistry and Biophysics, School of Medicine, Loyola University Medical Center, 2160 S. Firat Avenue, Maywood, Illinois, 60153; and Department of Microbiology, University of Tennessee, Knoxville, Tennessee, 37916

Stritch

Received December13, 1982 DNAgyraee (Topoiaomeraee II) has been purified from Peeudomonaa aeruginosa strain PAO. This enzyme is inhibited by novobiocin and nalidixic acid. DNA gyraee from P. aeruginoisa is resistant to a much higher level of na=dixic'acid than is Eecherichia coli DliA gyraee. This increased level of resistance z explain, at least in part, the higher levels of natural resistance exhibited by -P. aeruginoaa toward nalidixic acid. Naturally higher

occurring

concentrations

bacterial

species

strains

of Pseudomonasaeruginosa are

of the drug nalidixic Typical

(1).

aeruginosa for nalidixic

and Sugino,

et al.

Escherichia

in

II),

and that resistant

gyrase

4).

which

MI&

(MICs) of -P.

for other

Studies by Gellert,

coli is to inhibit strains

of --E.

the enzyme DNAgyrase

is more resistant

to nalidixic

(2)

nalidixic

(‘l’opoisomersase

coli contain a resistant

This paper demonstrates the presence in 5

Gram-

et al.

(3) have shown that the mechanismof action of

acid

enzyme (2,

concentrations

acid are 128 to >256 ug/ml while (1).

to

acid than most other Gram-negative

minimal inhibitory

negative species range from 4 to 8 pg/ml

resistant

form of

this

aer;lginosa of DNA

acid than is the enzyme from -E.

coli. MATERIALSAND METHODS Bacterial Strain and Growth Conditions. For this study, strain RN40 (5), a derivative of strain PAO, was used. This strain has an.MIC for nalidixic acid of 175 Dg/ml on Mueller-Hinton Medium. For the isolation of DNAgyrase, cultures were grown in Luria Broth as described by Scurlock and Miller (6). Purification method of

of DIVAGyrate. Cultures were grown, harvested, and lysed by the Scurlock and Miller (6). This procedure yielded 16 to 20 g (wet

0006-291X/83/020694-07$01.50/0 Copyright AI1 rights

0 I983

by Acudemic Press, in any form

of reproduction

Inc. reserved.

694

Vol. 110, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

weight) of cells. All subsequent operations were carried out at 4" C. Cell debris and DNAwere removed by centrifugation for 30 min at 35,000 X g. The supernatant fluid was decanted and treated with a 5% (w/v) solution of streptomycin sulphate prepared in water. One milliliter of the streptomycin solution was added for each 425 optical density units at 260 nm present. The mixture was stirred for 20 mia, and the precipitate which formed was separated by centrifugation for 20 min at 27,000 X g. The precipitate was discarded, and the supernatant fluid was dialyzed overnight against 1.5 liters of 50 mMTrisHCl buffer (pH 8.0) containing 0.1 mMdithiothreitol, 0.1 mM EDTA, and 10% (w/v) glycerol. This fraction was made 30% (w/v) in glycerol and layered onto a DEAE-cellulose column (2.5 x 12.5 cm) which was equilibrated with 50mMTrisHCl (pH 8.0) containing 0.1 mMdithiothreitol, O.lmM EDTA, and 30% (w/v) glycerol (Buffer A). The sample was followed by a 35 ml wash of Buffer A and a 300 ml gradient of 0.05 to 0.3 M NaCl prepared in Buffer A. The fractions which eluted between 0.1 and 0.2 M NaCl were pooled. Ammoniumsulphate was then added to the pooled fractions to a final concentration of 70% (w/v). The precipitate which formed upon stirring for 20 min was collected by centrifugation and dialyzed overnight against 1 liter of 0.03 M potassium phosphate buffer (pH 7.0) containing 0.1 mN dithiothreitol, 0.1 mMEDTA, and 30% (w/v) glycerol (Buffer B). The dialyzed solution was layered onto a column of Sephadex G-200 (1.5 x 48 cm) which had been equilibrated in Buffer B. The column was eluted with Buffer B. Fractions showing gyrase activity were pooled, concentrated with a Millipore Submersible Concentrator, and layered onto a hydroxyapatite column x 5 cm) which had been equilibrated in 10 mMpotassium phosphate buffer (0.5 (pH 7.0) containing 6 mM2-mercaptoethanol, 1 m&lEDTA, and 10% (w/v) glycerol (Buffer C). The column was eluted with a 0.1 to 0.7 M phosphate buffer solucontaining 6 mM2-mercaptoethanol, 1 mMEDTA and 10% glycerol. tion Each fraction (1.0 ml) was dialyzed against Buffer C and tested for gyrase activity. Peak activity eluted between 320 and 360 mMphosphate. This fraction was stored at -2O'C until used. Gyrase Assay. P. aeruginosa DNAgyrase activity was determined in a reaction which measurerthe conversion of relaxed, closed-circular DNA to the The reaction supercoiled form as demonstrated by agarose gel electrophoresis. mixture (100 j.11) contained 35 mMTris-HCl (pH 7.5), 6 mMMgCl , 1.5 mMdithiothreitol, 1.5 mMspermidine-HCl, 1.5 mMATP, 5 ug bovine serum albumin, 0.6 ug of relaxed, closed-circular DNA, and an enzyme sample of various concentrations. The DNA in the reaction was either relaxed, closed-circular $X174 (RFI) closed-circular ColEl DNA. The $,X174 (RFl) DNAwas purchased DNA or relaxed, from New England Biolabs while the ColEl DNAwas prepared from E. coli strain JC411(ColEl) as described by Tanaka and Weisblum (7). Relaxed, xosed-circular a nicking-closing forms of the DNAs were prepared from supercoiled DNAs using extract prepared from chicken reticulocytes as described by Gellert et al. The reaction mixture was incubated at 37°C for 60 min. The react?& (",ii stopped by addition of 5 ~1 of 0.25 M EDTA, and the solution was extracted with an equal volume of chloroform-isoamyl alcohol (24:l v/v). The aqueous phase and 0.025% was added to 5 ~1 of a solution of 5% sarkosyl, 25% glycerol, bromphenol blue, loaded onto a 0.8% agarose gel and electrophoresed at 65 volts The gel was then stained with a solution of 0.5 fig ethidium for 16 h (9). bromide/ml of water for 30 min. destained for 30 min in water, and photographed with a Polaroid MP-4 camera using Polaroid type 57 film and W filter (9). One unit of DNAgyrase converts 0.1 pg of relaxed DNA to the.supercoiled form in 30 min at 37°C.

DNA

In the experiments examining the inhibition of P. aeruginosa DNA gyrase by various drugs, gyrase activity was quantitated byycanning negatives of the SD3000 photographs prepared from the agarose gels with a Kratos-Schoeffel Spectrodensitometer equipped with a Hewlett-Packard 33qOA integrator as described by Sugino and Bott (IO). 695

Vol. 110, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Linear Determination of Molecular Height by Glycerol Gradient Centrifugation. glycerol gradients (20 to 40%) prepared in 30 mMpotassium phosphate buffer (pH in a Beckman SW60swinging-bucket rotor at 50,000 rpm 7) were centrifuged (260,000 X g) for 12 h in a Beckman L5-75 ultracentrifuge. The gradients were divided into 30 fractions and assayed for DNA gyrase activity. Molecular weight of P. aeruginosa DNA gyrase was determined from the relative sedimentation velocity using the fraction showing the highest DNAgyrase activity (6, 11). Aldolase, catalase, and thyroglobulin were used as sedimentation standards. Protein Determinations. Protein determinations were by the method of Lowry et al. (12) using BSA as a standard. Concentrations below 0.05 mg/ml were measured by ultraviolet absorbancy at 280 nm. RESULTSAND DISCUSSION Preparations of

of purified

15,000 units/mg protein

1).

Difficulty

extracts Mixing

with material

was due to an inhibitor

on the DEAE-cellulose

specific

(e.g.

preparation

was unstable

on relaxed,

-20°C.

supercoiling

activity weight Table

of the purification from earlier

has not

II.

Streptomycin

III.

from the DNA

away

or

for 2-3

of 1.

$X174 DNA.

and is inactivated aeruginosa

-P. Purification

Lysate

of

by heating to 70°C for DNA gyrase

Peeudomonae

was estimated

DA& Gyrase. PROTEIN (w/ml 1

ACTIVITY (lJnits/mg

protein)

9.8

NDa

29

6.0

ND

DEAE-Cellulose

6

7.1

13

IV.

Sephader

3

2.7

50

V.

Hydroryapatite

2

0.01

C-200

DNA gyrase

The weeks

The enzyme requires ATP and magnesium

29

aND:

a non-

been determined.

could only be retained

VOLUME (ml)

Cleared

yield.

indicated

Whether this is a specific

inhibitor

crude

and

fractions

which was purified

and activity

FRACTION

I.

of DNA gyrase in

Figure 1 demonstrated -P. aeruginosa DNAgyrase activity

closed-circular

The molecular

column.

an endonuclease)

when stored at

for

fractions

activity

a minimumof 1200-fold (Table

determining the activity

an accurate determination

purified

that this activity gyrase

and had been purified

in accurately

prevented of

aeruginosa DNAgyrase had a specific

-P.

activity

was

not

detectable

696

in

these

15,000

fractions

(see

text).

15 min. to

be

Vol. 110, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1. Assay of P. aeruginasa DNA gyraseactivity under different conditiooa. (a) Supercoizd @Xl74 (RFl) DNA (lower band) containing SOlIE opencircular DNA (upper band). (B) Relaxed, double-stranded $X174 DNA. (cl Relaxed, doable-stranded $X174 DNA incubated with 30 ~1 of purified gyrase under standard reaction conditions. (D-H) Relaxed, double-stranded $X174 DNA incubated with 30 ~1 of purified gyrase for 60 min under the following condi(D) ATP omitted; (E) Magnesium omitted; tions: (F) Standard reaction conditiona; (C) Nalidiric acid added (500 rig/ml); (H) Novobiocin added (1 dml). Figure

3.620.5

x

lo5

by

sedimentation

through

a 20

to

40%

(w/v)

glycerol

gradient

(6,

11).

The

sensitivity

novobiocin standard of

of

reaction

these

method

of Gellert

from

strain

--et al.

-P. aeruginosa

antibiotics

(Figure to

enzyme 50%

approximately

(Figure of

the 'j-fold

These

(8).

is

the

required

preparations

activity higher

than

the 697

from were

M.

used

aeruginosa

of nalidixic aeruginosa

-P.

MIC

and

in

of one or the

higher

the -P.

acid

incubated

Gellert)

the

other

the

same by the

comparative

by both

of these

and aE. coli

DNA

concentrations enzyme acid

gyrase, determined

to

for

of -P. aeruginosa

significantly

of

were

to be inhibited

sensitivity

to inhibit

nalidixic

was purified

obtained

The concentration

2).

enzyme

DNA gyrase

was found

similar,

to

concentrations

HN356 (recB21,

While

1).

are

coli

DNA gyrase

novobiocin acid

nalidixic

various

Escherichia

purposes.

gyrases

of purified

containing

inhibitors.

DNA gyrase

Samples

mixture

activity

inhibit

aeruginosa

was investigated.

specific

coli

&

than

the

necessary 600

for

of

this

-E. to

ug/ml,

is strain

Vol. 110, No. 2, 1983

I

I

BIOCHEMICAL

I

I

400

200

acid

RESEARCH COMMUNICATIONS

I

600

Nalidixic

AND BIOPHYSICAL

800

0.2

1c

( Pg/ml

0.4

0.6

Novobiocin

)

0.6

()Iglfnt

1

)

Sensitivity of DBA gyrase to nalidixlc acid and novobiocln. Standcoocentraard supercoiling assays containing relaxed 4A1'74 DNA and different Supercoiling activity tions of nalidixic acid or novobiocin were carried cut. wan quantitated by tracing photographic negatives of the stained gels as (A) Sensitivity of DNA gyraee to nalidixic described by Sugino and Bott (IO). DNA acid: (B) Sensitivity of DNA gyrase to novobiocin. (0) -P. aeruginosa gyrase; (0) --E. coli DNA gyrase. Figure

2.

(175 ,,g/ml). 200 &ml

The (3 and

the --E. coli Engle

of

study)

DNA

(13)

which

was 50% inhibited is

SO-fold

h ave suggested

that

acids

in --E.

replication-fork

this

inhibitory

DNA-associated

consistent

with

only

a small organism

coli

by a

higher

than

the

mechanism complex

inhibition fraction corresponds

to form

concentration

of

MIC (4 ug/ml)

of

by would could

a

shown

that

recA-dependent be extremely

inhibit at a drug

enzyme activity

to a concentration 698

inhibitory

action

drug-gyrase-DNA

They have

of growth of the

the --in vitro

is

migration.

can be relieved

replication

MIC of this

gyrase

strain.

inhibit

and a single

inhibit

coli

and oxolinic

Certainly

is

this

et al.

nalidixic which

--E.

cell

complexes

this

inhibition

repair sensitive growth.

concentration as is

seen

of nalicixic

of

mechanism.

to the This

mechanism

which in --E. coli acid

drug

which

would (the 2

Vol. 110, No. 2, 1983

vitro

BIOCHEMICAL

approximately

inactivates

AND BIOPHYSICAL

5% of the gyrase activity).

aeruginosa DNAgyrase requires greater inhibition, drug

is equilivalent

inactivates

to

inhibition

drug-gyrase

acid inhibition

in -P. of 5

aeruginosa is also

induced

aeruginosa

gyraae

(13) for --E. coli

that

the

extremely

gyraae.

aeruginoaa may simply be due to a higher aeruginosa DNA gyrase.

--in vitro

This would imply

dissociated.

as measured by sodium dodecyl

where complex formation with

detected by DNA cleavage.

to

the

increased

acid both --in vitro

the

E. coli --

(14,

In this

resistance

aulfate15,

16,

DNAgyraae was

Hence, it appears probable that the atability

drug-gyraae-DNA complex is much reduced in P. -

nalidixic

activity

and suggeats that the mechansim may be

cleavage of the DNAat the site of complex attachment

under conditions

leads

of

we have not been able to demonstrate drug-gyraae-DNA complex formation

-P.

17,18)

-P.

A concentration

This indicates

the drug-gyrase-DNA complexes would be more easily

context,

the

acid for 50%

5 to 10% of the gyrase

of growth in -P.

to that proposed by Engle, --et al.

Ki for nalidixic

with

approximately

interaction

The heightened resistance

that

of nalidixic

to the --in vivo MIC of this organism.

mechanism of --in vivo

similar

concentrations

While

the MIC of this organism is also much higher.

which --in vitro

sensitive

RESEARCH COMMUNICATIONS

of the

aeruginosa, and this instability

of this species and ita

DNA gyrase

to

and --in vivo.

ACKNOWLEDGEMENTS This work was aupported by Public Health Service grant AI-12759 from the National Inatitute of Allergy and Infections Disease. R. V. M. ia the recipient of Research Career Development Award AI-00449 from the Public Health Service. T. R. S. waa a predoctoral trainee of the Public Health Service, National Institute of General Medical Sciences (GM-07438). REFERENCES 1.

2. 3. 4. 5. 6.

Manual of Clinical Microbiology, 2nd Edition, pp. Sherris, J. C. (1974) 439-442, American Society for Microbiology, Washington, D. C. Gellert, M., Mizuuchi, K., O'Dea, M. H., Itoh, T., and Tomizawa, J. (1977) Proc. Natl. Acad. Sci. USA 74,4772-4776. K. N., and Cozzaarelli, 8. R. (1977) Sugino, A., Peebles, C. L., Kreuzer. Proc. Natl. Acad. Sci. USA 74,4767-4771. N. R. (1978) Higgins, N. P., Peebles, C. L., Sugino, A,. and Cozzarelli, Proc. Natl. Acad. Sci. USA 75,1773-1777. Miller, R. V., and Ku, C.-M. C. (1978) J. Bacterial. 134,375-383. R. V. (1979) Nucleic Acid Rea. 7,167-177. Scurlock, T. R., and Miller,

Vol. 110, No. 2, 1983

7.

8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Tanaka, T., and Weisblum, B. (1975) J. Bacterial. 121,354-362. Proc. Gellert, M., Mizuuchi, K., O'Dea, M. H., and Nash, H. A. (1976) Natl. Acad. Sci. USA 73,3872-3876 Hinkle, N. F., and Miller, R. V. (1979) Plaemid 2,387-393. Sugino, A., and Bott, K. F. (1980) J. Bacterial. 141,1331-1339. Miller, R. V., and Clark, A. J. (1976) J. Bacterial. 127,794-802. Lowry, 0. H., Roeebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265275. Ecgle, E. C., Manes, S. H., and Drlica, K. (1982) J. Bacterial. 149,92-98. Cozzarelli, N. R. (1980) Science 207,953-960. Gellert, M. (1981) Ann. Rev. Biochem. 50,879-910. Peebles, C. L., Higgins, N. P., Kreuzer, K. N., Morrison, A., Brown, P. 0 Sugino, A., and Cozzarelli, N. R. (1979) Cold Spring Harbor Symp. Q;Hnt. Biol. 43,41-52. Snyder, M., and Drlica, K. (1979) J. Mol. Biol. 131.287-302. Tse, Y.-C., Kirkegaard, K., and Wang, J. C. (1980) J. Biol. Chem. 255,5560-5565.

700