Journal of Antimicrobial Chemotherapy (1988) 21, Suppl. B.

1-18

Alteration of bacterial DNA structure, gene expression,

and plasmid encoded antibiotic resistance following
exposure to enoxadn

J. B. Conrtrigfat, D. A. Turowski and S. A. Soosteiir1

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Department of Biology, Marquette University, Milwaukee, WI53233 and
'Department of Associated Health Professions. Eastern Michigan University,
Ypsilanti, MI 48197, USA

Enoxacin inhibits growth of Escherichia coli K12 strains primarily by binding to the
GyrA subunit of DNA gyrase (topoisomerase II); strains with gyrA, but not gyrB,
inutations are less susceptible to the bactericidal effects of this agent. In sensitive
strains, enoxacin completely inhibits DNA synthesis within Smin and produces
drug-gyrase-DNA complexes at numerous sites throughout the E. coli chromosome,
as shown by the formation of linear DNA molecules after detergent treatment.
Enoxacin, even at subminimal inhibitory concentrations, induces the bacterial SOS
system, even in partially resistant gyrA strains. This drug also inhibits the induced
expression of the lacZ encoded /7-galactosidase, regardless of whether this gene is
located on the chromosome, a low copy number F plasmid or high copy number
Col El related plasmids. This inhibition of gene expression at subminimal
inhibitory concentrations is likely to be a factor, in addition to gyrase inhibition, in
the elimination of Col El plasmids and to the reduction in R plasmid conjugal
transfer. Enoxacin enhances the bactericidal effects of kanamycin in both in-vitro
and in-vivo models, suggesting that this quinolone may be effective in the treatment
of infections due to strains resistant to antibacterials as a consequence of plasmid
encoded resistance determinants.

Introduction
The quinolone antibiotics, including nalidixic acid, oxolinic acid and related
compounds, are major inhibitors of bacterial DNA topoisomerase II (DNA gyrase)
(Cozzarelli, 1980; Gellert, 1981) and produce a drug-gyrase-DNA complex as a
consequence of binding to the A subunit of the gyrase complex (Kreuzer & Cozzarelli,
1979; Sugino, Higgins & Cozzarelli, 1980). These antibiotics cause a major and
immediate reduction in the rate of DNA synthesis in vivo through their inhibition of
one or both of the gyrase present in the replication complex (Kaguni & Kornberg,
1984) and the gyrase present at 50 or more topological sites on the bacterial
chromosome (Crumplin & Smith, 1975; Snyder & Drlica, 1979). Cells exposed to
quinolones initiate a series of cellular responses which include filamentation, SOS
repair system induction, and changes in permeability (Gottesman, 1984; Dougherty &
Saukkonen, 1985; Tessman & Peterson, 1985) and which either singly or in
combination lead to cell death. Experiments showing that the bactericidal effect of
quinolones is reduced when protein or RNA synthesis is inhibited (Crumplin,
Kenwright & Hirst, 1984; Zeiler, 1985; Benbrook & Miller, 1986) support the
1
0305-7453/88/21B0O1 +18 $02.00/0 1988 The Britbh Society for Antimicrobial Chemotherapy
2 J. B. Coartrigfat et aL

hypothesis that either unbalanced growth or the synthesis of specific proteins is

required for cell killing by this class of antibacterials.
Other features of the cytopathic effects of gyrase inhibitors include a reduction in
conjugal transfer (Nakamura et al., 1976; Burman, 1977; Gill & Iyer, 1982; Michel-
Briand, Laporte & Bassignot, 1983) and elimination of bacterial plasmids
(Danilevskaya & Gragerov, 1980; Cejka, Holubova & Hubacek, 1982; Wolfson et al.,
1982; Hooper et al., 1984). The^e.changes in.the maintenance and transmissibility of
plasmids can be attributed to direct effects on plasmid topology (Lockshon & Morris,
1983; Pruss & Drlica, 1985), plasmid gene expression (Sanzey, 1979; Gomez-
Eichelmann, 1981), as well as selective inhibition of the gyrase B subunit (Wolfson et
al., 1982), or to a lesser extent, alteration of the gyrase complex through inhibition of

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the A subunit (Hooper et al., 1984; Weisser & Wiedemann, 1985).
We have examined the effects on enoxacin on several aspects of chromosomal and
plasmid DNA structure and expression.

Materials and methods

Bacterial strains used in these studies are listed in Table I. Strains C600, C600 rpsL,
KL163, JA200, and W1485 were from either the Coli Genetics Stock Center or Cold
Spring Harbor Laboratory. Strains were generously provided by individuals, as
indicated: GE94 (Weisemann, Funk & Weinstock, 1984) from G. Weinstock, MCI000
(Casadaban, Chou & Cohen, 1980) from M. Casadaban, RM100.RM101, and RM102
from R. Menzel. R plasmids R68.45 and R702 (Hedges & Jacob, 1974; Haas &
Holloway, 1976) from D. Noel, lacZ containing plasmids pMCl314 from
M. Casadaban, pUR288 from U. Ruether (Ruther & Muller-Hill, 1983), FtsLacl 14
from Cold Spring Harbor Laboratory (Chumley, Menzel & Roth, 1979). Other strains
used in these studies were derived in our laboratories, either by selection for
spontaneous mutants or by conjugal transfer.
Strains with an increased resistance to enoxacin (MIC ^ 0-4 mg/1) were recovered at
a rate of approximately 10"8, suggesting a single locus mutation. Isolated strains
MCI060-1 and C6003 were also resistant to 100 mg/1 nalidixic acid, as expected for
strains with a mutation in the gyrA gene (Taylor, Ng & Lior, 1985). In the case of
GE94-1, to confirm that the recA-lacZ fusion construction was still intact, enoxacin
resistant derivatives were incubated with mitomycin C and lacZ (/?-galactosidase)
activity assayed (Weisemann et al., 1984).
MICs were determined by serially diluting suitable drug concentrations in L broth
and inoculating 1 ml of each dilution with approximately 105 cells. The MIC was
defined as the lowest concentration of agent that inhibited visible bacterial growth in
18 h under these conditions. The fraction of plasmid free cells after growth in the
presence of enoxacin or other quinolones was determined by plating dilutions of cells
on MacConkey lactose medium and identifying the lactose nonfermenting colonies.
Representative Lac" colonies were shown to be sensitive to the appropriate antibiotics
and were lacking the 915 kb (pMC1314) or 8-5 kb (pUR288) lacZ plasmid, as
determined by electrophoretic separation and staining of DNA prepared by the
alkaline lysis method (Maniatis, Fritsch & Sam brook, 1982). The elimination of R
factor plasmids was determined by plating equal volumes of diluted cells on to LB
media or LB media containing tetracycline (15 mg/1). DNA synthesis was measured as
described (Engle, Manes & Drlica, 1982).
 Alterations in DNA and gene activity with enoxadn

Table I. Bacteria and plasmids

Enoxacin
Strain Genotype MIC (mg/1)

E. coli
C600 thi-1 thr-1 leuBS lacY 0-2
tonA21 supE44
C600-3 C6O0gyrA 2-0
119 C600rpsL 0-2
119-1 U9gyrA 10-2-0
W1485 wild type 0-2

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RM100 gyrA26O TnlO 2-0
RM101 TnlO 0-2
RM103 gyrB221 gyrB203 TnlO 0-2
GE94 WjecA lacZ) 0-2
GE94-1 WjecA lacZ) gyrA 1-0-2-0
MC1000 araD139, A(ara, leu) 7697, 0-3
MacX74, galU. galK, rpsL
MC1000-1 MC1060g>r,4 1-0-2-0
1314 pMC1314/MC1000 0-3
1314-1 pMC1314/MC1000-l 20
R68 R68.45/J53 0-2
R7O2 R702/J53 0-2
F114 F J 1 4 lac zzf-20:: Tnl0/MC1060 0-3
F l l recA VrecA* lacP z'/Alac-pro thi rifA rpsL 0-3
J53 pro' met~ rifA 0-3
JA2OO thr-1 leuB6 trpE63 recA56 005
thi-1 ara-14 lacYl galK2 galT22
xyl-5 mtl-1 k~ supE44 (glnVU)

S. typhimurium
627 F\H/pyrC rpsLl 0-2
Plasmids
pMC1314 lac 'zy Knf
pUR288 lacpoz" Apr
R68.45 Ap r Tc 1 Km' IncP
R702 Km' Smr Sur H ^ Tc1 IncP
Flac F u 114 lac zzf-20:: TnlO

Conjugal transfer of R factors to recipient cells was performed by the method of

Curtiss (1981) in which 10 log phase donor cells were mixed with recipient cells in
ratios ranging from 01 to 0-25 in a total volume of 2 ml in 125 ml Erlenmeyer flasks
maintained at 37C during the conjugation period. Following conjugation, cells were
diluted with 0-85% NaCl solution with 10% L broth and spread on either minimal or
complete medium with tetracycline to select for transconjugants.
The formation of drug-gyrase-DNA complexes was assayed by the electrophoretic
mobility of DNA in cell lysates. Mid log phase cells (1 ml) (A 600 ~ 0-4-0-6) were
collected by centrifugation, resuspended in 0-2 volume 50 mM glucose, 50 mM Tris-
HC1, lOmM EDTA, pH 8-0, containing 4 mg/1 lysozyme, and incubated in 1-5 ml
microcentrifuge tubes at room temperature for 5 min. Cells were lysed by the addition
of sodium dodecylsulphate (SDS) to a final concentration of 0-5%. The lysate was
4 J. B. Cum (right el aL

treated with 25 mg/1 proteinase K for 2 h at 37C; 5-10 /il aliquots of the treated lysate
were placed directly in wells in 0-7% agarose slab gels and electrophoresed in Tris-
EDTA-acetate buffer for 3-4 h at 1 V/cm. Under these conditions, 50-60 kb DNA
fragments are detected after electrophoresis of quinolone-treated, but not control, cell
samples. For the analysis of plasmid DNA following enoxacin treatment, mid log
phase (2 x 108) cells were treated for 30 min and concentrated three-fold by
centrifugation, and plasmids were isolated by either the alkaline lysis procedure
(Maniatis et al., 1982) or by the boiling method (Holmes & Quigley, 1981). Plasmid
DNA was precipitated by two volumes of ethanol and resuspended in 25 [d 10 mM
Tris-HCl, 01 mM EDTA, pH 8-0. Plasmid samples were analysed on agarose slab gels

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as described above for chromosomal preparations.
Induction of /7-galactosidase was performed by first incubating cells with enoxacin
for 30 min, followed by 1 DIM of the gratuitous inducer, isopropylthiogalactoside
(IPTG). In assays for quinolone-dependent induction of the recA system, cells were
incubated with enoxacin for 60 min. Cell density was measured at 580 nm and aliquots
of cells were treated with chloroform and SDS as described (Miller, 1972). /J-
Galactosidase was assayed by the time-dependent formation of o-nitrophenol at A 420 .
Enzyme activities are expressed in terms of /flnole/min/absorbancy unit of cells.

Results
SDS-induced chromosome breakage by quinolones
Cell lysates of quinolone-treated cells were prepared and the electrophoretic properties
of the chromosomal DNA were determined as described. The highly viscous
chromosomal DNA from lysates prepared with the nondenaturing detergent Triton-
XI00, and without proteinase K, did not enter the agarose gel under these conditions.
In contrast, SDS-treated lysates from cells incubated with enoxacin were less viscous
and migrated on agarose gels only slightly less than bacteriophage lambda DNA.
When samples were prepared from cells incubated in the presence of subminimal
inhibitory concentrations of enoxacin, a definite change in the average DNA mobility
was noted, with a substantial fraction of the DNA migrating with the mobility
characteristics of 50-70 kb linear fragments (Figure 1).
When oxolinic or nalidixic acid replaced enoxacin, a similar increase in the
electrophoretic mobility of chromosomal DNA was noted at both MIC and
subminimal inhibitory concentrations. This effect was limited to gyrA inhibitors since
no chromosomal cleavage was observed in extracts from cells incubated with
coumermycin Al or novobiocin. Moreover, normal SDS-induced chromosomal
breakage was observed in the quinolone-sensitive, gyrB strain. No chromosome
breakage was detected when quinolone-resistant, gyrA strains 14-1, KL163, and
RM100 were incubated with enoxacin at concentrations up to 5 mg/1.
The effect of short-term enoxacin treatment on plasmid DNA was examined by
electrophoretic analysis of pMC1314, prepared either by the SDS-alkalinc lysis
procedure or by the boiling method. In the case of this plasmid, there was no
detectable change in the quantity of plasmid DNA which was obtained nor in the
mobility of the plasmid molecules (Figure 2). Linear molecules could be detected in
some preparations, but as was shown in a previous study (Lockshon & Morris, 1983),
they always represented a small proportion of the total plasmid DNA. Changes in the
proportions of plasmid topoisomers after 10 min of enoxacin treatment were not
detected after electrophoresis in the presence of chloroquine (Figure 2(b)).
 Alterations in DNA and gene activity with eooxadn

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Figure 1. Effects of enoxadn on chromosome structure. Cells (strain 1314) were treated for 10 min with
the indicated concentrations of enoxadn and lysates prepared and dectrophoresed as described io Methods.
Lambda DNA it included as a molecular marker. Samples treated with enoxadn as foDows: (a) control; (b)
0-05 mg/1; (c) 0-1 mg/1; (d) 0-2 mg/1; (e) 0-4 mg/1; ( 0 1-0 mg/1; (g) 2-0 mg/1.

Figure 2. Effects of enoxadn on plasmid pMC1314. Ptaxmid DNA was prepared by the alkaline lysis
procedure from cells treated for 10 min with various concentrations of enoxadn and ekctrophoresed as
described in Methods. A, Same a Figure 1. oc. Open circles; ccc, covalently closed circles. B,
Electrophoresis in 7-5 mg/1 chloroquine of plasmids (prepared by method of Quigley & Holmes, 1981).
Samples treated as follows: a, control; b, 0-3 mg/1; c, 0-6 mg/1; d, 2-0 mg/1; e, 6-3 mg/1.
 J. B. Conrtrigfat et at.

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1-5 2-0
Enoxacin (mg/l)
Figure 3. Inhibition of DNA synthesis by enoxacin. Strains C600 rpsL ( ) and C600 rpsL gyrA (A) were
pretreated with the indicated concentrations of enoxacin for S min, followed by the addition of
*H-thymidine for 2 min. The amount of thymidine incorporation in the treated samples is expressed as a
fraction of incorporation in the untreated control.

Inhibition of DNA synthesis by enoxacin

The fact that SDS-induced chromosome breakage occurred at subminimal inhibitory
concentrations of enoxacin suggested that this agent could have similar effects on in-
vivo DNA synthesis. Both gyrA* and gyrA strains were incubated with varying
concentrations of enoxacin for two minutes and the amount of DNA synthesis
measured. As shown in Figure 3, partial inhibition of DNA synthesis in the sensitive
strain occurred at enoxacin levels as low as 005 mg/1 and more than 95% inhibition
was achieved with 0-5 mg/1. DNA synthesis in the resistant strain was not inhibited by
enoxacin concentrations as high as 2 mg/1.

Induction of recA dependent SOS system by enoxacin

Previous studies (Weisemann et at., 1984) have shown that recA: lacZ fusion strains
can be used to assay for the effect of UV light, mitomycin C and nalidixic acid on the
induction of the SOS system. Strain GE94 contains a fusion of the first part of the
recA gene to the lacZ gene, thereby providing a convenient assay for recA gene
induction. GE94 was grown in the presence of enoxacin for 1 h and the amount of f}-
galactosidase produced was measured by spectrophotometric assay. As shown in
Figure 4, there was little induction at subminimal inhibitory concentrations but a six-
fold increase at inhibitory concentrations. The extent of induction at 10 mg/1 enoxacin
was less than at lower concentrations. When induction was measured in GE94-1, a
gyrA derivative of strain GE94, it was found that there was only minimal induction of
 Alterations in DNA and gene actirtty with eooxadn

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2 -'

0-3 I 3 10
Enoxacin (mg/l)
Figure 4. Induction of recA gene by enoxacin. The recA-lacZ fusion strain GE94 (#) and its enoxacin
resistant derivative GE94-1 (A) were grown in the presence of the indicated concentrations of enoxacin for
1 h and the amount of ^-galactosidase activity measured. The arrows indicate the respective MIC for each
strain.

RecA synthesis until concentrations inhibitory to this strain were reached. Similar
results were also obtained when these strains were incubated in nalidixic or oxolinic
acid.

Effect of quinolones on plasmid maintenance

Strain MC1314 containing Lac+ Km' plasmid pMC1314 was used as an indicator
strain for monitoring the effects of enoxacin and other quinolones on plasmid
maintenance. In this strain, the plasmid is fairly stably maintained and no more than
12% of the cells lose the plasmid after 24 h growth. After 6 h exposure to enoxacin
more than 95% of the cells surviving treatment with 0 1 , 0-2, or 0-3 mg/l enoxacin were
Lac" (Figure 5). The extent of pMC1314 plasmid loss was also monitored after
treatment with either nalidixic or oxolinic acid. Both these antibiotics also induced
plasmid loss, but the fraction of plasmid free cells at 1/2 MIC after 24 h treatment
ranged from 17-63% (Table IT). With all three quinolones the extent of plasmid loss at
these concentrations was significantly less in the quinolone resistant MC1000-1 gyrA
derivative. Parallel experiments to determine the loss of pUR288 demonstrated that,
even though this plasmid was lost at a higher rate spontaneously, there was an
increased number of plasmid-free cells following treatment with various quinolones.
Unexpectedly, there was less pUR288 loss at higher enoxacin concentrations. This
contrasts to the pattern with pMC1314, where higher enoxacin concentrations resulted
in a higher fraction of plasmid free cells. In these experiments, representative Lac"
cells were separately shown to be lacking the pMC1314 or pUR288 plasmid, as based
 J. B. Coartright et aL

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Figure 5. Loss of pMC1314 during enoxacin treatment Strain 1314 was grown in the presence of sub MIC
of enoxacin and the fraction of plasmid free cells determined by plating on MacConkey medium. Cells were
treated with no ( ) , 0-1 mg/1 (O), or 0-2 mg/1 (A) enoxacin.

Table IL Plasmid elimination by gyrase inhibitors

Percent plasmid-free colonies

gyrA + gyrA
Quinolone (mg/1) pUR288 pMC1314 pMC1314

None 340 110 110

Nalidixic acid
0-3 9-6
0-6 47-0 170
1.2 65-0 360 350
Oxolinic acid
006 730 240
012 850 360 250
0-25 270 1000 28-0

Enoxacin
0.03 700 170
006 470 630
012 450 900 160
0-25 1000
0-50 290
100 280
200 730
 Alterations in DNA and gene acthity with enoxadn

Table ID. Effect of enoxacin on F u lacl 14 elimination

Percent plasmid free cells

Bacteria Enoxacin (mg/1) gyrA+ gyrA

E. colt 0 6-9 3-5

003 0-8
0-06 0-9
012 0-9
0-50 no growth 0-9
1-00 no growth 0-5
2-00 no growth 0-3

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S. typhinwium 0 0
01 22-7
0-2 0-7

on both the sensitivity to kanamycin and ampicillin, respectively, and the absence of
the 915 kb or 8-5 kb plasmid in cell extracts.
To test for the generality of this enoxacin effect on plasmid maintenance, Escherichia
coli strains containing plasmid FtsLac :: TnlO and Salmonella typhimurium containing
FtsLac :: TnlO were also grown in subminimal inhibitory concentrations of enoxacin.
After growth for 24 h, the fraction of plasmid free cells in the treated culture was
compared with that in the control culture (Table m). Flac was eliminated from about
12% of the MC1000 cells after 18 h growth but was only lost from 1-2% of the cells
grown at subminimal inhibitory concentrations. A similar effect with high enoxacin
concentrations was observed in FtsLac:: TnlO/MC1000-l grown in 2 mg/1 enoxacin.
When the stability of Flac was measured in 5. typhimurium 627, it was found that
more than 20% of the cells grown in the presence of 0-1 mg/1 enoxacin were plasmid
free; no significant loss was observed at higher enoxacin concentrations. Comparable
experiments were also carried out with E. coli strains harbouring the Tet1 R factor
plasmid R68.45 and R702. No elimination of these plasmids was detected at any of the
enoxacin concentrations tested.

Reduced R factor transfer in enoxactn-treated samples

Conjugal R factor transfer was measured in experiments in which donor strains with R
plasmids were mated in the presence of enoxacin. The two R plasmids studied here,

Table IV. Inhibition of transconjugation by enoxacin

No. of transconjugants
Enoxacin Percent
Donor Recipient Untreated (10 mg/1) reduction

R68 C600 8-6x10* 6-5 xlO 3 92-4

R702 C600 l-4x!0 7 3-5x10* 74-7
R68 C600-3 2-8 xlO 7 1-lxlO 7 60-2
R702 C600-3 3-5x10* 1-7x10* 53-2
10 J. B. Courtrigbt et aL

R68.45 and R702 each contain a gene for tetracycline resistance and the transmission
of this Tet resistance determinant to sensitive recipients was employed as the basis for
determining the number of transconjugants. In crosses with both donor and recipient
strains sensitive to enoxacin, there was more than a 90% reduction in the number of
transconjugants (Table IV). To eliminate a possible effect of enoxacin on recipients,
crosses of these R factor donors to gyrA recipients were also performed. With both R
factors studied, there was a substantial reduction in the number of transconjugants
obtained, indicating that the primary effect on the donor cells was due to enoxacin.

Altered gene expression during enoxacin treatment

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To measure how enoxacin might alter chromosomal or plasmid gene expression,
experiments were undertaken to determine the extent to which lacZ gene activity was
modified during treatment. Cells with lacZ gene on either the chromosome or a
plasmid were grown with varying concentrations of enoxacin for 30 min, induced with
IPTG for 30 min and assayed for the activity of /?-galactosidase. All sensitive strains
studied showed an inhibitory effect of enoxacin on the inducibility of the LacZ activity
at all concentrations studied. To allow for strain differences, the reduction in lacZ
inducibility was compared with the MIC for each strain. When compared in this
manner, the results indicated that inhibition of /?-galactosidase induction was less in
strains with a plasmid borne lacZ gene (Table V). However, with the exception of the
slight increase in inducibility at low concentrations, there was a uniform decrease in
lacZ inducibility at all concentrations tested. Since this difference in the inducibility
between chromosomal and plasmid lacZ sequences may be related to the copy number
and/or the superhelical density of the Co! El like replicons, induction studies with a
low copy number of plasmid would provide a means of determining if there were
fundamental differences in enoxacin effects on plasmid genes. When induction studies
were carried out with wild type and strain Fiac/MClOOO, containing 1-2 copies of a
150 kb F plasmid (Palchaudhuri, Maas & Ohtsubo, 1976), the extent of /?-
galactosidase induction was similar to that with strains with a chromosomal gene
(Table V).

Table V. Inhibition of LacZ induction by enoxacin

Percent of control after

1 h enoxacin treatment
Strain Lac Genes iMIC MIC 2xMIC
Chromosomal
W1485 lac i + poz + y + a + 70-8 53-2 41-8
JA200 lac i + poz + y"a + 92-5 80-3 74-6
RM100 (gyrA) lac i + poz + y + a + 1000 100-0 91-0

Plasmids
MC1000/Flacll4: :TnlO lac i + poz + y + a + 951 79-9 48-2
FllrecA/pUR288 lac p o / 1040 88-4 63-6
pLC20-30/JA20O lac i + poz + y + a + 1020 1000 93-4
 Alterations In DNA and gene activity with enoxadn 11

Novel type of antibiotic synergy in strains with a resistance plasmid

These combined results suggested that pretreatment of cells with enoxacin could result
in a reduction in the level of expression of other genes, including those of plasmid
encoded antibiotic resistance. As a consequence of this pretreatment, the level of
expression of a resistance determinant would be decreased and the sensitivity of the
surviving cells to this antibiotic would be correspondingly increased. This hypothesis
can be tested in strain 1314, which has a plasmid encoded gene for kanamycin
resistance. Thus, cells which are treated with enoxacin could also become more
susceptible to kanamycin.
The results of this experiment with strain 1314 are presented in Table VI. The

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samples which were not exposed to kanamycin showed a significant enoxacin
bactericidal effect at concentrations greater than 0-2mg/l, while there was only
minimal killing of this strain at concentrations less than 50mg/l kanamycin. An
example from this experiment is seen at antibiotic combinations involving enoxacin at
01 mg/1 and kanamycin at 10 mg/1. Even though this concentration of kanamycin only
reduced cell survival by 15%, the approximate fraction of plasmid free cells in this
strain, there was even a more marked reduction in viable cells at the enoxacin
concentrations tested. Further examples of synergy were also noted at higher
kanamycin concentrations.
The increased susceptibility of strain 1314 when incubated in vitro with both
enoxacin and kanamycin suggested that a similar combination of these antibiotics
could be studied in mice with infections of bacteria with resistance factors. For this
experiment, four groups (I-IV) of female CD-I mice (18-22 g) were infected by
intraperitoneal injection with 10 x the LD50 of strain 1314. Group I served as a
virulence control, group II was given enoxacin by the oral route immediately after
inoculation, group m was given kanamycin intraperitoneally in varying
concentrations 1 h after the bacterial inoculation, and group IV was given enoxacin
orally as in group II and kanamycin intraperitoneally after 1 h as in group III. The fate

Table VI. Synergy of enoxacin and kanamycin in vitro

Cell titre (cfu/ml)

<
Kanamycin Enoxaciri(mg/l)
Strain (mg/1) 0 01 0-2 0-3 0-4 0-5
7
MC1000 0 1-0x10' 4-5 x 10* l-3xl0 1-2x10' <300
200 3-5 xlO 7 2-0x10** 10x10** <300* <300
300 2-0x10* 2-0xl0 2 * 2-5 xlO 3 <300 <300
400 3-0 xlO 3 2-4xlOJ l-2xlO 2 <300 <300
MC1314 0 9-5 x 10' 4-2x10' 1-OxlO7 11 xlO* l-3xlO 3
10 8-2x10' 3-2x10' 5-3 x 10* 4-9x10** 5-OxlO3
50 4-6x10' 2-6 xlO 7 1-OxlO5* 4-4 xlO3* <300*
100 2-2x10' 2-4 xlO 7 31 x 10** <300 <300*
250 21 x 10* 1-0x10** <300* <300 <300*
500 1-4x10* 370 <300* <300*
Antibiotic combination* yielding l(H-fold lower cfu/ml are marked with an (*), indicating synergy by the
criterion of Hallander et al. (1982).
12 J. B. Coortright et aL

Table VII. Synergy of enoxacin and kanamycin in infected mice

NumbeT of
Concen- survivor
tration Percent survivors (number
Experiment Antibiotic mg/kg 24 h 48 h 72 h 96h tested)
I 0 0 0 0 0(10)
II enoxacin 0-8 20 0 0 0 0(10)
enoxacin 1-6 10 0 0 0 0(10)
enoxacin 20 84 38 8 4 1(25)

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ni kanamycin 6-3 12 0 0 0 0(8)
kanamycin 250 25 0 0 0 0(8)
kanamycin 100-0 38 12-5 12-5 12-5 1(8)
rv enoxacin (0-8 mg/kg)
kanamycin 6-3 29 15 15 15 1(7)
kanamycin 250 50 12-5 12-5 12-5 1(8)
kanamycin 1000 70 12-5 12-5 12-5 1(8)
enoxacin (1-6 mg/kg)
kanamycin 6-3 88 0 0 0 0(8)
kanamycin 25-0 63 25 25 25 2(8)
kanamycin 1000 25 12-5 12-5 12-5 1(8)
enoxacin (2-0 mg/kg)
kanamycin 6-3 70 63 50 50 4(8)
kanamycin 250 75 75 75 75 6(8)
kanamycin 1000 88 88 88 88 7(8)

of the injected bacteria was monitored in selected group II mice by plating on

MacConkey medium samples from either cardiac puncture or peritoneal Iavage to
determine the frequency of plasmid loss. The fraction of plasmid loss was 50% and
90% at 7 and 24 h postchallenge, respectively. All mice were observed for 96 h and
scored for morbidity and mortality.
Under these conditions, the LD J 0 for strain 1314 was 1-3 x 107 cfu/ml. As shown in
Table VII, the inoculation of 10xLD s o resulted in death in all group I mice within
24 h. There was no effect when enoxacin was administered at doses of 0-8 and
1 -6 mg/kg while a dose of 20 mg/kg resulted in survival of one of 25 mice. Kanamycin
at doses of 6-3 and 25 mg/kg did not protect against strain 1314; at 100 mg
kanamycin/kg only one of eight mice survived 96 h. In the group IV animals,
significant increases in survival time were seen at all three non-protective kanamycin
doses. The combination of 20mg enoxacin/kg with 100mg kanamycin/kg resulted in
88% survival of infected mice after 96 h.

Discussion
Enoxacin and other fluoroquinolones have recently been shown to act as efficient
inhibitors of DNA gyrase in vitro (Domagala et al., 1986) and are therefore likely to
 Alterations in DNA and gene activity with eooxacin 13

have effects on E. coli K.12 and other Gram-negative bacteria similar to those
described for nalidixic acid and other quinolones (CozzareUi, 1980; Crumplin et al.,
1984). The early cellular events following exposure to both fractionally inhibitory
concentrations of enoxacin and concentrations in excess of the MIC described in this
report clearly indicate that enoxacin affects several features of DNA synthesis, plasmid
maintenance, and gene expression that have not been fully documented for other
4-quinolones.
In the presence of enoxacin, there is an immediate reduction in E. coli DNA
synthesis, which occurs even at concentrations below the MIC. A decrease in the rate
of synthesis is also seen, but to a lesser extent and only at substantially higher

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concentrations, in a strain with a gyrA mutation. Similar immediate effects on DNA
synthesis also occurs when cells are inhibited by oxolinic acid (Engle et al., 1982) and
can be attributed to the involvement of gyrase in the E. coli replication complex
(Kaguni & Kornberg, 1984). The decreases in R plasmid conjugal transfer in the
presence of enoxacin can be attributed to direct inhibition of the DNA synthesis
required for transfer of the donor chromosome, although effects due to differential
gene expression or to the SOS response, as further explained below, cannot be
excluded.
In addition to the inhibition of DNA replication, there is also a rapid formation of
an apparent enoxacin-gyrase-DNA complex. This complex is readily cleaved by SDS,
but not by Triton X, into linear fragments with a length in excess of 50 kb. Similar
cleaving has been shown for chromosomes from nalidixic acid inhibited E. coli
(Crumplin & Smith, 1975; Snyder & Drlica, 1979). In the case of enoxacin, these
current studies have shown that there is detectable DNA cleavage at sub MIC
concentrations (005-0-2mg/1) for normal as well as gyrB mutant strains sensitive to
the effects of this agent. This evidence, when combined with the virtual absence of SDS
associated linear fragments in lysates from enoxacin treated strains with gyrA
mutations, suggests that this cleavage is caused by binding of the A subunit of DNA
gyrase to numerous chromosomal sites in addition to binding at the site of DNA
replication. As nearly all the chromosome is cleaved into 50-70 kilobase linear
fragments, this indicates that the involvement of DNA gyrase in chromosome topology
occurs at approximately 60 equidistant sites on the bacterial chromosome. The relative
importance of these topological sites to the overall rate of chromosome replication is
not fully known, but they may be as important as the gyrase reactions that occur at the
replication fork (Drlica, 1984).
This rapid formation of gyrase complexes with the bacterial chromosome contrasts
with the negligible effects of enoxacin or other quinolones on the physical structure of
the bacterial plasmids. After 10 min treatment, there is no major change in the number
of detectable linearized plasmids or in the distribution of plasmid topoisomers
(Figure 2). This result suggests that a significant change in chromosomal topology is
likely to be a major disruptive event in quinolone treated cells and that other
phenomena, such as plasmid elimination, are likely to be secondary effects of
diminished expression of bacterial chromosomal genes required for plasmid
maintenance. Further confirmation of this hypothesis can be noted in the minimal
effects that enoxacin has on plasmid gene expression (Table V), even though these
concentrations are in the range which yield the highest fraction of plasmid free cells
(Table II).
An additional comparison between the MIC and a cellular effect is seen during the
14 J. B. Coortrigbt et al.

induction by enoxacin of the SOS system. In this case, it seems likely that a signal is
generated by single strand DNA tracts in the vicinity of the drug-gyrase-DNA
complex. The evidence cited above indicates that such drug-gyrase complexes are
already being formed during incubation with low concentrations of enoxacin, as
suggested by the SDS associated linear DNA fragments that can be detected. In this
context, therefore, it is important to note that there is measurable induction of the RecA
component (Figure 4), and presumably other members of the damage-inducible
system, with enoxacin, but not with novobiocin or coumermycin A, at sub-MIC. Thus,
there is every reason to believe that this induction is directly related to inhibition of
gyrase action. In an enoxacin resistant gyrA derivative of strain GE94, there is no

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induction of the recA gene at enoxacin concentrations less than 1 mg/1 (Figure 4). This
correlates well with the observation that in strains with a gyrA mutation there is no
formation of SDS associated DNA linear fragments at these enoxacin concentrations.
These combined results are consistent with the hypothesis that the formation of gyrase-
DNA complexes generates a signal which induces the SOS repair systems.
In addition to the induction of the SOS system by enoxacin, treatment with other
quinolones (McPartland, Green & Echols, 1980; Weisemann et al., 1984), mitomycin C
and UV light leads to activation of a recA protease (Tessman & Peterson, 1985) and
the resulting induction of the damage inducible (SOS) genes as well as resident
prophage. Cells subjected to continued induction of the SOS system will produce large
quantities of the SfiA protein, an inhibitor of cell division (Huisman & D'Ari, 1981;
Drapeau, Gariepy & Boule, 1984), with the result that cell division does not occur and
cellular filaments are formed (Crumplin et al., 1984). Since the SOS response requires
RNA and protein synthesis, it is possible that this regulatory system may account for
the protective action of protein or RNA synthesis inhibition (Crumplin & Smith, 1975;
Crumplin et al., 1984; Benbrook & Miller, 1986) on nalidixic acid induced killing.
Moreover, the fact that both the recA441 and lexA(Ts) mutations are lethal at 42C
indicates that the induction of the SOS system can be lethal to a cell unless the
inducing stimulus is removed or the sfiA gene is inactivated by mutation. The
bacteriostatic action of high concentrations of nalidixic acid, and to a lesser extent that
of norfloxacin (Crumplin et al., 1984), may also be due to an inhibition or prevention
of the SOS induction.
Included among the manifestations of the SOS response is an increased susceptibility
to antibiotics and membrane active agents such as sodium cholate (Tessman &
Peterson, 1985). Although many features of this membrane effect have yet to be
described, it seems likely that SOS related changes in the bacterial membrane may be
implicated in the release of endotoxin from ciprofloxacin treated cells (Cohen &
McConnell, 1986) or in the increased membrane permeability seen during nalidixic
acid treatment (Dougherty & Saukkonen, 1985). This enhanced permeability may also
be a contributing factor in the synergistic interactions between enoxacin and
kanamycin (Table FV).
In contrast to the induction of the SOS system, enoxacin and other quinolones have
the more general property of inhibiting both chromosomal and plasmid gene
expression. In the current studies, inducibility of lacZ, the gene encoding /J-
galactosidase, served as a useful model for assaying for the effects of topological
changes caused by gyrase inhibition. Regardless of whether lacZ is located on the
chromosome, the Fiac plasmid or on a multicopy cloning vector, pretreatment of cells
with enoxacin significantly reduced the IPTG inducibility of this operon. This
 Alterations in DNA and gene actirtty with enoxadn IS

reduction is not seen in gyrA strains, indicating that this effect is due to a change in
DNA topology rather than a possible direct effect of enoxacin on RNA polymerase.
Even though the recA system, as a member of the SOS system, is induced by enoxacin
damage, this induction is decreased at high drug concentrations. It seems likely that
these transcriptional effects are due to changes in the superhelical densities of
chromosomal and plasmid molecules, resulting in truncated gene transcripts (Brahms
et al., 1985). In addition to decreases in the expression of the lacZ gene, quinolones
have been shown to cause substantial decreases in the level of many, but not all,
cellular genes (Drlica, 1984).
Changes in gene expression are likely to be a contributing factor, if not the primary
basis, for many of the cellular responses to quinolone treatment. It is apparent that a

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cell's ability to replicate and to express plasmid genes is critically important to those
bacteria whose antibiotic resistance is specified by plasmid genes. At the level of
replication, plasmids, such as pMC1314 and pUR288 with P15A or ColEl origins of
replication (Casadaban et al., 1980; Ruther & Muller-Hill, 1983) require both the
chromosomal polA gene product (Chang & Cohen, 1978) and also transcription of
primer RNA at the origin of replication (ori) (Messing, Staudenbauer & Hofschneider,
1972; Tomizawa, 1986). Inhibition of transcription or expression of these genes by
enoxacin should interfere with the replication of daughter chromosomes and would
lead to increases in the frequency of plasmid-free cells. Such a plasmid curing effect has
been shown to occur with several quinolones and with a variety of plasmids, albeit at
different efficiencies (Hooper et al., 1984; Weisser & Wiedemann, 1985). Moreover, the
same plasmids are eliminated with different frequencies in different host strains
(Table III), thereby indicating the importance of the bacterial genotype in controlling
plasmid maintenance and replication, possibly by means of the RecD protein (Biek &
Cohen, 1986). As indicated in earlier studies, these plasmid losses may also be caused
by inhibition of the GyrB subunit. Consequently, it is not entirely clear whether or not
the elimination of the P15A based plasmid (pMC1314) or the ColEl based plasmid
(pUR288) is due to antagonism of the B subunit by enoxacin or, as seems also likely,
due to an alteration in the expression of genes necessary for plasmid replication.
Recent studies of the effect of recD mutations on plasmid stability indicate that this
gene plays a central role in determining the relative stability of low copy and high copy
number plasmids. Strains with recD mutations lose ColEl-like replicons at a high rate
but these mutations have little effect on the stability of F and R plasmids (Biek &
Cohen, 1986). Since a similar difference is seen in the present studies with pMC1314
and Flac, it is quite possible that this differential effect is due to a decreased expression
or activity of the recD gene during enoxacin treatment. In view of the diversity of
plasmids that are eliminated, or retained, with differing efficiencies by quinolones
(Cejka et al., 1982; Hooper et al., 1984), future studies directed towards elucidating the
transcription and function of the recD product should help identify the relevant
cellular targets of this gene and to clarify the possible mechanisms involved in plasmid
maintenance (Clewell, Evenchik & Cranston, 1972; Sonstein & Baldwin, 1972; Hahn &
Ciak, 1976).
A second important consequence of the decrease in gene expression is that plasmid
genes will be affected to nearly the same extent as chromosomal genes. In this case, it
makes little difference whether or not the alteration in gene expression is brought
about primarily as a result of an overall change in plasmid topology (Lockshon &
Morris, 1983) and supercoiling (Brahms et al., 1985) or a change in the net
16 J. B. Coartright et aL

transcription of individual plasmid genes (this paper, Table III; Drlica, 1984; Gomez-
Eichelmann, 1981). Either of these mechanisms, in conjunction with the chromosomal
genes required for plasmid maintenance, will produce as a net result fewer plasmid
molecules per daughter cell or decreased expression of plasmid genes. We have used
this rationale for predicting that the combined use of enoxacin with an antibiotic,
whose resistance determinant is specified by a plasmid gene, should prove more
effective than either antibiotic used singly.
When these synergy experiments were done with both in-vitro and in-vivo model
systems, more bacterial killing occurs in vitro and more infected animals survive than
in experiments where each antibacterial is used singly. The explanation for this novel

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form of synergy, as derived from the known effects of quinolones, is that enoxacin
causes either plasmid elimination or a reduction in the expression of plasmid specified
neomycin phosphotransferase (for KanO- It therefore seems likely that at subminimal
inhibitory enoxacin concentrations there is (1) a partial inhibition of cellular DNA
synthesis, (2) induction of the bacterial SOS repair system, leading to increased
susceptibility or permeability to enoxacin and/or kanamycin (Tessman & Peterson,
1985), and (3) impaired plasmid function, including antibiotic resistance gene
expression as well as replication related transcription. These numerous cellular
responses can be attributed to the demonstrated ability of enoxacin to inhibit DNA
gyrase (Domagala et al., 1986). At the cellular level, this agent halts DNA synthesis
and forms drug-gyrase complexes at numerous sites on the bacterial chromosome. A
significant consequence of these multiple interactions is that enoxacin can be used in
vivo to diminish plasmid encoded kanamycin resistance, resulting in the success of
combined therapy in infected mice. As a consequence of any or all of these alterations,
it can be assumed that enoxacin is unlikely to select for the survival of cells carrying an
antibiotic resistance plasmid, even at sublethal doses. Clearly this type of synergy
occurs with strain 1314 and this approach may hold promise as an effective mechanism
for treating infections caused by organisms resistant to one or more antibiotics by
virtue of plasmid-borne determinants.

Acknowledgement
We thank the Warner-Lambert Corporation for their support of this research.

References
Benbrook, D. M. & Miller, R. V. (1986). Effects of norfloxacin on DNA metabolism in
Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 29, 1-6.
Biek, D. P. & Cohen, S. N. (1986). Identification and characterization of recD a gene affecting
plasmid maintenance and recombination in Escherichia coli. Journal of Bacteriology 167,
594-603.
Brahms, J. G., Dargouge, O., Brahms, S., Ohara, Y. & Vagner, V. (1985). Activation and
inhibition of transcription by supercoiling. Journal of Molecular Biology 181, 455-65.
Burman, L. G. (1977). R-plasmid transfer and its response to nalidixic acid. Journal of
Bacteriology 131, 76-81.
Casadaban, M. J., Chou, J. & Cohen, S. N. (1980). In vitro gene fusions that join an
enzymatically active /5-galactosidase segment to ammo-terminal fragments of exogenous
proteins: Escherichia coli plasmid vectors for the detection and cloning of translational
initiation signals. Journal of Bacteriology 143, 971-80.
Cejka, K., Holubova, I. & Hubacek, J. (1982). Curing effect of clorobiocin on Escherichia coli
plasmids. Molecular and General Genetics 186, 153-5.
 Alterations in DNA and gene actirtty with enoudn 17

Chang, A. C. Y. & Cohen, S. N. (1978). Construction and characterization of amplifiable

multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. Journal of
Bacteriology 134, 1141-56.
Chumley, F. G., Menzel, R. & Roth, J. R. (1979). Hfr formation directed by TnlO. Genetics 91,
639-55.
Clewell, D. B., Evenchik, B. & Cranston, J. W. (1972). Direct inhibition of Col El plasmid
DNA replication in Escherichia coli by rifampicin. Nature New Biology 237, 29-31.
Cohen, J. & McConnell, J. S. (1986). Release of endotoxin from bacteria exposed to
ciprofloxacin and its prevention with polymyxin B. European Journal of Clinical
Microbiology 5, 13-7.
Cozzarelli, N. R. (1980). DNA gyrase and the supercoiling of DNA. Science 207, 953-60.
Crumplin, G. C. & Smith, J. T. (1975). Nalidixic acid: an antibacterial paradox. Antimicrobial
Agents and Chemotherapy 8, 251-61.

Downloaded from http://jac.oxfordjournals.org/ at University of California, Santa Barbara on June 29, 2015
Crumplin, G. C , Kenwright, M. & Hirst, T. (1984). Investigations into the mechanism of action
of the antibacterial agent norfloxacin. Journal of Antimicrobial Chemotherapy 13, Supp. B,
9-23.
Curtiss, R. (1981). Gene transfer. In Manual of Methods for General Bacteriology (Gerhadt, P.
Ed.), pp. 243-65. American Society for Microbiology, Washington, DC.
Danilevskaya, O. N. & Gragcrov, A. I. (1980). Curing of Escherichia coli K12 plasmids by
coumermycin. Molecular and General Genetics 178, 233-5.
Domagala, J. M., Hanna, L. D., Heifetz, C. L., Hutt, M. P., Mich, T. F., Sanchez, J. P., et al.
(1986). New structure-activity relationships of the quinolone antibacterial using the target
enzyme. The development and application of a DNA gyrase assay. Journal of Medicinal
Chemistry 29, 394-404.
Dougherty, T. J. & Saukkonen, J. J. (1985). Membrane permeability changes associated with
DNA gyrase inhibitors in Escherichia coli. Antimicrobial Agents and Chemotherapy 28,
200-6.
Drapeau, G. R., Gariepy, F. & Boule, M. (1984). Regulation and SOS induction of division
inhibition in Escherichia coli K12. Molecular and General Genetics 193, 453-8.
Drlica, K. (1984). Biology of bacterial deoxyribonucleic acid topoisomerases. Microbiological
Reviews 48, 273-89.
Engle, E. C, Manes, S. H. & Drlica, K. (1982). Differential effects of antibiotics inhibiting
gyrase. Journal of Bacteriology 149, 92-8.
Gellert, M. (1981). DNA topoisomerases. Annual Review of Biochemistry 50, 879-910.
Gill, S. & Iyer, V. N. (1982). Nalidixic acid inhibits the conjugal transfer of conjugative
U incompatibility group plasmids. Canadian Journal of Microbiology 28, 256-8.
G6mez-Eicbelmann, M. C. (1981). Effect of nalidixic acid and novobiocin on pBR322 genetic
expression in Escherichia coli minicells. Journal of Bacteriology 148, 745-52.
Gottesman, S. (1984). Bacterial regulation: Global regulatory networks. Annual Review of
Genetics 18, 415-41.
Haas, D. & Holloway, B. W. (1976). R factor variants with enhanced sex factor activity in
Pseudomonas aeruginosa. Molecular and General Genetics 144, 243-51.
Hahn, F. E. & Ciak, J. (1976). Elimination of resistance determinants from R-factor Rl by
intercalative compounds. Antimicrobial Agents and Chemotherapy 9, 77-80.9.
Hallander, H. O., Dornbusch, K., Gezelius, L., Jacobson, K. & Karlsson, I. (1982). Synergism
between aminoglycosides and cephalosporins with antipseudomonal activity: Interaction
index and killing curve method. Antimicrobial Agents and Chemotherapy 22, 743-52.
Hedges, R. W., & Jacob, A. E. (1974). Transposition of ampicillin resistance from RP4 to other
replicons. Molecular and General Genetics 132, 31-40.
Holmes, D. S. & Quigley, M. (1981). A rapid boiling method for the preparation of bacterial
plasmids. Analytical Biochemistry 114, 193-7.
Hooper, D. C , Wolfson, J. S., McHugh, G. L., Swartz, M. D., Tung, C. & Swartz, M. N.
(1984). Elimination of plasmid pMGUO from Escherichia coli by novobiocin and other
inhibitors of DNA gyrase. Antimicrobial Agents and Chemotherapy 25, 586-90.
Huisman, O. & D'Ari, R. (1981). An inducible DNA replication-cell division coupling
mechanism in Escherichia coli. Nature 290, 797-9.
Jacob, A. E., Cresswell, J. M. & Hedges R. W. (1977). Molecular characterization of the P
group plasmid R68 and variants with enhanced chromosome mobilizing ability. FEMS
Microbiology Letters 1, 71-4.
18 J. B. Coortright et aL

Kaguni, J. M. & Kornberg, A. (1984). Replication initiated at the origin (oriQ of the
Escherichia coli chromosome reconstituted with purified enzymes. Cell 38, 183-90.
Kreuzer, K. N. & Cozzarelli, N. R. (1979). Escherichia coli mutants thermosensitive for
deoxyribonucleic acid gyrase subunit A: effects on deoxyribonucleic acid replication,
transcription and bacteriophage growth. Journal of Bacteriology 140, 424-35.
Lockshon, D. & Morris, D. R. (1983). Positively supercoiled plasmid DNA is produced by
treatment of Escherichia coli with DNA gyrase inhibitors. Nucleic Acids Research 11,
2999-3017.
McPartland, A., Green, A. L. & Echols, H. (1980). Control of recA gene RNA in Escherichia
coli: regulatory and signal genes. Cell 20, 731 -7.
Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning. Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY.

Downloaded from http://jac.oxfordjournals.org/ at University of California, Santa Barbara on June 29, 2015
Messing, J., Staudenbauer, W. L. & Hofschneider, P. H. (1972). Inhibition of minicircular DNA
replication in Escherichia coli by rifampicin. Nature New Biology 238, 202-3.
Michel-Briand, Y., Laporte, J-M. & Bassignot, A. (1983). Inhibition du transfert de plasmides
chez Escherichia coli, par l'acide oxolinique. Comptes Rendus des Seances de L'Academie des
Sciences. Serie III. Sciences de la Vie. Paris 296, 989-94.
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY.
Nakamura, S., Inoue, S., Shimizu, M., Iyobe, S. & Mitsuhashi, S. (1976). Inhibition of conjugal
transfer of R plasmids by pipemidic acid and related compounds. Antimicrobial Agents and
Chemotherapy 10, 779-85.
Palchaudhuri, S., Maas, W. K. & Ohtsubo, E. (1976). Fusion of two F-prime factors in
Escherichia coli: studied by electron microscope heteroduplex analysis. Molecular and
General Genetics 146, 215-31.
Pruss, G. J. & Drlica, K. (1985). DNA supercoiling and suppression of the leu-500 promoter
mutation. Journal of Bacteriology 164, 947-9.
Ruther, U. & Muller-Hill, B. (1983). Easy identification of cDNA clones. EMBO Journal 2,
Sanzey, B. (1979). Modulation of gene expression by drugs affecting deoxyribonucleic acid and
gyrase. Journal of Bacteriology 138, 40-7.
Snyder, M. & Drlica, K. (1979). DNA gyrase on the bacterial chromosome: DNA cleavage
induced by oxolinic acid. Journal of Molecular Biology 131, 287-302.
Sonstein, S. A. & Baldwin, J. N. (1972). Loss of the pcnicillinase plasmid after treatment of
Staphylococcus aureus with sodium dodecyl sulfate. Journal of Bacteriology 109, 262-5.
Sugino, A., Higgins, N. P. & Cozzarelli, N. R. (1980). DNA gyrase subunit stochiometry and
the covalent attachment of subunit A to DNA during DNA cleavage. Nucleic Acids
Research 8, 3865-74.
Taylor, D. E., Ng, L. K. & Lior, H. (1985). Susceptibility of campylobacter species to nalidixic
acid, enoxacin, and other DNA gyrase inhibitors. Antimicrobial Agents and Chemotherapy
28, 708-10.
Tessman, E. S. & Peterson, P. (1985). Plaque color method for rapid isolation of novel recA
mutants of Escherichia coli K12: New classes of protease-constitutive recA mutants. Journal
of Bacteriology 163, 677-87.
Tomizawa, J. (1986). Control of ColEl plasmid replication: binding of RNA I to RNA II and
inhibition of primer formation. Cell 47, 89-97.
Weisemann, J. M., Funk, C. & Weinstock, G. M. (1984). Measurement of in vivo expression of
the recA gene of Escherichia coli by using lacZ gene fusions. Journal of Bacteriology 160,
112-21.
Weisser, J. & Wiedemann, B. (1985). Elimination of plasmids by new 4-quinolones.
Antimicrobial Agents and Chemotherapy 28, 700-2.
Wolfson, J. S., Hooper, D. C, Swartz, M. N. & McHugh, G. L. (1982). Antagonism of the B
subunit of DNA gyrase eliminates plasmids pBR322 and pMGHO from Escherichia coli.
Journal of Bacteriology 152, 338-44.
Zeiler, H. J. (1985). Evaluation of the in vitro bactericidal action of ciprofloxacin on cells of
Escherichia coli in the logarithmic and stationary phases of growth. Antimicrobial Agents
and Chemotherapy 28, 524-7.