Cell Killing by the F Plasmid CcdB Protein Involves Poisoning

of DNA-Topoisomerase II Complexes
Philippe Bernard and Martine Couturier

In Escherichia coli. the miniF plasmid CcdB protein is rcsponsiblr for cell death when its
action is not prevented by polypeptide C’cdA. \Z’e report the isolation, localization.
scyuencing and properties of a bacterial mutant resistant to the cytotoxir activity of thf,
(‘(~111 protein. This tnut,ation is located in the gene rr~odinp t,he .A subunit of t,opoisomerasc
I I and producrs an Arg462 +C~J-s substitution in the amino acid sequenccl of the (:?-r;\
polypeptide, Hencca. the mutation was called gy.r.446,“. LYr show that in the wild-type strain.
ttr~ (‘cdl3 protein promotes plasmid linearization: in the gyr.44(i2 strain. this douhle-
stranded I)NA cleavage is suppressed. This indicates that the (%dH protein is responsible filr
gyrase-tnrtliated double-stranded DNA breakage. C’cdK, in the absencr of (WA. induces thrh
SOS pathway. SOS induction is a biological response to DSA-damaging agents. MT:r shon
t.hat thr gyrA4(i% mut.ation suppn~sses this SOS activation. indicating that SOS induct ion is
a cAonsrquenc:r of T)NA damages promoted by- t.he (‘cdK prot,ein 011 gy-asctI)X\‘A complrxc>s.
In additSion, WP observe that the CrdB” sensitive phenotype dominates over the resistant
phenotype>. This is better explained by the con\-crsion, in gyrA +/gyrA462 tnrrodiploicl
strains. of thr wild~tppe gyrase into a DNA-damaging agent. These results strongly suggflst
that t,he (IcdK protein. like quinolone antibiot,ics and a variet>- of antitumoral drugs. is a
11X.4 topoisomerasr IT poison, This is the first proteini(* I)oison-antipoison mecahanism that
has hren found to act rGa the 1)X=\ topoisomrrasr 11.
KP!/ IUW~S: miniF plasmid; topoinomerasc, I I poison: (IcdU prot,cin

1. Introduction (usuallv c+alled gyrase in bac+eria) is thought to

maintain suprrc~oiling at levels appropriate for
(‘los~d c+c*ular I)XA molecules in bact,erial cells survival.
are under negative superhelical tension. a feature Thr trtrarnerica A,K, I)NA gyrase. product of thr
that facilitates most DNA transactions. The free gyrrl (4X min) and gyrH (83 min) genes, is an rssen-
energy st,ored in ncgat,ively supercoiled DSA favors Cal cbnzytne t,ha.t catalyzes the ATT’-dependent
t’hr> I)NA reactions that depend on strand separa- negat,ive suprrtsoiling of DNA ((iellrrt rt CL/.. 1976:
t,ion. i.e. replic3tion, transcription, t~ransposition. Higgins rt (11.. 1978: Kreuzer it nl., 197X: Brown K-
and both homologous and site-specific rrcombina- (:ozzarelli, 1979: Morrison 82 (lozzarelli. 1!)79: Sugino
Con. I+‘urthermorc. negative supercoiling influences it ~1.. 1980: Mizuuchi rf (11.; 1980). Thr enzyme acts
the t,hrt.r-dimensional configuration of DNX and is a by eff’erting a transient double-strand nick in its
source of structural motifs that help regulat,ory and DNA substrate. passing the double helix through
structural prot~eins to select their specific target (for the break in the direction t,hat serves to decarease thr
reviews. see Drlic~a. 1990; Wang, 1991). Regulating linking number. and then resealing t.htk break.
DIL’A topology appears therefore to be of general During the breaking-rejoining reaction. the .5’ phos-
itnport ante for a balanced DNA metabolism. DEA phate t,ermini of the nicked I>SA strands are (*ova-
topoisomrrases are a unique class of ubiquitous lentSly linked to a tyrosine residue of the A subunit.
enzymes that are ablr to cont’rol supercoiling by of IJNA gyrase. This transient covalrntly linked
breaking and rejoining the phosphodiester backbone gyrasc>-1)NA intJrrmrdiate has been called t.hta csleav
of one or both I)?\‘A strands (the type 1 or 11 able c*oniplcx (for reviews, set’ Wang. 1985: Maxwell
topoisomerase, respectively). In Eschrricirin coli, a B (:ellert 1986: I,iu, 1989).
homeostatic: balance between the countervailing In prokaryotict and eukaryotic cells. the clravablt~
activities of topoisomerase I and topoisomerase TI t,opoisotrterase~l)?;A complexes are the molecular
 t,arget)s of potent t herapeutica agents. Including t IW
quinolone antibiotic*s. which act on bac.terial g~rase.
and anticancer agent’s (e.g. campt,othecin. acri;lines.
uc%inomycines. ellipticines, epipodophyllotoxins).
M hich target mammalian DNA t.opoisomerases I or
I I. Most have been shown t,o act by nl~ecificatl~~ and
reversibly blocQking the DNA-rejoining step of lope-
iaomrrases. resulting in trapping of t)he cleavable
complex (for a review. see I,iu, 1989).
All these observations emphasize thr importance
of t.opoisomerase poisons in acquiring a hasir under-
standing of t~opoisomerase biochemistry and
molecLular biology. as wtill as fi)r drvelopmrnta~l
therapeutics.
In this paper? we report the identitication of a
new t.ypr of topoisomerase IT poisoning agent: the
c:>-totoxic C’cdK protein of plasmid F. The F plasmid T,K rnrdiunl contains IO g of tryptone ((:ibco. I-K). 5 g
(‘cd K protein is responsible for cell death and induc- of yeast extract ((:ib(ao. I’K) and .5g of Xa(‘I per litrlr of
tion of the SOS pathway, when its action is not, wat’er. Antibiotics were used at the following final ronct’tl-
prtbvent.ed 1)y prot,ein CcdA (Karoui et ul.. 1983; Bex trat,ions (in pg:!ml): ampic+ilin (Ap). 50; tet.rac)-clinr (TV).
it al., 1983; Ogura $ Hiraga, 1983; Miki et ccl., 19843; 10; kanamycin (Km). 50: chloramphenicol ((‘In). 20: and
Sommer et al., 1985; Bernard & Couturier. 1991). nalidixics acid (Nal). 20. In vitro reactions with restriction
Hot,h proteins are encoded by the ccd locus of enzymes. phagr T1 l)SA ligase, and the Klenow fragment
plasmid F (ccd stands for control of cell deat,h). This of DKA polymrrasr T WPW as rec~ommrndt~d by the
manufacturers.
locus participates in stable maint’enance of plasmid
F (Ogura & Hiraga, 1983: Miki et al., 1984u): it
favors the plasmid-carrying cells by killing daughter
cells that have not inherited a plasmid copy. at c%ell
Most routine genetic manipulations were prrfornrrd as
division (Jaff6 et al., 1985). The proposed mechan-
described by Maniatis et nl. (1982). Purification of restric-
ism underlying this post-segregational killing of tion fragment,s srparatr~l L)y convrntional aparose gel
bacteria is differential decay of the activities of the electrophoresis (TAE buffer) was done with Gene (!lean
C’cdA and CcdB proteins, the half-life of active C’cdA (Bio 101). Plasmid DNA was purified as drticrihed by
protein being shorter than that of active C‘ctlB C’lewell & Helinski (1969). For small-scale preparations of
prot.ein. In newborn plasmid-free bacteria, persist- plasmid DXA. the method described hy Birnhoim &. Daly
ence of the cytotoxic CcdB protein would lead to (1979) was used. For resolution of plasmid DNA t,opo-
cell death (Jaff6 et al., 1985). ,\iliki rt nl. (1988) isomers by electrophoresis as described by Shure et al.
isolated a particular type of bacterial mutant resist- (1977). small-scale preparations of DBA were purified on
columns cont.aining Sephacryl S-400 (Pharma.cia). (IcdB-
ant to the killer activity of the CrcdB protein that
induced cleavage was revealed upon treatment of DXA
maps at, the yro&&’ locus. The GroES product) is a with SDS and protrinasr K. as described bj
“(shaperone” protein participating in the corrrc~t O’Conn’or & Malarny (1985). Cell s were made (Lompetent
folding of native protein (van Dyk ut al., 1989). It. is and transformed as described by T,ederberg & Cohen
likely that the GroES protein plays a role in the (1974). Transformation of E. coli by electroporation was
folding of the CcdH protein or of its target, allowing accomplished by the method of Dower et al. (19&C+), using
intermolecular association. a Bio-Rad gene pulser. Relative DX’A solution viscosity
In this paper. we describe the isolation, mapping, was determined using caapillary tubes and calculated
sequencing and properties of another bacterial according to the Poiseuille’s equation. as described by
mutant resistant to the cytotoxic activity of the Spencer (1972).
CcdB protein. We show that this mut.ation is
located in the gyTA gene (48 min), which encodes the (d) Inkrrupted mating
A subunit of the bacterial gyrase. In, uiivo. we
additionally show that the presence of the CcdB To allow recombination between chromosomic markers
of the recipient and donor cells. plasmid pCOS2.1, carry-
protein is responsible for plasmid DNA breakage.
ing the reed gene of Erzoinia chrysanthemi 3665 (which
On the other hand, plasmid DEA breakage is not promotes recombination in E. coli; C. Bertinchamps,
observed in the mutant strain that resists the cyto- unpublished results). was introduced int,o PBll (lh80)
toxic activity of the CcdB protein. These observa- bacteria. R,ecombinant recipients were selected for kana-
tions: together with a set of convergent results, mycin resistance conferred by the Ret ’ pCOS2.1 plasmid
strongly suggest that the CcdB protein is a poison and for acquisition of tetracycline resistance conferred by
for fG. coli topoisomerase II. the TnZO transposon of the donor cells. They were then
t.lsted for the ability to escape the killer activity mediated
by the CcdB protem. Hfr and F-cells were grown in LR
2. Materials and Methods medium to a density of 2 x lo8 cells/ml. The Hfr culture
(0.5ml) was mixed with the F- culture (45 ml) in a
(a) Bacterial strains and plasmids
prewarmed flask at 37°C with gentle shaking to allow
The E. coli K-12 strains used were: C600 thr-I. thi-1. formation of mating pairs. To interrupt mating. a sample
leuB6. lacY1, tonA2Z. supE44 (Appleyard. 1954): IV100 of the mixed culture was diluted IO@fold into saline and
 Cell Killing by the F Plasmid CcdH Protein 737

Table 1
Phasmids and plarmids

Description Source or referencr

minib’ hp’ plasmid cloned in the A631 vector (‘outurier rt u/. (I 979)
i.pSC138 with a amber mutation in the ccdrl grw Karoui rt nl. (1983)
@Cl38 copy number mutant Kex et al (19X6)
E.pWl38 double mutant Bernard & (‘outrrrirr
(1991)
J’lasmids
pA(‘Y(‘lX4 ‘I‘? and (‘m’ cloning vector derived from the P15A plasmid Chang & Cohen (197X)
1’1’1,1~2230 HarnHl 42.84 to Sol1 49 kb fragment of plTLI3201.5 cloned into the RanHI-Sal1 sites of Bernard 8: (‘outurirr
p;2(‘Y(‘l X4 (Cm’) (1991)
pl’LB2232 BornHI 42.84 to WI 49 kh ccdAnm22 fragment of pl’LB22l.i cloned int,o the BumHI-Sal1 Thin work
sites of pA(‘YC184 (Cmr)
pKK22:G:l Apr expression vector with the strong tar promoter Brosius 8r Holy I 1984)
l’l’LH2208 Xdpl 43.1 to BglII 46.92 kb fragment of plTLB201.5 cloned into t,he SnmI site of pKK223-3 Bernard & (‘outurier
(AI”) (1991)
S~/el 43.1 to HglII 46.92 kb fragment of pVLH2215 cloned into the SrnrcI site of pKK223-3 Bernard & (‘outrrriel
(W) (1991)
pKT979 pHR322 Tc’ cloning vector Talmadgr h Gilbert (1980)
pIlLR2007 &coRl f6 ecdAam22 fragment cloned into the EcoRI site of pKT279 (Tc’) Karoui et al. (1983)
pITI, 132015 Ir:coRI f5 fragment deleted of the 43.61 to 46.9 kb KpnI miniF sequence and cloned into Karoui et al (1983)
the EroRI site of pKT279 (Tc’)
1’171,1~2”1;i EcoRI f5 ccdAwn22 fragment deleted of the 43.61 to 469 kb KpnI miniF sequence and Bernard Kr (‘outurirl
cloned into the EcoRI site of pKT279 (Tc’) (1991)
p.K‘75-5X (‘onditionally replicating (thermosensitive) ColEI Ap’ cosmid cloning vector Collins & Briining (1978)
p~‘os2~1 Km’ cosmid pMMB34 with the gene WCA of Erwinia chrysanthemi 3665 This lahorator)
plTLUZI91 &coRI t5 fragment deleted of the AoriII (XhoI-HglII) and cloned into the fl’coR1 site of This lahorator~
pK1’279 (Tc’)
pI~L1?2291 pKT279 f5 ccddum’?% AoriII (Tc’) plasmid constructed by cloning the 4361 to 46.9 kb This work
Kpnl AoriIT fragment of plasmid pULH2191 into the KpnI site of plasmid pULB221.i.
plKlH220 Conditionally replicating (thermosensitive) ColEl Km’ cosmid constructed hy cloning a This work
Km’ cassette (Pharmacia) into the PstI restriction site of cosmid pJC7.5-58.
pl’LB2222 EcoRI f5 A&II fragment of pULB2191 cloned into the EcoRI site of pULB2220 (Km’) This work
pIU12224 EcoRI f5 ccddnm29 AoriII fragment of pUL132291 cloned into the EcoRI site of pULH2220 This work
(Jim’)
pI’LU2226 BnmHI EcoRV gyrA + fragment of PH30(i) cloned into the I.87 to 3.21 kh BanbHI-H&II
sites of’ p.i(‘Y(Z184 (Cmr) This work
pl~LIW%?T RmmHl BcoR\,- gyrA462 fragment of 1’831 (L) cloned into the l-87 to 3.21 kb BarnHI---
HincII sites of pA(‘YC184 (Cm’) This work
pL’LB2228 ClnI-C’/rcI gTlr.4 + fragment of pULB2226 cloned into the C’lal site of pACYCl84 (Cm’) This work
pl:LlW229 ClaI -C’/nl gyr.4462 fragment of pULB2227 cloned into the C’lnI site of pA(lYC184 (Cmr) This work

shaken vigorously The cultures were then plated on residues) together with its antagonist, the CcdA
s&rtirr medium. protein (72 amino acid residues). In order to identify
the bacterial target with which the cytot’oxic CcdR
(P) 1)X,4 sequenciny
protein interacts. we selected bacterial mutants
The PstT-By/II fragments were sequenced on resistant to its killing activit,y (CcdB’ phenotype).
pl-LB2228 and pI’LB2229 plasmids by the dideoxy The CcdB’ bacterial mutants were obtained by a
chain-termination procedure (Sanger et al.. 1977), using double selection using miniF hybrids able to synthe-
[c(-35S]dATP and 2 synthetic 18 nucleotide primers. size the CcdB protein but unable to produce the
Sequencing Sequenase Kit was obtained from the U.S. antagonistic CcdA protein (as a result of an amber
Biochemical f’orporation.
mutation in the ccdA gene, ccdAam22; Karoui et al.,
(f) Mrc~.wrement of SOS induction 1983).
The two CcdA-CcdB’ miniF hybrids used in
Induction of s$A gene t,ranscription was used as a
the selection were the iminiF phasmid
measure of SOS induction. s&4 induction was monitored
IlpSCl38ccdAam22cop5 (Bernard & Couturier, 1991)
by the amount of fl-galactosidase produced in lysogens by
a s&4 :: ZucZ fusion carried by a icIind prophage (Huisman and the conditionally replicating (thermosensitive)
& d’Ari. 1981). The specific activity of p-galactosidase was ColEl miniF plasmid pULB2224 (for construction,
determined as described by Miller (1972). see Materials and Methods). Independent cultures of
the sup’ I-lysogenic strain NlOO(AhSO), muta-
3. Results genized with 2-amino-purine (according to Miller,
1972) were infected at a multiplicity of three
(a) Isolation oJ” bacterial zmutants resistant to the by the CcdA-CcdB+ Ap’ AminiF phasmid
cytotoxic activity of the CcdR protein
(IpSC~13XccdAamZZcop5) and grown exponentially
The ccd locus of the miniF plasmid encodes the in LB broth supplemented with ampicillin to select
potent cell-killing CcdB protein (101 amino acid for t.he presence of the phasmid. iZfter this first
 A first genetic mapping of’ t.he ccrlH’ mutat ion was
performed by interrupted mat’ings bet ween a Fbrc, +
derivative of the f’Hl 1 (lhX0) (‘cdl%’ strain (stbe
Materials and Methods) and a series ot’ tlfr s:t.rains,
carrying TnlO transposon at detined Ioc,ations on
the chromosome. Tetracycline-resist’ant’ t,ra,t:sconju-
gant.s were selected and tested for their srnsitivit)>
or resistance to the (Lytotoxic (‘cdl3 protein (W f3’
recipient transconjugants \l;t’re obtainc~d I)>, mating
between HfrKL98 donor strain, having it,s itljec+.ion
Overnight cultures of the Iysogrnic i. strains were infected with point at. 52 minutes and tht, TnIO tratrsposotl
the ipSC138cop5 ((‘odA+CcdH ‘) or ~pS(Il~RecdAum”~eopS inserted at 37 minutes. and PBl 1 (,?h80) rccipirnt
((‘c~dA~(‘cdB+ in SZLI)~ strain) at a multiplicity lower than 1.
c,ells. On t,hr other hand. mating perform~~d with
After 20 min xdsorption. thr infected cells wvre plated on 13
agar plates supplemented with ampicillin. The A$ plasmidiza- HfrKL96 donor strain, having it,s injection l)oint at
tion efficiency was c&ulated its the ratio of thr number of Ap’ 45 minutes and the TnlO insertion a~ L’i minutes,
c4ones formed on ampicillin plates to thr number of infecting did not give rise, Tao (%dK” trarlsc.onjugallt,s. These
phagrs. (aross(‘s locate 1he ~c:dH’ mutation betweet) 15 and
52 minutes OII t)he gc>neticL tnap of’ E. roli.
The 45 to 52 minute genornic region c*ontains the
qyr,4 gene. This prompted us to analyze the proxi-
mity of t.hr c:cclB’ alIt+ witah a TnIO transposon
exposure to the cytotoxic (IcdH product. we inserted at 48 minutes nthar the !/!/“.-I gene
observed that most of the surviving bacteria were (zeG29X::TnIO). To do this, a 1’1 Iysatt’ was
not. resistant to its killing activit’y. The surviving prepared on the C’cdlY ~ri-2.9X::Tt110 st,rain and
ba,c:teria were mostly plasmid-free Ap’ bacterial used to infect a Kw+ derivativr of t,hr f’Rl 1 (JLhFIC))
mutants or bacteria bearing a CcdB- mut’ant C’cdH’ strain. As a c~ont,rol. the same 1’1 lysatts was
plasmid. used to infect a WC’31IO(i.) strain bearing a tlalitiixic*
In order to recover the rare CcdH’ bacterial resistant (Nal’) mut,atiott in the yyr.4 ytbnt.. In both
mutants among the survivors, we performed a transduct,ions. tc,l.rac.~~litle-resistant t rat1stiuc.t atlts
second exposure to the CcdB cytotoxic protein. The were selrc~ted and t estc>d for their sensitivity or
exponentially growing Ap’ cells (survivors) were resistance to t,he caytot.oxic. (‘c~il3 protein (for the
rendered competent and transformed with the PHI 1 (AhSO) t’ransductants) and sensit.ivity or resist-
(‘udA CcdK+ Km’ pULB2224 plasmid. In this ance to tlalidixic acid (for the \VSl 10(/i) t rallsdu(.-
second selection, the undesirable Ap’ or CcdB- sur- tants). LZ’C observed similar c~otrailsdric,t’ic,n
vivors were eliminated by the CcdB protein frequencbies of t’hr c~d/Y and nalS (gyr,-l ) alleles with
expressed by the pL’LB2224 plasmid and by ka,na- the Tn IO t ransposon (respectively, 46 o. atIt 48 o0
mycin present in the growth medium. c.otransduc~t,ion). A 1’1 tysate was thtbn 1)rv1)ared on
Note that, plasmid pULB2224 displays a potent the PI{1 1 (i.h80) Rev+ TV’ C’cdlI’ derivativ(~ strain
IncFT incompatibility phenotype (because it and was used to infec-1 the \Y3110(/.) straits. r\s a
contains repeat,ed sequences related to the col)y- result. Tc’ (‘c~lI3” PK30(~) and ‘I’(*’ (‘cdK’ l’tUl(i)
number control). It, thus prevents replication of the transductatrt.s were obtained.
resident lminiF plasmid used in the first selection.
(‘onsequently, the latter plasmid is diluted during
t.he following cell divisions.
In order to verify the resistant (IcdH’ phenotype
of t,he bacteria subjected to the double selection, the The gyr;t gene of E. c~li KlX chromosome is
survivors were cured of the conditionally replicating contained in an 8 kbt EcoKV- HamHI restriction
prLB2224 plasmid by exposure to the non-prrmis- fragment located around 4X.4 minutes on the
sive temperat,ure (42°C). The cured bacteria were physical map (Kohara it CLLI.,1987). In order to clone
then reinfect.ed w&h the C’cdA-~CcdB+ Ap’ AminiF the yyrA region of’ tht> wild-type T’K30(1) itnd
phasmid. mutated PBS1 (1) strains. their genomic DXAs were
This time, none of the surviving bacteria was restricted with EcoRC’ and HnmHI restrict’ion endo-
killed by the phasmid, indicating that the mutation nucleases and then ligated into the Hl.ncIT--RamHI
conferring the CcdB’ phenotype is located on the sites of plasmid vect’or pACYC184. The ligation
bacterial chromosome. By this procedure, we products were used to transform &rain OV6 (yyrA.,
isolated several independent bacterial mutants csupF,,: Hussain et al., 1987). and the recombina,nt
resistant to t’he cytotoxic activity of the (%:dK pro- plasmids wcrc selected for their ability to csomple-
tein. One of these, the PHI l(Ah80) strain, was tnent t>he growth defect of this strain at) 42’(‘. The
analyzed further. Tts colony-forming ability after
infect.ion wit,h the cyt.otoxic CcdA-CcdB+ Ap’ t Abbreviations used: kl). IO3 i~,ses or hnstl-l);tirs: I)b).
AminiF phasmid is shown in Table 2. basct-pair(s).
 Cell Killing by the F Plasmid CcdB Protein 739

Plasmid pULB2228 (derived from the CcdB”

strain) and plasmid pULB2229 (originated from the
CcdB’ strain) were used to transform CcdR” and
CcdR’ strains. The phenotypes of the resulting
Ad 8 merodiploids were determined according to their
ability t,o form colonies after transformation with a
CcdA -CcdR + plasmid (pULB2212). The results
SOll 7.2
pULBPro
presented in Table 3 show that ccdB”/ecdB” and
BornHI 6.9 (9.3 kb)
ecdB”/ccdB’ merodiploid bacteria do not give rise to
viable transformants, whereas ccdB’/ccdB’ merodip-
C/o1 65 loids vield viable transformants after transforma-
tion with plasmid pULB2212. Thus, the presence of
8gllI 6.2 the wild-type pULB2228 plasmid in the CcdB’
strain reverses the CcdB’ phenotype of the strain.
We conclude that the mutation conferring t.he
Kpnl Bgln
5.6
AvoI 4.3 CcdH’ phenotype is located in the 5.1 kb fragment
5-3 I
PSfI 4.9 carried by plasmid pULB2229, and that’ t’he sensi-
Figure 1. Structure of plasmid pULB2228. Plasmid tive phenotype dominates over the resistance
pULB2228 is a pACYC184 (fine line) with a 51 kb Cl& phenotype. This strongly suggests that t,he CcdH
ClaI insert containing a complete wild-type gyrA gtme. protein is more than a mere inhibitor of an essential
The structure of pULB2229 is similar, except that the function but a poison of that function.
51 kb fragment is derived from the CcdB’ strain PB31(1). In order to determine which part of this 51 kb
By reconstruction experiments between both plasmids, fragment is responsible for the CcdB’ phenotype,
we located the ccdR’ (gyrA462) mutation within the PstI- restriction fragments of plasmid pULB2228 were
BglII 49 to 53 kb restriction fragment. The open boxes replaced with equivalent restriction fragments of
represent a duplication of vector DNA. plasmid pULB2229, to reconstruct the locus. Then,
the resulting recombinant plasmids were tested for
resulting ‘ ‘GyrA +” recombinant plasmids derived their CcdR” or CcdB’ character. By this means, we
from the CcdBS (pULB2224) and CcdB’ strains observed that the mutation conferring the CcdB’
(pULB2226) were digested with a variety of restric- phenotype maps in the central part. of the gyrA gene
tion endonucleasesin order to check whether their in the small 380 bp PstI-BglII fragment (Fig. 1).
restriction patterns match those of the gyrA region
and of the pACYC184 vect’or. From each of the two (e) Sequencing of the ccdI3’ mutation
plasmids, a 51 kb fragment containing the entire
gyrA gene was cloned into the ClaI site of the We determined and compared the nucleotide
plasmid vector pACYC184, giving rise to plasmids sequencesof the 380 bp PstI-BgZII fragment of the
pULB2228 and pILB2229, respectively (Fig. 1). gyrA gene from plasmids pULB2228 (CcdBS) and
pULB2229 (CcdB’). The results revealed that the
central part of the wild-type gyrA gene (ccdB”)
(d) The ccdB’ mutation maps in the central part of differs from that of the mutated gyrA gene (ccdB’)
the gyrA gene and is recessivewith respect to the
by a point mutation: a C.G to T.A transition at
wild-type gyrA allele
position 1384, which produces the substitution of
In order to det’ermine whether the ccdB’ mutation Arg462 by a Cys residue in the amino acid sequence
maps in gyrA, we performed complementation and of the A subunit of gyrase. Henceforth. the ccdB
reconstruction experiments. mutation was called gyrA462.

Table 3
(‘omplementation analysis of the ccdB’ and ccdB’ chromosomal markers by plasmid pULB2228 (ccdl3”) and
plasmid p U LB2229 (ccdB’)

Rate of transformation

NlOO/ NlOO/ NlOO/ PBl l/ PBll/ Pull/

Relevant pACYC184 pULB2228 pULB2229 pACYCl84 pULB2228 pULB2229
Plasmids character ccdBS/vector ccdB”/ccdB” ccdB”/ccdlT ccdB’/vector ecdB/ccdB” ccdFT/cedB’

pKK223-3 Vector 1 OS-l.2 081.2 0.8-I .2 0.8-1.2 @H-1.2

pULB2208 CcdA +CcdB + 1 081.2 081.2 081.2 0.8-1.2 o+- I.2
pULB2212 CcdA-CcdB+ 1 <0901 < 090 1 OS-l.2 < 0901 08-1~2

Plasmids pKK223-3, pULB2208, and pULB2212 were used to transform I?. coli strains CcdR” NlOO and CcdB’ PBll, harboring
plasmid pACYC184, pULB2228 or pULB2229. Ampicillin resistance was used to select transformants. For each plasmid DNA, the
strain transformation rate was defined as the ratio of the number of transformants obtained with this strain to the number of
transformants obtained with NlOO/pACYC184.
 MJcbalso found that, the central part of hot h pencsh 1234567
examined hert‘ difiers from t.he wild-type gyr=l gcnc~
sequence determined by Swanberg Hr Wang (1987)
by a rirut’rai point, mutat,ion: a (‘.(i to (i.(' tl%l~sVf~~-

sion at position 1690.

It is worth noting that’ the amino acid suhstitu- G-
tion introduc& by the gyri146Y mutation lies in a
domain different from the site of yuinoione action cb
on subunit A (ver!. close to Tyr122: Yoshida et (11.. L-
1988). Tn agreement with this loca~iization. rve
observed that, a strain bearing a nalidixic resistant C-
(Nai’) mutation in the gyrA gene is sensitive to
(‘cdl< protein and reciprocally that the ~yr.-l-IG%
mutation does not’ confer resitance t.0 naiidixica acid.

(f) Tn vivo, the (‘cd13 protein induces thr hreakagr of

One of the most prominent ccilular effect of t,opo-

isomerase II poisons is a significant breakage of
double-stranded DNA. The breakage of DIVA. aft)rr
addition of the drug, is revealed only upon t,reat-
ment with a prot)ein denat’urant su& as alkali 01
SDS and results in a complex of topoisomerase TT
bound covaiently to cleaved DXA (Cellert. it rrl.,
1977: Sugino et nl.. 1977). We investigated whether
the CedB prot’ein is able to promote in civo &>avage
of piasmid I>XA in the absence of the (‘edA product.
Double-strand cleavage of piasmid I>SA leads to its
linearization. Therefore, t,o detect a putative ( !cd K-
induced DNA clea,vage, we analyzed linearization of
piasmid I>KA in the absence of (‘uJA protc~in. 1n
order to construct the strain and t)o avoid the lrt~hai
c*hara.c*trr of t.he CcdA~~CcdB’ plasmid. WV first
t,ransformed t,he yyril + NlOO(/l) strain bg the cond-
tionaliy replicating (thermosensitive) p1’LB2222 total I)NA preparations exfractetl from
plasmid, which carries a wild-type ccd operon. Thr C’cdA ( ‘cdB + bact,eria and from (‘cad A + (‘c*dH i
resulting Y lOO(i)/pr’LB2222 transformants were bacteria. We olwrved that total I)NA i)rf~part‘d in
further t)ransformed by plasmids pl‘LH2230 and the presence of activcl C’cdK protc~in (c*ondit,ion of
prLR2232. At 32°C:. the lethal chara.ct,er of plasmid piasmid DSA cleavage. lane 5) is ten times 1~s
pl’LH2232 is prevented h\- the presence of t)hr viscous than the total I)?iA prepared in t tit, ahsen(.f~
pCLB2222 (CcdA’CcdR’) piasmid. At 43’(‘, the of actirf> (‘cdl3 prot,ein (wdition of’ initd iviktiorl ot
conditionally replicating pl’LB2222 piasmid is CcdK by (‘cYl;\. lane 1-i. This ohsrrvation is ill1
segregated and onI> the resident pia~smitls. evidence that (.!cdB protein is rrsponsihlrh not onl!
pl’LK223O ((‘cdA+CedR’) LWd pl:I,K2232 for plasmid I)SA CleaVite? Ijut. also fi,r gf?nornic
((‘rdA~~(‘cdK+). arc conserved. On thch other Ilan(l. I>NA fragmc>ntation.
t’hc strain gyrA $62 1’131 l(Ah80) was dirrcatiy trans-
formed by piasmids pULB2230 and 1)1’7,132232
wit,tiout, prior transformation with plasmid
pI’I,H2222 (beca.use of its rrsistant. (%tlR’ phc~rro-
t~,pe). and thr working temperature wan always
37 “( ‘_ Figure 2 shows t,hat in thr absencr of (‘cd4 piasmid 1)K.A in gy/-A462 mutants. Strains gyr.-l ’
protein synt,hesis. piasmid I)NA is partially linear- PR30(~W) or gyr.446:! PR31 (i.) harboring a pKR322
ized in a yy7.4 + strain (lane 5). whereas no lineariza- piasmid were grown exponent~ialiy in l,li mrdium.
tion OWUI’S in a gyril4GB strain (lam 7) attd that T’lasmid DNA was extracted hy an alkali/SIX5 min-
when (.‘cdA protein is normallg synthesiactd. no prep met,hoti and plasmid topoisomcrs wc’rth t)hcn
plasmid I)NA linearization occaurs (lanes 2. 3. 1 arltl separated 1)~. rlectroptiorf~sis in apvsc eel
6). These results show that,. it, C.~CW.the (‘cdl3 pro- cont’aining different concentrations of chioroquinr.
tGn ipromotes gvrase-nrediat~rd tloul)ic~-st ruriti Figure 3 sho\-r-s that piasmid DNA ext rac+tc:d from
plasmid USA c+ravatgc gyrA + st,rain (lanes 1 aA 4) or gyrA40’P strain (lanes
Furthermore. in order t’o show that, the (‘c~iR 2 and 5) display a similar level of suprrh&city.
protjein is also rcq)onsible for t)he fragmentation of Contrarily. a pHR322 DNA prepared from the
the penomic- I)NA. we c>omparrd the visc*ocity of strain g?yYA3.3,, (after 30 min inruhation at rion-
 Cell Killing by the F Plasmid CcdB Protein 741

Figure 3. Effect of gyrA462 mutation on plasmid DNA Q

supercoiling. In cGvo supercoiling of resident pBR322
plasmid during exponential growth of gyrA+ PB30(1) and
yyrA462 PB3l (A) strains. At the mid-exponential phase of
growth. the bact’rria were lysed and the superhelical 0 3b 60. Go I20. I50
densit?; of plasmid DKA was analyzed by chloroquine (2 Time (mini
and 4pg/ml corresponding to lanes 1 and 2. and 4 and 5, (a)
respectively) agarose (0.806. w/v) gel electrophoresis.
Lane 3 and 6 show topoisomer distributions (at 2 and
4pgjml of chloroquine) of pBR322 DKA harboring a
different superhelical density (prepared from the gyrA43,,
strain incubated for 30min at the non-permissive
temperature, 43°C‘). The direction of migration is from
t)op t,o bottom: under the conditions of electrophoresis.
more negatively supercoiled DNA migrated more rapidly.
Lanes I and 4. gyrA &/pBR322: lanes 2 and 5. !lyrA468/
pBR322: lanes 3 and ti. gyrAL?,,/pRR322.

permissive temperature) clearly shows a lower level

of supercoiling (lanes 3 and 6). This allows us to
conclude that the gyrA462 mutation does not affect
the superhelicit!; of plasmid DXA.
60

(h) SOS induction Time (mln)

(bl
Like the quinolones. the cytotoxic CcdB protein is
Figure 4. SOS induction ($4) promoted bx the VcdH
a potent inducer of the SOS system (Karoui et al.. protein or by ultraviolet light in yyrrl + and gyrA4GP
1983: Sommer et nl., 1985: Gudas & Pardee? 1976; strains. (a) SOS induction by the CcdB protein in yyrA +
Karu $ Kelk, 1982). We investigated whether the and qyrd46% skains. GyrA* (circle) and gyrA462
yyril462 mutation abolishes induction of the SOS (triangle) strains were infected at a multiplicity of infer-
system. normally promoted by the CcdB protein. tion of 3. with lpSC138ccdAarr~22cop5 (filled symbols) 01
The s&d gene product, is an inhibit)or of cell division with Ipscl38cop:i (open spmbols). After 20 min of absorp-
that is rapidly induced by SOS-inducing treatments. tion. the infected cells were grown in LB broth supplr-
(Huisman &, D’Ari, 1981). We monitored induction mented with ampicillin and periodicall?; assayed for
of t,he SOS response with a @A::lacZ gene fusion, production of P-galactosidase as described h?; Millet
(1972). (b) SOS induction by ultraviolet light in yyrA +
which makes /?-galactosidase an artificial SOS func-
and gyrA462 strains. gyrA+ (circle) and gyrilJ62 (trian-
t)ion. rxtremrl- sensitive to SOS-inducing gles) strains were exposed (filled symbols) or not (open
treatment,s. symbols) to ultraviolet light (lO,J m-‘). The dells were
The result,s (Fig. 4) show that the CcdA -CcdB+ grown in LB and periodirally assayed for J)rodu(*tion of
lminiF phasmid (E,pSC138ccdAamZZcop5) fails to fi-galactosidasr.
induce ~$4 in a gyrA462 s&4 ::lacZ recipient cell
(YB41). whereas ?@4 is fully induced in a
gyril +,sjL4::lacZ reclplent cell (PB40). On the other In the absence of CcdA protein, the (IcdB protein is
hand, the SOS syst’em of both &rains is fully responsible for cell death, activation of the SOS
induced after irradiation with ultraviolet light. pathwaS, reduction of DPI;A synt,hesis. cell filamen-
Thus, the yyrA462 mutation abolishes tation and formation of anucleate cells (Karoui et
CcdB -protein-mediated induction of the SOS al.. 1983; Kex et al., 1983; Ogura & Hiraga. 1983;
system. hut has no effect on ultraviolet light-trig- Miki pt ul., 1984b; Mori et al., 1984; JaffG uf al.. 1985:
gered SO8 induct ion. Sornmer et al., 1985: Bernard & Couturier. 1991).
Tn order to investigate the molecular mechanism
by which the CcdB protein kills cells. we isolated a
4. Discussion InactArial mutant (CcdB’) resist,ant to its lethal
The F sex factor plasmid contains a pair of genes, effect. Hfr crossing, Pl transduction, reconstruction
ccdA and ccdB. involved in plasmid maintenance. and complementat’ion experiments indicated that
 (:,vr.~t~ is an essential enzyme that int roduws
rityiit ivtk supercwils into f)NA f)y passing ;I f)NLA
helix t~flrtjugh i1 trsnsirnt dtjulJl+str>~tltf fjrtL>lk (fi~r
r(Lvits\vs. SW LVilllg. 1985: hlaXWtAll & (:t511rrt. 1986).
f)uring t ht fJr.t~aking-rejoiiiing stef). fwtfl f)N.-\
SI rands iirtl t*ovalentIy linktatf 10 tflr .A suf)rtrliis of
t lw tt~trarnrric~ ,\,I<, 1)K.A gyrasr. Thrw cxjvalentl~
fjritlgtvl IINX-gyr;tw t~onlfilext~s. c~allrtl c*lr;t\~al~lf~
c~otnfJltws. au fJrtwrtn~~~f to f,r t.fw Icta>- cYJvitlcnt
illlt~r.lrlt~ffi;ltt~ ilr t IIt, tofJt~isotnc~i~asr St rilllti-fJilSSitlp
rtw4 iorl fjat,h\z ay. Thrw twxyrrrt~ f>SA inter,-
nlcYiiittPs ;ll’t’ t fit, 1:lrget,S Of ii \.iLriet?. Of’ tof)oiso-
tnt~t%st~ fjoisorls. which are fJot.clrlt i~,lt,ifJ;tc.tt,riif illltf
;illt.itllmOrill drugs (for ;t revirw. St‘? f,iu. I!#!)).
A ftxl~urt~ of’ tof~oisomt~rst~ If fjoisons. whit*h inttav
tiartt \vitfj tfjtl fjrt’~LktLgr-rt~ullitjtj rewtion of fof)oiko-
tll(‘rilst’ I I. is Ihat ~‘XfJOSUrx’ of’t.fw c~lrav~rfJlP c’nzymc~-
f)?;&tilWg ~~Otllf)l1~X t0 it St 1’011g protrGn tl(‘Tlitt llr;\rlt
such ;LB SIXS or alkali leads to t.fw format iojj of’
tlollfJlt,-st,rarjtf 1)N.A fjrwks (f,iu. f9X9). \\‘tb t bus
invwtigatt~tf wtjrtht~r thti (‘(~113 fjrottaitr is ilfjlt. to
f)romoit ~IoII~J~~~-sI r;lnd fJrta>iks II~KJII t r<h;l,tmellt wit fl
ii strong fuwtriti dt~naturitnt (SIN).
Our rrsults show t fiat. j/4 cGl,o and in tflr wiltf-tyfw
wfl, t.htt (‘c:dK fwottkin is wsf)onsil-)I(& for f&mid An illttwsting poxsiljilit~~ Houltl tw 1 Ii;11 (‘t~lf%
1)iS.l lintwieation. whr~rws in t)fw g!/~.-f46% xt rain. protriti inlerferrs fjrt+rt~ntia.fly with illI c~tlxytrlk~-
wf1it.h resists ttiv c*yt~otoxit~ activity of tflv (‘cdl3 f)NA t~omf~lt~s in\-ol\-etl in chromosomt~ st~gwptiolt.
f)rotein. no f~li~~st~liti 1)N.A finrarization is ofwrvetl. Inderd. t~fl til;tmrntatiot~ (t~longatrcl t~t~lls \\itlr ii
This i~ltlic~ates t hut the C‘t~if< f~rot,rin fxomotrs localizrd mass Of’ 1)N.A iitlfl il W~lU(~~Vl IXttJ 0i’ 1)X:\
gyrasc,-mediated tfouhlc-st,rarrtf~(f f)SA f)rrakege. syrit his). and fi)rmat ion 01’ ~rnut~lt~itlt~ twills
‘f’he (‘cdl5 prot~c~in was shown to fxomotc thr fuwmotetf t)y (‘c*dl< pro1r*in (.laffG (4 (I/.. I!&%) ~YJ~II~I
intlnc’tion of the SOS pathway (Karoni rf II/.. fR83: result t’rorn iAn inafJilit\ to t1rc~~~trnal.t~ (tofjologic~al
Sommw rt (11.. f 9%). Tnduction of’thr SOS system is rcsolutioti) or to posit IOII (tofJtr~r;~fJlJic~~l svgrty+
il, fjiologit~al resfjonsc to f)N.&daniaging qt’llls (for t ion) t fjta tli1lJ~l~tf’l’ f’tlrot1josot1lf‘. .IfJ[JiLI’f’“l I!. tf~ft~~
a rwiew. ser LVafker. 1984). fSi~il(jnt~ rt ~1. (19%) ti\-tb t~h~~ot~iosor11t~ styrt~gal ion (I’ar f~lit~trot~~fw) Iins
tiaw sfiown that iiiduction of tflv SOS syst,em fj> fwrrl 0lJser~vtYi for IJot h pyrxs-;” iltltl f)Ni\ 1ofjoistb
t hrs (‘cdl< f)rotrin requires the un\vinding of IINA 1~) meraht I \’ rllllt iitlts. srlggwt irlg t II;Ll Ijo1 ll typf’ II
t fit, Rrt~fK’ fwlivaw. These authors f’urthtar f)ropowd tofJoisorlit,l,ases i*W ~tC’C’(‘SSiit’~ tiJr t flih St C’fJ (s:f tY.li &
t fiat, t.rappitig of’ a gyrasr-f)SA twtnfjles fry (‘t*tlR I)rlic*;r. I!#&: Hussain rt r/l.. 1987: K;;tto c,f c/l.. I989.
fjrotcsin may c*ausr the fiwtnation ot’ tl~jufjlt,-st,rarld 1990). ~lort~ovt~r. it has IJt~t~Jl stlo\r-11 t tJ;t1 1 fltb fJiiI4 i-
I)sA breaks from which the Ret~fX c~nxymt~ may t.ion wgiori of f~l~srnitl fjS( ‘IO1 is ij sfjt~c~ific IJiridirig
initiate I)SA unwinding. I)NA unwinding tJy the site for f)SA gyraw (\VafrIr & Kc~rnfwrg. IWX) iLIlt
fG~f3(’ rnzytnt~ tnay gc~nerittr single-stranded f)SA. that I)SA sufjrrht~livit?~ ih t*tLntralIy involvtbcl irj t ht.
constituting an SOS signal that ;it%i\:attls tht> fi~ec*A partit,ioning mwhanism (Millt~r PI ~1.. 1990;
fjrotein. fka1tcq$? rt </I.. 199 I ).
In this work. WC’ show t’hat the SOS in(luvtion
normally promoted IJV the (‘cdl3 fjrotein is
supressed fry the yyrA&&2 mutat ion, whereas ultra-
violet light-triggered SOS induct)ion is unaffwtrd fry
this mutation. WV may cwwlude that thtx ( ‘cdB
 Cell Killing by the F Plamnid ClcdN Protrin i43

Tyrl 22: Yoshida vt al.. 1988). This helical domain ident,itication of a new JO5 kilodalton sJie(+s. E’MRO
contains a heptad repeat of teucine residues J. 2, 1853~-1861.
(residues 447 to 489: Yang & Ames. 1988), a Bcx. F.. J’ikrard. P.. Desmyter, A.. Dreze. I’.. (‘olet, >I. &
“leucine zipper motif’ identified in several DXA- Couturier, M. (1986). Mini-F E protein: the carboxy-
terminal fd is essential for E gene repression and
binding proteins and involved in protein dimer-
mini-F copy number cont,rol. .I. MO/ Hiol. 189.
ization (Landschulz et r~f., 1988). Secondary struc- %!%303.
ture prediction. using the Garnier algorithm Rirnbcmn. H. (‘. XL Daly. .J. (1979). A rapid alkaline
(Carnier of /II., 1978) and the MY> software extraction procedure for screening recombinant
(Devereux et rrl., 1984). reveals that an Arg462 to plasmid DNA. SUCZ. Acids Elss. 7. 1513 1523.
(“ys substitution apparently does not destroy t,he Brosius. ,J. B Holy. A. (1984). Regulation of ribosomal
x-helix structure of this domain, but rather creates a RSA promoters with a synthetic lw operator. f’ror..
more nearly perfect, helical configuration in the Sat. z4cad. Sci.. T’.S.A. 81. 692C+69:~3.
mutated than in the wild-type Gyr=\ protein. A Krown. I’. 0. & Oozzarelli. S. R. (1979). .I sign inversion
weak perturbation of secondary structure is con mechanism for enzvmatic supercoiling of DNA.
Sirnw. 206. 1081~1083.
&tent with our finding that the gyrA462 mutation.
C’ssadaban. YJ. ,I. (1976). Transposition and fusion of t,he
in rive, does not affect the suprrhrlicity of ptasmid lnc genes to select,ed promotters in E coli using
I)XA. bacteriophage Lambda and Mu. ./. .Wo/. Hiol. 104.
Tn addition to t’heir value as new powerful investi- *?-cl-555.
gat,ive tools to probe the function and mechanism of (‘hang. A (1. Y. & (‘ohen. Si. S. (1978). (‘onstruction and
t~opoisomrrasrs, prot,einic topoisomerasr poisons are charactrrisation of amplifiable multicopy J)SA
potential therapeutic agents. Tn part>icular. elucida- cloning vehicles derived from the 1’1.5.~ cryptic
tmion of their mode of action may lead to the design miniplasmid. .J. Racteriol. 134. II41 1156.
of antibiotics and antitumoral polypept’ides that. (Xewell. I). B. & Helinski, 1). R. (1969). Supercoiled
using appropriat,e vectors. could he directed to circ.ular JJNA-protein complex in E. roli: purification
and induced conversion to an 0Jien c.ircnlar I),h;A
spe&ic cellular target’s,
form. t’roc. &Vat. Acad. Sci.. I..S..-l. 62. 115!J&1166.
Further biochemical and genetic analyses of the (‘oIlins, J. & Burning. H. ,J. (1978). Plasmid useable as
mechanism by which the CcdR protein leads to cell gene-cloning vectors in an in vitro pwkaging by
death and hou its action is prevented by Ccd.L\ coliphagtb i. Gene. 4. 85.m107.
protfG arv underway. (‘outurier. XI.. .lanssens. J.. 1+x. ii’., J)rsrnvtrr. f\. 8r
Jhmnevalle. I. (1979). ( ‘onstruc+ion in vitro of a
“phage-plasmid“ vhimerae: a ncx\vtool to analp the
IVr are very grateful to Michel Faelen and Genevieve
mechanism of F plasmid maint~rnanc~r. Mol. Cen.
Marnhaut for helpful discussions in the (*ourse of this
work. and to JtrnG Thomas and Franqoise J(ex for critical Gmet. 169. 113-I 16.
reading of this tnanuscript. We express special thanks t,o J)rvrrrux. .I.. Haeberli. I’. & Smithies. 0. (1984). A
J’hilippe (iabant and Laurence Van Melderen for romprehrnsive set of sequence analysis Jirograms for
the VAX. Sucl. Acids Rrs. 12. 387-595.
sequencing the q?yrA&TZ mutation. and to Lucie Dexmet
Dower, LV. J.. Miller. tJ. F. & Itagsdale. (‘. \V. (1988). High
for numerous strains and advice. We also thank Marc
(‘olet and Robert Herzog for the computer prediction of efficiency transformation of E. CC& bv high voltage
protein secondary. structure. This work was supported by elrc’troJ)oration. 51~1. dcids Hrs. 16. 612776145.
J)rlic~a. K. (19!+(J). Bacterial topoisomrrasirs and the
grant,s from the Institut pour I’Encouragement de la
control of DSA superroiling. 7’rwrls (;enpt. 6.
Rrchrrche Scirntifique dans 1’Industrie et 1’Agriculture.
133-437.
the Fonds h-at&al de la Recherche Scientifiyue. the
Englr E. C’., Jlanes. S. H. B J)rlicna. K. (I!%?).
Actions de la Rrchrrcahr C‘oncertee and the J<anyue natio-
nalr dr Belgiqw. IXfferential effects of antibiotics inhibiting gvrase.
J. i3nctwiol. 149. 92-98.
(:arnicar. .J.. Osguthorpe, 1). ,I. $ Robson 1). (l!J78).
Analysis of the accuracy and impli~~ations of simple
methods for predicting the secondary structure of
References
globular proteins. J. Mol. Hiol. 120. 97-I 20.
AJipleyard. It. K. (1954). Segregation of new Jysogenic* (:rllrrt. >I.. AJizuuchi. Ii.. O’J>ea. .\I. B Nash. H. (1!176).
t,ypes during growth of the doubly lysogemc strain 1)X-i\ gyrasr: an enzyme that introduces superhelical
derived from E’. coli K12. Genetics, 39. 44W52. turns into I)?riA. Proc. ,\:nt. <3rccd. Sri. I*.$.=1 73.
Bailone. A., Sommer, S. 8: Devoret’. R. (1985). Mini-F 3X72-387H.
plasmid-induced SOS signal in E. coli is RecBC Grllert. 51.. Jlizuuchi. K.. O’Dea. JJ. H., Itoh. 7’. &
dependent. Pror. LYat. Acad. s-i.. I:.s.‘4. 82. Totnizaua. .I. I. (1977). Xalidixic arid resistance: a
597~3~5977. s~~ind genetic character involved in I)NA gyrase
Beaucage. S. I... Miller (‘. A. & Cohen, S. S. (1991). a,tivity.~I’roc. Xat. Acad. Sci., I..S.=l. 74. 4772-4776.
Ciyrasr-dependent stabilization of pKC101 plasmid Cudas, I,. .I. & Pardee. A. 13. (1976). J)SA synthesis
inheritance by transcriptionally active promoters. inhibition and the induc%ion of’ protrm S in E. coli.
EMHO J. 10. 2583-2588. J. Mol. Hiol. 101, 459-&Y?.
Bernard. P. & (‘outurier. $4. (1991). The 41 carboxy- Cottesman. JJ. 8~ Tarmolinsky. M. (196X). The integration
terminal residues of the miniF plasmicl CcdA protein and excision of the bacteriophage lambda genome.
are sufficient to antagonize the killer activity of the (‘old ~Spring Harbor 9yw~p. Quant.. Hiol. 33, 735-747.
CcdB protein. Mol. Gin, Genet. 226, 297-304. .Jaffi, A.. Ogura. T. & Hiraga. S. (1985). Effects of the ccd
Bex. F.. Karoui. H Rokeach? L., Drkze. I’.. Garcia, L. 8r function of the F plasmid on bacterial growth.
C’outurier. M. (1983). Mini-F encoded Jlroteins: .J. Hactrriol. 163. 841b849.
Hill. (‘. \Y. & Harnish. H. R’. (1981). Jnversions hrtwt~en
rihosomal RNB genes of E. c~li. I’ror. Snt. rlcrrc!.
A?;., (‘.#.A 78. 7069-7072. Jliki, ‘I’.. Orita. ‘I’.. Furunc~. hf. 6 Horluc.hl. ‘I’. ( I!)%+).
Higgins. Iv. f’.. J’eebles. C. I,.. Sugino. .-\. & (‘ozzarc5iIl. (‘ontrol of’ (~11 division I)\- sex factor 1’ in /Csrhiric,hicl
N. R. (1978). f’urification of subunits of’ E. toll 1)SA c~li. I I I, f’articipatiorr (if the yr0E.S (n/r,p/l) p-tlr* 01
gyrase and reconstitution of enzymatic activity. the host bacteria. .J. Mol. Hiol. 201. 327 -348.
I’ror. Saf. Acnd. Sri.. 17.S.A. 75, 1773-1777. R;liller. (‘. A.. I%raucagr. S. I,. Nr (‘ohen. S. S. (1990). Role
Hsiang, Y’. H.. Lihou. M. (:. 8r Liu. L. F. (1989). Arrest of of f)KA srrprrheiic+t,g in f>artitioning of’ the pS(‘JOJ
replication forks by drug-stabilized topoisomerase plasmi(l. (‘PI/. 62, 127--13X
I-DEA cleavable complexes as a mechanism of cell Yfiller. .J. H. (I 972). Exprrin~r~ts irr Nolwulnr (kndics,
killing hv camptothecin. Cancer Res. 49, 5077-5081. (‘old Sfjring Harhor T,aboratory Press, (‘oltl Sflring
Huisman, 0. & L)‘Ari. R. (1981). An inducible DSAi Harbor. SY.
replication-cell division mechanism in k:. coli. Suture Mizuuchi. Ii.. Fisher. I.. hf.. 0‘f)ra. JI. H. & (;rllert. ICI.
(London). 290. 597-799. (1980). 1)X:2 ggrase action involves the introduction
Hussain. K.. Elliott. fC. ,J. & Salmond. C. P. C’. (1987). of’ transient double-strand breaks into D,h;A. I’roc.
The ParW mutant of E. roli also rarries a gyr,-l., Sat. .4wd. ,Sci.. I’.S..1. 77. 1X47--1X.51.
mut’ation. The compfet,e sequence of gyrA. Mol. Mori. H.. Ogura. T. & Hiraga. S. (1984). f’rof)hagc i
Microbid. 1. 259-273. induc~tion (.aused 1,~ mini-F f)lasmitl genes. .Mo/. (+PI~.
Karoui. H.. f<ex, F.. f>r&ze. P. 8i Couturier, M. (1983). GPnrf. 196. IX:?-19X
HnmZ. a mini-F rnutation which is lethal to host, cell Morrison, .\. K- (‘ozzarrlli. S. I<. (1979). Site-sprc~ifit
and promotes WCA -dependent induct’ion of lamhdoid cleavagr of J)XiA 1)~ 15. co/i f)R’A gyrasr. (‘I~//. 17.
fjrophage. EiMHO .I. 2, 1863%1868. 175m 1x4.
Karu. A. E. & Belk. E. D. (1982). Induction of’ h’. coli O’Connor. JI. K. bt Malam,v. 11. H. (1985). Mapping of
rrc;l protein via recBC and alternate pathways: f)XA gvrase cleavage sites irz oiro. Oxolonic, acid
cfuantitation hp enzyme-linked immunosorhent assa? induced cleavages in plasmid pHR322. ,J. .Wol. Hiol.
@LISA). Mol. &w. &net. 185. 275-282. 181, M--Z,O.
Kato. ,J.. h’ishimura. Y. &r Suzuki, H. (1989). E. coli pard Ogura. 1’. & Hiraga, S. (I!)%). Mini-F plasmid gene that
is an allele of t,he gyrB gene. Nol. Gen. &nut. 217. couplt~ host cull division to plasmid proliferation.
178-1X1. I’roc. .Vrr/. .4 cad. SC;., C‘.S. .4 80. 4784-4788.
Kat,o. .I.. Xishimura. \‘.. Imamura. It., Niki. H., Hiraga. Sanger. F.. Sic*klrn. S. & (‘or~lson. A. R. (1977). f)SA
S. 8: Suzuki. H. (1990). New topoisomerase rssent,ial sryurncaing with c.hairl-tc~rrnillatirlg inhibitors I’roc.
for chromosome segregation in E’. coli. C’rll. 63. ~Vat. .4 rud. Sri., I ‘.S.,4 74. 5463-5467.
393-404. Schnaitman. .\. (‘. B McI)onaltl. (:. 21. (1!#4). Regulation
Kohara. Y.. Akyama. K. & Isono. K. (1987). The phvaical of outer membrane protein synthesis irl /G. coli K-12:
rnap of’ the whole E. coli chromosome: application of delrt~iori cut’ orn~~(’ affects rxf)ression of the OmpF
a new strategy for rapid analysis and sorting of a protrin. .J. Izkteriol. 159. A.%-563.
large genomic library. Cl&. 50. 495-508. Shurc, M. f’ullr~~blank. I). E. X; \‘inograd. .J. (1977). The
Kreuzer, K. X. $ Cozzarelli, S. R. (1979). E. co/i f)roblettis of eukaryotic, and prokaryot,ic, f)?jA
mutants thermosensitive for deoxyribonucleic acid flackaging and in \-iv0 c*onforrnation posed b?
replication, transcription, and bacteriophage growth. superhelix densit?- hrterogrnrGt~-. Su~l. .-f~irl.s /ips. 4.
.I. Hncteriol. 140. 424-435. I f83- I205.
Kreuzer, K. N.. JlrEnt,ee, K.. Geballr, A. I’. & (‘ozzarelli. Sommer. S’.. I~ailone. .\., K- f)t~vorct. r<. (f985). SOS
S. R. (1978). Lambda transducing phages for the induction by thermosensitivr replic~at.ion mutjants of
wld gene of Eschrrichia coli and conditional lethal miniF plasmid. .Vol. (:~II. Grnrt. 198. 4% 464.
na14 mutations. Mol. Cm. Grnrt. 167. 129-137. Spencer .I. H. (1972). The fjhysics and chemistry of f)NA
Landschulz. W. H.. ,Johnson. P. F. & McKnight. S. I>. and RNA In Studiex irr I’hyysics and (‘hrmisfry,
(1988). The leurine zipper: a hypothet,ical structure ~-01 9. pf) 101 120. \V 13. Saunders (‘omf)an,v.
(‘ommon to a new class of DKA binding proteins. f’hiladrlphia. London. Toronto.
Sci~ncr. 240. 1759%1764. Steck. T. I< &Y 1)rlic.a. K. (1984). f3ac+rrlal chromosome
Lederherg. E. M. d (‘ohen. S. X;. (1974). Transformation segrrgatiotl. rvidrnc~e for 1)X.\ gyrase involvemr~nt in
of ~Salmonellrr typhimurium b> plasmid dec~atcnation. (‘~11. 36. IOXl~~JOX8.
tlrox~rihonurlri~~ arid. .J. Bacterio2. 119. 1072-- 1074. Sugino. *-!.. I’erhlrs. C’.. Kreuzer. K. & (‘ozzarrlli. S. I<.
Liu. r,. F. (1989). DNA topoisomerasr poisons as (1!)77). .\Irt*hariism of acatiofr of nalidixic, a(%]:
antitumor drugs. ;Innu. Rw. Biochem. 58. 351-375. puritication of E. roli ttcllrl pent f)roducat ant1 its
Maniatis. T.. Fritsch. E. F. B Sambrook. ,J. (1982). relationshi], to f)NA gyrase and a no\-rl nicking-
J~oletular C’loniug: ‘4 Lat~oratory Xand. (‘old closing c~nz~rrie. I’m- .Vclf. .Jmtl. Sri.. C’.S.:l 74.
Spring Harbor T,ahoratorv Press. (‘oltf Spring 4i6-477 I
Harbor. SY. Sugino. ;\.. fiigpins. N. I’. & (‘ozzarelli. X. R. ( f9XO).
Maxwell. .I. B (:rllert. M. (1986). Mechanistic aspects of 1)X.-! gj’rasr stoichiomrtr.~ and t,hr cv)valrnt
f)XA topoisomerases. Advan. Protein (‘hem. 38. attachment of subunit ,I to 1)X-A during f)NA
69-107. cleavage. Sucl. Acids Rus. 8, 3865-3874.
Miki. T.. Yoshioka, K. & Horiuchi. T. (1984~). Control of Swanberg. S. & !Vang. ,J. (1987). (‘Joning and seyuencaing
(VII division fly sex factor F in Escherichia coli. I. Thea of the E. w/i yyrd gene encoding for the A subunit of
42.84 -43.6 F segment couples cell division of the host Dh’A g>-rase. .I. Mol. Biol. 197, 729-736.
bacteria with replication of plasmid TINA. J. Mol. Talmadgr. K.. 8: Gilbert. \V. (1980). C’onstruction of
Hiol. 174 . 60%62’,.
( ( plasmid vectors with unique PstT cloning sites in a
Miki. T.. (‘hang. %. T. & Horiuchi. T. (19846). (‘ontrol of signal sequence coding region. Gene. 12. 23,&2hf
wII division h?- sex factor F in Escherichia coli. II. van Dyk. T. K., Gatenhy, A. A. & LaRossa. R. A. (1989).
J(fentification of genes for inhibitor protein and Demonstration by genetic. suppression of interaction
 Cell Killing by the F Plasmid CcdB Protein 745

of GroE products with many proteins. ,Vature Wanner, B. L. (1986). Novel regulatory mutants of the
(London), 342. 451453. phosphate regulon in E. coli. J. Xol. Riol. 191,39958.
Wahle. E. & Komberg. A. (1988). The partition locus of Yang, Y. h Ames, G. F. L. (1988). DXA ggrase binds to
plasmid pSClO1 is a specific binding site for DKA the family of prokaryotic repetitive extragenic
gyrase. EMBO ,I. 7> 1889-1895. palindromrc sequences. Proc. *Vat. .-hrl. hi., U.S.A.
U’alker. G. C. (1984). Mutagenesis and inducible responses 85. 8850-8854.
to deoxyribonucleic acid damage in E. coli. Micro6ioZ. Yoshida. H.. Kojima, T.. Yamagishi. J. T. 61 Xakamura,
Keu. 48, 6Ck-93. S. (1988). Quinolone-resistant mutations of the gyyrA
Wang. *J. (1985). DXA topoisomerases. 4n.w~. Rert. gene of E. co/i. Mol. Gen. Tenet. 211. l-7
Biochrm. 54. 66,5-97.
Wang, *J. (1991). DSA topoisomerases: Why so many!
.I. Hiol. ~h.rm. 266. 6659-6662.

Edited by M. Gottecman