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 douhlestranded 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,

1. Introduction (‘los~d c+c*ular I)XA molecules in bact,erial cells are under negative superhelical tension. a feature that facilitates most DNA transactions. The free energy st,ored in ncgat,ively supercoiled DSA favors t’hr> I)NA reactions that depend on strand separat,ion. i.e. replic3tion, transcription, t~ransposition. and both homologous and site-specific rrcombinaCon. I+‘urthermorc. negative supercoiling influences the t,hrt.r-dimensional configuration of DNX and is a source of structural motifs that help regulat,ory and structural prot~eins to select their specific target (for reviews. see Drlic~a. 1990; Wang, 1991). Regulating DIL’A topology appears therefore to be of general itnport ante for a balanced DNA metabolism. DEA topoisomrrases are a unique class of ubiquitous enzymes that are ablr to cont’rol supercoiling by breaking and rejoining the phosphodiester backbone of one or both I)?\‘A strands (the type 1 or 11 topoisomerase, respectively). In Eschrricirin coli, a homeostatic: balance between the countervailing activities of topoisomerase I and topoisomerase TI

I I poison:

(IcdU

prot,cin

(usuallv c+alled gyrase in bac+eria) is thought to maintain suprrc~oiling at levels appropriate for survival. Thr trtrarnerica A,K, I)NA gyrase. product of thr gyrrl (4X min) and gyrH (83 min) genes, is an rssenCal cbnzytne t,ha.t catalyzes the ATT’-dependent negat,ive suprrtsoiling of DNA ((iellrrt rt CL/.. 1976: Higgins rt (11.. 1978: Kreuzer it nl., 197X: Brown K(:ozzarelli, 1979: Morrison 82 (lozzarelli. 1!)79: Sugino it ~1.. 1980: Mizuuchi rf (11.; 1980). Thr enzyme acts by eff’erting a transient double-strand nick in its DNA substrate. passing the double helix through the break in the direction t,hat serves to decarease thr and then resealing t.htk break. linking number. During the breaking-rejoining reaction. the .5’ phosphate t,ermini of the nicked I>SA strands are (*ovalentSly linked to a tyrosine residue of the A subunit. of IJNA gyrase. This transient covalrntly linked gyrasc>-1)NA intJrrmrdiate has been called t.hta csleav able c*oniplcx (for reviews, set’ Wang. 1985: Maxwell B (:ellert 1986: I,iu, 1989). In prokaryotict and eukaryotic cells. the clravablt~ 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. ellipticines, uc%inomycines. 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 lopeiaomrrases. 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 understanding 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 (‘cd K protein is responsible for cell death and induction of the SOS pathway, when its action is not, prtbvent.ed 1)y prot,ein CcdA (Karoui et ul.. 1983; Bex it al., 1983; Ogura $ Hiraga, 1983; Miki et ccl., 19843; Sommer et al., 1985; Bernard & Couturier. 1991). Hot,h proteins are encoded by the ccd locus of plasmid F (ccd stands for control of cell deat,h). This 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 division (Jaff6 et al., 1985). The proposed mechanism underlying this post-segregational killing of bacteria is differential decay of the activities of the C’cdA and CcdB proteins, the half-life of active C’cdA protein being shorter than that of active C‘ctlB prot.ein. In newborn plasmid-free bacteria, persistence of the cytotoxic CcdB protein would lead to cell death (Jaff6 et al., 1985). ,\iliki rt nl. (1988) isolated a particular type of bacterial mutant resistant to the killer activity of the CrcdB protein that maps at, the yro&&’ locus. The GroES product) is a “(shaperone” protein participating in the corrrc~t folding of native protein (van Dyk ut al., 1989). It. is likely that the GroES protein plays a role in the folding of the CcdH protein or of its target, allowing intermolecular association. In this paper. we describe the isolation, mapping, sequencing and properties of another bacterial mutant resistant to the cytotoxic activity of the CcdB protein. We show that this mut.ation is located in the gyTA gene (48 min), which encodes the A subunit of the bacterial gyrase. In, uiivo. we additionally show that the presence of the CcdB protein is responsible for plasmid DNA breakage. On the other hand, plasmid DEA breakage is not observed in the mutant strain that resists the cytotoxic activity of the CcdB protein. These observations: together with a set of convergent results, strongly suggest that the CcdB protein is a poison for fG. coli topoisomerase II.

2. Materials and Methods (a) Bacterial The E. coli K-12 leuB6.

lacY1,

tonA2Z.

strains

and plasmids

strains used were: C600 thr-I. thi-1. supE44 (Appleyard. 1954): IV100

T,K rnrdiunl contains IO g of tryptone ((:ibco. I-K). 5 g of yeast extract ((:ib(ao. I’K) and .5g of Xa(‘I per litrlr of wat’er. Antibiotics were used at the following final ronct’tltrat,ions (in pg:!ml): ampic+ilin (Ap). 50; tet.rac)-clinr (TV). 10; kanamycin (Km). 50: chloramphenicol ((‘In). 20: and nalidixics acid (Nal). 20. In vitro reactions with restriction enzymes. phagr T1 l)SA ligase, and the Klenow fragment of DKA polymrrasr T WPW as rec~ommrndt~d by the manufacturers.

Most routine genetic manipulations were prrfornrrd as described by Maniatis et nl. (1982). Purification of restriction fragment,s srparatr~l L)y convrntional aparose gel electrophoresis (TAE buffer) was done with Gene (!lean (Bio 101). Plasmid DNA was purified as drticrihed by C’lewell & Helinski (1969). For small-scale preparations of plasmid DXA. the method described hy Birnhoim &. Daly (1979) was used. For resolution of plasmid DNA t,opoisomers by electrophoresis as described by Shure et al. (1977). small-scale preparations of DBA were purified on columns cont.aining Sephacryl S-400 (Pharma.cia). (IcdBinduced cleavage was revealed upon treatment of DXA with SDS and protrinasr K. as described bj O’Conn’or & Malarny (1985). Cell s were made (Lompetent and transformed as described by T,ederberg & Cohen (1974). Transformation of E. coli by electroporation was accomplished by the method of Dower et al. (19&C+), using a Bio-Rad gene pulser. Relative DX’A solution viscosity was determined using caapillary tubes and calculated according to the Poiseuille’s equation. as described by Spencer (1972). (d) Inkrrupted

mating

To allow recombination between chromosomic markers of the recipient and donor cells. plasmid pCOS2.1, carrying the reed gene of Erzoinia chrysanthemi 3665 (which promotes recombination in E. coli; C. Bertinchamps, unpublished results). was introduced int,o PBll (lh80) bacteria. R,ecombinant recipients were selected for kanamycin resistance conferred by the Ret ’ pCOS2.1 plasmid and for acquisition of tetracycline resistance conferred by 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 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 prewarmed flask at 37°C with gentle shaking to allow formation of mating pairs. To interrupt mating. a sample of the mixed culture was diluted IO@fold into saline and

Cell Killing

by the F Plasmid

737

CcdH Protein

Table 1 Phasmids

and plarmids

Description

Source

minib’ hp’ plasmid cloned in the A631 vector i.pSC138 with a amber mutation in the ccdrl @Cl38 copy number mutant E.pWl38 double mutant J’lasmids pA(‘Y(‘lX4 1’1’1,1~2230 pl’LB2232 pKK22:G:l l’l’LH2208

pKT979 pIlLR2007 pITI, 132015 1’171,1~2”1;i p.K‘75-5X p~‘os2~1 plTLUZI91 pI~L1?2291 plKlH220 pl’LB2222 pIU12224 pI’LU2226 pl~LIW%?T pL’LB2228 pl:LlW229

shaken s&rtirr

The (P) 1)X,4

cultures

were

then

plated

on

sequenciny

The PstT-By/II fragments were sequenced on pl-LB2228 and pI’LB2229 plasmids by the dideoxy chain-termination procedure (Sanger et al.. 1977), using [c(-35S]dATP and 2 synthetic 18 nucleotide primers. Sequencing Sequenase Kit was obtained from the U.S. Biochemical f’orporation. (f)

Mrc~.wrement

of SOS induction

Induction of s$A gene t,ranscription was used as a measure of SOS induction. s&4 induction was monitored by the amount of fl-galactosidase produced in lysogens by a s&4 :: ZucZ fusion carried by a icIind prophage (Huisman & d’Ari. 1981). The specific activity of p-galactosidase was determined as described by Miller (1972).

3. Results (a)

Isolation

cytotoxic The potent

(‘outurier

grw

‘I‘? and (‘m’ cloning vector derived from the P15A plasmid HarnHl 42.84 to Sol1 49 kb fragment of plTLI3201.5 cloned into the RanHI-Sal1 sites of p;2(‘Y(‘l X4 (Cm’) BornHI 42.84 to WI 49 kh ccdAnm22 fragment of pl’LB22l.i cloned int,o the BumHI-Sal1 sites of pA(‘YC184 (Cmr) Apr expression vector with the strong tar promoter Xdpl 43.1 to BglII 46.92 kb fragment of plTLB201.5 cloned into t,he SnmI site of pKK223-3 (AI”) S~/el 43.1 to HglII 46.92 kb fragment of pVLH2215 cloned into the SrnrcI site of pKK223-3 (W) pHR322 Tc’ cloning vector &coRl f6 ecdAam22 fragment cloned into the EcoRI site of pKT279 (Tc’) Ir:coRI f5 fragment deleted of the 43.61 to 46.9 kb KpnI miniF sequence and cloned into the EroRI site of pKT279 (Tc’) EcoRI f5 ccdAwn22 fragment deleted of the 43.61 to 469 kb KpnI miniF sequence and cloned into the EcoRI site of pKT279 (Tc’) (‘onditionally replicating (thermosensitive) ColEI Ap’ cosmid cloning vector Km’ cosmid pMMB34 with the gene WCA of Erwinia chrysanthemi 3665 &coRI t5 fragment deleted of the AoriII (XhoI-HglII) and cloned into the fl’coR1 site of pK1’279 (Tc’) pKT279 f5 ccddum’?% AoriII (Tc’) plasmid constructed by cloning the 4361 to 46.9 kb Kpnl AoriIT fragment of plasmid pULH2191 into the KpnI site of plasmid pULB221.i. Conditionally replicating (thermosensitive) ColEl Km’ cosmid constructed hy cloning a Km’ cassette (Pharmacia) into the PstI restriction site of cosmid pJC7.5-58. EcoRI f5 A&II fragment of pULB2191 cloned into the EcoRI site of pULB2220 (Km’) EcoRI f5 ccddnm29 AoriII fragment of pUL132291 cloned into the EcoRI site of pULH2220 (Jim’) 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) RmmHl BcoR\,- gyrA462 fragment of 1’831 (L) cloned into the l-87 to 3.21 kb BarnHI--HincII sites of pA(‘YC184 (Cm’) ClnI-C’/rcI gTlr.4 + fragment of pULB2226 cloned into the C’lal site of pACYCl84 (Cm’) ClaI -C’/nl gyr.4462 fragment of pULB2227 cloned into the C’lnI site of pA(lYC184 (Cmr)

vigorously medium.

ccd locus cell-killing

oJ” bacterial zmutants resistant activity of the CcdR protein

to the

of the miniF plasmid encodes the CcdB protein (101 amino acid

or referencr

rt u/. (I 979)

Karoui rt nl. (1983) Kex et al (19X6) Bernard & (‘outrrrirr (1991) Chang & Cohen (197X) Bernard 8: (‘outurirr (1991) Thin work Brosius 8r Holy I 1984) Bernard & (‘outurier (1991) Bernard & (‘outrrriel (1991) Talmadgr h Gilbert (1980) Karoui et al. (1983) Karoui et al (1983) Bernard Kr (‘outurirl (1991) Collins & Briining (1978) This lahorator) This lahorator~ This work This work This work This work

This work This work This work This work

residues) together with its antagonist, the CcdA protein (72 amino acid residues). In order to identify the bacterial target with which the cytot’oxic CcdR protein interacts. we selected bacterial mutants resistant to its killing activit,y (CcdB’ phenotype). The CcdB’ bacterial mutants were obtained by a double selection using miniF hybrids able to synthesize the CcdB protein but unable to produce the antagonistic CcdA protein (as a result of an amber mutation in the ccdA gene, ccdAam22; Karoui et al., 1983). The two CcdA-CcdB’ miniF hybrids used in were iminiF phasmid the selection the IlpSCl38ccdAam22cop5 (Bernard & Couturier, 1991) and the conditionally replicating (thermosensitive) ColEl miniF plasmid pULB2224 (for construction, see Materials and Methods). Independent cultures of I-lysogenic strain NlOO(AhSO), mutathe sup’ genized with 2-amino-purine (according to Miller, 1972) were infected at a multiplicity of three by the CcdA-CcdB+ Ap’ AminiF phasmid (IpSC~13XccdAamZZcop5) and grown exponentially in LB broth supplemented with ampicillin to select for t.he presence of the phasmid. iZfter this first

Overnight cultures of the Iysogrnic i. strains were infected with the ipSC138cop5 ((‘odA+CcdH ‘) or ~pS(Il~RecdAum”~eopS at a multiplicity lower than 1. ((‘c~dA~(‘cdB+ in SZLI)~ strain) After 20 min xdsorption. thr infected cells wvre plated on 13 agar plates supplemented with ampicillin. The A$ plasmidization efficiency was c&ulated its the ratio of thr number of Ap’ c4ones formed on ampicillin plates to thr number of infecting phagrs.

exposure to the cytotoxic (IcdH product. we observed that most of the surviving bacteria were not. resistant to its killing activit’y. The surviving ba,c:teria were mostly plasmid-free Ap’ bacterial mutants or bacteria bearing a CcdBmut’ant plasmid. In order to recover the rare CcdH’ bacterial mutants among the survivors, we performed a second exposure to the CcdB cytotoxic protein. The exponentially growing Ap’ cells (survivors) were rendered competent and transformed with the (‘udA CcdK+ Km’ pULB2224 plasmid. In this second selection, the undesirable Ap’ or CcdB- survivors were eliminated by the CcdB protein expressed by the pL’LB2224 plasmid and by ka,namycin present in the growth medium. Note that, plasmid pULB2224 displays a potent IncFT incompatibility phenotype (because it contains repeat,ed sequences related to the col)ynumber control). It, thus prevents replication of the 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 survivors were cured of the conditionally replicating prLB2224 plasmid by exposure to the non-prrmissive temperat,ure (42°C). The cured bacteria were then reinfect.ed w&h the C’cdA-~CcdB+ Ap’ AminiF phasmid. This time, none of the surviving bacteria was killed by the phasmid, indicating that the mutation conferring the CcdB’ phenotype is located on the bacterial chromosome. By this procedure, we isolated several independent bacterial mutants resistant to t’he cytotoxic activity of the (%:dK protein. One of these, the PHI l(Ah80) strain, was analyzed further. Tts colony-forming ability after infect.ion wit,h the cyt.otoxic CcdA-CcdB+ Ap’ AminiF phasmid is shown in Table 2.

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:sconjugant.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 point at. 52 minutes and tht, TnIO tratrsposotl inserted at 37 minutes. and PBl 1 (,?h80) rccipirnt c,ells. On t,hr other hand. mating perform~~d with HfrKL96 donor strain, having it,s injection l)oint at 45 minutes and the TnlO insertion a~ L’i minutes, did not give rise, Tao (%dK” trarlsc.onjugallt,s. These (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 proximity of t.hr c:cclB’ alIt+ witah a TnIO transposon inserted at 48 minutes nthar the !/!/“.-I gene (zeG29X::TnIO). To do this, a 1’1 Iysatt’ was prepared on the C’cdlY ~ri-2.9X::Tt110 st,rain and used to infect a Kw+ derivativr of t,hr f’Rl 1 (JLhFIC)) C’cdH’ strain. As a c~ont,rol. the same 1’1 lysatts was used to infect a WC’31IO(i.) strain bearing a tlalitiixic* resistant (Nal’) mut,atiott in the yyr.4 ytbnt.. In both transduct,ions. tc,l.rac.~~litle-resistant t rat1stiuc.t atlts were selrc~ted and t estc>d for their sensitivity or resistance to t,he caytot.oxic. (‘c~il3 protein (for the PHI 1 (AhSO) t’ransductants) and sensit.ivity or resistance to tlalidixic acid (for the \VSl 10(/i) t rallsdu(.tants). LZ’C observed similar c~otrailsdric,t’ic,n frequencbies of t’hr c~d/Y and nalS (gyr,-l ) alleles with the Tn IO t ransposon (respectively, 46 o. atIt 48 o0 c.otransduc~t,ion). A 1’1 tysate was thtbn 1)rv1)ared on the PI{1 1 (i.h80) Rev+ TV’ C’cdlI’ derivativ(~ strain and was used to infec-1 the \Y3110(/.) straits. r\s a result. Tc’ (‘c~lI3” PK30(~) and ‘I’(*’ (‘cdK’ l’tUl(i) transductatrt.s were obtained.

The gyr;t gene of E. c~li KlX chromosome is contained in an 8 kbt EcoKV- HamHI restriction fragment located around 4X.4 minutes on the physical map (Kohara it CLLI.,1987). In order to clone the yyrA region of’ tht> wild-type T’K30(1) itnd mutated PBS1 (1) strains. their genomic DXAs were restricted with EcoRC’ and HnmHI restrict’ion endonucleases and then ligated into the Hl.ncIT--RamHI sites of plasmid vect’or pACYC184. The ligation products were used to transform &rain OV6 (yyrA., csupF,,: Hussain et al., 1987). and the recombina,nt plasmids wcrc selected for their ability to csompletnent t>he growth defect of this strain at) 42’(‘. The t Abbreviations basct-pair(s).

used:

kl). IO3 i~,ses or hnstl-l);tirs:

I)b).

Cell Killing

Ad

SOll

8

7.2

BornHI

pULBPro

6.9

C/o1

by the F Plasmid

(9.3 kb)

65

8gllI

6.2

Kpnl

Bgln

5.6

5-3

I PSfI

AvoI

4.3

4.9

Figure

1. Structure of plasmid pULB2228. Plasmid pULB2228 is a pACYC184 (fine line) with a 51 kb Cl& ClaI insert containing a complete wild-type gyrA gtme. The structure of pULB2229 is similar, except that the 51 kb fragment is derived from the CcdB’ strain PB31(1). By reconstruction experiments between both plasmids, we located the ccdR’ (gyrA462) mutation within the PstIBglII 49 to 53 kb restriction fragment. The open boxes represent a duplication of vector DNA.

CcdB

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 merodiploids were determined according to their ability t,o form colonies after transformation with a CcdA -CcdR + plasmid (pULB2212). The results presented in Table 3 show that ccdB”/ecdB” and ecdB”/ccdB’ merodiploid bacteria do not give rise to viable transformants, whereas ccdB’/ccdB’ merodiploids vield viable transformants after transformation with plasmid pULB2212. Thus, the presence of the wild-type pULB2228 plasmid in the CcdB’ strain reverses the CcdB’ phenotype of the strain. We conclude that the mutation conferring t.he CcdH’ phenotype is located in the 5.1 kb fragment carried by plasmid pULB2229, and that’ t’he sensitive phenotype dominates over the resistance phenotype. This strongly suggests that t,he CcdH protein is more than a mere inhibitor of an essential function

but a poison

‘ ‘GyrA +” recombinant plasmids derived from the CcdBS (pULB2224) and CcdB’ strains (pULB2226) were digested with a variety of restriction endonucleasesin order to check whether their restriction patterns match those of the gyrA region and of the pACYC184 vect’or. From each of the two plasmids, a 51 kb fragment containing the entire gyrA gene was cloned into the ClaI site of the plasmid vector pACYC184, giving rise to plasmids pULB2228 and pILB2229, respectively (Fig. 1). (d) The ccdB’ mutation maps in the central part of the gyrA gene and is recessivewith respect to the wild-type gyrA allele In order to det’ermine whether the ccdB’ mutation maps in gyrA, we performed complementation and reconstruction experiments.

of that

function.

In order to determine which part of this 51 kb fragment is responsible for the CcdB’ phenotype, restriction fragments of plasmid pULB2228 were replaced with equivalent restriction fragments of plasmid pULB2229, to reconstruct the locus. Then, the resulting

resulting

739

Protein

recombinant

plasmids

were

tested

for

their CcdR” or CcdB’ character. By this means, we observed that the mutation conferring the CcdB’ phenotype maps in the central part. of the gyrA gene in the small 380 bp PstI-BglII fragment (Fig. 1). (e) Sequencing

of the ccdI3’ mutation

We determined and compared the nucleotide sequencesof the 380 bp PstI-BgZII fragment of the gyrA gene from plasmids pULB2228 (CcdBS) and pULB2229 (CcdB’). The results revealed that the central part of the wild-type gyrA gene (ccdB”) differs from that of the mutated gyrA gene (ccdB’) by a point mutation: a C.G to T.A transition at position 1384, which produces the substitution of Arg462 by a Cys residue in the amino acid sequence of the A subunit of gyrase. Henceforth. the ccdB mutation

was called

gyrA462.

Table 3 (‘omplementation analysis

of the ccdB’ and ccdB’ chromosomal plasmid p U LB2229

markers

by plasmid

pULB2228

(ccdl3”) and

(ccdB’)

Rate of transformation

Plasmids pKK223-3 pULB2208 pULB2212

Relevant character Vector CcdA +CcdB + CcdA-CcdB+

NlOO/ pACYC184 ccdBS/vector 1 1 1

NlOO/ pULB2228 ccdB”/ccdB” OS-l.2 081.2