JOURNAL OF BACTERIOLOGY, Dec. 1990, p. 6973-6980 Vol. 172, No. 120021-9193/90/126973-08$02.00/0Copyright C 1990, American Society for Microbiology

Recombination at ColEl cer Requires the Escherichia coli xerCGene Product, a Member of the Lambda Integrase Family of

Site-Specific RecombinasesSEAN D. COLLOMS, PETER SYKORA,t GEORGE SZATMARI,t AND DAVID J. SHERRATT*

Institute of Genetics, University of Glasgow, Church Street, Glasgow GIl 5JS, Scotland

Received 25 July 1990/Accepted 11 September 1990

Site-specific recombination at the plasmid ColEl cer site requires the Escherichia coli chromosomal genexerC. The xerC gene has been localized to the 85-min region of the E. coli chromosome, between cya and uvrD.The nucleotide sequences of the xerC gene and flanking regions have been determined. The xerC gene encodesa protein with a calculated molecular mass of 33.8 kDa. This protein has substantial sequence similarity to thelambda integrase family of site-specific recombinases and is probably the cer recombinase. The xerC gene isexpressed as part of a multicistronic unit that includes the dapF gene and two other open reading frames.

Multimerization of multicopy plasmids by homologousrecombination in Escherichia coli can lead to a decrease intheir heritable stability. The natural plasmid ColEl containsa 280-bp stability region, cer, at which host-encoded proteinsact to bring about monomerizing site-specific recombination(36, 39). Two copies of cer in a direct repeat within a singleplasmid molecule are the only ColEl sequences needed forthis site-specific recombination (39).

Site-specific recombination reactions require an enzyme,the recombinase, which catalyzes the cutting and rejoiningof the participating DNA molecules. All recombinases so farcharacterized show amino acid sequence similarities toeither the resolvase-invertase family or the lambda integrasefamily of site-specific recombinases (3, 16, 31, 33). To isolatethe cer recombinase, we made mutants defective in cersite-specific recombination (36). Two E. coli chromosomalgenes, argR and pepA, which were absolutely required forsite-specific recombination at cer were found (35, 37). TheargR gene product, the arginine repressor, binds to cer in thepresence of its corepressor, L-arginine, about 100 bp fromthe crossover region (37).A variant cer site which recombines independently of

ArgR and PepA has been found, although the directionalityof recombination at this site is affected by their presence(38). We believe that neither ArgR nor PepA is the cerrecombinase because neither is required for recombinationat this variant cer site and neither shows amino acid se-quence similarity to any known recombinase. We believeinstead that PepA and ArgR have an accessory role in the cersite-specific recombination reaction.Here we report the cloning of a third gene, xerC, that is

also required for cer site-specific recombination. We showthat this gene encodes a protein of 33.8 kDa which hassequence similarity to the lambda integrase family of recom-binases. This gene is part of a multicistronic transcriptionunit which maps at 85 min on the E. coli chromosome andwhich includes the gene dapF, encoding diaminopimelateepimerase.

* Corresponding author.t Present address: Institute of Molecular and Subcellular Biology,

Comenius University, Bratislava, Czechoslovakia.t Present address: Departement de Microbiologie et Immunolo-

gie, Universitd de Montrdal, Montreal, Quebec, Canada.

MATERIALS AND METHODS

Bacterial strains. All strains are derivatives of E. coli K-12AB1157 (4). DS941 is AB1157 recF lacIq lacZ AM15. DS953is a sup° derivative of DS941. DS941 argR (DS941xerA9: :fol) has a Tpr gene inserted into the chromosomalargR gene (12). DS941 pepA7 is a spontaneous mutant ofDS941 initially selected as being resistant to the toxicpeptide Val-Leu-NH2 (26). Other derivatives of DS941 aredescribed in the text.

Plasmids and bacteriophage. Diagrammatic representa-tions of pKS455 and pSDC110 are shown in Fig. 1. pCS202is a Cmr Tcr lambda dv-based reporter plasmid containingtwo directly repeated cer sites that deletes Tcr on cer-mediated recombination. pMAK101, pMAK102, andpVMK42 are described by Aldea et al. (2) and were the giftof S. Kushner. pSDC102, pSDC104, and pSDC123 containpMAK101-derived fragments (see Fig. 3) in the vectorpTZ18R (Pharmacia). pSDC112 contains the 1232-bp XerC+fragment from pSDC104 in a lambda dv-based Cmr Kmrvector. pSD105 contains the same 1,232-bp XerC+ fragmentin the expression vector pBAD (a derivative of pKK223-3;Pharmacia). Bacteriophage Plkc was used for generalizedtransduction. The suicide vector, lambda NK467 (Ab221rex: :Tn5 Oam29 Pam8O [7]), was used to deliver Tn5.

Bacterial growth media and conditions. L broth (19) wasused for routine growth of E. coli and was supplementedwith agar and antibiotics when appropriate. Medium forDS941 dapF was supplemented with 20 ,ug of diaminopimelicacid (a mixture of LL, DD, and meso isomers; Sigma) per ml.

Bacterial transformation and mutagenesis. Plasmid trans-formation of CaCl2-treated competent cells was done by themethod of Cohen and Hsu (5). Mutagenesis was carried outby incubating DS953 with lambda NK467 for 30 min in Lbroth at 37°C. Mutagenized cells, selected for Kmr, weretransformed with pKS455 and selected for Apr. Colonieswere screened for Cmr by replica plating. The plasmid-bornemutant xerC2 allele (Fig. 2) was used to replace the chromo-somal copy of xerC by the method of Flinn et al. (12).

In vitro DNA manipulations. DNA manipulations wereperformed essentially as described by Maniatis et al. (25).Sequencing reactions were carried out by a modification ofthe chain termination reaction (32) with reagents from theSequenase kit (United States Biochemical Corp.). Frag-ments derived from pMAK101 by restriction enzyme cleav-

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Ar

Ap

X><ctk e

ariV

cerl

rCm

B cer

N 'NL. Ia, , L ,

>0~'Z,

cer

pKS455

p456

Apb cer

pSDCI 10

=p 1pl I I dimer

p I I IAp

cer

or/f.rFIG. 1. cer-mediated site-specific recombination in Xer' and Xer- E. coli strains. (A) DS941 E. coli Xer' and Xer- strains were

transformed with reporter plasmid pKS455, containing two directly repeated cer sites flanking a Cmr determinant. cer-mediatedrecombination yields plasmid p456, containing a single cer site and lacking the Cmr determinant. Plasmid DNA was isolated and run on a 1.2%agarose gel. (B) DS941 E. coli Xer' and Xer- strains were transformed with pSDC110, containing two variant cer sites (cer*, the type IIhybrid [Sau3A-TaqI fragment], which allows intermolecular as well as intramolecular site-specific recombination without ArgR and PepA[38]). Intramolecular cer site-specific recombination on pSDC110 yields plasmid pill. Plasmid DNA was isolated and run on a 1.2% agarosegel.

age and exonuclease III deletion (17) were cloned intoM13mpl8 and mpl9 (40) for sequencing. DNA from in vivorecombination experiments was purified from cells by themethod of Holmes and Quigley (18) and run on 1.2% agaroseTris-acetate gels (25).

N-terminal sequencing of XerC. Partially purified XerCwas run on a sodium dodecyl sulfate-polyacrylamide gel(22). XerC was eluted from a gel slice and precipitated withacetone (14). The purified material was sequenced by Edmandegradation for 20 cycles on an Applied Biosystems 477Aautomated protein sequencer.

Nucleotide sequence accession number. The sequence re-ported here has been submitted to the GenBank and EMBLdata bases under the accession number M38257.

RESULTS

E. coli xerC, a new gene involved in site-specific recombi-nation at plasmid ColEl cer. To ascertain whether other E.coli genes in addition to argR and pepA are involved in cersite-specific recombination, we selected further mutantsdeficient in cer site-specific recombination. The mutagenesisand selection procedure used was that described for theselection of earlier mutants (36). DS953 was mutagenizedwith TnS by use of the defective phage lambda NK467 as adelivery system. Mutagenized cells were transformed withplasmid pKS455, which contains two cer sites flanking acopy of the chloramphenicol acetyltransferase gene (Fig.1A). In a wild-type host this gene is deleted by site-specificrecombination, leaving the transformant chloramphenicol

sensitive. Mutants with mutations in genes required for cersite-specific recombination can be selected because theyremain resistant to chloramphenicol after transformationwith pKS455.One such mutagenesis produced a mutant with a novel

leaky Xer- phenotype. This mutant retained a mixture ofunresolved pKS455 and the deleted replicon (p456) aftertransformation with pKS455 (Fig. 1A) and remained resis-tant to chloramphenicol. The leaky Xer- phenotype wasalso seen when the mutant was transformed with other 2-cerplasmids. The phenotype was not complemented by plas-mid-borne copies of either argR or pepA; therefore, themutation defined a new xer complementation group, xerC.The phenotype of the xerC mutant could be distinguishedfrom that of argR and pepA mutants in another way;recombination at a derivative cer site (here called cer*),created by site-specific recombination between ColEl cerand the cer analog of plasmid CloDF13 (parB), was indepen-dent of the argR and pepA genes but required the xerC gene(Fig. 1B) (38).

Isolation and mapping of xerCL. DNA hybridization anal-ysis with Southern blots showed that the mutant strain(DS953 xerCl) carried a single chromosomal copy of TnS(data not shown). This copy of TnS was shown to be tightlylinked to xerC by P1 transduction into DS941. The mutantgene was cloned into pBR322 from an EcoRI digest of DS941xerCl chromosomal DNA by selecting for Tn5-encodedkanamycin resistance. The resulting plasmid (pSDC100)contained an 8.3-kbp fragment of E. coli chromosomal DNAwith a copy of TnS (5.8 kbp) within it. Comparison of the

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RI B BI I I

H p

S

cya95 kDa

P PII

Bg BgI I

RI

I IS Pv

or1235 orI28827kDa 27kDa

cyvaXY dapf xerC17 kDa 30 kDa 34 kDa

1/uvr7.276 kDa

Y30 . 13 YS Y2xerCol XerC2::Tn5 ::kan

pVMK42 XerC

pMAK102 XerC

pMAKI01 XerC+

pSDC102 XerC+

pSDC104 XerC+

pSDC123 XerC

FIG. 2. Map of the 85-min region of the E.coli chromosome (coordinates 3,716 to 3,724 kbp on the map of Kohara et al. (21; see alsoreferences 2 and 29). The top of the figure shows the restriction map of the 8.3-kbp EcoRI fragment: B, BamHI; Bg, BgIII; R1, EcoRl; H,HindIII; S, Sall; P, PstI; Pv, PvuII. Below this, the genes contained within the fragment are indicated. The molecular masses shown were

calculated from DNA sequence data. All of the genes are transcribed in the same (left to right) direction (1, 9, 10, 30). Y30, Y13, Y17, andY2 are all insertion mutations that yielded in-frame lacZ translational fusions obtained with Mu dII PR13 (29). Tn5 indicates the site ofinsertion that gave rise to the xerCl mutation. kan indicates the Sall restriction site at which a kanamycin resistance gene was introduced intothe chromosome to yield the xerC2 mutation. The extent of chromosomal DNA present in various plasmids is indicated at the bottom of thefigure. These plasmids were assayed for complementation of the xerC- phenotype of DS941 xerCl by examining the resolution products ofthe plasmid pCS202 on agarose gels. The inserts in pSDC102, pSDC104, and pSDC123 are in the correct orientation for xerC to be transcribedfrom the lac promoter of pTZ18R.

restriction map of pSDC100 with Southern blot data con-

firmed that the correct xerC-linked TnS insertion had beencloned. A restriction map of the wild-type xerC gene andsurrounding DNA was deduced from the restriction map ofpSDC100. Comparison of this deduced map with the com-

plete restriction map of the E. coli chromosome (21) showedthat xerC maps to 85 min. Further refinement of this mapposition was made possible by comparison with the detailedmap of the ilv-metE-udp region of the E. coli chromosome(2), accurately locating the site of insertion of TnS in DS953xerCI to between the genes for adenylate cyclase (cya) andSOS-inducible DNA helicase II (uvrD) (Fig. 2).

Defining the wild-type xerC gene. The entire ilv-metE-udpregion of the E. coli chromosome has been cloned intomulticopy plasmids (2). S. Kushner kindly supplied us withplasmids covering the cya-uvrD region. One of these,pMAK101, complemented the xerCl mutation, allowingrapid resolution of the reporter plasmid pCS202 in xerCmutant strains. A 3.6-kbp HindIII-BglII fragment frompMAK101 was subcloned into pTZ18R to produce

pSDC102, which was also found to complement the xerClmutation. Exonuclease III deletion (17) ofpSDC102 from theHindIII end produced the XerC+ plasmid pSDC104, whichcarries a 1,232-bp chromosomal fragment. The sequence ofthis fragment and neighboring regions was determined inboth directions by the Sanger dideoxynucleotide chain ter-mination reaction (Fig. 3). The exact insertion point of TnSin DS953 xerCl was also determined.The minimal complementing fragment contains an open

reading frame which could encode a protein of 298 aminoacids. The site of Tn5 insertion in DS953 xerCl was outsidethe minimal complementing fragment and 5' of the xerC openreading frame. The leaky phenotype of the xerCl mutationwas probably the result of a reduction in the transcription ofxerC brought about by the polar upstream TnS insertion. Todetermine whether xerC is absolutely required for cer site-specific recombination, we cloned a kanamycin resistancegene into the Sall site of a plasmid-borne copy of the xerCgene, disrupting the xerC open reading frame to yield xerC2(Fig. 2). The mutant gene was used to replace the wild-type

I kb

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a 0 0 a L0

1 1 1 1 II I

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dapF orf235 PstI1101 TATATGACTGGCCCGGCGGTACATGTCTACGACGGATTTATTCATCTATGAAGCAACCA6GGGA^GAACTGCAGGAAACACTCACGGAGCTTGATGACCG

Y M T G P A V H V Y D G F I H L *M K Q P G E E L a E T L T E L D D R

1201 GGCGGTTGTCGATTATCTGATTAAAAATCCTGAGTTTTTTATCCGTAATGCGCGCGCAGTAGA^GCGATACGTGT@CCACATCCGGTACGCGGCACCGTTA V V D Y L I K N P E F f I R N A R A V E A I R V P H P V R ¢ T V

1301 TCGTTGGTCGAGTGGCACATGGCCCGCGCACGTAATCATATTCATGTTCTGtAGMAGAGACATGGCGCTGTTGATGGAACAGGCTATCGCCAACGAAGCCS L V E W H M A R A R N H I H V L E E N M A L L M E 0 A I A N E G L

PstI140 1 TGTTTTATCGCCTACTCTACCTGCAGCGCAGTCTCACCGCCGCCAGCAGTCTCGACGATATGCTGATGCGCTTTCACCGCT GATCTCGGCCT

F Y R L L Y L 0 R S L T A A S S L D D M L M R F H R W A R D L G L

lS01 GGCAGGTGCGAGTCTGCGCCTGTTTCCGa^TCGCTGGCGCTTAGGTGCGCCGTCGAACCACACTCATCTGGCATTAAGCCGTCAGTCTTTCQAACCGCTGA G A S L R L F P D R W R L G A P S N H T H L A L S R C S f E P L

_T-n5 epSDC1041601 CGTATTCAGCGTTTGGGGCAGGMACAGCACTAiTCTTGGGCCGCTTAACGGACCAGAGCTGCTGGTGGTGCTACCGhAGCAAGCGGT¢GGATCGGTGG

R I Q R L G Q E 0 H Y L G P L N G P E L L V V L P E A K A V G S V A

1701 CGATGTCGATGCTGGGAAGCGATGCTGATTTGGGTGTCGTGCTGTTTACCAGTCGCGATGCCGTCACTATCAACAGGGCAAGGAACGCAGTTACTTCAM S M L G S D A D L G V V L f T S R D A S H Y 0 Q G Q G T Q L L H

_Y13 xerC1801 TGAAATTGCGCTGATGTTGCCGGAGCTTCTGGAGCGTTGGARTTGAACGCGTATGACCGATTTACACACCGATGTAGAACGCTACCTACGTTATCTGAGCG

E I A L M L P E L L E R W I E R V *M T D L H T D V E R Y L R Y L S V

p6&DC123190 1 TGGAGCGCCAGCTTAGCCCGATMACCCTGCTTAACTACCAGC(IrAG'CTTGAGGCGATCATCAATTTTGCCAGCGAAACGGCCTGCAAAGCTGGCAGCA

E R C L S P I T L L N Y Q R a L E A I I N F A S E N G L C S W1 0 Q

2001 ATGTGATGTGACGATGGTGCGCATtTTTGCTGTACGCAGtCGCCGTAAAGGGCTGGGAGCAGCAAGTCTGGCGTTACGGCTTTCTGCGCTACGTAGCTTTC D V T M V R N f A V R S A R K G L G A A S L A L R L S A L R S f

_Y17 SaII2101 TTTGACTGGCTGGTCAGCCAGAACGAACTCAAAGCTAACCCGGCGAAAGGTGTTTCOXACCGAAAGCGCCGCGTCATCfGCCGAAAACATCGACGTCG

F D W L V S 0 N E L K A N P A K G V S A P K A P R H L P K N I D V D

2201 ACGATATGAATCGGCTGCTGGATATTGATATCAATGATCCCCTCGCTGTACGCGACCGTGCAATGCTGGAAGTGATGTACGGCGCGGGTCTGCGTCTTTCD M N R L L D I D I N D P L A v R D R A- M L E V -M Y G A -G L R. L S

2301 TGAGCTGGTZGxGCTGGATATTAAACACCTCGACCTGGAGTCTGGTGAAGTGTGGGTTATGGGGAAGGCAGCAAAGAGCGCCGCCTGCCGATT¢GTCOCE L V G L D I K H L D L E S G E V W V M G K G S K E R R L P I G R

2401 AACGCTGTGGCGTGGATTGAGCACTGG:CTTGATTTGCGCGACCTGTTTGGTAGCGAAGACGACGCGCTTTTTCTGTCGAAACTGGGCAAGCOTATCTCCGN A V A W I E H W L D L R 0 L F G S E D D A L F L S K L G K R I S A

2501 CGCGTAATGTGCAGAAACGCTTTGCCGAATGGGGCATAAAACAAGGGCTGAATATCACGTTCA.TCCGCATAAATTACGTCACTCGTTCGCCACGCATATR N V Q K R F A E W G I K a G L N N H V H P H K L -R H S F A t HM

2601 GCTGGAGTCGAGCGGCGATCTTCGTGGTGTGCAGGAGCTGCTGGGTCATGCCAACCTCTCCACCACGCAAATCTATACTCATCTTGATTTTCAACACtTTL E S S G- L R G V. 0 E L L G H A N L S T T Q I Y T tl L D f a H L

or1 2382701 GCCTCGGTGTACGATGCGGCGCATCCACGCGCCAAACGGGGGAAATAATGCGTTTTTACCGGCCTTTGGGGCGCATCTCGGCGCTCACCTTTGACCTGGA

A S V Y D A A H P R A K R G K *

M R F Y R P L G R I S A L T F D L D

2801 TGATACCCTTTACGATAACCGTCCGGTGATTTTGCGCACCGAGCGAGAGGCGCTTACCTTTGTGCAAATTATCATCCGGCGCTGCGCAGCTTCCAGAATD T L Y D N R P V I L R T E R E A L T f V a N Y H P A L R S F Q N1 9L101 Y2

2901 GAAGATCTGCAACGCCTGCGvCCAGGCGGTACGGGAAGCGGAACCCGAGATTTATCACGACGTGACGCGCTGGCGTT'TTCGTTCGATTGAACAAGCGATGCE D L a R L R a A v R E A E P E I Y H D V T A W R F R S I E a A M L

3001 TCGACGCCGGGCTGAGTGCC -AAGAAGCCAGTGCAGGCGCACACGCAGCAATGATCAACTTTGCCAAATGGCGCAGCCGAATCGACGTCCCGCAGCAAACD A G L S A E E A 8 A G A H A A M r N F A K W R S R I D V P Q Q T

3101 TCACG AGTGGCACACCTGGC CCGCAGA TA ATCACTAACTGGCGCCCAGCCTGAGCTGTTTGGTTGAGGTGTtATTTTAAGH D T L K Q L A K K W P L V A I T N G N A Q P E L F G L G D Y F E

* ~~~~~~~~~~89L11I3201 tTTGT~sGCTGCGCtGTGCCCGCACGG TCAAAACCGTTCAGCGATATGTACTTTTTGGCTGCGOAAAACTCAACGTGCCGATCGWGATCTTAC

F V L R A G P H G R' S K P F S D M Y F L A A E K L N V P I ¢ E I L t1

3301 ATGTTGGGGCGATCTCACCACTGACGTcGGtGGGGWATTCGCAGCGGAATGCAGGCTI'GTTGGATCAGACCAAAATQGCMTCTGATGCACCTGV G D D L T T D V G G A I R S G M Q A C W I R P E N G D L M Q T W1

.-35 Pi -10 Lex3401 GGACAGCCGTTTACTGCCGCCATCTGGoAATTTCCCGGTTGGCATCTCTGACCTCGCTGATATAATCAGCAAATCTGTATATATACCCAGCTTTTTiCGG

D S R L L P H L E I S R L A S L T S L I*-35 P2 -1D uvrD

3501 AGGGCGTTGCGCTTCTCCGCCCAACCTATT~TTACGCGGCGGTGCCAATGGACGTTTCTTACCTGCTCGACAGCCTTAATGACA~AACAG=GAGCGGTM D V S Y L L D S L N D IC R E A

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chromosomal copy of xerC in DS941. This mutant strain isviable (although it produces smaller colonies than the wildtype) and is totally deficient in cer site-specific recombina-tion (Fig. 1).The xerC gene is part of a multicistronic unit. The gene for

diaminopimelate epimerase, dapF, has also been found tomap between cya and uvrD on the E. coli chromosome (29).The sequence that we have determined to flank xerC at the 5'end overlaps with the published 3'-flanking sequence ofdapF (30). Taken together, the sequence data show thatthere is an open reading frame (orf235) between dapF andxerC which could encode a protein of 235 amino acids (Fig.3). Downstream from xerC, where our sequence overlapswith the published uvrD sequence (9-11), there is anotheropen reading frame (or238) which terminates just before theuvrD promoter P1. This open reading frame could encode aprotein of 238 amino acids (Fig. 3). The close proximity ofthese four open reading frames suggests that they could beexpressed from one mRNA and that they might be transla-tionally coupled.

C, Richaud supplied us with four strains carrying mini-MuMu dII PR13 insertions in the dapF-xerC region. Theseinsertions are lacZ translational fusions to the four openreading frames (Fig. 2 and 3) (29). P1 transduction was usedto move these insertions into DS941 for further study. Thetwo insertions upstream of xerC-orf235 Y13 and dapFY30-yielded a leaky Xer- phenotype very similar to that ofthe xerCI mutation (Fig. 4). This phenotype was probablycaused by polar effects of the insertions on the transcriptionof xerC. The Y17 insertion, which is within the xerC openreading frame, yielded a totally Xer- phenotype. All of theseinsertion mutations were complemented by the 1,232-bpminimal xerC clone (Fig. 4). The Y2 insertion, which isdownstream of xerC within or238, yielded no detectablephenotype. All four mini-Mu insertions yielded similar beta-galactosidase activities (1 to 5% of the fully derepressedlevel of expression from a chromosomal lacZ gene), suggest-ing that the four genes are expressed at comparable levels.

FIG. 3. DNA sequence of the dapF-uvrD region of the E. colichromosome. The sequences of dapF (30) and of uvrD (9-11) havebeen previously reported. The sequence presented here joins dapFand uvrD. The inferred protein sequence for or1235, xerC, andor1238 is shown, as are those for the C terminus of dapF and the Nterminus of uvrD. The positions of the in-frame mini-Mu insertions(Y13, Y17, and Y2), as determined by DNA sequencing, are shown.The position of the TnS insertion in xerCl is indicated, as is the Sansite at which the Kmr cassette was inserted to produce the xerC2mutation. Conserved domains 1 and 2 found in the lambda integrasefamily of site-specific recombinases are underlined within xerC; thetotally conserved H, R, and Y residues are in domain 2. The XerC+plasmid pSDC104 (Fig. 2) carries the 1,232-bp fragment from theposition marked at nucleotide 1675 to the BglII site at nucleotide2907. The XerC- plasmid pSDC123 (Fig. 2) carries the fragmentfrom the position marked at nucleotide 1945 to the BglI site atnucleotide 2907. Two potential promoters for uvrD, P1 and P2, anda LexA-binding site are shown (9-11). Nucleotide 1101 is enumer-ated as 1101 by Richaud and Printz (30), who presented thesequence as far as nucleotide 1306. There are two minor differencesbetween the sequence presented here and that presented by Richaudand Printz (30): T at 1275 is TT in reference 30, and CC at 1277 isCCC in reference 30. We have checked our sequence data (fromboth strands) very carefully in this region and believe that theversion presented here is the correct one. The sequence determinedin this work runs from the PstI site at 1169 to the BglII site at 3297.The sequence from the PvuII site at 3117 is presented in reference10. Nucleotide 3292 is enumerated as 1 in reference 11.

xrerCplasmid

C)nr- C)C)r

Cn _ _ C\, e') _ -4

xerCpSDC1 1 2

- ~pKS455

- p456

FIG. 4. Complementation of Xer- insertions in dapF, orf235,and xerC by a fragment containing only the xerC open readingframe. The DS941 derivatives indicated were transformed eitherwith pKS455 alone or with pKS455 and pSDC112. Plasmid DNAwas isolated and run on a 1.2% agarose gel. pKS455 is a reporterplasmid, and p456 is its plasmid resolution product. pSDC112contains the same minimal xerC+ fragment as pSDC104 in a lambdadv replicon compatible with pKS455. The xerC gene is in the correctorientation to be transcribed from the lac promoter in pSDC1l2.

We have not detected any regulation acting on the expres-sion of any of these fusions in exponentially growing cul-tures.XerC belongs to the lambda integrase family of site-specific

recombinases. The translated protein sequence of XerCcontains two regions which are homologous to the twoconserved domains of the lambda integrase family of site-specific recombinases (Fig. 5) (3). Domain 2 of the XerCsequence has three totally conserved amino acids, histidine,arginine, and tyrosine, as well as other less conserved aminoacids. The XerC sequence has 32% amino acid identity to theE. coli proteins FimB and FimE in an alignment coveringabout 160 amino acids. These two proteins are involved ininverting a segment of the E. coli chromosome to switchfimbrial antigens (20). Within conserved domain 2, the XerCsequence shows considerable similarity (66% identity) to anintegraselike inferred protein sequence from plasmid R46(15).A search of protein data bases (17,425 proteins) with the

best-local-similarity algorithm of Smith and Waterman (34)as modified by Collins et al. (6) revealed no convincinghomology to the inferred protein sequences of Orf235 and0rf238.The xerC gene was placed under the control of the

isopropyl-i-D-thiogalactopyranoside-inducible Pta, promoterby cloning the gene into the expression vector pBAD,creating plasmid pSDC105. On induction with isopropyl-p-D-thiogalactopyranoside, a protein with an apparent molec-

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consensus

XerCR46 Orf3

FimE

FimB

Tn4430 TnpITn554 TnpATn554 TnpB

F:D Prot

P1 CreP22 Int

Lambda Int

Domain _I1

elay tG RisEll L didlRAMLEVMYGAGLRLSELVGLDIKHLDLERLFAQLLYGTGMRISEGLQLRVKDLDFDYCLILLAYRHGMRISELLDLHYQDLDLNYCLTLLCFIHGFRASEICRLRISDIDLKYAIATLLAYTGVRISEALSIKMNDFNLQKLILMLMYEGGLRIGEVLSLRLEDIVTWATMTMIVQECGMRISELCTLKKGCLLEDKMLLATLWNTGARINEALALTRGDFSLATAGVEKALSLGVTKLVERWISVSGVADDKSVVEFALSTGLRRSNIINLEWQQIDMQRLAMELAVVTGQRVGDLCEMKWSDIVDG

Domain 2

consensus H LRHs at 1 e G- ir vq 11GH n-i tt- YthXerC HKLRHSFATHMLESS-GDLRGVQELLGHAN-LSTT-QIYTH

R46 Orf3 HTLRHSFATALLRSG-YDIRTVQDLLGHSD-VSTT-MIYTHFimE HMLRHACGYELAERG-ADTRLIQDYLGHRN-IRHT-VRYTAFimB HMLRHSCGFALANMG-IDTRLIQDYLGHRN-IRHT-VWYTA

Tn4430 TnpI HQLRHFFCTNAIEKG-FSIHEVANQAGHSN-IHTT-LLYTNTn554 TnpA HMLRHTHATQLIREG-WDVAFVQKRLGHAHVQTTL-NTYVHTn554 TnpB HAFRHTVGTRMINNG-MPQHIVQKFLGHES-PEMT-SRYAH

F:D Prot HTFRHSYAMHMLYAG-IPLKVLQSLMGHKS-ISST-EVYTKP1 Cre HSARVGAARDMARAG-VSIPEIMQAGGWTN-VNIV-MNYIRP22 Int HDLRHTWASWLVQAG-VPISVLQEMGGWES-IEMV-RRYAH

Lambda Int HELRSLSA-RLYEKQ-ISDKFAQHLLGHKS-DTMA-SQY-RFlp HIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTH

FIG. 5. Alignment of the XerC protein sequence to sequences ofother site-specific recombinases of the lambda integrase family.Only the two most conserved regions (domains 1 and 2) are shown,although XerC, FimB, FimE, and TnpI can be aligned in the regionbetween domains 1 and 2. The regions of XerC shown are thoseunderlined in Fig. 3. The consensus line shows residues present in atleast four of the proteins in lowercase type, those present in at leasteight of the proteins in uppercase type. Gaps (dashes) have beenintroduced to maximize the homology. The sequence of R46 Orf3 isfrom Hall and Vockler (15). The sequences of the chromosomallyencoded E. coli recombinases FimB and FimE are from Dorman andHiggins (8) and Klemm (20). TnpI is from the B. thuringiensistransposon Tn4430 (24). The sequences of the Tn554 transpositionproteins TnpA and TnpB are from Murphy et al. (27). The D proteinis a recombinase from the E. coli F factor (23). The sequences of theP1, P22, lambda, and Flp recombinases are taken from the data ofArgos et al. (3).

ular mass of 32 kDa appeared in cells carrying this plasmid.This result is in good agreement with the predicted molecularmass of XerC, 33.8 kDa. XerC overproduced from pSDC105was purified from a sodium dodecyl sulfate-polyacrylamidegel, and the N-terminal sequence was determined on anautomated peptide sequencer. The sequence determinedagrees totally with that predicted for the first 20 residues ofXerC.

DISCUSSIONThe E. coli xerC gene is the third unlinked chromosomal

gene to be identified as necessary for site-specific recombi-nation at ColEl cer. Together, ArgR, PepA, and XerCmaintain ColEl and related plasmids in a monomeric state,thus ensuring their stable inheritance (35-37, 39). The workreported here provides the DNA sequence of xerC andimplicates xerC as the gene encoding the cer recombinase.This work also provides the DNA sequence spanning the gapbetween the published dapF (30) and uvrD (9-11) sequences.There are two minor differences between the sequencepresented here and that presented previously (30). We havechecked our sequence data carefully and believe that thesequence presented in Fig. 3 is correct.The xerC gene appears to be part of a multicistronic unit

that has dapF as the first gene and xerC as the third gene.The second and fourth genes (orf235 and or1238) are ofunknown function but appear to be translated at levelssimilar to those of dapF and xerC.

Insertion mutations 5' of the xerC open reading frame,within either the dapF or the orJ235 open reading frame,substantially reduce site-specific recombination at cer. Thedefect caused by at least one of these (a Tn5 insertion inor1235 to yield xerCl) is not complemented by plasmidpVMK42, which carries the complete dapF and or1235reading frames but only part of xerC. All of the insertionmutations upstream ofxerC are, however, complemented bya plasmid carrying only the intact xerC open reading frame,suggesting that upstream insertions reduce cer recombina-tion only by reducing transcription ofxerC and that the dapFand orJ235 gene products are not themselves required for cersite-specific recombination. An insertion in or1238 (down-stream of xerC) does not abolish recombination at cer,showing that the product of this gene is not required for cerrecombination. It is known that dapF has its own promoter(30), and it seems probably that dapF, or1235, xerC, andorJ238 are all transcribed on the same mRNA from thispromoter.

Strains carrying insertions within the xerC open readingframe are totally defective in recombination between cersites. They are also totally defective in recombination at avariant cer site which does not require ArgR or PepA forrecombination. This variant cer site is functionally muchsmaller than the wild-type cer site, requiring at most only 50bp around the crossover region (38), suggesting that XerCacts at or near the crossover site of cer and (taking intoaccount the similarity to other known recombinases) isalmost certainly the cer recombinase.The XerC protein has substantial amino acid sequence

similarity to the lambda integrase family of site-specificrecombinases, especially at the two most conserved do-mains. The second of these domains contains the absolutelyconserved tyrosine residue that is known to become linkedto the recombination site DNA in lambda integrase (28) andin the FLP protein of the yeast 2ixm plasmid (13). The otherparts of domain 2, including the other two absolutely con-served residues, might also be elements of the recombinaseactive site (3). Given this similarity to the lambda integrasefamily, it seems likely that XerC catalyzes recombination atcer by a mechanism similar to that of these other recombi-nases. This mechanism would involve making two staggerednicks within the cer crossover region, generating transient5'-protruding ends and 3' phosphodiester protein-DNA link-ages to tyrosine 275 of XerC.

In the region between the two conserved domains, theXerC sequence is most similar to the sequences of the E. colichromosomally encoded FimB and FimE (20) proteins and tothe TnpI protein of Bacillus thuringiensis Tn4430 (24). Withinconserved 38-amino-acid domain 2, the XerC sequence isclosest to that of a possible recombinase from plasmid R46(15). The significance of these similarities is not known.We believe that, as well as serving to stabilize ColE1 and

related plasmids by site-specific recombination, xerC prob-ably has some other role in E. coli. This belief is supportedby the observation that xerC mutants have some alteredgrowth characteristics, which are complemented in trans byxerC. The cellular role of xerC might involve acting at someE. coli chromosomal recombination site. However, by usingboth a sequence comparison to cer and a functional (dimerresolution) assay, we have not been able to detect any suchsite in the cya-xerC-uvrD chromosomal region. We have not

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xerC AND ColEl STABILITY 6979

been able to assign any functions to the two open readingframes (or1235 and or9238) which appear to be cotranscribedwith dapF and xerC. However, since these four genesappear to be cotranscribed, their functions might in someway be related.We are undertaking further work to purify the XerC

protein to study its in vitro DNA-binding properties and itspossible recombinase activity. These investigations shouldhelp to clarify the role of ArgR and PepA in cer site-specificrecombination. Additionally, we are investigating the cellu-lar role of xerC in E. coli.

ACKNOWLEDGMENTS

We thank the Molecular Palaeontology group in the Departmentof Geology, University of Glasgow, for N-terminal sequencing ofXerC and John Collins at the University of Edinburgh for searchingof protein data bases. We also thank Catherine Richaud for supply-ing strains and plasmids carrying mini-Mu insertions in the xerCregion, Sidney Kushner for supplying plasmids covering the xerCregion, and Chris Boyd for supplying pBAD. Eternal love andgratitude are extended to Liz Morrell and Marshall Stark for criticalreading of the manuscript.

This work was supported by the Medical Research Council andthe Leverhulme Trust.

LITERATURE CITED1. Aiba, H., K. Mori, M. Tanaka, T. Ooi, A. Roy, and A. Danchin.

1984. The complete nucleotide sequence of the adenylate cy-clase gene of Escherichia coli. Nucleic Acids Res. 12:9427-9440.

2. Aldea, M., V. Maples, and S. Kushner. 1988. Generation of adetailed physical and genetic map of the ilv-metE-udp region ofthe Escherichia coli chromosome. J. Mol. Biol. 200:427-438.

3. Argos, P., A. Landy, K. Abremski, J. B. Egan, E. Haggard-Ljungquist, R. H. Hoess, M. L. Kahn, B. Kalionis, S. V. L.Narayana, L. S. Pierson Ill, N. Sternberg, and J. M. Long. 1986.The integrase family of site-specific recombinases: regionalsimilarities and global diversity. EMBO J. 5:433-440.

4. Bachmann, B. J. 1972. Pedigrees of some mutant strains ofEscherichia coli K12. Bacteriol. Rev. 36:525-557.

5. Cohen, S. N., and L. Hsu. 1972. Non chromosomal antibioticresistance in bacteria: genetic transformation of Escherichia coliby R factor DNA. Proc. Natl. Acad. Sci. USA 69:2110-2114.

6. Collins, J. F., A. F. W. Coulson, and A. Lyall. 1988. Thesignificance of protein sequence similarities. Comput. Appl.Biosci. 4:67-71.

7. de Bruijn, F. J., and J. R. Lupski. 1984. The use of transposonTn5 mutagenesis in the rapid generation of correlated physicaland genetic maps of DNA segments clones into multicopyplasmids-a review. Gene 27:133-149.

8. Dorman, C. J., and C. F. Higgins. 1987. Fimbrial phase variationin Escherichia coli: dependence on integration host factor andhomologies with other site-specific recombinases. J. Bacteriol.169:3840-3843.

9. Easton, A. M., and S. R. Kushner. 1983. Transcription of theuvrD gene of Escherichia coli is controlled by the lexA repressorand by attenuation. Nucleic Acids Res. 11:8625-8640.

10. Finch, P. W., and P. T. Emerson. 1983. Nucleotide sequence ofthe regulatory region of the uvrD gene of Escherichia coli. Gene25:317-323.

11. Finch, P. W., and P. T. Emerson. 1984. The nucleotide sequenceof the uvrD gene of Escherichia coli. Nucleic Acids Res.12:5789-5799.

12. Flinn, H., M. Burke, C. J. Stirling, and D. J. Sherratt. 1989. Useof gene replacement to construct Escherichia coli strains carry-ing mutations in two genes required for stability of multicopyplasmids. J. Bacteriol. 171:2241-2243.

13. Gronostajski, R. M., and P. D. Sadowski. 1985. The FLPrecombinase of the Saccharomyces cerevisiae 2pLm plasmidattaches covalently to DNA via a phosphotyrosyl linkage. Mol.Cell. Biol. 5:3274-3279.

14. Hager, D., and R. R. Burgess. 1980. Elution of proteins fromSDS-polyacrylamide gels, removal of SDS and renaturation ofenzymatic activity: results with the sigma subunit of E. coliRNA polymerase, wheat germ topoisomerase and other en-zymes. Anal. Biochem. 109:70-86.

15. Hall, R. M., and C. Vockler. 1987. The region of the IncNplasmid R46 coding for resistance to beta-lactam antibiotics,streptomycin/spectinomycin and sulphonamides is closely re-lated to antibiotic resistant segments found in IncW plasmidsand Tn2l-like transposons. Nucleic Acids Res. 15:7491-7501.

16. Hatfull, G. F., and N. D. F. Grindley. 1988. Resolvases andDNA-invertases: a family of enzymes active in site-specificrecombination, p. 357-396. In R. Kucherlapati and G. R. Smith(ed.), Genetic recombination. American Society for Microbiol-ogy, Washington, D.C.

17. Henikoff, S. 1987. Unidirectional digestion with exonuclease IIIin DNA sequence analysis. Methods Enzymol. 155:156-165.

18. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method forthe preparation of bacterial plasmids. Anal. Biochem. 114:193-197.

19. Kennedy, C. K. 1971. Induction of colicin production by hightemperature or inhibition of protein synthesis. J. Bacteriol.108:10-19.

20. Klemm, P. 1986. Two regulatory fim genes, fimB and fimE,control the phase variation of type I fimbriae in Escherichia coli.EMBO J. 5:1389-1393.

21. Kohara, Y., K. Akiyama, and K. Isono. 1987. The physical mapof the whole Escherichia coli chromosome: application of a newstrategy for rapid analysis and sorting of a large genomic library.Cell 50:495-508.

22. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

23. Lane, D., R. de Feyter, M. Kennedy, S. H. Phua, and D. Semon.1986. D protein of miniF plasmid acts as a repressor of tran-scription and as a site-specific resolvase. Nucleic Acids Res.14:9713-9728.

24. Mahillon, J., and D. Lereclus. 1988. Structural and functionalanalysis of Tn4430: identification of an integrase-like proteininvolved in the co-integrate resolution process. EMBO J.7:1515-1526.

25. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

26. Miller, C. G., and G. Schwartz. 1978. Peptidase-deficient mu-tants of Escherichia coli. J. Bacteriol. 135:603-611.

27. Murphy, E., L. Huwyler, and M. do Carmo de Freire Bastos.1985. Transposon TnS54: complete nucleotide sequence andisolation of transposition-defective and antibiotic-sensitive mu-tants. EMBO J. 4:3357-3365.

28. Pargellis, C. A., S. E. Nunes-Duby, L. Moitoso de Vargas, and A.Landy. 1988. Suicide recombination substrates yield covalentlambda Int-DNA complexes and lead to the identification of theactive site tyrosine. J. Biol. Chem. 263:7678-7685.

29. Richaud, C., W. Higgins, D. Mengin-Lecreulx, and P. Stragier.1987. Molecular cloning, characterization, and chromosomallocalization of dapF, the Escherichia coli gene for diaminopime-late epimerase. J. Bacteriol. 169:1454-1459.

30. Richaud, C., and C. Printz. 1988. Nucleotide sequence of thedapF gene and flanking regions from Escherichia coli K12.Nucleic Acids Res. 16:10367.

31. Sadowski, P. 1986. Site-specific recombinases: changing part-ners and doing the twist. J. Bacteriol. 165:341-347.

32. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

33. Sherratt, D. 1989. Tn3 and related transposable elements:site-specific recombination and transposition, p. 163-184. InD. E. Berg and M. M. Howe (ed.), Mobile DNA. AmericanSociety for Microbiology, Washington, D.C.

34. Smith, T. F., and M. S. Waterman. 1981. Identification ofcommon molecular subsequences. J. Mol. Biol. 147:195-197.

35. Stirling, C. J., S. D. Colloms, J. F. Collins, G. Szatmari, and

VOL. 172, 1990

on April 16, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

6980 COLLOMS ET AL.

D. J. Sherratt. 1989. xerB, an Escherichia coli gene required forplasmid ColEl site-specific recombination, is identical to pepA,encoding aminopeptidase A, a protein with substantial similarityto bovine lens leucine aminopeptidase. EMBO J. 8:1623-1627.

36. Stirling, C. J., G. Stewart, and D. J. Sherratt. 1988. Multicopyplasmid stability in Escherichia coli requires host-encoded func-tions that lead to plasmid site-specific recombination. Mol. Gen.Genet. 214:80-84.

37. Stirling, C. J., G. Szatmari, G. Stewart, M. C. M. Smith, andD. J. Sherratt. 1988. The arginine repressor is essential forplasmid-stabilising site-specific recombination at the ColEl cer

locus. EMBO J. 7:4389-4395.38. Summers, D. K. 1989. Derivatives of ColEl cer show altered

topological specificity in site-specific recombination. EMBO J.8:309-315.

39. Summers, D. K., and D. J. Sherratt. 1984. Multimerisation ofhigh copy number plasmids causes instability: ColEl encodes adeterminant for plasmid monomerisation and stability. Cell36:1097-1103.

40. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. ImprovedM13 phage cloning vectors and host strains: nucleotide se-quences of the M13mpl8 and pUC19 vectors. Gene 33:103-119.

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