Homologues of helicase genes priA and ruvAB of Borrelia burgdorferi, the lyme borreliosis agent

© iNSTITUT PASTEUR/ELSEVIER

Res. Microbiol.

Paris 1998

1998, 149, 235-245

Horno|ogues of he|icase genes priA and ruvAB of Borrelia burgdorferi, the Lyme borreliosis agent C. Boursaux-Eude (!) D. Margarita (~), J. Belfaiza (2) I.G. Old (1) and I. Saint Girons (1)(*) (!) Unitd de Bactdriologie Mol#culaire et Mddicale, lnsti~ut Pasteur, 75724 Paris Cedex 15, and (2) Faealtd des Sciences d'EI-Jadida, UniversitE Chouaib Doukkali (Morocco)

SUMMARY A DNA library of strain HB19 from Borrelia burgdoderi sensu stricto, an agent of Lyme borreliosis, was constructed in the cosmid pLA2917. Genes involved in initiation of DNA replication and resolution of recombination intermediates (Holliday junctions) were found on a 23-kbp region up to 0.7 kbp of the "left" extremity of the linear chromosome in representative species of B. burgdofferi sensu lato. The potential ruvB gene, located at 22 kbp from the left telomere, was identified by the similarity of its deduced amino acid sequence to RuvB (helicases) of other bacteria. B. burgdofferi ruvB is part of an operon which comprises the homologues of ruvA, queA and proB. Expression of the B. burgdofferi ruvB and ruvA genes renders a wild-type Eschefichia coil sensitive to UV light and mitomycin, indicative of negative complementation. priA, which encodes the potential recognition factor for the primosome assembly site, was found at 15 kbp from the left telomere. RuvB and PriA sequences have motifs characteristic of helicases: a DExH box and an ATP binding site.

Key-words: Borrelia burgdorferi, Helicase, Gene priA, Gene ruvAB; Telomere, Replication, Recombination, Prirnosome.

Borrelia burgdorferi is the agent of Lyme

however, those of the linear plasmids consist of covalently closed ends (Hinnebusch and Barbour, 1991 ).

borreliosis (Barbour and Hayes, 1986). The genome of this spirochaete is original in that it is essentially linear, comprising a small 1-Mbp linear chromosome with several linear and circular plasmids (Barbour and Garon, 1987; Baril et al., 1989; Ferdows and Barbour, 1989). The structures of the B. b u r g d o r f e r i chromosomal telomeres are not yet known;

There are models for the replication of linear genomes such as animal viruses in which it is suggested that helicases may play a role (DeLange and McFadden, 1990). DNA hellcases can be divided into two sets which play essential roles in DNA replication or recombination and repair (Kornberg and Baker, 1991). Genetic r e c o m b i n a t i o n depends upon the

INTRODUCTION

Submitted November 10, 1998, accepted February 20, 1998. (*) Address for correspondence.

236

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RecA protein to promote homologous recombination and recombinational repair in vivo. In vitro, the Escherichia coli RecA protein catalyses the formation of an intermediate called a Holliday junction, in which the two interacting duplexes are held together. The resolution of this intermediate into recombinant products requires the product of the ruvA, ruvB and ruvC genes (for a review see West, 1996). E. coli ruv mutants are sensitive to mitomycin C, UV light and various c h e m i c a l m u t a g e n s (Otsuji et al., 1974). Another set of bacterial helicases such as PriA (formerly replication factor Y or protein n') and DnaB unwind DNA at the replication fork (Lebowitz and McMacken, 1986; Lee and Marians, 1987). PriA was first discovered as a protein essential for the conversion of single-stranded ~X174 DNA to the duplex replicative form in vitro. PriA binds to a specific hairpin (primosome assembly site or PAS) on ~X174 DNA coated with single-stranded DNA binding protein. PriA and primosome assembly play a role in E. coli DNA replication (for a review, see Zavitz and Marians, 1991) different from that of the " o r i C type primosome". Our ultimate goal was to identify the structures of the chromosomal telomeres from B. burgdorferi. As a first step towards this objective, we characterized the genes close to the "left" extremity. In this paper, we report the isolation and nucleotide sequence of 23 kbp which includes two helicase gene homologues, priA and ruvB, ruvA (specificity factor for RuvB), and also queA (S-adenosylmethionine tRNA ribosyltransferase isomerase), truA (pseudouridine synthase) and pfbB (pyrophosphate-dependent phosphofructokinase). The transcriptional organization of ruvA and ruvB was analysed and complementation of E. coli ruv mutants was explored.

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AL. MATERIALS

AND METHODS

Bacterial strains B. burgdorferi sensu stricto HB19 (Barbour et aL, 1983), of human origin and at passage 3, was used as source of DNA for the construction of the cosmid library. The ability of the clonal population of HB19 to infect CB17 SCID mice was confirmed by A. Barbour (personal communication). E. coli strains (table I) were grown in LB broth or on LB agar plates (Sambrook et al., 1989) with ampicillin (100 Ixg/ml) or isopropylthio-13-D-galactoside (IPTG) at 0.5 mM or mitomycin (0.2•0.5 ~tg/ml) when needed.

Construction of a B. burgdorferi cosmid library. Nucleotide sequence of the 23-kbp left telomeric region Total DNA from strain HB 19 was prepared as described (Frey et al., 1983) from 500 ml of culture at l0 s bacteria/ml. DNA was partially digested with Sau3A and purified by sizing on a 10 to 40 % sucrose density gradient. The 17-30-kbp long DNA fragments (1.4 p.g) were ligated into the BamHldigested and alkaline-phosphatase-treated cosmid vector pLA2917 (1.6 ~tg) (Allen and Hanson, 1985) which had been purified on a "Wizard" DNA cleanup system (Promega, USA). The ligated mix was packaged into phage lambda particles as described by the supplier and used to infect E. coli HB101. The library was kept as individual clones in microtitre plates at -80°C. Fragments from the chosen recombinant cosmids were cloned into a pGEM-7Zf(+) multicopy plasmid cloning vector (Promega, France) which has convenient sites for the construction of sets of nested deletions (Erase-a-base system, Promega, France). Nucleotide sequencing of the subsequent inserts was performed by dideoxynucleotide chain-termination reactions (Sanger et al., 1977) on double-stranded templates with T7 DNA polymerase (Pharmacia, France), 0 ~ ( 3 3 p ) dATP and synthetic oligonucleotides. Nucleic acid and deduced amino acid sequences were analysed by "BLAST" and "FASTA" programs from the GCG (Genetics Computer Group) package (Devereux, 1991) and CLUSTAi., software (Higgins and Sharp, 1988).

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RuvAB AND PriA HELICASES FROM B. BURGDORFERI Cloning of PCR fragments A 2-kbp fragment containing both ruvA and ruvB was cloned into pCR-Script Amp SK(+) (Stratagene, USA), after PCR. The forward and reverse primers were A48 (5'-ATC CAT TAG GAA CTT TT-3', 251 bp upstream of the ATG from ruvA) and A43 (5'-CTA ACT TTT GAG TTA TT-3', downstream from the ruvB structural gene within queA) from recombinant plasmids carrying the D23 and 19 inserts, respectively (fig. 1). The recombinant plasmid with the lac promoter in the same orientation as the ruvA and ruvB genes was kept (insert R2). D I 3 was like D23, but the genes were under the control of the lac promoter of the pGEM-7Zf(+) vector. TM

RNA isolation and RT-PCR Total cellular RNA was extracted from B. burgdorferi in exponential growth using "Tri Reagent" (Sigma, France). The RNA was incubated at 37°C for 1 h in the presence of RNase-free DNase (0.3 U/~tg, Pharmacia, France) and further purified using the "RNeasy" kit from Qiagen (Germany). To perform coupled reverse transcription and PCR amplification (RT-PCR), the "Access" RT-PCR system (Promega, France) was used following the instructions of the manufacturer. A sample of each reaction was analysed on a 2% agarose gel with TAE buffer (Tris-acetate 0.04 M, EDTA 0.001 M, pH8).

237

strain 212) (Davidson et al., 1992) bordered by a BssHII site and the left telomere was used as a probe to screen the B. burgdorferi cosmid library by colony hybridization (Grunstein and Hogness, 1975). Four out of nine selected recombinant cosmids had inserts of approximately 20 kbp with several restriction fragments in common. CosmJd Db9 was kept for further study. After digestion of Db9 with EcoRI, the resulting six fragments (fig. 1) were cloned into pGEM7Zff+) (Promega, France). Analysis of the nucleotide sequence of the inserts of the six recombinant plasmids derived from Db9 enabled the design of the potential genes p r e s e n t in the left t e l o m e r i c region (fig. IA). The fragment I12 located closest to the telomere (at 0.7 kbp) comprises several ORFs without any significant similarity with the content of the available data libraries. We had previously analysed insert D4, which comprises the udk gene at 15 kbp (Boursaux-Eude et al., 1997) and in the following paragraphs, we analysed the inserts I2, D23 and I9, which are located at 13 and 15-23 kbp from the left telomere.

PriA and RuvB contain structural motifs characteristic of helicases

Complementation of E. coli ruv mutant Growth was in LB broth or on LB agar plates (Sambrook et al., 1989) with carbenicillin (100 txg/ml) and IPTG. E. coil ABl157 containing B. burgdorferi ruvAB or ruvB constructs (insert R2 and D13 respectively, fig. 1) or pCR-Script SK(+) or pGEM-7Zf(+) vectors as controls were exposed to a calibrated 254-nm UV source for 5 or 10 s. Appropriate dilutions of exposed and unexposed cells were plated on the same medium as above. The surviving fraction was counted after UV irradiation and overnight incubation in the dark at 37°C. TM

RESULTS AND DISCUSSION

Gene order within the left telomeric region of the linear chromosome of B. burgdorferi B. burgdorferi sensu stricto strain HB 19 was used as a source of DNA for the construction of a cosmid library. A 6.9-kbp BsF fragment (from

An open reading frame of 1,980 bp was identified as the potential coding region for PriA, the recognition factor for the primosome assembly site. This ORF begins at one extremity of fragment I2, continues into fragment D4 (fig. l) and is oriented away from the left telomere and opposite udk (Boursaux-Eude et al., 1997). The molecular mass of the priA gene product was calculated to be 77,316 Da. We found significant similarity between B. burgdorferi PriA and the published sequences of PriA from E. coli (28 % identity), Haemophilus influenzae (28 % identity) and PriA (URF2) from Rhodospirillum rubrum (26% identity) (Falk et al., 1985; Fleischmann et al., 1995; Lee et al., 1990; Nurse et al., 1990) (fig. 2). The priA gene had previously been mapped (Casjens et al., 1995) at the left extremity of the chromosome in 20 representatives of B. burgdorferi sensu lato.

C. BOURSA UX-EUDE ET AL.

238

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D 13 insert (6.4 kbp) Fig, 1, A) Location of the potential genes originating from the 22,760-bp left telomeric region of the B. burgdorferi linear chromosome with the direction of transcription indicated by arrowheads. The linear chromosome ends at approximately 0.7 kbp from the left of the figure. The vertical bars below the arrowheads are situated in the middle of the gene. The names of the 6 recombinant inserts carrying the genes or ORFs are boxed by EcoRl sites. Accession number AJ224476 has been attributed to the sequence of the 112 insert, while AJ224474 corresponds to the D5 insert with the truA gene. The sequence data of the potential genes udk, priA and queA, ruvB, ruvA have been assigned Genbank accession numbers X97449 and Y08885, respectively (inserts 12, D4, 19 and part of D23). AJ224475 corresponds to the rest of the D23 insert with the pfpB homologue. B) Inserts R2 and D13, the construction of which is described in "Materials and methods", are indicated. Direction of transcription is as above.

At one extremity of the 6.5-kbp EcoRI fragment (D23, see fig. 1), an ORF of 1,044 bp was identified as the potential coding region for RuvB, a helicase. A putative ribosome-binding site AGGA was located 7 bp upstream of the ATG start codon. The molecular mass of the ruvB gene product was calculated to be 38,981 Da. B. burgdorferi RuvB had a significant similarity of up to 50% when compared to nine eubacteria belonging to different phyla,

namely Gram-negative, thermophiles, cyanobacteria, Gram-positive, Mycoplasma (all accession numbers are noted and six of the representative sequences are aligned in figure 3). At the N-terminus of B. burgdorferi PriA and RuvB (figs. 2 and 3), there is a motif characteristic of an ATP binding site with the "P loop" consensus (GA)XXXXGK(ST) (Saraste et al., 1990). RuvB from 6 bacterial species showed the motif GPPG(LV)GKT, while PriA from 4 bacterial spe-

RuvAB AND PriA HELICASES FROM B. BURGDORFERI

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The P loop and DExH motive are boxed at positions 157-164 and 255-258 from the B. burgdorferi ruvB sequence, respectively• The SATP motif is boxed at positions 291-294. The cysteine-rich zinc-binding motif C(X)2C(X)sC(X)2C(X)I4C(X)2C(X)gC(X)2C is indicated by boxes around the cysteines from positions 369 to 412 of the B. burgdorferi priA sequence• The following is also valid for fig. 3. Identical residues are indicated by stars, similar residues by dots. A hyphen indicates a gap.

cies showed the motif G(IV)(PT)GSGKT. B. burgdorferi PriA is different at the second and third position of the latter motif (boxed in figs. 3 and 2, respectively). The second motif, shared by RuvB and PriA, is the DExH motif characteristic of helicases (Ouzounis and Blencowe, 1991) which is found in the first half of the molecule (second boxes in figs. 2 and 3). In addition, PriA possesses

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RRIRDIAQVTGSLVITSDIVSIGLEMLRIDGEGLDEQDRNILRSLILKFNGGPVGVDTLAISVGETADSLEDFYEPY RRVRDFAEVKHDGTISADIAAQALDMLNVDAEGFDYMDRKLLLAVIDKFFGGPVGLDNLAAAIGEERETIEDVLEPY RRVRDYADVRNGGIISINVAKQALSMLDVDDAGFDYLDRKLLSAVIERFDGGPVGLDNLAAAIGEERDTIEDVLEPY RRVRDFAEVRGQGDITRVIADKALNLLDVDERGFDHLDRRLLLTMIDKFDGGPVGIDNLAAALSEERHTIEDVLEPY KRLFEQKIVN--KKIDLAALFKSLMIYKN---GLQSIDVQYLKALNGQYE--PQGIKSICSMLGIDKSTVENKIEPF KRVRDMLTVVKADRINTDIVLKTMEVLNIDDEGLDEFDRKILKTIIEIYRGGPVGLNALAASLGVEADTLSEVYEPY o*o

B.burg. E.coll H.infl. P,aeru, M.pneu. T,mari.

.,

75 73 73 74 56 69

k * * * o * * *

.

PLPKFTLIGATTKPGKVTSPLYARFGITARFELYSEIELVEIIKRNSLILNIEIEEDAAFLLARSSRGTPRIANRLL DLPPFTLIGATTRAGSLTSPLRDRFGIVQRLEFYQVPDLQYIVSRSARFMGLEMSDDGALEVARRARGTPRIANRLL DLPPFTLVGATTRAGSLTSPLRDRFGIVQRLEFYSVEDLTSIVARSAGCLNLELEQQAAFEVARRSRGTPRIANRLL DLPPFTLVGATTRAGMLTNPLRDRFGIVQRLEFYNVEDLATIVSRSAGILGLEIEPQGAAEIAKRARGTPRIANRLL KVNPFTWIGATTQFGKIINPLEDRFGMILNIDYYSNQEIERIVSIYGEQMELELKPEEITQITQHSKQTPRIAIRIV DIQPFTLVGATTRSGLLSSPLRSRFGIILELDFYTVKELKEIIKRAASLMDVEIEDAAAEMIAKRSRGTPRIAIRLT •

B.burg. E.coli H.infl. P.aeru.

PLAS PLAN PLAN PLAN PLAR PLAH

~EIH DEIH ~EIH ~EIH ~EIH

•

B.burg. E.coli H.infl.

]PPGLGK~ ]PPGLGKq ]PPGLGKq ~PPGLGK~ ]PPGVGK~ ]PPGLGK~

MKDENSISFLSSNENYLYD-KSENELRPKV-FEDFKGQVNVKETLSIFIRASKERDEALDHVFLS MIEAD--RLISAGTTLPED-VADRAIRPKL-LEEYVGQPQVRSQMEIFIKAAKLRGDALDHLLIF MIEAD--RIISGQAKVDED-VIDRAIRPKL-LADYVGQPQVREQMDIFIKAAKLRQDALDHLLIF MIEPD--RLISAVSGRERDEQLDRAIRPLK-LADYIGQPSVREQMELFIHAARGRQEALDHTLIF MKLQIKPPNNFAEFVGKQEIINQIQLSIKASRINKAQLDHILLY ..... MSEFLTPERTVYDS--GVQFLRPKS-LDEFIGQENVKKKLSLALEAAKMRGEVSDHVLLA

306 304 304 305 280 300 .

347 336 335 352 307 334

*

Fig. 3. Alignment of B. burgdorferi RuvB with RuvB from H. influenzae (P44631), E. coli (AE000280), Pseudomonas aeruginosa (D83138), Mycoplasma pneumoniae (AE000028), Mycobacterium leprae (U00011) and Thermus maritima (U38840). Alignments with the sequences of Mycobacterium tuberculosis (Z77724), Mycoplasma genitalium (U39720), Synechocystis PCC 6303 (U38892) and Thermus aquaticus (U22817) are not shown. The P loop and DExH motif are boxed at positions 64-71 and 115-118 from the B. burgdorferi RuvB sequence, respectively. See also legend, figure 2.

ruvB is the second gene of an operon comprising at least four genes: ruvA, ruvB, queA and

pfpB 45 bp upstream from ruvB, we found the beg4"nning of another ORF which continued into the EcoRI fragment 19 adjacent to D23 (fig. 1). This ORF was a ruvA homologue (encoding a

specificity factor for RuvB) and consisted of 197 codons. The molecular mass of the ruvA gene product was calculated to be 22,702 Da. A significant identity was found between B. burgdorferi RuvA and RuvA from Haemophilus influenzae (44%), E. coli (42%), Pseudomonas aeruginosa (40%) and Mycobacterium leprae (37%) (Benson et al., 1988; Fleischmann et al.,

RuvAB AND PriA HELICASES FROM B. BURGDORFERI 1995; Hishida et al., 1996; Philipp et aL, 1996; Shinagawa et aL, 1988) (data not shown). The proximity of ruvA and ruvB suggested that these genes might be arranged in an operon. We tested the potential cotranscription of ruvA and ruvB using RT-PCR. A band of the expected size (690 bp) was found between ruvA and ruvB (fig. 4, lane 2). In addition, the potential queA gene (encoding S-adenosylmethionine tRNA ribosyltransferase isomerase) (Slany et al., 1993) overlaps the end of ruvB by 35 bp. This gene is followed by a potential pfpB gene which could encode a pyrophosphate-dependent phosphofructokinase (Carlisle et al., 1990). We checked whether the mRNA continued onwards from ruvB. Bands of the expected size were found between ruvB and queA (657 bp) and between queA and pfpB (612 bp) (fig. 4, lanes 4 and 6). These transcripts were not formed when reverse transcriptase was omitted from the reaction mix (fig. 4, lanes 3, 5 and 7), confirming that the RTPCR products were derived from mRNA and not DNA. The organization of B. burgdorferi ruvA and ruvB in an operon can be compared to the organization of E. coli ruvA and ruvB in a single LexAregulated operon (Benson et al., 1988; Shinagawa et al., 1988). However, no SOS boxes could

24~

be identified in the upstream region of the B. burgdo~feri ruv operon, and the latter is much larger, since it contains at least two additional genes. Large operons have been found in B. burgdorferi for genes involved in motility of the bacterium (Ge et aL, 1977) and this feature is perhaps related to its small genome size (Baril et aL, 1989; Ferdows and Barbour, 1989). In contrast to the situation found for E. coli and B. burgdorferi, the ruvB gene is a single transcription unit in thermophiles (Tong and Wetmur, 1996) and in the spirochaete T r e p o n e m a p a l l i d u m , which is responsible for syphilis (S. Norris, personal communication). The ruvA and ruvB genes are adjacent in H. influenzae and M. leprae, but their organization as an operon has not been demonstrated experimentally (Fleischmann et al., 1995; Philipp et al., 1996). The ruvB gene was also located in the left telomeric region of the chromosome (at 22 kbp from the extremity) for representatives of several Borrelia species, B. burgdorferi sensu stricto HB19, 212, B31, B. garinii 20047, B. afzelii VS461 and B. japonica HO14 (data not sho~n), as expected, since theft genetic maps had been found to be very similar (Casjens et al., 1995; Ojaimi et al., 1994).

Comparison of the left telomeric region of the chromosomes of B31 and HB19 strains

1234567

Fig. 4. RT-PCR products after agarose gel electrophoresis. A sample of each reaction was analysed on a 2 % agarose gel with TAE buffer (0.04 M Tris-acetate; 0.001 M EDTA pH8). The amplified products span the intergenic regions as follows: lane 2, ruvA-ruvB, lane 4, ruvB-queA, lane 6, queA-pfpB, lanes 3, 5 and 7, corresponding negative controls without reverse transcriptase. Lane 1 : • X 174 markers restricted by Haelll (bp).

While this paper was in preparation, the preliminary sequence data of almost the whole linear c h r o m o s o m e of B. b u r g d o r f e r i strain B31 (970,715 nucleotides) was made available by TIGR (ftp://ftp.tigr, org/pub/data/b_burgdorferi). The available sequence is not annotated; it is reported by TIGR to contain errors and is oriented with the right extremity corresponding to our left extremity. Comparison of the sequence closest to the telomere from strain HBI9 (112 fragment, fig. 1A) with the corresponding sequence from strain B31 revealed that there were 51 differences for 8,652 nucleotides. The priA structural gene from strain HB19 is 99% identical to nucleotides 897970 to 895988 from strain B31 (http://www.ncbi.nlm.nih.

242

C. BOURSAUX-EUDE ET AL.

gov/cgi-bin/BLAST/nph-tigrbl) with four differences. The differences at nucleotides 372, 377, 453 and 1663 correspond to positions 897621, 897626, 897584 and 896274 and lead to one ~o acid change from leucine to proline. This difference remains to be confirmed when the def~tive B31 sequence from TIGR is deposited in the data banks. The ruvA and ruvB structural genes from strain HB 19 are 99% identical to nucleotides 887977 to 888516 and 888615 to 889658, respectively, from strain B31 (http://www.ncbi.nlm. nih.gov/cgi-bin/BLAST/nph-tigrbl). The differences at positions 51, 156, 308 and 342 for ruvA correspond to nucleotides 888027, 888132, 888284 and 888318. The differences at positions 213, 657, 846 and 939 for ruvB correspond to nucleotides 888827, 889271, 889460 and 889553. These differences do not lead to any changes in the amino acid sequence. The identity at 99 % of the ruvA, ruvB and pr/A sequences for the two strains confirmed the sequence conservation within the san~e B. burgdorferi sensu stricto species.

Sensitivity of wild.type E. coli to mitomycin C and UV due to expression of B. burgdorferi ruvA and ruvB genes To assess the functionality of the B. burgdorferi Ruv proteins we attempted to complement E. coli ruv mutants. Use was made of ruvB or ruvA or (ruvA, ruvB) E. coli mutants (table I)

transformed by the construct bearing both B. burgdorferi ruvB and ruvA genes (insert R2; see fig. 1). No positive complementation (assayed by restoration of resistance to mitomycin C in the presence of IPTG) was detected. Although B. burgdorferi RuvB (and RuvA) are similar to other bacterial RuvB (and RuvA) regarding their amino acid sequence, the absence of complementation is not surprising, since spirochaetes, although members of the eubaeteria (Woese, 1987), are phylogenetically distant from Enterobacteriacae. Only partial complementation was obtained in the case of RuvB from thermophilic eubacteria (Tong and Wetmur, 1996). Surprisingly, expression of B. burgdorferi ruvB (and/or ruvA) in wild-type mitomycin-Cand UV-resistant E. coil renders the latter sensitive. When transformed by pCR-Script (R2) (fig. 1B), the corresponding wild-type strain AB 1157 (table I) was more sensitive to mitomytin and UV than was the control AB1157 transformed with the plasmid vector pCR-Script alone (table II). Similar results were obtained when A B l I 5 7 was transformed with pGEM (D13) recombinant plasmid where the ruvB gene (followed by queA, pfoB and other genes; fig. 1B) is under the control of plac (table II). These results suggest that B. burgdorferi ruvB and ruvA gene products may interfere with the activity or formation of the complex multimeric structure of E. coli RuvAB (Parsons et al., 1995). It has been shown that overproduction of E. coli RuvA has a profound negative effect in E. coli, amplified by the presence of an SOS-regulated

Table I. Characteristics of E. coli strains used in this study. Strain

Genotype

Reference

ABlI57

F- thi-I his-4 A(gpt proA)62 argE3 thr-1 leuB6 ara-14 kdgK51 rfbDl lacYl galK2 xyl-5 mtl-I supE44 tsx-33 rpsL31

(Bachmann, 1996)

H124

Like AB 1157, but ruvB4

(Otsuji et al., 1974)

N2057

Like AB1157, but ruvAtO::TnlO with a polar effect on ruvB

(Sharpies, 1990)

N3881

Like AB1157, but ruvA200 eda-51::TnlO

(Sargentini and Smith, 1989)

RuvAB AND PriA HELICASES FROM B. B U R G D O R F E R I

243

Table I L Effects of B. burgdorferi ruvB or ruvA ruvB expression on survival to UV light of E. coli AB 1157 transformed by different constructs.

B. burgdorferi insert

ruvA

ruvB

5"

pCR-Script pCR-Script

O R2

+

+

0.2 0.02

0.03 0.006

pGEM-7f(+) pGEM-7f(+)

O D13

-

+

0.15 0.03

0.009 0.002

Plasmid vector

Expression of:

Relative survival 10"

Experiments were reproducible and numbers correspond to the means of 2 to 3 experiments.

E. coil promoter (Sharpies, 1990). Here, we demonstrate that overproduction of B. burgdorferi RuvB, or of both RuvA plus RuvB, has a modest negative effect. Similar results of negative complementation were obtained with B. burgdorferi flgB, which renders wild-type E. coli non-motile (Ge et al., 1997).

Le g ~ n e ruvB p o t e n t i e l , situE h 22 k b p du tElom~re gauche a EtE identifiE grace/l la similitude de la sequence en acides amines de son produit avec celle de RuvB (hElicase) d'autres bactEries. Le g~ne ruvB de B. burgdorferi fait pattie d'un opEron comprenant les g~nes homologues de ruvA, de queA et de pfbB. L'expression des g~nes ruvB et ruvA de B. burgdorferi chez Escherichia coli de type sauvage rend la bactErie sensible h la lumi~re ultraviolette et la mitomycine, ce qui indique une complEmentation negative.

Acknowledgements

Le g~ne priA, qui spEcifie le facteur de reconnaissance potentiel du site d'as~emblage du primosome, est situ6 h 15 k p b du t E l o m ~ r e g a u c h e . Les s e q u e n c e s R u v B et P r i A o n t des m o t i f s caractEristiques d'hElicases: une boite DExH et un site de fixation de I'ATP.

This work was supported by the Pasteur Institute. It represents a portion of a thesis submitted by C. Boursaux-Eude, recipient of a fellowship from the CANAM, to the University of Paris VI for the PhD degree. We thank the following individuals for providing strains: A. Barbour, F. Biville, E. Turlin and R.G. Lloyd; we are grateful to G. Baranton for his support. We also thank S. Casjens for communicating the HB 19 physical map prior to publication.

Homologues des g~nes sp~cifiant des h~licases, pr/A et ruvAB de Borrelia burgdorferi, agent de la borr~iiose de Lyme Une banque d ' A D N de la souche HB19 de Borrelia burgdorferi sensu stricto, agent de la borrEliose de Lyme, a EtE construite dans le cosmide pLA2917. Des g~nes impliquEs dans la mise en route de la r e p l i c a t i o n de I ' A D N et la r e s o l u t i o n des intermEdiaires de recombinaison (jonctions Holliday) ont EtE trouvEs dans une region de 23 kpb situEe ~ moins de 0,7 kpb de l'extrEmitE gauche du c h r o m o s o m e linEaire de diverses esp~ces de B. burgdorferi sensu lato.

Mots-cl~s : Borrelia burgdorferi, HElicase, G~ne priA, G~ne ruvAB; TElom~re, REplication, Recombinaison, Primosome.

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