Peptostreptococci account for 20 to 40% of all anaer-obes recovered from human clinical specimens (18, 20).Finegoldia magna (formerly Peptostreptococcus magnus)is recognized as the most pathogenic species of pep-tostreptococci, since it is frequently isolated from varioussites of infections, including abscesses, intra-abdominalsepses and diabetic foot infections (19, 32). Whenrecovered, F. magna is usually mixed with other bacteria.However, the fact that it is often isolated as a pure culturesuggests that F. magna has higher pathogenicity thanother species (18, 20).

Despite this clinical importance, virulence factorgenes and the genome structure of the F. magna have notbeen extensively investigated. Moreover, taxonomy ofpeptostreptococci based on DNA sequences has onlycommenced recently. On the basis of this new classifi-cation, only Peptostreptococcus anaerobius, the typespecies of peptostreptococci, remained within the genusPeptostreptococcus. The remainders of the species were

divided into five new genera, Finegoldia, Micromonas(erratum), Peptoniphilus, Anaerococcus and Gallicola(12, 21). A phylogenic tree based on the 16S rRNAsequences revealed that Peptostreptococcus anaerobiuswas related to group XI (containing Clostridium difficileor Clostridium sordellii) of the clostridia (8, 12).

We previously constructed the physical map of F.magna ATCC 29328 DNA as a first step towards eluci-dating the genomics of peptostreptococci and reportedthat this strain has a single circular chromosome of 1.9Mb, containing four rrn operons, and a single megaplas-mid of 200 kb (30). In this study, we constructed abacterial artificial chromosome (BAC) library of F.magna ATCC 29328 DNA to further facilitate genomeanalysis of F. magna. This library was constructed in thepBeloBAC11 vector (16), which combines a simplephenotypic screen for recombinant clones with the stablepropagation of large inserts (27). We attempted to obtaincomplete coverage of F. magna ATCC 29328 DNA with

Editor-Communicated Paper

Bacterial Artificial Chromosome Library of Finegoldia magna ATCC 29328 for Genetic Mappingand Comparative Genomics

Takatsugu Goto, Kozo Todo, Kazuaki Miyamoto, and Shigeru Akimoto*

Department of Microbiology, Wakayama Medical University, Wakayama, Wakayama 641–0012, Japan

Communicated by Dr. Jun Sakurai: Received October 1, 2003. Accepted October 14, 2003

Abstract: We constructed a bacterial artificial chromosome (BAC) library of Finegoldia magna ATCC 29328DNA to facilitate further genome analysis of F. magna. The BAC library contained 385 clones with an aver-age insert size of 55 kb, representing a 10.1-fold genomic coverage. Repeated DNA hybridization usingprimer sets designed on the basis of BAC-end sequences yielded nine contigs covering 95% of the chro-mosome and two contigs covering 98% of the plasmid. The contigs were localized on the physical map ofF. magna ATCC 29328 DNA. A total of 121 BAC-end sequences revealed 103 unique genes, which had notbeen previously reported for F. magna. The homolog ORF of albumin-binding protein (urPAB), one of theknown virulence factors from F. magna, was sequenced and localized on the physical map. Homology analy-sis of 121 BAC-end sequences revealed that F. magna is most closely related to clostridia, particularlyClostridium tetani. This close relationship is consistent with the recent classification of peptostreptococci basedon 16S rRNA sequence analysis. The BAC library constructed here will be useful for the whole genomesequencing project and other postgenomic applications.

Key words: Finegoldia magna, Peptostreptococcus magnus, BAC-end sequence, Albumin binding protein

1005

Microbiol. Immunol., 47(12), 1005–1016, 2003

Abbreviations: BAC, bacterial artificial chromosome; CM,chloramphenicol; kb, kilo base; LB, Luria-Bertani; Mb, megabase; ORF, open reading frame; PFGE, pulsed-field gel elec-trophoresis; TBE, Tris/Borate/EDTA; TE, Tris/EDTA.

*Address correspondence to Dr. Shigeru Akimoto, Depart-ment of Microbiology, Wakayama Medical University, 811–1Kimiidera, Wakayama, Wakayama 641–0012, Japan. Fax:81–73–448–1026. E-mail: akimotos@wakayama-med.ac.jp

a minimal set of BAC clones. We also performedgenome sequencing analysis using BAC-end sequencesto compare genome structures among closely relatedspecies.

Materials and Methods

Bacterial strains and culture conditions. Finegoldiamagna ATCC 29328 strain was purchased from theAmerican Type Culture Collection and was grown anaer-obically at 37 C in liquid or on agar plates using GifuAnaerobic Medium (GAM) (Nissui) for 13–24 hr.Escherichia coli strain DH10B [F araD139 ∆(ara-leu)7697 ∆lacX74 galU galK rpsL deoR φ80dlacZ∆M15endA1 nupG recA1 mcrA ∆(mrr hsdRMS mcrBC)] car-rying pBeloBAC11 was kindly provided by Dr. H.Shizuya, Department of Biology, California Institute ofTechnology, Pasadena, Calif., U.S.A. TransforMaxTM

EC100TM Electrocompetent E. coli (Epicentre), carry-ing recombinant pBeloBAC11, was cultured in Luria-Bertani (LB) medium or on LB agar plates at 37 C for 24hr. Where required, 12.5 µg/ml chloramphenicol (CM),25 µg/ml isopropyl β-D-thiogalactopyranoside (IPTG)and 50 µg/ml 5-bromo-4-chloro-3-indolyl β-D-galac-topyranoside (X-gal) were added.

Preparation of high molecular weight DNA from F.magna, partial digestion and size selection. High mo-lecular weight DNA from F. magna ATCC 29328 for thelibrary was prepared according to a standard protocol (2)with some modifications. F. magna ATCC 29328 wasgrown in 600 ml of GAM broth under anaerobic condi-tions at 37 C for 13 hr. Cells were harvested by cen-trifugation and washed twice in 200 ml of TE buffer [1mM Tris-HCl, 0.1 mM EDTA (pH 8.0)]. The pellet wasresuspended with 2 ml of distilled water and mixed withan equal volume of molten 1% (w/v) Agarose (Bio-Rad) of low melt preparative grade, pipetted into plugmolds, and allowed to cool. The resulting plugs wereincubated at 37 C for 29 hr in buffer A [10 mM Tris-HCl,10 mM NaCl, 1 mM EDTA (pH 8.0)] containing 1 mg/mlachromopeptidase (Wako). The plugs were transferred tobuffer B [1% (w/v) N-laurylsarcosine, 0.5 M EDTA (pH8.0)] containing 0.68 mg/ml proteinase K (Roche) andincubated at 50 C for 24 hr. This step was repeatedonce. Plugs were washed with TE buffer at 4 C for 16 hr,followed by inactivation of proteinase K at 50 C for 30min with 4 mM Pefabloc (Roche). Plugs were washedtwice with TE buffer for 30 min on ice and stored at 4 Cin 50 mM EDTA (pH 8.0). Partial HindIII digestionwas used to prepare large DNA fragments from theplugs. The plugs were washed in two changes with 50ml of 0.5TBE buffer [45 mM Tris-borate, 1 mM EDTA(pH 8.0)] per four plugs overnight at 4 C and subjected to

pulsed-field gel electrophoresis (PFGE) to remove impu-rities, which can interfere with the digestions. PFGE wasperformed on a 1% (w/v) Pulsed Field CentrifiedAgarose gel (Bio-Rad), using a CHEF DR III apparatus(Bio-Rad) in 0.5TBE buffer at 14 C, with a ramp timeof 5 sec at 2 V/cm for 15 min. Four plugs from whichimpurities had been removed were equilibrated in 1.2 mlof 1HindIII buffer for 30 min on ice. The buffer wasthen removed and replaced by ice-cold HindIII buffer(500 µl per plug) containing 10 U of HindIII (Promega).After incubation at 4 C for 12 hr, the plugs were trans-ferred to a 37 C air incubator for 2 hr. Digestion wasstopped by replacing with 2.5 ml of 50 mM EDTA (pH8.0). The plugs were equilibrated in 10 ml of 0.5TBEbuffer per four plugs for 30 min on ice and subjected toPFGE on a 1% (w/v) SeaPlaque agarose gel (BMA),using a CHEF DR III apparatus in 0.5TBE buffer at 14C, with a ramp time from 5 to 35 sec at 6 V/cm for 18 hr.Agarose gel slices from 50 to 125 kb (fraction I) and 125to 200 kb (fraction II) were excised from the gel and usedfor ligation into the cloning vector.

Ligation and transformation. The cloning vectorpBeloBAC11 was purified from E. coli DH10B carryingthe vector according to a standard protocol (2) withminor modifications. Ligation and transformation wereperformed using a previously published protocol (2)with slight modifications. Purified pBeloBAC11 (5 µg)was completely digested with 10 U of HindIII (Promega)at 37 C for 30 min. The digested plasmid (2.5 µg) inHindIII buffer, containing 10 U of HK phosphatase(Epicenter) and 5 mM CaCl2, was dephosphorylated at 30C for 1 hr. The reaction mixture was drop-dialyzedagainst 1TE containing 50 mM NaCl using VS 0.025µm VSWP membranes (Millipore) for 30 min at roomtemperature, followed by inactivation by heating at 65 Cfor 30 min. Each 0.4 g of agarose gel slice, containingeither fractions from 50 to 125 kb or 125 to 200 kb,was washed in two changes with 50 ml of TE buffer con-taining 50 mM NaCl overnight at 4 C. Gel slices weretransferred to a 200 µl of NaI solution (Wako) and melt-ed at 50 C for 15 min following which they were drop-dialyzed against TE buffer containing 50 mM NaCl,using VS 0.025 µm membranes at 45 C. After dialysisfor 30 min, 4 U of β-agarase (BMA) was added to a 400µl of gel solution and the dialysis was continued forone hour. Each gel solution was incubated on ice for 2 hrand was centrifuged at 16,000g for 10 min at 4 C toremove undigested agarose. The concentration of DNAin the supernatant and the dephosphorylated vector wasdetermined by conventional agarose gel electrophoresis.Then 25 to 50 ng of each size-selected insert DNA wasligated into 12.5 to 25 ng of HindIII-digested, dephos-phorylated pBeloBAC11 vector in a 1:10 molar ratio,

1006 T. GOTO ET AL

using 40 U of T4 DNA ligase (New England Biolabs) at16 C for 4 hr, following which it was drop-dialyzedagainst 0.2TE buffer, using VS 0.025 µm membranesat room temperature for 1 hr.

Three-microliter aliquots of the ligation mixture wereused for electroporation of 20 µl of TransforMAXTM

EC100TM Electrocompetent E. coli (Epicenter) and 57 µlof 10% (v/v) glycerol in a GenePulser II (Bio-Rad),with settings of 1.25 kV/mm, 25 µF, and 100 Ω. Afterelectroporation, cells were resuspended in 500 µl ofSOC medium, incubated at 37 C for 45 min, and thenplated on to LB agar plates containing 12.5 µg/ml CM,25 µg/ml IPTG and 50 µg/ml X-gal. The plates wereincubated at 37 C for 24 hr.

Isolation of BAC DNA and determination of insertsize. Recombinant BAC DNAs were isolated using astandard alkaline lysis method (25) with minor modifi-cations. To estimate the insert size, BAC DNA wasdigested with NotI and analyzed by PFGE using a 1%(w/v) Pulsed Field Centrified Agarose gel (Bio-Rad) in0.5TBE buffer at 14 C, with a ramp time from 5 to 15sec at 6 V/cm for 17 hr.

DNA hybridization. For dot-blot hybridization, eachDNA sample (100 ng) isolated from all BAC clones inthe library was blotted onto nylon membranes (Gene-Screen Plus, NEN). F. magna ATCC 29328 DNA as apositive control and pBeloBAC11 vector DNA as a neg-ative control were blotted onto the same membranes.Blotted membranes were denatured with 0.5 M NaOH for1 min and neutralized with 1 M Tris/HCl (pH 7.5) for 1min.

For Southern hybridization, preparation of gel blocksfor PFGE, restriction digestion of the genomic DNAand PFGE analysis were performed as described previ-ously (30). After PFGE, the restriction fragments weretransferred to a nylon membrane using a capillary trans-fer method (25).

All membranes for DNA hybridization were incu-bated at 37 C for 1 hr in a prehybridization buffer (DIGEasy Hyb, Roche) and incubated at 37 C overnight inDIG Easy Hyb containing each digoxigenin (DIG)-labeled probe. Most probes were synthesized using aPCR DIG probe synthesis kit (Roche) with each extract-ed BAC DNA serving as a template. The primers forprobe synthesis were designed from the BAC-endsequences determined in this study or from known DNAsequences of F. magna. A probe for urPAB, a homologof protein PAB, was synthesized using genomic DNAfrom F. magna ALB1B strain (5, 6) as a template. In dot-blot hybridization using SgrAI fragments of F. magna asprobes, four separated SgrAI fragments (SgA, SgB, SgCand SgD) were recovered from the PFGE gel and labeledby random priming method using a BcaBEST DIG label-

ing kit (TaKaRa). HindIII-digested BAC DNAs werelabeled in the same way. Hybridizing signals weredetected using a DIG Luminescent Detection kit (Roche).

BAC-end sequencing. BAC DNA for end-sequencingwas obtained using the standard akaline lysis method (2,3) with minor modifications or using QIAGEN Plas-mid Midi Kit (Qiagen). Sequencing reactions were per-formed using a BigDye Terminator Cycle Sequencing Kit(Applied Biosystems) with approximately 1 µg of BACDNA and 10 pmol primer (T7 or SP6) per reaction.The thermal profile was 96 C for 5 min followed by 35cycles of 96 C for 30 sec, 55 C for 5 sec and 60 C for 4min. Homology search of each BAC-end sequence wasperformed using BLASTX algorithms (1) at the NCBIdatabase (www.ncbi.nlm.nih.gov/BLAST/BLAST.cgi).

Results and Discussion

Construction of F. magna ATCC29328 BAC LibraryPartial HindIII fragments of F. magna ATCC 29328

DNA in the size range of 50 to 200 kb were ligatedinto pBeloBAC11 vector and transformed into E. coli.Cloning of fraction I (50 to 125 kb) and fraction II (125to 200 kb) gave transformants of 210 and 175, respec-tively. A total of 385 BAC clones (designated as BF1through BF385) were obtained from the ligations. Toestimate insert sizes of BAC clones, DNAs were isolat-ed from 211 (50 from fraction I and 161 from fraction II)of 385 clones and were subjected to PFGE analysis ofNotI-cleaved BACs. Analysis for the presence of insertsshowed that 95% of clones had an insert of the appro-priate size. Insert sizes estimated by PFGE analysisranged from approximately 20 to 120 kb (Fig. 1, a and b).The average insert sizes of each 50 clones, which wererandomly selected from fraction I and fraction II, were 56and 53 kb, respectively. Overall average size of 385BAC clones was estimated to be approximately 55 kb(Fig. 1b). From the sum of the average insert size, weestimated that 385 BAC clones contain approximately 21Mb of the F. magna genome. Our previous macrore-striction analysis of F. magna DNA with ApaI, PmeI,SgrAI and I-CeuI showed that F. magna ATCC 29328has a chromosome of approximately 1.9 Mb and a plas-mid of 200 kb (30). Assuming DNAs of this size, ourlibrary should provide a 10.1-fold coverage of the ATCC29328 DNA. Theoretically, the probability that thelibrary contains any particular 1-kb gene on the genomewas calculated to be 99.9% (7). It was reported that BAClibraries of other bacteria, such as Bacillus cereus (23),Mycobacterium tuberculosis (3), Pseudomonas aerugi-nosa (9) and Methanosarcina thermophila (10), hadgenome coverage of 5.75, 70, 9.5 and 8.0 fold, respec-tively. Comparing with these data, our BAC library

1007BAC LIBRARY OF FINEGOLDIA MAGNA ATCC 29328

would be satisfactory for genome analysis.

Even Distribution of BAC Clones on the Physical MapTo investigate whether BAC clones distribute evenly

on the physical map of F. magna ATCC 29328 DNA, wescreened 385 BAC clones by dot-blot hybridizationusing probes of four SgrAI-cleaved genomic fragments(SgA, SgB, SgC and SgD). The SgA, B, C and Dprobes identified 110 (29%), 93 (24%), 57 (15%) and 39(10%) positive clones out of 385 BAC clones, respec-tively. We estimated previously that the sizes of SgA, B,C and D account for 32, 27, 22 and 8% of the genome,respectively (30). For each SgrAI fragment, percent ofpositive clones in all BACs is similar to that of genomecoverage, suggesting that the BAC clones are distributedevenly on the physical map of F. magna ATCC 29328DNA.

Construction of BAC Contigs on the Physical Map byDNA Hybridization

We constructed BAC contigs and placed them on thephysical map of F. magna ATCC 29328 DNA in thefollowing three steps:

(i) As starting points for the construction of contigs,we selected five ApaI sites and four rrn operon loci onthe map. We previously determined sequences of fiveApaI-linking clones (pAL3, pAL4, pAL7, pAL10 andpAL12) and 16S ribosomal RNA sequences on the chro-mosome (30). Accordingly, we screened 385 BAC

clones by dot-blot hybridization using probes designedfrom these sequences. The dot-blot screening usingprobes of pAL3, pAL4, pAL7, pAL10 and pAL12 gavepositive clones of 17, 2, 19, 7 and 14, respectively. The16S rRNA-specific probe also identified a total of 16 pos-itive clones. We classified these 16 clones into fourgroups, which represent four rrn operon (rrnA, rrnB,rrnC and rrnD) which we reported previously (DDBJaccession numbers AB109769-AB109772) (31). rrnA,B, C and D had positive clones of 3, 4, 8 and 1, respec-tively. The average number of positive clones in thesehybridizations was 8.3 per screening. This value is ingood agreement with our estimation that our library hasa genomic coverage of 10.1-fold. One representativeclone carrying the longest insert was selected from thepositive clones in each hybridization and used for thenext step.

(ii) We examined the elongation of BAC contigs fromnine starting clones with both end-sequences (approxi-mately 600 bp) being determined. Then we designed aprimer pair within each end-sequence to obtain a PCRproduct of approximately 300 bp. Each DIG-labeledPCR probe was synthesized using these primers, with anextracted BAC DNA being used as a template. Subse-quently, each probe was hybridized to the dot-blottedmembranes. Cycles of hybridization and new probedesign were repeated until the end probe failed to iden-tify a new positive clone, namely, until the elongation ofeach contig stopped at both ends.

1008 T. GOTO ET AL

Fig. 1. Estimation of insert sizes by NotI-PFGE analysis of F. magna ATCC 29328 BAC library. (a) Typical photograph of a PFGE gel.PFGE was performed using 1% Pulsed Field Centrified Agarose gel (Bio-Rad) in 0.5TBE buffer at 14 C, with a ramp time from 5 to15 sec at 6 V/cm for 17 hr. Lanes: 1, BF211; 2, BF212; 3, BF213; 4, BF214; 5, BF215; 6, BF216; 7, BF217; 8, BF218; 9, BF219; 10,BF220; 11, BF221; 12, BF222; 13, BF223; 14, BF224; 15, BF225; 16, BF226; 17, BF227; 18, BF228; 19, BF229; 20, BF230; 21, BF231;22, BF232; 23, BF233; M, Lambda Ladder PFG Marker (BioLabs). (b) Size distribution of 100 BAC clones which were randomly select-ed (50 clones per each fraction). Insert sizes were estimated by PFGE analysis of NotI-cleaved clones.

(iii) Where required, the orientation and detailed posi-tion of each contig on the map were determined. For thispurpose, Southern hybridizations were performed toconfirm linkage and to estimate lengths of overlappingregions between BAC clones. An example is shown inFig. 2. In this case, BAC clone BF324 DNA was used asa probe. A 6.2-kb hybridizing band (lane 1, Fig. 2b) indi-cates that the 6.2-kb HindIII fragment overlappedbetween BF264 and BF324. A 3.1-kb hybridizing band(lane 3, Fig. 2b) indicates that the 3.1-kb HindIII frag-ment alone overlapped between BF111 and BF324.Similar experiments in this manner were performed toobtain more reliable results for contig positions. Inaddition, a total of 23 BAC-end probes were individual-ly used for hybridization to the PFGE membrane ofmacrorestriction fragments (ApaI, PmeI, I-CeuI andSgrAI digests) of F. magna ATCC 29328 DNA to con-firm the contig positions (See legend of Fig. 3). Byrestriction analysis, location of four I-CeuI sites andfive ApaI sites were fixed on the four BAC clones (BF89,BF111, BF170, and BF185) and the five BAC clones(BF217, BF225, BF232, BF268, and BF320), respec-tively. These results gave more detailed contig posi-tions.

After three steps, finally, nine contigs covering 95% ofthe chromosome and two contigs covering 98% of theplasmid pPEP1 were localized on the physical map of F.magna ATCC 29328 DNA (Fig. 3). Nine gaps on thechromosome and two gaps on the plasmid remained asnon-covering regions. We estimated that total gap size onthe chromosome and the plasmid was 95 and 4 kb,respectively. In the Treponema pallidum BAC library(28), it was suggested that ORFs coding for compo-nents of protein complexes, such as DNA-dependentRNA polymerase or ribosome, were difficult to clone inE. coli because of their interference with E. coli com-ponents. The T. pallidum library also suggested thatthe proximity to rRNA gene clusters could lead to regionsbeing impossible to clone (28). These explanations mayaccount for the missing F. magna clones.

The Genetic Map of F. magna ATCC 29328 Based onBAC-End Sequencing

A total of 121 end sequences (yielding a totalsequence of approximately 74 kb) out of 134 BAC-endloci were determined and localized onto the physicalmap of F. magna ATCC 29328 (Fig. 3, Table 1). Theaverage guanine-plus-cytosine (GC) percentage of121 end sequences was 37%. This value was a littlehigher than the GC content (32%) of the ATCC 29328strain reported previously by DNA-DNA hybridizationmethods (13). This difference may reflect either ourlimited sequences or the relatively low accuracy ofDNA-DNA hybridization methods. Determined 121BAC-end sequences were subjected to the homologysearch using BLASTX algorithms (1). Ninety-fiveunique BAC-end sequences were deposited in the DNAData Bank of Japan (DDBJ) under accession numbersAG266856 through AG266950. BAC-end sequencing ofthis material revealed 103 genes that had not been pre-viously reported for F. magna (Table 1).

The following proteins have recently been the focus ofresearch interest as potential virulence factors for F.magna-induced infectious diseases: a human serum albu-min (HSA)-binding protein termed protein PAB (5, 6), animmunoglobulin light chain-binding protein, protein L(14, 15), and proteolytic enzymes (17, 22). The partialsequence of an urPAB protein homolog, which is one ofthese virulence factors (5, 6) of F. magna, was found byour BAC-end sequencing (See BAC-end locus no.107 inTable 1). To obtain the entire sequence of this homolog,we performed dot-blot screening of our library usingthe PCR probe designed from the partial sequence. Twopositive clones (BF61, BF184) which should have theentire ORF were identified from our library. The com-plete sequence of this ORF and its flanking regions wasdetermined by genomic walking using BF184 DNA as a

1009BAC LIBRARY OF FINEGOLDIA MAGNA ATCC 29328

Fig. 2. Confirmation of linkage and estimation of lengths ofoverlapping regions between BAC clones. (a) HindIII-cleavedBAC clones. Extracted DNA of overlapping clones (BF264,BF324, BF111) were digested with HindIII and subjected toPFGE. PFGE were performed using 1% SeaKem GTG agarose(FMC) in 0.5TBE buffer at 14 C, with a ramp time of 0.1 sec at9 V/cm for 4 hr. Lanes: 1, BF264; 2, BF324; 3, BF111; M, 1 kbDNA Step ladder marker (Promega). (b) Southern hybridizationanalysis of Fig. 2a using BF324 DNA as a probe. BF324 DNAwas labeled by random priming using a BcaBEST DIG labelingkit (TaKaRa) and extracted BF324 DNA, which contain regionsboth vector and insert, as a template.

template and deposited in DDBJ (accession no.AB114038). The coding sequence of the urPABhomolog consists of 870 nucleotides and encodes a 290amino-acid protein (32 kDa) with a predicted N-terminal22-amino-acid signal peptide. The overall amino acididentity of our urPAB homolog protein compared to theurPAB protein from the ALB1B strain (345 amino-acid)was 45%. Specifically, a high degree of homology wasobserved within the following regions: signal sequence(89%), unknown function domain (68%), wall-span-ning region (46%) and transmembrane region (100%).This novel homolog may be useful in determining theevolution of PAB proteins by module shuffling among F.magna strains (5, 6).

We could infer the location of the putative origin ofreplication (oriC) on the F. magna ATCC 29328 chro-mosome, since the dnaN-recF cluster was found byBAC-end sequencing of 366R (See locus no. 1 in Table1). This inference is based on the fact that the rnpA-rpmH-dnaA-dnaN-recF-gyrB-gyrA cluster in the oriCregion is well conserved among many Gram-positive

organisms (24). The presence of such a cluster in F.magna was confirmed by dot-blot hybridization using thegyrB probe (data not shown). We assumed that the difsite, which is located 180˚ away from oriC (29), is locat-ed around locus no. 55 in F. magna (Table 1). About81% of the 86 predicted ORFs on the chromosome of F.magna are transcribed in the same direction as DNAreplication. Similar percentages of 82 and 83% were cal-culated from the complete genome sequences of Clostrid-ium tetani (4) and Clostridium perfringens (26), respec-tively. This bias of gene orientation appears to be acommon feature of low-GC Gram-positive organisms(11).

Comparative Genomics among Species Closely Relatedwith F. magna

Table 2 shows the BLAST hits in the homologysearches in order of the organisms in which the highestdegree of conservation was observed. These resultsindicated that F. magna is most closely related toclostridia, including C. tetani, C. perfringens and

1010 T. GOTO ET AL

Fig. 3. Localization of BAC contigs onto the physical map of F. magna ATCC 29328 DNA. The shaded boxes represent the linearizedmap of the chromosome (a) and plasmid (b). Horizontal bars of the various lengths indicate insert DNA of BAC clones (Scales show thelength of DNA in kb). Italic number under the bar shows BF number of BAC clones. Clone numbers without parenthesis constitute theminimal set covering genomic DNA. Clone numbers with parenthesis are not included in the minimal set but their BAC-end sequenceswere determined. Numbers 1 to 134 show the loci of BAC-end sequences on the physical map and they are the same as those in Table1 and Fig. 4. The roman letter a to z and the greek letter α to ι indicate the restriction fragments of F. magna genome. The hatched boxesunder the roman numerals represent the genome coverage of BAC contigs. The downward arrowhead shows the location of a gap. A,I, P and S indicate the restriction sites of ApaI, I-CeuI, PmeI and SgrAI, respectively. ori indicates the putative origin of replication. rrnA,B, C and D indicate the rRNA operon. From the results of the Southern hybridization to F. magna genome fragments separated by PFGE,locus no. 4, 9, 11, 12, 18, 21, 23, 35, 38, 47, 54, 64, 70, 79, 80, 84, 103, 119, 120, 124, 125, 132 and 134 were mapped onto the regionsof a, c-d, e, d-e, f-g, f-g, g-h, i, h-i-j, p, r, t, u, x, y-z, z, γ-δ, η, η, η, η, θ, and just between η and ι in F. magna genome, respectively.

1011BAC LIBRARY OF FINEGOLDIA MAGNA ATCC 29328

Table 1. Homology searches of BAC-end sequences locarized on the physical map of F. magna ATCC 29328

Locus no.a) End of cloneb) Dir.c) Function of best BLAST hitd) Sourcee) E-value f ) Accession no.g)

putative ori1 366R DNA polymerase III, DnaN, beta subunit CT 2e-13 AG266856

366R conserved hypothetical protein TT 5e-13 AG266856366R RecF protein BAN 2e-07 AG266856

2 114F or R hypothetical protein PF 4e-04 AG2668573, 4 366F, 80F aminopeptidase CP 6e-99 AG266858

5 114R or F not done6 260F not done7 170F 50S ribosomal protein L3 CP 4e-44 AG266859

7–8 rRNA (rrnD) AB1097728 266R not done9 170R cell wall surface anchor family protein SPN 4e-05 AG266860

10 266F ABC transporter BH 9e-39 AG26686111 14R ketoisovalerate oxidoreductase, vorB subunit PAB 1e-37 AG26686212 215R 2-oxoisovalerate-ferredoxin oxidoreductase, alpha subunit AF 5e-38 AG26686313 256F no homology14 375R oligopeptide ABC transporter BH 1e-40 AG26686415 14F DNA polymerase IV CT 5e-33 AG26686516 316R not done17 256R hypothetical protein Nsp 3e-16 AG26686618 215F homoserine dehydrogenase CT 2e-12 AG266867

215F aspartate-semialdehyde dehydrogenase CP 6e-12 AG26686719 375F DNA polymerase I CP 3e-33 AG26686820 359R homoserine dehydrogenase BH 3e-38 AG26686921 316F no homology22 359F DNA polymerase I, polA CA 9e-30 AG266870

359F ribose 5-phosphate epimerase LI 2e-26 AG26687023 252F GTPase, YYAF B. subtilis ortholog CA 1e-49 AG26687124 340R excinuclease ABC subunit C HM 3e-45 AG26687225 252R pyruvate:ferredoxin oxidoreductase and related TT 4e-65 AG266873

2-oxoacid:ferredoxin oxidoreductases, alpha subunit26 240F pyruvate ferredoxin oxidoreductase CP 1e-66 AG26687427 340F tRNA/rRNA methyltransferase CT 2e-18 AG26687528 217R adenine phosphoribosyltransferase CP 2e-23 AG26687629 240R deoxyribose-phosphate aldolase CT 1e-40 AG266877

29–30 pAL10 uncharacterized protein CA 5e-66 AAK7959130 321F transcriptional regulator, marR family CP 8e-09 AG26687831 11R not done32 217F peptidyl-tRNA hydrolase TT 3e-25 AG26687933 321R parvulin-like peptidyl-prolyl isomerase TT 4e-05 AG26688034 29R peptidyl-prolyl cis-trans isomerase CT 1e-24 AG266881

35, 36 11F, 29F hypothetical protein PF 4e-04 AG26688237 185R no homology38 46R methionyl-tRNA synthetase TA 4e-22 AG266883

46R PHP superfamily hydrolase CA 3e-24 AG26688338–40 rRNA (rrnC), 39 (232F) contain partial sequence of rrnC AB109771

40 185F not done41 46F Asp-tRNAAsn/Glu-tRNAGln amidotransferase B subunit TT 9e-55 AG266884

41–42 pAL3 uncharacterized protein YqeB (pAL3) EC 1e-57 Q4680842, 43 33R,18F DNA integration/recombination protein CT 8e-27 AG266885

44 232R not done45 33F ABC transporter ATP-binding protein OI 7e-13 AG26688646 26R CBS-domain containing protein, YHDP B. subtilis CA 1e-65 AG266887

ortholog, hemolysin-related protein47 18R hypothetical protein PF 9e-05 AG26688848 113F or R cell wall-associated hydrolase TT 8e-24 AG26688949 26F selenophosphate synthetase EA 5e-62 AG266890

1012 T. GOTO ET AL

Table 1. (continued)

50 105F conserved hypothetical protein SE 3e-11 AG266891105F hypothetical protein TF 5e-04 AG266891

51 113R or F not done52, 53 348F, 277R metal-dependent phosphoesterases, PHP family TT 2e-14 AG266892

54 270F transcription terminator, NusA CA 9e-58 AG26689355 105R triosephosphate isomerase TT 2e-44 AG26689456 348R integral membrane protein, ComEC LI 3e-10 AG26689557 24R 30S ribosomal protein S1 CT 8e-34 AG26689658 277F flavoproteins TT 4e-10 AG266897

277F cytidylate kinase TT 1e-11 AG26689759 270R 50S ribosomal protein L32 TM 2e-07 AG26689860 230F no homology61 24F tRNA/rRNA methylase SPN 1e-29 AG26689962 13R not done63 230R hypothetical protein HI 5e-28 AG26690064 13F no homology65 284F or R not done66 59R no homology67 384R pullulanase BD 2e-35 AG26690168 125R uracil permease CP 5e-57 AG26690269 284R or F hypothetical protein FN 1e-38 AG26690370 89F hypothetical protein BM 5e-06 AG266904

89F molybdopterin biosynthesis protein CP 1e-09 AG26690471 59F V-type sodium ATP synthase subunit C CP 1e-29 AG26690572 384F V-type sodium ATP synthase subunit I CT 8e-37 AG26690673 125F conserved hypothetical protein TT 1e-40 AG266907

73–74 rRNA (rrnB) AB10977074 89R cysteine proteinase, cell surface protein MM 2e-06 AG266908

75, 76 349F, 308R conserved hypothetical protein CP 4e-22 AG266909349F, 308R ABC transporter CP 4e-17 AG266909

77 349R elongation factor, SelB EA 7e-27 AG26691078 212F not done79 308F elongation factor, SelB EA 4e-05 AG26691180 212R conserved hypothetical protein OI 2e-05 AG26691281 193R alanine racemase CT 4e-31 AG26691382 223R ATP-dependent protease Clp, ATPase subunit TT 2e-54 AG26691483 210R trigger factor CP 5e-20 AG26691584 193F origopeptide ABC transporter TT 1e-73 AG26691685 210F no homology86 264F NADH-dependent butanol dehydrogenase BS 5e-27 AG26691787 223F no homology88 324F heme biosynthesis (nirJ-2) family protein CA 2e-58 AG26691889 264R PTS system, fructose-specific IIABC component FN 1e-52 AG26691990 111R ATP dependent helicase LI 5e-12 AG26692091 324R no homology92 197R ABC transporter, outer membrane protein PAE 4e-63 AG266921

92–93 rRNA (rrnA) AB10976993 197F no homology94 111F no homology95 39F peptidoglycan anchored protein, LPXTG motif protein CT 2e-04 AG26692296 225F conserved protein CT 0.006 AG26692397 39R not done98 224R no homology

98–99 pAL7 response regulator CA 1e-18 AAK78270pAL7 histidine kinase SPY 2e-13 AAB92598

99 225R ABC transporter SM 2e-38 AG266924100 268F ABC transporter SA 6e-09 AG266925101 224F pyridoxine biosynthesis protein FN 4e-64 AG266926

1013BAC LIBRARY OF FINEGOLDIA MAGNA ATCC 29328

Table 1. (continued)

102 61F transcriptional regulator, GntR family FN 1e-33 AG266927102–103 pAL12 predicted coding region MJ0003 MJ 3e-13 AAB97990103, 104 239F, 307R ATP-dependent exoDNase (exonuclease V), alpha TT 3e-36 AG266928

subunit-helicase superfamily I member105 268R chaperone protein DnaJ Ssp 2e-07 AG266929106 239R nitroreductase family protein CA 8e-11 AG266930107 307F urPAB protein precursor FM 5e-06 AG266931108 61R multidrug resistance related protein SS 0.003 AG266932109 320R type I restriction-modification system specificity subunit MM 1e-28 AG266933

(methylase, HsdM)109–110 pAL4 phosphotransferase enzyme II A component YP 3e-27 Q46808

110 1F hypothetical protein EC 1e-39 AG266934111 320F IgG-binding Protein A precursor SX 6e-27 AG266935112 9R Ca-transporting ATPase CP 1e-07 AG266936113 1R no homology114 260R phosphate acetyltransferase BAP 0.079 AG266937115 80R conserved hypothetical protein CT 2e-12 AG266938116 9F glucose-inhibited division protein CT 8e-09 AG266939117 209F no homology118 360F or R single-stranded DNA-specific exonuclease CG 0.052 AG266940119 96R no homology120 42R no homology121 96F hypothetical protein pXO1-20-Bacillus anthracis BAN 0.002 AG266941

virulence plasmid pXO1122 360R or F antirestriction protein, DNA primase homolog TT 4e-12 AG266942123 42F N-acetylmuramoyl-L-alanine amidase CT 2e-08 AG266943124 12R hypothetical protein PF 0.019 AG266944125 209R no homology126 55R internalin-like/N-acetylmuramoyl-L-alanine amidase CT 5e-11 AG266945127 12F not done128 116F putative cell surface protein, LPXTG motif LI 3e-10 AG266946129 216R hypothetical protein PF 0.093 AG266947130 55F GrpE protein BAP 0.035 AG266948131 216F NADH dehydrogenase TP 0.016 AG266949132 116R Bacillus subtilis YabE protein homolog lmo0186 LM 0.003 AG266950133 380R no homology134 380F no homology

a) ‘Locus no.’ are the same as those in Figs. 3 and 4.b) ‘End of clone’ indicates BAC clone no. and its end-sequence. F and R indicate sequencing using T7 and SP6 primer in the vector,

respectively. Exceptionally, pAL clones indicate ApaI-linking clones described in our previous report (30).c) Dir. indicates the direction of transcription, namely to the right () or to the left ().d) Homology search of each BAC-end sequence was performed using BLASTX algorithms.e) ‘Source’ indicates species with the best BLAST hit. These names are abbreviated as follows: AF, Archaeoglobus fulgidus; BAN, Bacil-

lus anthracis; BAP, Buchnera aphidicola; BD, Bacillus deramificans; BH, Bacillus halodurans; BM, Bacillus megaterium; BS,Bacillus subtilis; CA, Clostridium acetobutylicum; CG, Corynebacterium glutamicum; CP, Clostridium perfringens; CT, Clostridiumtetani; EA, Eubacterium acidaminophilum; EC, Escherichia coli; FM, Finegoldia magna; FN, Fusobacterium nucleatum; HI,Haemophilus influenzae; HM, Heliobacillus mobilis; LI, Listeria innocua; LM, Listeria monocytogenes; MJ, Methanococcus jannaschii;MM, Methanosarcina mazei; Nsp, Nostoc sp.; OI, Oceanobacillus iheyensis; PAB, Pyrococcus abyssi; PAE, Pseudomonas aeruginosa;PF, Plasmodium falciparum; SA, Streptococcus agalactiae; SE, Staphylococcus epidermidis; SM, Streptococcus mutans; SPN, Strep-tococcus pneumoniae; SPY, Streptococcus pyogenes; SS, Sulfolobus solfataricus; Ssp, Synechococcus sp.; SX, Staphylococcus xylosus;TA, Thermus aquaticus; TF, Thermobifida fusca; TM, Thermotoga maritima; TP, Tetrahymena pyriformis; TT, Thermoanaerobacter teng-congensis; YP, Yersinia pestis.

f ) Homologs with an e-value of more than 0.1 were excluded and were shown as ‘no homology’ in column 4.g) ‘Accession no.’ indicates the DDBJ accession number of each BAC-end sequence. Exceptionally, AG266931 codes the complete

ORF gene which determined by gene walking from the end-sequence. Each of AG266858, AG266882, AG266885, AG266892,AG266909 and AG266928 codes single sequence assembled from two BAC-end sequences.

Clostridium acetobutylicum, and to Thermoanaerobac-ter tengcongensis, an anaerobic eubacteria. The recentclassifications of anaerobic cocci based on their 16SrRNA sequences revealed that the former genus Pep-tostreptococcus was closely related to the genus Clostrid-ium and the genus Ruminococcus (8, 12). Thus, ourresults were in good agreement with these classifica-tions. Since C. tetani had most BLAST-top hits, wecompared the global genome structure of F. magnaATCC 29328 and C. tetani E88. Fifty-four homologs,including homologs which were not necessarily besthit, were found in C. tetani E88 by BLASTX searches.

Figure 4 shows the location of the homologous genes inthe chromosomes of F. magna ATCC 29328 and C.tetani E88. This comparison indicates that the majorityof homologous genes in F. magna appear to be locatedinversely in C. tetani. However, some of the F. magnagene clusters are present as similar gene clusters in C.tetani. For example, gene pairs such as locus numbers11–12, 18–20, 32–34, 71–72, 82–83 and 115–116 wereconserved between the two species. Thus, it is possiblethat the genome backbones of the two species are close-ly related to some extent but are nevertheless distinct.

1014 T. GOTO ET AL

Table 2. Comparison of BAC-end sequences of F. magna ATCC 29328 with sequences of other organisms

Organism Total number of homologs with the best BLAST hit a)

Clostridium tetani 16Thermoanaerobacter tengcongensis 15Clostridium perfringens 14Clostridium acetobutylicum 9Plasmodium falciparum 5Fusobacterium nucleatum, Listeria innocua 4Bacillus halodurans, Eubacterium acidaminophilumb) 3

Bacillus anthracis, Buchnera aphidicola, Escherichia coli2

Methanosarcina mazei,b) Oceanobacillus iheyensis, Streptococcus pneumoniae

Bacillus subtilis, Finegoldia magna,b) Haemophilus influenzae, Listeria monocytogenes,Pseudomonas aeruginosa, Streptococcus agalactiae, Staphylococcus epidermidis, 1Streptococcus mutans, Streptococcus pyogenes and other species

a) Homologs with e-value of more than 0.1 in BLASTX search were excluded. b) The genome of these organisms is not completely sequenced yet.

Fig. 4. Genomic location of homologous genes in F. magna ATCC 29328 and C. tetani E88. The map of F. magna ATCC 29328 was con-structed by BAC-end sequencing in this study. For C. tetani E88 map, its complete genome sequence (4) was utilized to compare withF. magna map. The homolog numbers on each map are the same as locus numbers of Fig. 3 or Table1. Scale shows the length of DNAin kb.

ConclusionsThe BAC library of F. magna ATCC 29328 DNA

consisted of 385 clones representing a 10.1-fold geno-mic coverage. Nine contigs covering 95% of the chro-mosome and two contigs covering 98% of the plasmidwere localized on the physical map. BAC-end sequenc-ing revealed 103 novel genes of F. magna and that F.magna was most closely related to clostridia. A homologof albumin binding protein, a candidate virulence factorof F. magna, was also sequenced and mapped on thechromosome. We believe that our library will serve as agood starting tool towards further elucidation of thepeptostreptococci genome. This library will also beuseful for assemblies of sequences generated by thewhole genome shotgun project and for the other postge-nomic applications such as expression of pathogenicity-related F. magna genes.

We are grateful to Mieko Tamura, Yoshimi Tsuchiya andAkio Honda for technical assistance. This work was supported inpart by Grant-in-Aid for Young Scientists (B) (MEXT-15790226)from the Ministry of Education, Culture, Sports, Science andTechnology, Japan and in part by Research for the Future Programfrom the Japan Society for the Promotion of Science (JSPS-RFTF00L01411) and in part by funds from the Yakult Bio-Sci-ence Foundation.

References

1) Altschul, S.F., Gish, W., Miller, W., Myers, E.W., andLipman, D.J. 1990. Basic local alignment search tool. J.Mol. Biol. 215: 403–410.

2) Birren, B., Mancino, V., and Shizuya, H. 1999. Bacterial arti-ficial chromosomes, vol. 3, p. 241–295. In Birren, B., Green,E.D., Klapholz, S., Myers, R.M., Riethman, H., andRoskams, J. (eds), Genome analysis, Cold Spring HarborLaboratory, Cold Spring Harbor, New York.

3) Brosch, R., Gordon, S.V., Billault, A., Garnier, T., Eiglmeier,K., Soravito, C., Barrell, B.G., and Cole, S.T. 1998. Use of aMycobacterium tuberculosis H37Rv bacterial artificial chro-mosome library for genome mapping, sequencing, and com-parative genomics. Infect. Immun. 66: 2221–2229.

4) Brüggemann, H., Bäumer, S., Fricke, W.F., Wiezer, A.,Liesegang, H., Decker, I., Herzberg, C., Martínez-Arias, R.,Merkl, R., Henne, A., and Gottschalk, G. 2003. The genomesequence of Clostridium tetani, the causative agent of tetanusdisease. Proc. Natl. Acad. Sci. U.S.A. 100: 1316–1321.

5) de Château, M., and Björck, L. 1994. Protein PAB, a mosa-ic albumin-binding bacterial protein representing the first con-temporary example of module shuffling. J. Biol. Chem. 269:12147–12151.

6) de Château, M., and Björck, L. 1996. Identification of inter-domain sequences promoting the intronless evolution of abacterial protein family. Proc. Natl. Acad. Sci. U.S.A. 93:8490–8495.

7) Clarke, L., and Carbon, J. 1976. A colony bank containing

synthetic Col E1 hybrid plasmids representative of the entireE. coli genome. Cell 9: 91–99.

8) Collins, M.D., Lawson, P.A., Willems, A., Cordoba, J.J.,Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H., andFarrow, J.A.E. 1994. The phylogeny of the genus Clostridi-um: proposal of five new genera and eleven new speciescombinations. Int. J. Syst. Bacteriol. 44: 812–826.

9) Dewar, K., Sabbagh, L., Cardinal, G., Veilleux, F.,Sanschagrin, F., Birren, B., and Levesque, R.C. 1998.Pseudomonas aeruginosa PAO1 bacterial artificial chromo-somes: strategies for mapping, screening, and sequencing 100kb loci of the 5.9 Mb genome. Microb. Comp. Genomics 3:105–117.

10) Diaz-Perez, S.V., Alatriste-Mondragon, F., Hernandez, R.,Birren, B., and Gunsalus, R.P. 1997. Bacterial artificial chro-mosome (BAC) library as a tool for physical mapping of thearchaeon Methanosarcina thermophila TM-1. Microb. Comp.Genomics 2: 275–286.

11) Fraser, C.M., Gocayne, J.D., White, O., Adams, M.D.,Clayton, R.A., Fleischmann, R.D., Bult, C.J., Kerlavage,A.R., Sutton, G.G., Kelley, J.M., Fritchman, J.L., Weidman,J.F., Small, K.V., Sandusky, M., Fuhrmann, J.L., Nguyen,D.T., Utterback, T., Saudek, D.M., Phillips, C.A., Merrick,J.M., Tomb, J., Dougherty, B.A., Bott, K.F., Hu, P.C.,Lucier, T.S., Peterson, S.N., Smith, H.O., and Venter, J.C.1995. The minimal gene complement of Mycoplasma geni-talium. Science 270: 397–403.

12) Hill, K.E., Davies, C.E., Wilson, M.J., Stephens, P., Lewis,M.A.O., Hall, V., and Brazier, J. 2002. Heterogeneity with-in the gram-positive anaerobic cocci demonstrated by analy-sis of 16S-23S intergenic ribosomal RNA polymorphisms. J.Med. Microbiol. 51: 949–957.

13) Huss, V.A.R., Festl, H., and Schleifer, K.H. 1984. Nucleicacid hybridization studies and deoxyribonucleic acid basecompositions of anaerobic, Gram-positive cocci. Int. J. Syst.Bacteriol. 34: 95–101.

14) Kastern, W., Holst, E., Nielsen, E., Sjöbring, U., and Björck,L. 1990. Protein L, a bacterial immunoglobulin-bindingprotein and possible virulence determinant. Infect. Immun.58: 1217–1222.

15) Kastern, W., Sjöbring, U., and Björck, L. 1992. Structure ofpeptostreptococcal protein L and identification of a repeatedimmunoglobulin light chain-binding domain. J. Biol. Chem.267: 12820–12825.

16) Kim, U.J., Birren, B.W., Slepak, T., Mancino, V., Boysen, C.,Kang, H.L., Simon, M.I., and Shizuya, H. 1996. Constructionand characterization of a human bacterial artificial chromo-some library. Genomics 34: 213–218.

17) Krepel, C.J., Gohr, C.M., Walker, A.P., Farmer, S.G., andEdmiston, C.E. 1992. Enzymatically active Peptostrepto-coccus magnus: association with site of infection. J. Clin.Microbiol. 30: 2330–2334.

18) Mascini, E.M., and Verhoef, J. 2000. Anaerobic cocci, p.2570–2573. In Mandell, G.L., Bennett, J.E., and Dolin, R.(eds), Principles and Practice of Infectious Diseases, 5thed, Churchill Livingstone, Philadelphia.

19) Murdoch, D.A., Mitchelmore, I.J., and Tabaqchali, S. 1994.The clinical importance of Gram-positive anaerobic cocci iso-lated at St Bartholomew’s Hospital, London, in 1987. J.

1015BAC LIBRARY OF FINEGOLDIA MAGNA ATCC 29328

Med. Microbiol. 41: 36–44.20) Murdoch, D.A. 1998. Gram-positive anaerobic cocci. Clin.

Microbiol. Rev. 11: 81–120.21) Murdoch, D.A., and Shah, H.N. 1999. Reclassification of

Peptostreptococcus magnus (Prevot 1933) Holdeman andMoore 1972 as Finegoldia magna comb. nov. and Pep-tostreptococcus micros (Prevot 1933) Smith 1957 asMicromonas micros comb. nov. Anaerobe 5: 555–559.

22) Ng, J., Ng, L.-K., Mayrand, D., and Dillon, J.-A.R. 1998.Aminopeptidase activities in Peptostreptococcus spp. arestatistically correlated to gelatin hydrolysis. Can. J. Microbiol.44: 303–306.

23) Rondon, M.R., Raffel, S.J., Goodman, R.M., and Handelsman,J. 1999. Toward functional genomics in bacteria: analysis ofgene expression in Escherichia coli from a bacterial artificialchromosome library of Bacillus cereus. Proc. Natl. Acad. Sci.U.S.A. 96: 6451–6455.

24) Salazar, L., Fsihi, H., de Rossi, E., Riccardi, G, Rios, C.,Cole, S.T., and Takiff, H.E. 1996. Organization of the originsof replication of the chromosomes of Mycobacterium smeg-matis, Mycobacterium leprae and Mycobacterium tubercu-losis and isolation of a functional origin from M. semgmatis.Mol. Microbiol. 20: 283–293.

25) Sambrook, J., and Fritsch, E.F. (eds), 2001. MolecularCloning, 3rd ed, Cold Spring Harbor Laboratory, ColdSpring Harbor, New York.

26) Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K.,Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara,S., and Hayashi, H. 2002. Complete genome sequence ofClostridium perfringens, an anaerobic flesh-eater. Proc. Natl.Acad. Sci. U.S.A. 99: 996–1001.

27) Shizuya, H., Birren, B., Kim, U.J., Mancino, V., Slepak,T., Tachiiri, Y., and Simon, M. 1992. Cloning and stablemaintenance of 300-kilobase-pair fragments of human DNAin Escherichia coli using an F-factor-based vector. Proc.Natl. Acad. Sci. U.S.A. 89: 8794–8797.

28) Smajs, D., McKevitt, M., Wang, L., Howell, J.K., Norris, S.J.,Palzkill, T., and Weinstock, G.M. 2002. BAC library of T.pallidum DNA in E. coli. Genome Research 12: 515–522.

29) Steiner, W.W., and Kuempel, P.L. 1998. Cell division isrequired for resolution of dimer chromosomes at the diflocus of Escherichia coli. Mol. Microbiol. 27: 257–268.

30) Todo, K., Goto, T., Miyamoto, K., and Akimoto, S. 2002.Physical and genetic map of the Finegoldia magna (for-merly Peptostreptococcus magnus) ATCC 29328 genome.FEMS Microbiol. Lett. 210: 33–37.

31) Todo, K., Goto, T., Honda, A., Tamura, M., Miyamoto, K.,Fujita, S., and Akimoto, S. Comparative analysis of the fourrRNA operons in Finegoldia magna. System. Appl. Micro-biol., in press.

32) Wren, M.W. 1996. Anaerobic cocci of clinical importance.Br. J. Biomed. Sci. 53: 294–301.

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