Physical and genetic map of the Finegoldia magna (formerlyPeptostreptococcus magnus) ATCC 29328 genome

Kozo Todo, Takatsugu Goto, Kazuaki Miyamoto, Shigeru Akimoto �

Department of Microbiology, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-0012, Japan

Received 6 February 2002; accepted 18 February 2002

First published online 15 March 2002

Abstract

A physical and genetic map of Finegoldia magna (formerly Peptostreptococcus magnus) ATCC 29328 chromosome was constructed. Theorder of rare cleavage restriction fragments was determined by double digestion with the restriction enzymes I-CeuI, SgrAI, ApaI andPmeI, cross-hybridization and ApaI-linking clones. The size of the circular chromosome of F. magna was estimated to be 1.9 Mb. Thisstrain also had a 200-kb megaplasmid. The chromosome contained four rrn operons, and the orientation of two rrn operons was oppositeto the others. Fragment analysis of Peptostreptococcus anaerobius ATCC 27337T chromosome suggested that its size was much smallerthan that of F. magna ATCC 29328. 2 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Micro-biological Societies.

Keywords: Physical map; Plasmid; Albumin binding protein; Peptostreptococcus ; Finegoldia magna (formerly Peptostreptococcus magnus)

1. Introduction

Peptostreptococci are the second most common groupof anaerobes. They account for 20^40% of all anaerobesisolated from clinical sources and are involved in a widevariety of clinically signi¢cant infections, such as septicarthritis or diabetic foot infections [1]. Finegoldia magna(formerly Peptostreptococcus magnus), which has reporteda G+C content of 32^33% [2], is the most common speciesof Gram-positive anaerobic cocci in human clinical speci-mens. However, there is little known about the genomicsof this genus except for rRNA gene sequences. Since datafrom 16S rRNA sequencing studies have demonstratedthat the genus Peptostreptococcus is phylogenetically inco-herent, Murdoch and Ezaki recently proposed reclassi¢ca-tion of peptostreptococci [3^5]. According to the new clas-si¢cation, the type species, Peptostreptococcus anaerobius,

remained within the Peptostreptococcus genus and the restof the species of Peptostreptococcus were divided into ¢venew genera, Finegoldia, Micromonas (erratum), Peptoniphi-lus, Anaerococcus and Gallicola.The aim of this study was to construct a physical map

of F. magna ATCC 29328 as a ¢rst step towards the ge-nomics of peptostreptococci. Genome sizes of three othermembers of the former Peptostreptococcus group, thenumbers of I-CeuI fragments and putative numbers ofthe rrn operons in these organisms were also analyzedand compared with those of ATCC 29328.

2. Materials and methods

2.1. Bacterial strains, plasmids and culture conditions

The bacterial strains used in this study were F. magnaATCC 29328, Peptostreptococcus micros ATCC 33270,Peptoniphilus asaccharolyticus (formerly Peptostreptococ-cus asaccharolyticus) ATCC 29743, and P. anaerobiusATCC 27337T. For constructing the library of ApaI-link-ing clones, Escherichia coli JM109 was used as a hoststrain of plasmid pGEM-7Zf(+) (Promega). Peptostrepto-coccus (formerly) strains were grown in tubes with GifuAnaerobic Medium (Nissui) overnight at 37‡C. P. micros

0378-1097 / 02 / $22.00 2 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 5 6 9 - 4

* Corresponding author. Tel. : +81 (73) 441-0640;Fax: +81 (73) 448-1026.

E-mail address: akimotos@wakayama-med.ac.jp (S. Akimoto).

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were cultured in an anaerobic chamber (5% CO2, 6% H2,89% N2) for 3 days. E. coli was grown in LB medium at37‡C supplemented as required with 100 Wg ml31 ampicil-lin.

2.2. Pulsed-¢eld gel electrophoresis (PFGE) analysis

Cultures were harvested, embedded in low melting pointagarose and digested with rare cleavage restriction en-zymes, ApaI, I-CeuI, PmeI, SgrAI, AscI, NotI, S¢I, PacIand SwaI, following a standard protocol [6]. One agaroseplug contained cells derived from 100^1000 Wl of overnightculture. Plugs were treated with achromopeptidase (1000U ml31 ; Wako) in 10 mM Tris^HCl, 10 mM NaCl, 1 mMEDTA at 37‡C overnight, followed by lysis with protein-ase K (20 U ml31 ; Wako) in 1% N-laurylsarcosine 0.5 MEDTA at 50‡C overnight [6]. PFGE was carried out in aCHEF DRIII (Bio-Rad) with 1% Pulsed-Field Certi¢edAgarose (Bio-Rad) in 0.5UTBE bu¡er (45 mM Tris^bo-rate, 1 mM EDTA, pH 8.0).

2.3. DNA manipulation

DNA fragments were extracted from the unstained andexcised gels using NaI solution and silica matrix (Gene-pure; Nippon Gene). Extracted fragments were DIG-la-beled with a random primer extension method using BcaBEST DIG labeling kit (Takara) and used as probes forcross-hybridization. Primers used in this study were: 27f(5P-AGAGTTTGATCMTGGCTCAG-3P) and 1100r (5P-GGGTTGCGCTCGTTG-3P) for rrs (16S rRNA gene)-speci¢c probes, and 1948f (5P-GTAGCGAAATTCCT-TGTCG) and 2654r (5P-CCGGTCCTCTCGTACT-3P)for rrl (23S rRNA gene)-speci¢c probes. The forwardprimer for rrl was downstream of the I-CeuI site [7]. Prim-ers for pab were designed based on the sequence reported[8,9]. Probes for these genes were synthesized using PCRDIG probe synthesis kit (Roche). Genomic DNA used astemplate for PCR was isolated by lysing cells with achro-mopeptidase for 30 min and 0.5% SDS for 5 min, ex-tracted with phenol and dialyzed against 10 mM Tris^HCl (pH 8.0), 1 mM EDTA. For sequencing, PCR prod-ucts were ligated to the T/A vector pGEM-T, and pUC-derived universal primers (Promega) were used. Bothstrands of at least two separate PCR clones were se-quenced.

2.4. Isolation of ApaI-linking clones

Genomic DNA, isolated from ATCC 29328 as describedabove, was digested with HindIII, and self-ligated with T4DNA ligase. The ligated fragments were then digestedwith ApaI and ligated to the ApaI site of pGEM-7Zf(+).The linking library was maintained in E. coli JM109. Re-combinant clones were isolated by blue^white selection.The sequences were searched for homology by BLASTX.

3. Results and discussion

3.1. Restriction fragment analysis and estimation ofgenome size

Since the G+C content of the genome of F. magnaATCC 29328 was reported to be 31.6% [2], restrictionenzymes with GC rich recognition sites and those whichrecognize and cleave 8-bp sequences were mainly tried,seeking for ones suitable for mapping. PacI and SwaIproduced over 20 fragments. By contrast, AscI, NotI andS¢I did not cleave the chromosomal DNA. As a result ofthese trials, we have chosen ApaI, PmeI and SgrAI. Theseenzymes generated 14, 11, and four fragments of the chro-mosomal DNA, respectively. The intron-encoded endonu-clease I-CeuI, which cleaves rrl genes, also generated fourfragments, suggesting that the chromosome has four rrnoperons (Figs. 1B and 2A). Each fragment was designatedwith the ¢rst letters A, P, Sg, and I, respectively. A secondletter was added in alphabetical sequence according todecreasing size. Fragments generated by double digestionwere designated with the ¢rst letters, SgI to SgrAI/I-CeuIand PI to PmeI/I-CeuI (Table 1). AA, AC, AF, PD, PGand IA proved to contain two or more fragments whenresolved in optimal conditions. Since the sums of fragmentsizes obtained from these digestions were consistently1.9 Mb, we estimated that the size of the F. magnaATCC 29328 chromosome was 1.9 Mb.

Fig. 1. PFGE separation of the F. magna ATCC 29328 genome.A: Lane 1, lambda ladder PFG marker; lane 2, undigested genomeDNA was electrophoresed on 1% agarose in 0.5UTBE at 6 V cm31

with switch time 5^35 s for 20 h. B: Restriction fragments of F. magnaATCC 29328 were electrophoresed on 1% agarose in 0.5UTBE at 6 Vcm31 with switch time 5^75 s for 18 h. Lane 1, lambda ladder PFGmarker; lane 2, SgrAI digests ; lane 3, SgrAI/I-CeuI digests; lane 4,I-CeuI digests. C: Lane 1, fragments generated by partial digestion withI-CeuI were separated on 1% agarose in 0.5UTBE at 6 V cm31 withswitch time 70 s for 15 h and 120 s for 11 h; lane 2, yeast chromosomePFG marker.

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3.2. Existence of a 200-kb megaplasmid in F. magnaATCC 29328

PFGE of undigested genomic DNA or the I-CeuI digestof F. magna ATCC 29328 produced a faint band of about200 kb (Fig. 1A,B). The rrs-speci¢c probe did not hybrid-ize with this band. These results indicated that this frag-ment did not have a rrn operon, suggesting the presence of

a megaplasmid in this strain. The 200-kb DNA band wasextracted, labeled and used for Southern hybridization(Fig. 2). It hybridized with a single 200-kb band ofApaI, ApaI/I-CeuI and I-CeuI digests. In contrast, it hy-bridized with two bands of I-CeuI/PmeI, PmeI and ApaI/PmeI digest. Low-range PFGE analysis of PmeI fragmentsindicated that the 48-kb band was a doublet. These resultsstrongly suggested that this band was derived not from the

Fig. 2. Presence of a 200-kb megaplasmid in F. magna ATCC 29328. Southern blot of ApaI/I-CeuI/PmeI single and double digestion fragments was hy-bridized with the DNA excised from the 200-kb band. ApaI, ApaI/I-CeuI, I-CeuI did not cleave the DNA, indicating the presence of plasmid DNA.A: Lane 1, lambda ladder PFG marker; lane 2, ApaI digests ; lane 3, ApaI/I-CeuI digests; lane 4, I-CeuI digests ; lane 5, I-CeuI/PmeI digests ; lane 6,PmeI digests; lane 7, ApaI/PmeI digests. B: Southern hybridization results. PFGE conditions of Fig. 1A were on 1% agarose in 0.5UTBE at 6 V cm31

with switch time 5^35 s for 20 h.

Table 1Sizes of restriction fragments of single and double digestion of F. magna ATCC 29328 genomes

ApaI PmeI SgrAI I-CeuI SgrAI/I-CeuIc PmeI/I-CeuIc

Fragments of the chromosomea

AA(1) 312 PA 436 SgA 679 IA(1) 595 SgIA 568 PIA 317AA(2) 307 PB 324 SgB 574 IA(2) 572 SgC 467 PIB 264AB 262 PC 268 SgC 464 IB 432 IB 434 PIC 225AC(1) 182 PD(1) 219 SgD 163 IC 276 SgIB 160 PD(2) 220AC(2) 175 PD(2) 213 SgIC 133 PID 196AD 128 PE 129 SgID 126 PIE 172AE 103 PF 115 SgIE 14 PE 129AF(1) 84 PG(1) 75 SgIF 7 PF 115AF(2) 78 PG(2) 69 PG(1) 75AF(3) 73 PH 52 PG(2) 69AF(4) 71 PI 11 PH 53AG 45 PIF 48AH 33 PIG 45AI 19 PI 21

PIH 11Total 1873Q 33 1910Q 38 1881Q 17 1875Q 14 1909Q 16 1867Q 43

Fragments of plasmid pPEP1b

pPA 112 pPA 112pPB 48 pPB 48pPC 46 pPC 46

aFragment sizes are in kb and the means and standard deviations were calculated from at least three experiments.bPlasmid pPEP1 was not cleaved by ApaI, SgrAI and I-CeuI.cFragments obtained with double digestion are shown. New fragments generated were labeled with the ¢rst letters, SgI and PI, respectively.

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chromosomal DNA, but from an independent circularplasmid DNA, which was V200 kb in size and wascleaved only with PmeI into three fragments. We desig-nated the megaplasmid as pPEP1.

3.3. Construction of the I-CeuI physical map of F. magnaATCC 29328

Two approaches were combined to assemble the I-CeuIphysical map of F. magna ATCC 29328. These included (i)analysis of partial digestion with I-CeuI and (ii) cross-hy-bridization with fragments generated by SgrAI and PmeI.I-CeuI partial digestion produced bands of 1.6, 1.45, 1.3,1.02 and 0.86 Mb (Fig. 1C). Since the sizes of IA(1)^ICand IA(2)^IC fragments were equal to the size 0.86 Mb ofpartial digestion, and no fragment was observed around0.70 Mb (the expected size of IB^IC fragment), the orderof the I-CeuI fragments was considered to be IA(1)^IB^IA(2)^IC^(IA(1)) (Fig. 3). Subsequently, cross-hybridiza-tion was performed to verify the order. SgrAI and PmeIfragments of the genomic DNA were excised from gels,labeled and used for Southern hybridization. These probeswere used to determine the overlapping fragments (Table2). For example, the PC probe hybridized with IA(1) andIB. Therefore, the linkage IA(1)^IB was con¢rmed. All ofthe cross-hybridization results agreed with the order of theI-CeuI fragments described above.

3.4. Ordering the SgrAI, PmeI and ApaI fragments on theI-CeuI map

SgrAI fragments were aligned as SgC^SgB^SgA^SgD^(SgC) on the I-CeuI map based on cross-hybridizationresults. They were located more accurately upon the

I-CeuI map by analyzing double digestions of SgrAI/I-CeuI (Fig. 1B). Eight fragments obtained with double di-gestion SgrAI/I-CeuI are shown in Table 1. SgD was de-termined to overlap with IC. SgD was then proved tocontain an rrn operon and to be cleaved into 160 kb(SgIB with rrs) and 7 kb (SgIF with rrl) with I-CeuI bymeans of the hybridization with rrs- and rrl-speci¢cprobes. In a similar way, other fragments generated withSgrAI, PmeI and ApaI were aligned upon the I-CeuI andSgrAI map. In addition to those methods, ApaI-linkingclones were established to construct the ApaI map. Se-quences approximately 600 bp each before and aftereach ApaI site were determined. Then we designed primersapproximately 250 bp each away from the ApaI site to get500-bp PCR products from the template DNA of ATCC29328. When hybridized with the PCR products, ¢ve link-ages were con¢rmed (Table 3). The linkages are shownwith the numbers of ApaI-linking clones in Fig. 3. How-ever, the order of AD^AF(2), PG(1)^PH and PE^PG(2)remained unresolved, since no clone linked them and theywere involved in larger SgrAI/I-CeuI fragments.

3.5. The orientations and distribution of rrn operons

The orientations of rrn operons were determined usingSouthern hybridization of SgrAI/I-CeuI and PmeI/I-CeuIdouble digests with rrs- and rrl-speci¢c probes. The geneswere located on the opposite sides of the I-CeuI site. SinceI-CeuI cleaves rrl (position 1924^1928, E. coli numbering),the primers downstream of the I-CeuI site were chosen forthe rrl probe. The rrs probe hybridized with PIE, whichwas the smaller fragment generated from PA with I-CeuIdouble digestion, and the rrs probe hybridized with PIB,the larger fragment generated from PA. Therefore, theorientation of the rrn operon in PA was determined tobe from IA(2) to IC. Similarly, the orientations of theother three rrn operons were determined (Fig. 3). Two

Fig. 3. Physical and genetic map of the F. magna ATCC 29328 genome.The arrows indicate the orientations of rRNA operons. The numbers onthe outside of the circle show those of the ApaI-lining clones.

Table 2Cross-hybridization of F. magna ATCC 29328 genomic DNA with ex-cised fragments and hybridization with rrs- and rrl-speci¢c probes toApaI, PmeI, I-CeuI and PmeI/I-CeuI fragments

Probes Fragments hybridized

SgA AA(1), AF: PA, PD(1), PD(2): IA(2), ICSgB AA(1), AC(1), AD, AF: PC, PD(2), PE, PF, PG(2): IA(1), IBSgC AB, AC(2), AE: PB, PG(1): PH, IA(1)SgD AA, AC: PA, PB: ICPA AA(2), AC(2), AF(3or4) : PA, PIB, PIE: IA(2), ICPB AA(1), AB, AC(2), AE: PB, PIA, PIH: IA(1)PC AD, AF(1), AG: PC, PIC, PIF: IA(1), IBPD(2) AA(1), AC: PD(2), PID, PIG: IA(2), IBPE AC(1) : PE: IBPF AF(3 or 4), AH: PF: IArrs AA(1), AA(2), AC(2), AG: PA, PB, PC, PD(2): PIC, PID,

PIE, PIH: IA(2), IB, ICrrl AA(1), AA(2), AC(2), AG: PA, PB, PC, PD(2), PIA, PIB, PIF,

PIG: IA(1), IB, IC

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of them were opposite to the others and the rrn operons ofthis strain were scattered around the chromosome. Theseresults were not the same as Clostridium species that have10^12 rrn operons clustered close to the origin of replica-tion [11].Protein L, albumin binding protein (pab) and ur-pab are

the only three genes of F. magna previously reported.Based on the previous reports, we have done PCR trialsusing three sets of primers, cloning and sequencing, butfailed to detect those genes, although F. magna ATCC29328 was reported to have an albumin binding activity.The pab in this strain might be divergent or absent.

3.6. Comparison with the genome size and PFGE analysisof I-CeuI fragments of other formerly groupedpeptostreptococci

The genome sizes and I-CeuI fragments of three othermembers of the former peptostreptococci group were an-alyzed and compared with those of the F. magna ATCC29328 genome to check whether the di¡erences reported in[3^5] were re£ected in the genome sizes and the numbersof rrn operons. P. micros ATCC 33270, P. anaerobiusATCC 27337T and P. assacharolyticus ATCC 29743 pro-duced four, ¢ve and three I-CeuI fragments, respectively.P. assacharolyticus ATCC 29743 also has a faint 160-kbband suggesting the existence of a plasmid (data notshown). From the summation of the I-CeuI fragments,the chromosome sizes of these strains were estimated tobe 1.7, 1.3 and 2.0 Mb, respectively. Thus, the chromo-some of P. anaerobius ATCC 27337T was much smallerthan those of F. magna ATCC 29328, P. assacharolyticusATCC 29743 and P. micros ATCC 33270. These datasupport reclassi¢cation of formerly grouped peptostrepto-cocci other than P. anaerobius.

4. Conclusions

In conclusion, a physical map of the F. magna ATCC29328 genome was constructed. The circular chromosomewas estimated to be 1.9 Mb in length and to contain fourrrn operons. This strain also has a 200-kb megaplasmid.By sequencing the ApaI-linking clones, several putativegenes were located on the physical map. Among the for-mer peptostreptococci group, the size of the P. anaerobius

ATCC 27337T genome was much smaller than those ofother species. These data support the reclassi¢cation ofpeptostreptococci based on recent 16S rRNA sequenceanalyses. The physical map of the F. magna ATCC29328 will lead to more insights into the genomic structureof the peptostreptococci, which has been scarcely studiedand poorly understood.

Acknowledgements

We are grateful to Yoshimi Hirota for technical assis-tance. This work was supported in part by funds from theYakult Bio-Science Foundation.

References

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[3] Murdoch, D.A. and Shah, H.N. (2000) Micromonas micros comb.nov. (basonym Peptostreptococcus micros) and Finegoldia magnacomb. nov. (basonym Peptostreptococcus magnus). in Validation ofpublication of new names and combinations previously e¡ectivelypublished outside the IJSEM, List no. 75. Int. J. Syst. Evol. Micro-biol. 50, 1415^1417.

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[8] de Cha“teau, M. and Bjo«rck, L. (1994) Protein PAB, a mosaic albu-min-binding bacterial protein representing the ¢rst contemporary ex-ample of module shu¥ing. J. Biol. Chem. 269, 12147^12151.

[9] Kastern, W., Sjo«bring, U. and Bjo«rck, L. (1992) Structure of Pepto-streptococcal protein L and identi¢cation of a repeated immunoglob-ulin light chain-binding domain. J. Biol. Chem. 267, 12820^12825.

[11] Keis, S., Sullivan, J.T. and Jones, D.T. (2001) Physical and geneticmap of the Clostridium saccharobutylicum (formerly Clostridium ace-tobutylicum) NCP 262 chromosome. Microbiology 147, 1909^1922.

Table 3Results of homology search of DNA sequences of ApaI-linking clones by BLASTX

Clones Homologous protein Score Accession

pAL3 Uncharacterized protein YqeB of E. coli K12 1e357 Q46808pAL4 Phosphotransferase enzyme II A component of Yersinia pestis 3e327 CAC91371pAL7 Response regulator of Clostridium acetobutylicum 1e318 AAK78270

Histidine kinase homolog ScnK of Streptococcus pyogenes 2e313 AAB92598pAL10 Uncharacterized protein of C. acetobutylicum 5e366 AAK79591pAL12 Methanococcus jannaschii predicted coding region MJ0003 3e313 AAB97990

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