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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2003, p. 6888–6898 Vol. 69, No. 110099-2240/03/$08.00�0 DOI: 10.1128/AEM.69.11.6888–6898.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Sequencing and Characterization of pBM400 fromBacillus megaterium QM B1551

Michael D. Scholle,† Christen A. White, Muthusamy Kunnimalaiyaan,‡ and Patricia S. Vary*Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115

Received 26 June 2003/Accepted 18 August 2003

Bacillus megaterium QM B1551 plasmid pBM400, one of seven indigenous plasmids, has been labeled witha selectable marker, isolated, completely sequenced, and partially characterized. A sequence of 53,903 bp wasgenerated, revealing a total of 50 predicted open reading frames (ORFs); 33 were carried on one strand and17 were carried on the other. These ORFs comprised 57% of the pBM400 sequence. Besides the replicon regionand a complete rRNA operon that have previously been described, several interesting genes were found,including genes for predicted proteins for cell division (FtsZ and FtsK), DNA-RNA interaction (FtsK, Int/Rec,and reverse transcriptase), germination (CwlJ), styrene degradation (StyA), and heavy metal resistance(Cu-Cd export and ATPase). Three of the ORF products had high similarities to proteins from the Bacillusanthracis virulence plasmid pXO1. An insertion element with similarity to the IS256 family and severalhypothetical proteins similar to those from the chromosomes of other Bacillus and Lactococcus species werepresent. This study provides a basis for isolation and sequencing of other high-molecular-weight plasmids fromQM B1551 and for understanding the role of megaplasmids in gram-positive bacteria. The genes carried bypBM400 suggest a possible role of this plasmid in the survival of B. megaterium in hostile environments withheavy metals or styrene and also suggest that there has been an exchange of genes within the gram-positivebacteria, including pathogens.

Bacillus megaterium is a spore-forming bacterium found insoil, seawater, sediments, rice paddies, dried food, honey, andmilk (56). The economic importance of B. megaterium includesits production of vitamin B12 and penicillin amidase, its abilityto express foreign proteins without degradation, and its use inAIDS diagnostics (56; C. Ginsburgh, D. Spaulding, G. Robey,M. A. Shivakumar, O. R. Vanags, L. Katz, and J. L. Fox, Abstr.Int. Conf. AIDS, abstr. 674, 1989). Recently, a strain of B.megaterium has been found that produces plasmid-borne oxe-tanocin, a potential antiviral agent (35). Plasmidless strains areused in industrial and research settings as hosts for efficientexpression of foreign proteins. Most B. megaterium strains con-tain more than four plasmids (56), yet only a few of theirplasmid genes are known. B. megaterium strain QM B1551 hasan array of seven plasmids that have been shown to harborgermination, sporulation, megacin, and rRNA genes (23, 24,51; D. M. Stevenson and P. S. Vary, Abstr. 11th Int. SporesConf., abstr. 60, 1992).

Many plasmids in Bacillus and other gram-positive bacteriaare cryptic in nature. While the small rolling circle replicatingplasmids of gram-positive bacteria have been extensively stud-ied (20, 22), only a few of the plasmid genes or replicons of thelarge theta plasmids have been characterized (for reviews, seereferences 10 and 16). Plasmids usually provide the host cellwith a selective advantage, such as antibiotic resistance (17),

resistance to toxic heavy metals (49), or degradation of unusualcompounds (14, 44). In addition, some plasmids produce bac-teriocins (for review, see reference 19). Bacteriocin (megacin)genes have been reported to be carried by a 47-kb plasmid ofB. megaterium 216 (43), a 45-kb plasmid of ATCC 19213 (59),and a plasmid in strain QM B1551 (23). Gram-positive bacteriamay also harbor plasmids that carry virulence genes, i.e., theinsecticidal toxins of B. thuringiensis and B. sphaericus (46) andthe anthrax toxins produced by B. anthracis from plasmidspXO1 and pXO2 (36, 42).

B. megaterium QM B1551 carries over 11% of its cellularDNA as stable plasmid DNA under laboratory conditions with-out known selection (23). The seven indigenous plasmids are5.4, 9.1, 26.3, 54, 71, 108, and 165 kb (23) and are designatedpBM100 to pBM700, respectively. A plasmidless derivative ofQM B1551, strain PV361, has been isolated (58). The com-plexity of the array of plasmids in B. megaterium offers a uniqueopportunity to study plasmid interactions, replication, compat-ibility, plasmid-borne genes, and possible horizontal transfer.It also presents a challenge for the isolation and sequencing ofplasmids with no known selectable markers and widely varyingcopy numbers within a multiplasmid array. When strain QMB1551 of B. megaterium is cured of its seven indigenous plas-mids, there are few obvious phenotypic effects under labora-tory conditions besides a defect in germination (52) and pro-duction of a megacin (23). QM B1551 is known to be veryefficient at sporulation and germination (12). The plasmidlessstrain PV361 can grow normally on rich media, sporulate nor-mally, and germinate on rich media but cannot germinate onsingle germination triggers because of a plasmid-borne germi-nation gene (52). The plasmids are dispensable under labora-tory conditions, yet they are fairly stable upon subculturing.

To begin to understand plasmid interactions and the role of

* Corresponding author. Mailing address: Department of BiologicalSciences, Northern Illinois University, DeKalb, IL 60115. Phone: (815)753-7421. Fax: (815) 753-0461. E-mail: [email protected].

† Present address: Argonne National Laboratory, Bioscience Divi-sion, Argonne, IL 60439.

‡ Present address: Department of Surgery, K4/638 ComprehensiveCancer Center, University of Wisconsin—Madison, Madison, WI53792.

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plasmid genes in B. megaterium plasmid DNA, we cloned andsequenced fragments containing six of seven compatible rep-lication origins. The two smallest plasmids have been com-pletely sequenced and identified as rolling circle replicatingplasmids with replicon similarities to the pTX14-3 plasmid ofB. thuringiensis and the pXO1-89 plasmid gene of B. anthracis(G. Baisa, Y. Zhou, and P. Vary, unpublished data). The re-maining four sequenced replicons on clones I, II, III, and IVare iteron-type theta replicons from the larger plasmids andare very similar in both Rep proteins and iterons (all five largeplasmid replicons cross-hybridize) (24, 51, 57; M. Kunni-malaiyaan and P. Vary, unpublished data). The 12.3-kb repli-cating clone II fragment and its adjacent rRNA operon (6.3kb), located just upstream from this replication clone, havebeen sequenced and characterized from pBM400 (54 kb) (24).To date, this is the only rRNA operon found in nonessentialDNA, and its presence has considerable evolutionary signifi-cance. Since B. megaterium produces many unusual enzymesand is often found in contaminated environments along withdegradative-plasmid-bearing pseudomonads (56), we aregreatly interested to see if any similar metabolic genes areplasmid-borne. The complete sequencing of pBM400 and thelarger cryptic plasmids of B. megaterium will enable compara-tive plasmid genomics and the study of plasmid horizontaltransfer while expanding our understanding of plasmid genet-ics in gram-positive bacteria.

This paper presents the recombinational labeling, isolation,and sequencing of the remaining 35 kb of pBM400. It reportsthe initial description of several interesting plasmid-bornegenes, including a possible integrase, an insertion sequence(IS) element, unusual metabolic genes, and several large in-verted and direct repeats (DRs).

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. The strains used for thisstudy are shown in Table 1. Escherichia coli was grown in Luria-Bertani (LB)broth with appropriate selection at 37°C (45). B. megaterium was grown at 30°Cin supplemented nutrient broth (SNB) or MC minimal glucose broth with ap-propriate selection as previously described (11). Antibiotics (Sigma-Aldrich, St.Louis, Mo.) used were kanamycin (Bacillus, 5 �g/ml; E. coli, 20 �g/ml), ampicillin(E. coli, 100 �g/ml), and chloramphenicol (Bacillus, 5 �g/ml; E. coli, 34 �g/ml).

Plasmid DNA isolation and library construction. Plasmid DNA was routinelyisolated by the alkaline lysis method and was visualized by agarose gel electro-phoresis as previously described (51). The relative size of pBM400 was routinelycompared to the known size of the largest E. coli V517 plasmid (�55 kb). Largequantities of plasmid DNA were isolated by the method of Lovett and Keggins(29) after growth to an optical density at 600 nm of 1.0 in MC broth. Purificationof plasmid DNA by CsCl-ethidium bromide gradient centrifugation and libraryconstruction were performed according to the methods of Sambrook et al. (45).Three restriction enzyme libraries as follows were constructed from the pBM400plasmid: (i) a partial Sau3A library in pGEM3-Z f(�) (Promega, Madison, Wis.)was used for shotgun cloning and sequencing in E. coli DH10B, and (ii) EcoRIand (iii) PstI fragment libraries in pJM103 (39) were used for closing sequencegaps. B. megaterium was transformed by protoplast fusion based on the methodof Von Tersch and Carlton (59) and regenerated in regeneration medium (43,59) as described by English and Vary (11).

PCR. PCR was performed by using Promega Taq polymerase (Promega) orcDNA polymerase (Clontech, Palo Alto, Calif.) according to the manufacturer’sspecifications with a Perkin-Elmer (Foster City, Calif.) 480 thermocycler. Prim-ers derived from the sequence of clone II that were specific for pBM400 and usedfor the detection of integration of the kanamycin gene insertion were as follows:5�-CGGGAAGATGGCAAAT-3� and 5�-GCCTGGCAACGTCCTACTC-3�.

Southern hybridization. Southern hybridization and probe labeling was doneby use of the NEBlot phototope kit (New England Biolabs, Beverly, Mass.)according to the manufacturer’s instructions. The resulting membrane was pro-cessed and exposed to X-ray film for 3 to 12 min.

DNA sequencing. DNA sequencing was performed in the core DNA sequenc-ing facilities at Northern Illinois University (DeKalb, Ill.) and IntegratedGenomics (Chicago, Ill.) by use of the DYEnamic ET terminator cycle sequenc-ing kit (Amersham Biosciences, Piscataway, N.J.) and a 373 DNA sequencer(Applied Biosystems, Foster City, Calif.) or AB3700 sequencer (Perkin-Elmer).Universal primers were utilized for end sequencing of Sau3A, EcoRI, and PstI

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Genotype or description Source or reference

StrainsB. megaterium

QM B1551 Wild-type (7p�) B. megaterium J. C. Vary, Universityof Illinois—Chicago

PV361 Plasmidless (7p�) QM B1551 48QM B1551/pSM1 Wild-type (7p�) B. megaterium plus pSM1 This studyPV627 PV361 containing only pBM400::kan This study

E. coliDH5� F� �80dlacZ�M15 �(lacZYA-argF) U169 deoR recA1 endA1

hsdR17(rK�mK

�) phoA supE44 � thi-1 gyrA96 relA1Gibco BRL

DH10B F� mcrA �(mrr-hsdRMS-mcrBC) �80dlacZ �M15 �lacX74 deoR recA1endA1 araD139 �(ara, leu)7697 galU galK � rpsL nupG

Gibco BRL

V517 Eight plasmids used as supercoiled standard 30

PlasmidspJM103 Apr in pUC19, Cmr from pC194 (integrative) (3.7 kb) 39pHV33 Apr Tcr from pBR322, Cmr from pC194 (7.3 kb) 28pGEM3-Zf(�) lacZ, Apr, F� origin (3.7 kb) PromegapDG792 pMTL23 containing Kmr cassette 15pLTV1 Tn917, Emr Tcr Cmr, lacZ, ori (E. coli), pE194ts ori (B. megaterium) 7pKM31 2,152-bp XbaI fragment from pBM400 in pJM103 (5.8 kb) This studypKM58 1,045-bp XbaI-EcoRI pBM400 fragment in pJM103 (4.7 kb) This studypKM59 1.5-kb ClaI kanamycin gene (pDG792) in pKM58 (6.3 kb) This studypSM1 4-kb EcoRI oriE194ts (pLTV1) in pKM59 (10.3 kb) This studypSM3 8.7-kb EcoRI insert from pBM400 in pJM103 with ftsZ This study

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library clones. Sequence gaps were closed by primer walking and PCR, withcustom primers from MWG Biotech (High Point, N.C.). Sequences were assem-bled by use of Sequencher (Gene Codes, Ann Arbor, Mich.). Approximately 120oligonucleotides were designed for PCR and primer walking of pBM400 to closegaps and increase the overall coverage to an average of 3.

Construction of the integrative vector and integration. A list of all plasmidsutilized in vector construction is summarized in Table 1. An integrative plasmidwas constructed that carried a kanamycin gene flanked by DNA homologous topBM400, as shown in Fig. 1. The plasmid pKM31 harbors a 2.1-kb XbaI-to-KpnIsubclone from the distal 3� end of clone II that is specific for pBM400 (24). Thisplasmid was cleaved with EcoRI to remove one of two ClaI sites, ligated, andtransformed into E. coli DH5� cells. The resultant plasmid, pKM58, was cleavedwith ClaI, and a kanamycin cassette from pDG792 (15), also digested with ClaI,was inserted (pKM59). Plasmid pLTV1 (7) was cut with EcoRI, and the 4-kbfragment harboring the temperature-sensitive replication origin (pE194ts) wasligated into pKM59 at the EcoRI site. The resulting temperature-sensitivepBM400-specific integrative plasmid for B. megaterium was designated pSM1(Fig. 1). QM B1551 containing pSM1 was grown at 46°C in SNB-kanamycin, withsubculturing at 6 and 14 h. Cultures were serially diluted, plated on SNB plates,grown overnight at 37°C, and replica plated on SNB-chloramphenicol and SNB-kanamycin plates to test for integration of the labeled fragment by doublecrossover and loss of pSM1.

Computer analysis. Identification of open reading frames (ORFs) and homol-ogy searches utilized the BLAST series of programs (2) provided by the NationalCenter for Biotechnology Information (http://www.ncbi.nih.gov) and GeneMark.hmm (http://opal.biology.gatech.edu/GeneMark/hmmchoice.html). DNAand amino acid sequences were then analyzed by using PCGENE (Intelligene-tics, Inc.), as well as the following Internet sites: MultiAlin (http://prodes.toulouse.inra.fr/multalin/multalin.html) for multiple alignments, EINVERTED(http://bioweb.pasteur.fr/seqanal/interfaces/einverted.html) for inverted repeats

(IRs), REPuter (http://bibiserv.techfak.uni-bielefeld.de/reputer/) for degenera-tive repeats, and Promoter Predication by Neural Network (http://www.fruitfly.org/seq�tools/promoter.html). GC analysis was done by use of Artemis softwareprovided on the Internet by the Sanger Center (www.sanger.ac.uk/Software/ACT/). Access to several Internet sites was made through the site of A. Kropin-ski, Queen’s University, Ontario, Canada (http://molbiol-tools.ca).

Nucleotide sequence accession number. Sequence data for the entire 53,903 bpof pBM400 have been submitted to GenBank databases under the accessionnumber AF142677. The previously reported 18.6 kb corresponds to 8,003 bpbefore the BglII start site (nucleotide 45,900) to nucleotide 10,630 in the com-pleted plasmid sequence.

RESULTS

Construction of an integrative-replicative vector. To effec-tively study pBM400 and obtain its sequence, it was necessaryto isolate it from the plasmid array. This was done by con-structing an integrative plasmid using the distal KpnI-XbaIfragment of the 12.3-kb clone II that was specific for pBM400,as shown in Fig. 1 and described in Materials and Methods.The introduction of a temperature-sensitive replication originthen allowed an efficient two-step process of integration, inwhich B. megaterium was first transformed with pSM1 and thengrown at 46°C to select for homologous recombination andcuring of the vector. The transformation of QM B1551 bypSM1 with selection for Kmr colonies yielded a frequency of 10

FIG. 1. Construction of an integrative-replicative vector. The figure shows the parental clone containing an EcoRI-KpnI clone II fragmentspecific to pBM400 (A), removal of a 1-kb fragment containing a ClaI site (in bold) (B), incorporation of a 1.5-kb kanamycin gene from pDG792into the remaining ClaI site (C), and insertion of the 4-kb fragment with the origin of replication from pE194ts to yield pSM1 (D).

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transformants/�g of DNA (the positive control, pHV33,yielded 150 transformants/�g of DNA). The resulting transfor-mants were restreaked onto antibiotic plates to confirm thepresence of pSM1 in QM B1551. Several transformants werethen subcultured at 46°C in SNB-kanamycin, and a single col-ony that was Kmr and Cms, designated QM B1551/pBM400::kan, was isolated.

Isolation of a B. megaterium strain containing onlypBM400::kan. Plasmid DNA from QM B1551/pBM400::kanwas separated by electrophoresis, and a band corresponding toa size of approximately 54 kb was purified from agarose gels.Plasmidless strain PV361 was transformed with pBM400::kanDNA and selected for Kmr. Plasmid DNA was purified byCsCl-ethidium bromide gradient centrifugation from one ofthe transformants, designated PV627, and the presence of onlyone plasmid and its correct size were verified by agarose gelelectrophoresis and hybridization. Plasmid DNA was probedwith both a pBM400-specific probe (KpnI-XbaI fragment frompKM31) and a kanamycin gene probe (pDG792 1.5-kb ClaIfragment), as shown in Fig. 2. As expected, the kanamycin geneprobe hybridized to the bands corresponding to a 54-kb plas-mid in PV627 and QM B1551/pSM1, but not to QM B1551plasmid DNA or chromosomal DNA from plasmidless PV361.

The pBM400 probe from clone II produced a signal, as ex-pected, for plasmid bands corresponding to approximately 54kb, but not for an E. coli V517 plasmid standard or B. mega-terium chromosomal DNA. These results verified pBM400 asthe source plasmid for clone II and showed that double-cross-over integration of the kanamycin gene had occurred. It wasinteresting that in QM B1551/pSM1, a band approximately 10kb above 54 kb suggested that it contained a single-crossoverintegration as well as pSM1, which was confirmed by a chlor-amphenicol gene probe (data not shown). However, it is clearfrom the hybridization reactions with PV627 and QMB1551/pBM400::kan that the double crossover did occur dur-ing subculturing at 46°C.

Sequence and analysis. Isolation of pBM400 allowed restric-tion enzyme mapping, subcloning, and complete sequencing asdescribed in Materials and Methods. As shown in Table 2 andFig. 3, pBM400 is a circular plasmid of 53,903 bp with a totalof 50 possible ORFs, 34 of which encode �100 amino acids,based on GeneMark and BLAST findings. Of the 50 ORFs, 39have a Fickett score of �77% (PC Gene analysis). The criteriaused for including possible ORFs were two or more of thefollowing: Fickett scores of �70%, good Shine-Dalgarno sites,presence of an upstream putative promoter, and similarity with

FIG. 2. Southern hybridization with pBM400 and kanamycin gene probes to detect the integrated kanamycin gene. (A) Southern hybridizationwith a kanamycin gene DNA probe. Lane 1, E. coli V517 supercoiled standard; lane 2, QM B1551; lane 3, 54-kb plasmid pBM400::kan (fromPV627); lane 4, QM B1551 containing pSM1 and a possible single crossover of pSM1 into pBM400; lane 5, pBM400::kan in the QM B1551 plasmidarray following growth at 46°C; lane 6, PV361 chromosomal DNA. (B) Agarose gel electrophoresis of the same gel. (C) Southern hybridizationwith the pBM400-specific probe.



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proteins in the databases. There were 16 predicted proteinswith similarity to proteins with known functions, 23 with sim-ilarity to proteins of unknown function, and 11 with no simi-larity to known proteins. The plasmid also contains an rRNAoperon with 18 previously described adjacent tRNA genes(24). Of the 50 putative proteins, 33 are coded for by onestrand and 17 by the other. The entire putative coding se-quence may account for up to 57% of the total pBM400 se-quence. This is consistent with other studies that show a gen-eral tendency for plasmids to have large intergenic regions (36,61). Analysis of the base composition of pBM400 reveals anoverall G�C content of 36.5%, similar to the reported B.megaterium G�C content of 37 to 39% (41).

Possible functions of plasmid genes. Genes for replication,cell division, recombination, heavy metal resistance, and trans-position were found on pBM400. The replicon region(RepM400, ORFB, and ORFC) and the surprising presence ofan rRNA operon have been described and functionally char-acterized previously (24). RepM400 alone is sufficient for rep-lication, but ORFB is required for stability. The function ofORFC has not been determined. Recent database searchesconfirmed that the three replicon proteins do not resemble anyother gene products except those from the pBM500 replicon ofthe same plasmid array. RepM400 is also very similar toRepM300 and RepM700 (57; Kunnimalaiyaan and Vary, un-published data). ORF6 seems to be a conserved gene productencoded by plasmids from many gram-positive bacteria and ispresent on at least five other QM B1551 plasmids. It is similarto possible DNA binding proteins encoded by other plasmids,including ORF2C by B. subtilis plasmids pTA1060 andpTA1040 (33), Ptr by p1414 (54), and ORF 68 by B. anthracisvirulence plasmid pXO2 (36).

The predicted ORF22 protein was highly similar to a cad-mium-copper ATPase of the oral bacterium Fusobacteriumnucleatum that is a component of heavy metal transport acrossthe bacterial membrane. The adjacent ORF (ORF20) productwas also similar to a hypothetical protein in the same positionin the same organism. The cadmium-copper ATPase encodedby pBM400 was also similar to the first cadmium ATPasefound for gram-positive organisms, encoded by Staphylococcusplasmid pI258 (49). Cadmium resistance genes have been re-ported for other gram-positive bacterial plasmids, includingpXU5 in Staphylococcus aureus (55), pND302 in Lactococcuslactis (26), and other soil bacilli (18). However, initial tests forcopper and cadmium resistance in B. megaterium QM B1551yielded ambiguous results (data not shown). Another cation-transporting ATPase gene (ORF19) is located just upstreamon pBM400. Plasmid pBM400 also carries a possible zinc-binding dehydrogenase gene (ORF24) in the same region.

Interestingly, the predicted ORF25 protein showed signifi-cant similarity to a styrene monooxygenase from Pseudomonasthat is involved in the biodegradation of styrenes. In Pseudo-monas fluorescens ST, four genes, designated styA, styB, styC,and styD, are required for the oxidation of styrene to phenyl-acetic acid (5, 32). The protein encoded by pBM400 is similarto the styA gene product, which forms epoxystyrene from sty-rene (5). Chiral epoxides are valuable intermediates in severaloptically active drugs. Homologues to other genes for styrenedegradation were not found on pBM400, but there seem to beseveral pathways for styrene utilization found in different or-rr

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ganisms, including one that has only the monooxygenase withoxidation to �-phenylethanol (50). Styrene utilization oftenrequires a consortium of organisms. To our knowledge, therehas been only one report of styrene utilization in Bacillus, bythermophilic isolates that were not further identified (50). Therole of the monooxygenase is also of interest since three otherP450 cytochrome oxygenases are present in strains of B. mega-terium and have been intensely studied (40, 47).

Possible cell division proteins FtsZ and FtsK (SpoIIIE),encoded by ORFs 44 and 27, respectively, were not expected to

be carried by a plasmid. FtsZ is a key division protein that hasbeen shown to form the signature Z-ring at the septum duringcell division in E. coli (1). In B. subtilis, FtsZ is required forboth vegetative septation and asymmetric septation duringsporulation (4). Although FtsZ is essential for normal celldivision, excess FtsZ has the same effect as its absence, so thatits presence on a multicopy plasmid is puzzling. The ORF44product is similar to FtsZ from two Archaea, Pyrococcus hori-koshii and Methanocaldococcus jannaschii, but not to B. subtilisor E. coli FtsZ. However, a motif for GTPase activity of FtsZ

FIG. 3. Circular map of pBM400. The positions of genes are shown relative to a BglII start point of pBM400. Putative sigma A promoters (bentarrows), IRs (blue arrows), DRs (gray arrows), and possible rho-independent terminators are shown. The rRNA operon is shown in blue; thereplicon region is shown in red. Names for gene products with similarity to proteins with known functions in the databases are in bold, and theORFs are green. ORFs for gene products with similarity to known proteins but with unknown functions are yellow. Those with no similarity in thedatabases are white. Large bent arrows are sites of five tandem putative promoters. The 39-bp IRs flanking the transposase (ORF18) are red andare labeled IS. The large 127-bp IRs flanking the Int/Rec ORF (14) and 79-bp repeats (1) near the replicon are also red. ORFs shown on theoutside of the circle are read in the forward direction (clockwise) and those on the inside are read in the reverse direction. All of the restrictionsites of EcoRI and PstI and relevant BglII, KpnI, ClaI, and XbaI restriction sites are shown.



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was found in pBM400 FtsZ (GGGSGGG) that was similar tothe functional motif GGGTGTG reported for E. coli (9). A1.1-kb PCR probe was therefore made to recognize plasmidFtsZ and was hybridized to DNA from B. megaterium, E. coli,and B. subtilis as shown in Fig. 4. This probe hybridized only toplasmid DNA from QM B1551 and not to B. subtilis, E. coli, orgenomic B. megaterium DNA (PV361). Since the plasmidlessstrain PV361 divides normally, there must be an unrelatedgene for FtsZ on the chromosome as well. To test this, a 1.1-kbPCR probe was also made for the B. subtilis FtsZ gene (25)with the following primers: forward, 5�-CATTCGGCAGATTAGGAGG-3�, and reverse, 5�-GATTTTGTCCTTTACATTAGC-3�. There was evidence of hybridization to appropriate PstIfragments (11.4 and 5.8 kb) and an EcoRI fragment (10.8 kb)in the B. subtilis genomic lanes. There was also faint binding toPV361 and QM B1551 in the chromosomal DNA lanes at 4.8kb, but not to plasmid DNA (data not shown).

FtsK is associated with cell division and chromosomal trans-location in E. coli. Like FtsZ, FtsK is essential for cell divisionand is localized at the Z-ring. Liu et al. (27) have shown thatthe N terminus of the E. coli FtsK protein is required for celldivision, while the C terminus is involved in chromosomalsegregation. FtsK is an ATP-dependent DNA translocasewhich also activates XerCD-dependent recombination at thedif site to resolve chromosomal dimers. No FtsK gene has beenfound in B. subtilis, but a smaller protein, SpoIIIE, is a DNAtranslocase involved in mother cell-prespore transport acrossthe division septum (60). The pBM400 ORF27 product is small(265 amino acids) and most resembles an FtsK gene productfrom Thermoanaerobacter tengcongensis. Both the 787-amino-

acid B. subtilis SpoIIIE and ORF27 have similarity only to thecarboxy-terminal chromosomal segregation region of 1,329-amino-acid E. coli FtsK.

Other genes carried by pBM400 include those for ORF26,coding for a protein similar to a cell wall hydrolase, CwlJ, fromB. subtilis that is one of two proteins necessary to hydrolyze thespore cortex during germination. A cell wall lytic enzyme hasbeen purified and characterized from B. megaterium KM byFoster and Johnstone (13), but to our knowledge the gene hasnot been sequenced. CwlJ has recently been determined tolocalize to the spore coat of B. subtilis (3, 8). In addition to thegermination and cell division genes, several ORFs showedsimilarity to B. subtilis genomic gene products of unknownfunction (YcnI, YcgR, and YpiB). Four small ORFs, ORF46to -50, were also found within the rRNA operon. Small geneswithin the rRNA operon have been shown to be expressed inE. coli, but their functions are unknown (6).

Possible virulence plasmid gene exchanges. Three ORFproducts had significant similarities to proteins from the B.anthracis virulence plasmid pXO1. ORF34 was 60% similar toB. anthracis pXO1-07, encoding a reverse transcriptase, whileORF40 was similar to pXO1-103, encoding an integrase-re-combinase (see below). The ORF41 product was similar topXO1-101, which has been reported to have considerable nu-cleotide homology with the virulence factor bceT from B.cereus (37). ORF41 has a motif similar to the HTH-XREgroup of transcriptional regulators that include the lambdarepressor.

Possible mobile elements. A putative insertion sequence(IS) element was found in which 39-bp IRs (74% identical)

FIG. 4. Hybridization of pBM400 FtsZ (ORF44) with genomic and plasmid DNA of E. coli, B. subtilis, and B. megaterium. A 1.1-kb PCRproduct from ORF44 (FtsZ) of pBM400 was used as a probe to test for similarity to genes on E. coli, B. subtilis, and B. megaterium chromosomes.The primers were forward, 5�-AGGTTGAGGTACCATGATAGGTA-3�, and reverse, 5�-ATACTAAAAAGAATTCACGATTCTG-3�. Lanes 1,11, and 19, biotinylated 1-kb standard; lanes 2, 3, and 4, E. coli DH5� total DNA cut with PstI, EcoRI, and ClaI, respectively; lanes 5, 6, and 7,B. subtilis total DNA cut with PstI, EcoRI, and ClaI, respectively; lanes 8, 9, and 10, PV627 plasmid DNA cut with PstI, EcoRI, and ClaI,respectively; lanes 12, 13, and 14, QM B1551 total DNA cut with PstI, EcoRI, and ClaI, respectively; lanes 15, 16, and 17, PV361 (plasmidless) totalDNA cut with PstI, EcoRI, and ClaI, respectively; lane 18, pSM3 cut with EcoRI.



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flank ORF18. ORF18 codes for a protein that is highly similarto transposase ISEf1 of vancomycin-resistant Enterobacter fae-calis V583 (38) and to transposases from IS1201 of Lactoba-cillus helveticus and IS905 of Lactococcus lactis. Both IS1201and IS905 belong to the IS256 family of transposons (31),which have 24- to 41-bp IRs. IS1201 is a 1,387-bp IS thatcarries a single ORF coding for a putative transposase (53).While the pBM400 IS, designated ISBm400, has a similartransposase, its 39-bp IRs (5�AACTGAGAAAAATTAATACATTTAGTTTTTCCATTTCTT 3� and 5�AAGAGAATGAAAAACTAGTTGAATTAATTATTTTAAATT 3�) (labeled ISin Fig. 3) have no significant homology to any known IS ele-ment. It is also large (2,653 bp) but contains no obvious ORFother than that for the transposase.

ORF40 exhibited 48% similarity to the integrase-recombi-nase gene (pX01-103) of B. anthracis virulence plasmid pXO1.Plasmid pXO1 carries two putative integrase-recombinaseORFs similar to Methanobacterium thermoautotrophicum Int/Rec and B. subtilis RipX, the homolog of E. coli XerD requiredfor chromosomal dimer resolution (36). ORF40 of pBM400 ismore similar to the pXO1 RipX integrase-recombinase and is

flanked by large IRs of 127 bp (Fig. 3) with 79% identity (seebelow). The possible pBM400 integron also contained twoother predicted ORFs (38 and 39) with unknown functions. Itsintervening sequence was about 1.5kb. On pXO1, the inte-grase-recombinase gene is flanked by IRs and is part of aregion with several IS elements, integrases, and transposaseswithin the 44.8-kb pathogenicity island. The presence onpBM400 of integrase-recombinase and transposase genesflanked by IRs strongly suggests the possibility of DNA mobil-ity.

GC Analysis. To test the possibility of horizontal transfer,the entire pBM400 sequence was analyzed by use of Artemissoftware (see Materials and Methods). Horizontal transfer cansometimes be identified by one or more anomalies in GCcontent, GC deviation, and dinucleotide usage (21). As can beseen in Fig. 5, the rRNA operon showed typical high GCcontent (52%). Most of the rest of the plasmid was near 36.5%GC, except for a few regions that were very AT-rich, includingthe possible integron, the copper export gene, and the styrenemonooxygenase. The copper and integrase regions alsoshowed Karlin signature differences. There was a smaller GC-

FIG. 5. Whole plasmid GC analysis of pBM400. The entire plasmid was analyzed for possible alien genes or regions (pAs) by use of Artemissoftware for three different parameters: GC content, GC deviation, and Karlin signature difference. The line in the GC content graph is at 36.5%.Possible regions of sequence anomalies in one or more graphs are labeled. rrn, rRNA operon; Cu, copper export gene region; Sty, styrenemonooxygenase region; Int, integron; 2C, ORF2C (ORF6); IS, ISBm400.



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rich region near or within the IS region, which also exhibits aKarlin signature peak. (The transposase gene is 40.4% GC.)The FtsK gene is 44% GC, but it is in a region with no obviousanomalies. Interestingly, the conserved plasmid ORF2C genehas the highest Karlin signature difference peak and a smallregion of GC deviation.

DNA sequence structure. Several other IRs and DRs wereevident. Overall, there were 25 DRs, ranging from 18 to 39 bp,which had 3 kb of intervening DNA. IRs and DRs of �14 bpare shown in Fig. 3. In addition, a large IR (79 and 80 bp; no.1) flanking 640 bp has previously been described upstreamfrom the replication gene in a region of almost 2,500 bp withno obvious ORFs (24). Large rho-independent terminatorswere found downstream of ORF6, ORF23, ORF28, ORF29,ORF42, and ORF44. There were also four IRs that did notresemble rho-independent terminators. An area consisting of�13 DRs, some overlapping, was present within the 18 tRNAgenes and probably reflects their secondary structure. A regionin and around ORF29 also contained four DRs in close prox-imity to each other.

Potential sigma A promoters were found based on sequencesimilarity to the sigma A consensus of B. subtilis: �10,TATAAT, and �35, TTGACA (34). There were five possiblepromoter sequences found in close proximity to each otherupstream of the small ORF21, the product of which has nosimilarity to those in the databases. Five adjacent putativepromoters were also found upstream of ORF41, coding for theputative integrase-recombinase. Overall, there were 76 se-quences that showed significant similarity to the Bacillus sigmaA consensus sequence. However, actual promoter predictionsfrom sequences have not proved reliable.

DISCUSSION

To sequence large low-copy-number cryptic plasmids withina varied plasmid array is a challenge. Since attempts to labelthe pBM400 plasmid by transposition were not successful,pBM400::kan was constructed by recombination and trans-formed into the plasmidless strain PV361. This method ofinserting an antibiotic resistance gene into a high-molecular-weight plasmid of QM B1551 to allow isolation and completesequencing was successful. Several genes were carried bypBM400 that were unexpected. In addition to the rRNAoperon, a gene for a possible styrene monooxygenase andORFs with products similar to proteins involved in cell division(FtsZ and FtsK) were found. The dynamic state of the plasmidwas suggested by the presence of a possible integrase-recom-binase, a reverse transcriptase, an ISBm400 with a transposaseof the IS256 family, and two very large IRs, one with 79- to80-bp repeats flanking a 2.5-kb region near the replicon andanother with 127 bp flanking the Int/Rec ORF. Horizontaltransfer was suggested by the presence of putative alien genesor regions including ISBm400 and the integron as well as thepredicted styrene monooxygenase and copper export ORFs.ORFs were found for possible degradative or heavy metalresistance, DNA translocation, dimer resolution, and germina-tion proteins, and several encoded products similar to hypo-thetical proteins of unknown function on the genomes of B.subtilis, B. halodurans, and L. lactis (and presumably, to somealso present on the unsequenced B. megaterium genome).

However, no obvious mob genes or genes involved in plasmidtransfer were observed. The types of genes found on pBM400suggest a possible role for this plasmid in the survival of B.megaterium in hostile environments where heavy metals orstyrene are present and possible advantages by increased genedosage of cell division, germination, and rRNA genes forgrowth and sporulation. They also suggest that considerableexchange of genes has occurred within the gram-positive bac-teria, and perhaps Archaea, and include ORFs with productssimilar to those encoded by the pathogenicity island of pXO1.The lack of clear phenotypes in the plasmidless strain is partlyexplained by the data. Many of the genes discovered on thisplasmid could not easily be distinguished by phenotype, wouldbe expressed under unusual conditions, or had genomic coun-terparts, thus making their presence or absence difficult todetect. Sequencing of the larger plasmids of QM B1551 greatlyadds to our understanding of this plasmid gene pool in thebacilli. This study also provides a basis for isolation and se-quencing of other high-molecular-weight plasmids in the QMB1551 family of plasmids by recombinational labeling, so thatwe may better understand the role of megaplasmids of B.megaterium and of other gram-positive bacteria.

ACKNOWLEDGMENTS

We thank Scott Grayburn in the NIU Core Facility and IntegratedGenomics (Chicago, Ill.) for automated sequencing and advice. Wealso thank Barbara Ball for graphics support.

This research was supported in part by NIH grant IR 155GM59599-01/G12401R.

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