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Characterization of a Theta Plasmid Replicon with Homology to All Four Large Plasmids ofBacillus megateriumQM B1551
PLASMID
40, 175–189 (1998) PL981359
ARTICLE NO.
Characterization of a Theta Plasmid Replicon with Homology to All Four Large Plasmids of Bacillus megaterium QM B1551 David M. Stevenson,1 Muthusamy Kunnimalaiyaan, Kerstin Mu¨ller,2 and Patricia S. Vary3 Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115 Received August 12, 1997; revised April 2, 1998 A replicon from one of an array of seven indigenous compatible plasmids of Bacillus megaterium QM B1551 has been cloned and sequenced. The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551. The cloned 2374-bp HindIII fragment was sequenced and contained two upstream palindromes and a large (.419-aminoacid) open reading frame (ORF) truncated at the 39 end. Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF. The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication. The ORF product was shown to act in trans. A small region with similarity to the B. subtilis chromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins. Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream. The predicted ORF protein of .48 kDa was basic and rich in glutamate 1 glutamine (16%). There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF. The data suggest that this is a theta replicon with an expressed rep gene required for replication. The replicon contains its iterons within the gene and has no homology to reported replicons. It is the first characterization of a B. megaterium replicon. © 1998 Academic Press Key Words: plasmid; theta replication; sporulation; Bacillus megaterium; Bacillus subtilis; Bacillus thuringiensis.
a bacteriocin, called a megacin (Keiselburg et al., 1984), and a gene required for spore germination on all single germinants (Stevenson et al., 1993). Most of the research on plasmid replication has been done in Escherichia coli [see recent reviews (Helinski et al., 1995; Kahn, 1997)], but progress has been made in characterizing some of the gram-positive plasmid replicons including those from Streptococcus, Staphylococcus, and Bacillus (Janniere et al., 1993; Novick, 1989). Unlike E. coli and most gramnegative bacteria, several small plasmids in the gram-positive bacteria replicate by a rolling circle mechanism (RCM)4 [although two small RCM plasmids have been reported in gram-
Bacillus megaterium is a gram-positive sporeforming bacterium used in many industrial applications and as a cloning host for expression of intact foreign proteins [see reviews by Vary (1992, 1994)]. Most B. megaterium strains isolated from soil as well as those used widely in industry contain numerous plasmids. We have previously reported that strain QM B1551 carries more than 11% of its cellular DNA as an array of seven indigenous plasmids (renamed pBM100 –pBM700) of sizes 5.5, 8.8, 20, 47, 71, 108, and 165 kb, respectively (Keiselburg et al., 1984), but their function in the cell remains elusive. Phenotypes found to be associated with these plasmids include the production of 1 Current address: Department of Biological Sciences, Dartmouth College, Hanover, NH 03755. 2 Current address: Institut for Klinische Mikrobiologie und Immunologie, Wasserturmstrasse, 391054 Erlangen, Germany. 3 To whom correspondence should be addressed. Fax: (815) 753–1753. E-mail: [email protected].
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Abbreviations used: RCM, rolling circle mechanism; Em, erythromycin; Cm, chloramphenicol; Nm, neomycin; Ap, ampicillin; PCR, polymerase chain reaction; IPTG, isopropyl-b-D-thiogalactoside; ORF, open reading frame; IHF, integration host factor; Ter, termination sequence; aa, amino acids. 0147-619X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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negative Helicobacter (del Solar et al., 1993)]. A survey of 40 gram-positive bacterial RCM plasmids showed the average size to be 4 kb, with the largest 8.8 kb. Theta plasmids, on the other hand, were found to range in size from 3.8 to 95 kb (Espinosa et al., 1995; Janniere et al., 1993; Kiewiet et al., 1993). Little is known about the large plasmids of gram-positive bacteria, and descriptions of their theta replicons are new [see reviews (Janniere et al., 1993; Novick, 1989)]. Most of these theta plasmids are from Streptococcus, Lactococcus, and Enterococcus. Of the very few Bacillus replicons described, most are from plasmids of B. thuringiensis carrying the cry insecticidal genes. Theta plasmids have been classified into classes A–D depending on the presence of a Rep protein, iterons, DnaA boxes, and dependence on Pol I proteins (Bruand et al., 1993; Helinski et al., 1995; Janniere et al., 1993; Novick, 1989). The gram-positive bacterial plasmid pAMb1 family (including pIP501 and pSM19035) defined class D, which has a dispensable oriA structure and dependence on Pol I (Bruand et al., 1993; Janniere et al., 1993). A possible class E replicon with no Rep protein gene or requirement for Pol I, but an oriA region, has been described by characterizing the 3.1-kb replication region from a 55-kb B. subtilis plasmid pLS20 (Meijer et al., 1995). Our aim in studying the seven compatible cryptic plasmids of B. megaterium is to gain a clearer understanding of their function and their maintenance. Plasmids provide a nonlethal system to study plasmid and host replication including the proteins involved, the regulation of replication, partitioning, and stability. In addition, most of the cloning vectors used in Bacillus have been from other genera and replicate by a RCM. These vectors are unstable in B. subtilis, an important cloning host (Janniere et al., 1993). Only three theta replicons—pHT1030 from B. thuringiensis, pIP501 and pAMb1 from Enterococcus faecalis— have so far been used for vector construction (Arantes and Lereclus, 1991; Behnke et al., 1981; Janniere et al., 1990). Characterization of replicons from optimally adapted indigenous plasmids of Bacillus, especially those with theta
replicons, should lead to the construction of more stable vectors for this genus. The array of seven compatible plasmids of strain QM B1551 provides a unique system. By studying it in detail, it is possible that the role and possible interaction of the plasmids within the cell can be better understood. As a first step, we report here the isolation of a replicon that was found by screening a HindIII plasmid DNA library from B. megaterium QM B1551. Further characterization demonstrated that it is most probably a theta replicon and exhibits some distinctive characteristics. The Sequence Accession number in GenBank is AFO15255. MATERIALS AND METHODS Bacterial Strains, Plasmids, and Growth Conditions Strains and plasmids used in this study are listed in Table 1. B. megaterium was grown in SNB or MC broth or plates with appropriate selection at 30°C as described previously (English and Vary, 1986). Antibiotics and concentrations used for Bacillus were erythromycin (Em) 5 mg/ml, chloramphenicol (Cm) 5 mg/ml, and neomycin (Nm) 5 mg/ml for plates and 1 mg/ml for broth. Sporulation was assayed microscopically and/or by changes in colony morphology after 70 h incubation at 30°C in SNB. E. coli and B. subtilis were routinely grown in LB medium (Sambrook et al., 1989) with appropriate selection [100 mg/ml ampicillin (Ap) for E. coli.]. An E. coli–Bacillus shuttle vector pHT315 (Arantes and Lereclus, 1991) was used for gene library construction. It maintains about 15 copies in B. subtilis and has both B. thuringiensis and E. coli replicons. It carries an ApR gene for E. coli and an EmR gene for Bacillus. Plasmid pBEST501(Itaya et al., 1989) was derived from pGEM4 and has a NmR cassette that expresses in Bacillus, but lacks an origin capable of replicating in Bacillus. Plasmid pYZ11 is a compatible derivative of the RCM 5.5-kb plasmid (pBM100) from B. megaterium QM B1551. Plasmid pGEM7Zf(1) (Promega) was specifically designed for the Erase-a-Base system. Plasmid pGEMT-easy (Promega) is
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B. megaterium PLASMID REPLICON TABLE 1 Strains and Plasmids Used in This Study Strain/plasmid Strains B. megaterium QM B1551 PV361 PV622 PV623 B. subtilis 168 1A1 1A43 1A226 E. coli DH5a
Plasmids pHT315 pBEST501 pJM103 pGEM7Zf(1) pGEM-T easy pYZ11 pDS100 pDS110 pDS571 pDS611 pDS6163 pDS61100 pDS61133 p133JM pKM55 a b
Genotype or description
Source or Reference
Wild-type (7p1)a Plasmidless (7p2) QM B1551 derivative (gerP) PV361/pDS100 PV361/pDS110
J. C. Vary, UIC Sussman et al., (1988) This study This study
trpC2 recA1 trpC2 hisH2 pheA1 polA5 trpC2
BGSCb BGSC BGSC
F9 endA1 hsdR17 (r-m-) supE44 thi-1 recA1 gyrA96 relA1 D(lacZYA-argF) U169 deoR (f80)dlac D(lacZ M15)
Promega
AmpR, EmR shuttle vector (Bacillus, E. coli) B. thuringiensis ori1030, pUC19 lacZ, 6.51 kb AmpR in pGEM4, NmR from pUB110 Bacillus integrative vector, 4.15 kb AmpR in pUC19, CmR from pC194, integrative vector, 3.72 kb AmpR f1ori lacZ::PrT7, PrSP6, 3.0 kb AmpR f1ori lacZ::PrT7, PrSP6, 3.02 kb linear with T overhang pBM100 TetR pUC19 AmpR, 10.3 kb 2.4-kb Clone I in pHT315 2.4-kb Clone I in pBEST501 2.4-kb Clone I in pGEM7Zf(1) D1 in pGEM7Zf(1) D63 in pGEM7Zf(1) D100 in pGEM7Zf(1) D133 in pGEM7Zf(1) Clone I D133 in pJM103 93-bp iteron PCR fragment in pYZ11
Arantes and Lereclus (1991) Itaya et al. (1989) Perego et al. (1988) Promega Promega Y. Zhou, unpublished This study This study This study This study This study This study This study This study This study
Contains all seven indigenous plasmids. Bacillus Genetic Stock Center, Ohio State University.
pGEM5Zf(1) linearized with EcoRV with added T nucleotides for PCR cloning. DNA Isolation and Plasmid Library Construction Small-scale plasmid DNA isolations were prepared using alkaline lysis procedures for Bacillus (Zaghloul et al., 1985) and E. coli. (Sambrook et al., 1989). Large-scale Bacillus plasmid DNA isolations were prepared by the method of Lovett and Keggins (1979) following
cell growth to OD660 5 0.75 in MC broth. DNA was additionally purified by CsCl– ethidium bromide gradient centrifugation (Sambrook et al., 1989). A QM B1551 plasmid library was constructed by digesting QM B1551 plasmid DNA with HindIII and ligating the resulting fragments into pHT315 digested with HindIII and dephosphorylated. The ligation mixture was transformed into E. coli DH5a and plated on LB(1) Ap 1 IPTG 1 X-gal. More than 1300 white transformants were individually picked
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and incubated for 3 h as separate minicultures in 250 ml LB broth 1 Ap. Glycerol was added to 15%, and the minicultures were stored at 270°C. Transformation and Plasmid Stability B. megaterium was transformed by protoplast fusion (Von Tersch and Carlton, 1983) and regenerated in RHAF medium as described (English and Vary, 1986). B. subtilis was transformed by the method of Cutting and Van der Horn (1990) and E. coli by the method of Sambrook et al. (1989). To test for plasmid stability, cells were grown at 30°C in SNB broth with appropriate antibiotic to an OD660 5 0.5, then diluted 1:100 into SNB broth without antibiotic and also plated on SNB agar. The subculture was grown for 6 h, again subcultured, and plated, continuing at 6-h intervals for 72 h. All dilution plates were replicated after overnight incubation to SNB 6 antibiotic to determine the presence of plasmid. Sequencing the 2.4-kb Fragment Nested deletions of the 2.4-kb HindIII fragment cloned into pGEM7Zf(1) and transformed into strain DH5a were generated with the Erase-a-Base kit (Promega) following the manufacturer’s directions. DNA was sequenced with [a-32P]dATP (1 mCi/reaction) using the USB Taq cycle sequencing kit (United States Biochemical). Primers were both synthesized oligonucleotides and universal primers. DNA and amino acid sequences were analyzed with the computer program PCGENE (Intelligenetics, Inc., A. Bairock, University of Geneva, Geneva, Switzerland). Computer searches were performed using the BLAST programs (Altschul et al., 1990) provided by GenBank at the National Center for Biotechnology Information, Bethesda, Maryland. BLASTN was used to search for nucleotide similarity and BLASTX for similarity to proteins. The amino acid sequence of the ORF was also searched against protein databases using BLASTP.
Southern Hybridizations and PCR The Polarplex chemiluminescent blotting kit (New England Biolabs) was employed for Southern hybridizations following the manufacturer’s suggestions. DNA was blotted onto nylon membrane, crosslinked using UV Stratalinker (Stratagene, Inc.), and then hybridized at 68°C. Chemiluminescent blots were exposed to Fuji X-ray film for 2 to 30 min, depending on signal strength. A fragment containing the tandem direct repeats was generated by PCR using sense and antisense primers at nucleotides 1568–1585 and 1643–1660. PCR was carried out for 30 cycles of 95°C for 1 min, 45°C for 2 min, and 72°C for 1 min. The 93-bp product was cloned into the pGEM-T easy vector (Promega) according to the manufacturer’s directions and confirmed by sequencing. The insert (iterons) were then recloned into a stable vector at the EcoRI site of pYZ11. RESULTS A HindIII library of QM B1551 plasmid DNA was constructed in shuttle plasmid pHT315 and transformed into DH5a as described under Materials and Methods. Plasmid DNA was isolated from pooled E. coli clones and transformed into B. megaterium PV361. A 2.4-kb fragment was detected as a clone (pDS100) that dramatically decreased sporulation in B. megaterium. The recombinant colonies containing the clone lysed on plates within 2–3 days. To determine which of the seven plasmids was the source of the fragment, the entire 2.4-kb fragment was used as a probe against QM B1551 plasmid DNA and hybridized under stringent conditions. As can be seen in Fig. 1, hybridization was observed with all four large plasmids of QM B1551 (lane b). There was no homology with chromosomal DNA since no hybridization occurred with total DNA from plasmidless strain PV361 (lane e). Sequencing of the 2.4-kb Fragment Plasmid pDS571 was constructed by cloning the 2.4-kb HindIII fragment into pGEM7Zf(1), and a series of nested deletions were made to
B. megaterium PLASMID REPLICON
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FIG. 1. Southern hybridization of QM B1551 plasmid and PV361 total DNA using the HindIII 2.4-kb fragment as probe. (1) Agarose gel electrophoresis of DNA; (2) Southern blot of the gel probed with the 2.4-kb fragment. (A) The 2.4-kb HindIII fragment from pDS100; (B) QM B1551 CsCl–EtBr plasmid preparation. (C) l HindIII digest, (D) QM B1551 plasmids digested with HindIII; (E) PV361HindIII-digested total DNA (plasmidless strain).
sequence the 2.4-kb fragment as described under Materials and Methods. The complete DNA sequence is shown in Fig. 2. The 2374-bp fragment contained a large ORF, which encompassed more than half the clone (1257 bp), that could code for an incomplete basic polypeptide of 419 amino acids with a predicted molecular weight of .48 kDa. Within the ORF sequence were four tandem 12-bp direct repeats, three of which are identical (starting at bp 1592), the fourth differing by only two bases. Upstream from the ORF were two palindromic regions. The palindrome starting at position 531 was potentially the most thermodynamically stable, with a projected DG at 25°C of 238.8 kcal. It contained a 27-nucleotide inverted repeat with a two-nucleotide loop. Part of this region (552–584) showed 82% (26/29 bp) similarity to a portion of the Type II membrane binding region within the B. subtilis chromosomal
origin, oriC, as shown on the sequence (Fig. 2) and aligned in Fig. 5B. The second potential stem –loop structure, starting at position 687, was a small 9-bp G 1 C-rich inverted repeat with a 6-bp A 1 T-rich loop and a deduced DG of 222.4 kcal. The region surrounding the palindromes was A 1 T rich and contained 13 locations with runs of A . 5 and five runs of T $ 5. There was a possible s A promotor with a perfect consensus 210 at 836–841 (TATAAT) and a 235 at 809– 118 (TTATTGAAT) (consensus 5 TTATTGAAA). Several possible DnaA boxes were also observed and are marked. Interestingly, at least four putative integration host factor (IHF) boxes and a sequence with some homology to the termination sequences (Ter) of the B. subtilis chromosomal origin were found (Figs. 2 and 5). Searches of the database produced no significantly similar proteins, motifs, or nucleotide sequences. How-
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FIG. 2. DNA sequence of the 2374-bp fragment. Repeats (both direct and inverted) are indicated by thin arrows. Relevant restriction sites and possible s A promoter are indicated and asterisks mark a putative Shine–Dalgarno (SD) sequence. A sequence homologous to a region of oriC of the B. subtilis chromosome is indicated with a thick shaded line above the sequence. Possible IHF boxes are indicated with a single underline. Putative DnaA boxes are indicated with thicker arrows above the sequence, and differ by 1–2 bp from the consensus TTATCCACA. A sequence homologous to B. subtilis chromosomal terminators is marked by a double underline. Labeled small arrows mark the start and direction of DNA retained for deletions.
ever, the ORF was found to be somewhat similar to several plant gliadin proteins, apparently because of the large numbers of glutamine codons (16% glutamate 1 glutamine).
Replication by the 2.4-kb Fragment The presence of tandem direct repeats and homology with oriC of B. subtilis led to the hypoth-
B. megaterium PLASMID REPLICON
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FIG. 3. Agarose gel electrophoresis of pDS110 transformants showing accumulation of highly polymerized plasmid DNA and loss of largest plasmid. (A) QM B1551/pDS110; (B) QM B1551 (wt); (C) PV361/pDS110; (D) PV361/pDS110 PstI digest; (E) l HindIII linear DNA standard.
esis that the fragment contained an origin of replication. To test this, the 2.4-kb HindIII fragment was cloned into pBEST501 containing only an E. coli replicon, to construct pDS110, which was then transformed into both QM B1551 and PV361. Greater than 3200 transformants were obtained in plasmidless PV361, showing that the fragment, indeed, contained a functional origin of replication. However, transformation of QM B1551 with an equal amount of pDS110 DNA produced no transformants. Using 20 times more DNA yielded only 18 transformants. Plasmid DNA from two of the transformants was isolated. The CsCl– ethidium bromide-purified plasmid profile of one of these is shown in Fig. 3, lane A. The QM B1551/pDS110 transformant had lost at least the 165-kb plasmid. Comparison with QM B1551 plasmids (lane B) showed that it retained the 5.5- and 8.8-kb plasmids, but had highermolecular-weight bands in addition to probably the 20-, 47-, and 71-kb plasmids. Because of the
highly polymerized DNA present, it is difficult to assign the bands specifically. When PV361 was transformed with pDS110 (lane C), the DNA was found to exist in highly polymerized forms. Digestion of the DNA from a PV361 transformant by PstI resolved all the forms into one linear 6.6-kb band (lane D). These data suggest that the cloned fragment may be incompatible with the 165-kb plasmid (a band present at 108 kb molecular weight could be the 108-kb plasmid or highly polymerized DNA) and also explained the difficulty in transforming this strain. Deletion Analysis to Define the Minimal Replicon and Sporulation Effect To find the region of the fragment responsible for replication and the observed inhibition of sporulation, nested deletions as well as restriction enzyme deletions were made and subcloned into integrative vectors pJM103 or
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FIG. 4. Deletion analysis. Lines represent the DNA present in the deletion. The diagram is drawn to scale. The restriction sites were derived from the sequence and have been verified by digestions. Sporulation and replication were assayed as described under Materials and Methods.
pBEST501 (for replication) or pHT315 (for sporulation assays). All constructions were transformed into PV361. The results are shown in Fig. 4 along with relevant restriction sites. Sporulation was assessed by comparing the degree of colony clearing on plates after 5 days as compared with the Spo1 control (PV361/pHT315) with microscopic verification. It is apparent from the deletion analysis that neither the region upstream of the ORF alone (deletions 114 and 53) nor the region containing most of the ORF (deletion 133) supported replication. However, deletions 63 and 100 delineated the upstream region required for replication. Deletion 63, which replicated, retained 536 bp upstream to the ORF, the putative promoter, and most, but not all, of the upstream palindrome (see Fig. 2). Deletion 100, with 291 bp upstream to ORF, retained the putative 210, but not the 235 promoter region, and did not replicate. Interestingly, deletions 1 and 63, which lacked an additional 161 nucleotides at the carboxyl end (XbaI site), were still capable of replicating. No deletions other than 1 and 63 supported replication, indicating that at least most of the ORF and the region upstream (between 63 and 100) were required for replication. Sporulation inhibition roughly correlated with replication.
Plasmid Stability Cells containing the 2.4-kb fragment in pBEST501 were subcultured at 6-h intervals with appropriate controls, maintaining the cultures vegetatively through 38 generations without selective pressure. The plasmid carrying the 2.4-kb fragment (with the incomplete ORF) in pBEST501 was unstable. About 18% of the viable cells had lost the plasmid within 10 generations, and 98% had lost the plasmid within 38 generations. Cultures with a control plasmid lost less than 5% after 38 generations. Host Range and Requirement for Pol I or RecA To test whether the B. megaterium plasmid replicon was functional in B. subtilis and whether it required Pol I (like several theta plasmids) or RecA (required for single-stranded DNA conversion in some RCM plasmids), pDS100 was transformed into B. subtilis wildtype, polA, and recA mutant strains. Similar numbers of transformants were observed with each of the three strains and a control plasmid (data not shown), demonstrating that the B. megaterium replicon could function in B. subtilis and that the replicon did not require DNA polymerase I or RecA for replication.
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B. megaterium PLASMID REPLICON TABLE 2 Incompatibility of Direct Repeats No. transformants/mg DNA
Donor plasmid
Recipient strain
Both donor and recipient selection
Donor selection
160 350
90 —
A. Transformation pKM55 (repeats) pKM55
a
PV361/pDS110 PV361
B. Stability of transformed plasmids Percentage of plasmid retained Donor
Recipient
Both
5 100
66 100
4 100
PV361/pDS110/pKM55 PV361/pDS110/pHT315
a pKM55: 93-bp direct repeat PCR fragment in pYZ11(RCM,TcR); pDS110: 2.4-kb clone I fragment in pBEST501 (NmR); pHT315: EmR B. thuringiensis replicon.
Complementation of the orf Product If a diffusible product is produced by the orf it should complement a defective orf in trans. Clone p133JM, which does not replicate in PV361 because of a deletion in the orf (D133) at the 59 end (Figs. 2 and 4), was transformed into PV361 containing a functional orf (pDS110). The resultant transformants were selected on donor antibiotic plates and replicated to plates selecting for both plasmids. In three experiments, an average of 28 transformants/mg DNA were recovered (340 in pHT315 control) selecting for the donor plasmid. All these contained both plasmids. Presence of both plasmids was verified by testing several colonies for both plasmids, restricting the isolated plasmid DNA with HindIII.
was transformed into PV361 containing either pDS110 or pHT315 as a control. The transformants were selected for the donor plasmid (TcR), incubated overnight, and then replica plated, as shown in Table 2A. A few transformants were then tested for incompatibility by streaking on SNB, inoculating into SNB broth and incubating 6 h, then diluting and plating on SNB plates, all without selection. After overnight growth, colonies were replica plated to SNB and SNB plus donor, recipient, and donor 1 recipient selection (Table 2B). In the experiment shown, 996 colonies were tested. As can be seen, the cloned direct repeats could not be sustained in the same cell and the number of cells carrying pDS110 was also reduced. DISCUSSION
Demonstration of Incompatibility of the Direct Repeats To test incompatibility, a fragment containing the tandem direct repeats was generated by PCR as described under Materials and Methods. The 93-bp insert was cloned first into pGEM-T easy vector, then subcloned into the EcoRI site of pYZ11 to construct pKM55. Plasmid pKM55
A 2374-bp fragment from a HindIII library of plasmid DNA of B. megaterium QM B1551 has been shown to function as a replicon. The hybridization of the replicon to all four, large compatible plasmids suggests that they are homologous. This homology and the lack of a selectable phenotype precluded the assignment of the replicon to its plasmid. However, trans-
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formation into QM B1551 caused the loss of the 165-kb plasmid and perhaps the 108-kb plasmid, suggesting the possibility that one of these is the source of the replicon. The replication driven by the cloned origin is probably theta replication for several reasons. First, it was derived from a large plasmid. Janniere et al. (1993) reported that no RCM plasmid in gram-positive bacteria has been found greater than 8.8 kb in size, although recently pNG2, a 14.4-kb RCM plasmid from Corynebacterium diphtheriae, has been reported (Zhang et al., 1994). The homologous B. megaterium plasmids are much larger. Second, regions of the sequence are similar to other theta replicons, including a sequence similar to the chromosomal origin of B. subtilis (a theta replicon), a region of tandem direct repeats, a typical oriA region, possible DnaA boxes, and a sequence similar to a chromosomal (theta) Ter sequence. Third, no DNA or amino acid similarity was found to known RCM plasmids or their Rep proteins, in contrast to most grampositive RCM plasmids. Fourth, the direct repeats function as an incompatibility locus typical of theta plasmids. Fifth, there is similarity to a group of large, homologous, thetareplicating Lactococcus plasmids (Baum and Gilbert, 1992; Seegers et al., 1994). These plasmids have very homologous replicons with iterons, upstream palindromes, and large (.400 aa) ORFs coding for Rep proteins that bind to the iterons. It has been reported that the iterons of these compatible plasmids differ from one another by two bases (Seegers et al., 1994). Sixth, there is a region that Filutowicz and co-workers (1994) have observed in gram-negative theta plasmids immediately downstream from the iterons with the consensus CCACAGGNNNAA. The 2.4-kb origin sequence immediately downstream from the direct repeats (1636 –1651) was CCAgAacGNNNnnAA. Whether this is significant or coincidental cannot be determined since the function of the region is not known for any plasmid. Seventh, no requirement for RecA was observed, which is observed for some RCM plasmids. While these observations are not direct
proof of theta replication, they are consistent with known theta-replicating plasmids. The minimal replicon size is between 1385 and 1675 bp. Deletion 63 containing 578 bp upstream to the ORF replicated, while a further deletion of 300 bp closer to the ORF (deletion 100), which lacked the 235 sequence of a possible sigma A promoter, did not replicate (Fig. 4). Deletion clones 63 and 100 defined the minimal replicon. It is significant that deletion clone 100 (Fig. 4), which lacked part of the putative promoter, could not replicate. The 235 to 210 spacing is one nucleotide further than the optimal spacing, but this has been found in B. subtilis not to be as important as the base composition (Mountain, 1989). In front of the ORF is a good ribosomal binding site that agrees closely with the consensus ribosomal binding site of B. megaterium (Fliss and Setlow, 1985), further suggesting that the gene needs to be translated for replication to occur. The ability of the 2.4-kb clone to act in trans, replicating a 59 deleted ORF, demonstrated that the ORF is expressed and capable of replicating another plasmid. It also strongly suggests it codes for a Rep protein. The size of the ORF is similar to that reported for most theta Rep proteins (Janniere et al., 1993; Helinski, 1995). The carboxyl end does not seem to be essential for replication since a further deletion of 53 amino acids from the carboxyl end was still functional. We have not been able to confirm the location of the promoter as yet. However, the complementation tests showed that a product from the ORF does work in trans and therefore is expressed. Similar difficulty in determining a start site for the Rep protein of R6K has been encountered (Filutowicz et al., 1994). Unusual features of this replicon are the position of the tandem direct repeats inside the ORF, the small number and size of direct repeats compared with many other gram-positive and -negative bacterial replicons, and the lack of similarity of the ORF to any other prokaryotic proteins. Janniere et al. (1993) surveyed several replicons from gram-positive hosts with various tandem direct repeats, almost all of which flank the Rep protein gene and are 17–22 bp. When the 2.4-kb fragment was transformed
B. megaterium PLASMID REPLICON
into wild-type QM B1551, it was incompatible with at least the 165-kb plasmid (Fig. 3). It was then necessary to demonstrate that the direct repeats, the site of binding for a Rep protein in many theta replicons, were the site of incompatibility. Indeed, when a 93-bp fragment containing the direct repeats was introduced into a cell already harboring the whole replicon (pDS110), incompatibility was observed. These direct repeats, therefore, should be the equivalent of the iterons described previously for many theta plasmids (Helinski et al., 1995; Janniere et al., 1993; Seegers et al., 1994). There are a few examples of iterons internal to the Rep protein ORF. The internal iteron arrangement is similar to the lambda phage origin, where four tandem 19-bp direct repeats are within the O gene, a DnaA homologue (Furth and Wickner, 1983). Internal interons have recently been reported in B. thuringiensis homologous plasmids p43, p44, and p60, which have two direct repeats and two sets of 19-bp inverted repeats within the RepA protein gene (Baum and Gilbert, 1992). The large lactococcal plasmids (Seegers et al., 1994) have homologous replicons, but their eleven 22-bp direct repeats are upstream from the rep gene. No DNA or amino acid similarity of the B. megaterium replicon to any of the above replicons was found. The sequence of the replicon (Fig. 2) had other interesting features. Several potential DnaA boxes were near the direct repeats, similar to theta replicons of gram-negative bacteria (Filutowicz et al., 1994). All the DnaA boxes have one or two mismatches from the consensus TTATCCACA, which is common (Kiewiet et al., 1993). It has been reported that when the chromosomal origin, oriC, from B. subtilis was cloned, it caused instability (Moriya et al., 1988; Seiki et al., 1981; Yoshikawa and Wake, 1993), attributable to the DnaA boxes (Moriya et al., 1988). When the number of boxes was decreased or the sequences were mutagenized, transformation was possible. However, the transformants were often slow-growing, probably because of interference with chromosome replication. Interference with chromosomal DNA replication by the B. megaterium plasmid origin might explain the inhibition of sporula-
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tion, since completion of a round of DNA replication is required in the initiation of B. subtilis sporulation (Mandelstam and Higgs, 1974). In addition, four possible sites were found and are shown in Fig. 5A aligned with the binding site consensus for IHF from gram-negative bacteria (Freundlich et al., 1992). Goodrich and co-workers (1990) statistically analyzed 27 E. coli IHF functional sequences and reported that there was considerable deviation in binding sites adjacent to and within the consensus sequence. But in the sequence AATCAAnnAnTTA, the capitalized bases are found in more than 80% of the sites and the boldface positions occur 95–100% of the time. As shown in Fig. 5A, all those positions are conserved in the bp 1522 site, and all but one of the conserved sites are present in the bp 1339, 1846, and 2332 sites. The possible IHF site starting at bp 1522 is a good candidate for a functional site since it is upstream from the direct repeats and near two putative DnaA boxes. Binding studies must be done to test whether the sites are functional. Interestingly, three of the possible IHF binding sites had a DNA box directly downstream within 9 –12 bases (Fig. 5A), consistent with a model in which DnaA and IHF bind and bend DNA to promote Rep protein binding at the iterons (Filutowicz et al., 1994). TF1 from B. subtilis phage SP01 has homology to bacterial HU proteins and the E. coli IHF (Andera et al., 1994; Grove et al., 1997) and has been extensively studied, but to our knowledge, there is no report of an equivalent Bacillus protein. The B. megaterium origin sequence also contained a region with some homology to the oriC (Fig. 5B) and Ter regions of the B. subtilis chromosome (Fig. 5C, G. Wake, personal communication). In the replicon, TerBm is upstream from the ORF between the two palindromes. Regions homologous to Ter sequences of the B. subtilis chromosome have been reported on the Bacillus theta plasmid pLS20 replicon (Meijer et al., 1995). As can be seen, the homology of the TerBm is weaker and must be tested for function before any conclusions can be made. The cloned 2.4-kb fragment caused the formation of multimeric DNA, as do some RCM plasmids (Janniere et al., 1993; Nugent, 1989). Al-
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FIG. 5. Possible homologous IHF, oriC, and Ter I sequences in the 2.4-kb fragment (A) Alignment of putative IHF boxes found in the origin sequence with the consensus sequence. aFreundlich et al. (1992). bGoodrich et al. (1990). (B) Alignment of oriC of the B. subtilis chromosome with a palindromic region of the plasmid replicon. (C) Alignment of a possible termination (Ter) sequence from the 2.4-kb fragment with the Ter sequences of the B. subtilis chromosome and pLS20. cR. G. Wake (personal communication). dMeijer et al. (1995).
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though the bands as shown in Fig. 3 (lanes A and C) were faint and somewhat diffuse, the upper bands increased sequentially approximately 6–7 kb, suggesting that they represented multimeric forms of pDS110, confirmed by restriction digestion (lane D). Subclones of theta plasmid pAMb1 from E. faecalis accumulate highly polymerized DNA unless a 2.3-kb region directly downstream from the RepE protein is present (Swinfield et al., 1991). This region includes a gene with homology to the recombinases of INV–RES invertase class and resolvase of Tn917 and pIP404, and is required for stability (Janniere et al., 1993). Lack of such a downstream region in the B. megaterium 2.4-kb fragment could be a possible reason for the instability observed as well as the highly polymerized DNA. Highly polymerized DNA formation has also been observed in another large (49-kb), theta-replicating Lactococcus plasmid, pRT2030 (Hill et al., 1991). Attempts to clone a larger fragment containing the complete ORF and adjacent elements have been complicated by the homology of the origin clone with the four other large plasmids. Of eight hybridizing clones from another gene library, none had the sequence of this clone (Stevenson, unpublished data). Since theta-replicating plasmids have been classified into classes A–D by whether they contain a typical oriA structure and/or have a functional Rep protein, or require DNA polymerase I (Filutowicz et al., 1994; Pansegrau et al., 1994), the B. megaterium replicon was transformed into B. subtilis mutants to test both its host range and dependence on Pol I or RecA (needed by some RCM plasmids) and better define its replicon. Plasmid pDS110 successfully replicated in B. subtilis 168, as well as polA and recA mutants. This suggests that the replicon requires neither DNA polymerase I, nor RecA (another test suggesting it is not a RCM plasmid), and can replicate in a related species. Structurally, it has the typical oriA of several gram-negative bacterial plasmids, that is, tandem direct repeats, a GC-rich area (position 1672–1696) followed by an AT-rich region (82% A 1 T at position 1696 –1744). However, it differs from these in location of oriA within a large ORF. Computer searches have shown that the origin has no obvious similarity with any
known Rep proteins in class A, nor those of the Class E pLS20. In summary, the replicon described above has homology with three other plasmids from the same cell, is unstable in its cloned, minimal form, and posesses direct repeats within the Rep gene that cause incompatibility with its replicon. The product of the ORF is probably a Rep protein, which can act in trans. It is one of few theta plasmid replicons described in Bacillus and also has the capability to replicate in B. subtilis. The B. megaterium replicon may, therefore, define a new class of plasmid replicons. ACKNOWLEDGMENTS This work was supported in part by Grant 1R15 GM49440-01 from the National Institutes of Health (P.S.V.) and NATO Grant CRG.940770 (P.S.V.). The authors thank Dr. R. Tewari and Yansheng Zhou for critical reading of the manuscript and Gary Baisa for assisting in some of the plasmid constructions.
REFERENCES Altschul, S. F., Gish, W., Miller, W., Meyers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403– 410. Andera, L., Spangler, C. J., Galeone, A., Mayol, L., and Geiduschek, E. P. (1994). Interrelations of secondary structure stability and DNA-binding affinity in the bacteriophage SP01-encoded type II DNA-binding protein TF1. J. Mol. Biol. 236, 139 –150. Arantes, S. F., and Lereclus, D. (1991). Construction of cloning vectors for Bacillus thuringiensis. Gene 108, 115–119. Baum, J. A., and Gilbert, M. P. (1992). Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids. J. Bacteriol. 173, 5280 –5289. Behnke, D., Gilmore, M. S., and Ferretti, J. J. (1981). Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamicin/ kanamycin resistance determinant. Mol. Gen. Genet. 182, 414 – 421. Bruand, C., Le Chatelier, E., Ehrlich, S. D., and Janniere, L. (1993). A fourth class of theta-replicating plasmids: The pAMb1 family from gram-positive bacteria. Proc. Natl. Acad. Sci. USA 90, 11668 –11672. Cutting, S. M., and Van der Horn, P. B. (1990). Genetic analysis. In “Molecular Biological Methods for Bacillus” (C. R. Harwood and S. M. Cutting, Eds.), pp. 66 –78. Wiley, Chichester, England. Del Solar, G., Moscoso, M., and Espinosa, M. (1993).
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Rolling circle-replicating plasmids from gram-positive and gram-negative bacteria: A wall falls. Mol. Microbiol. 8, 789 –796. English, J. D., and Vary, P. S. (1986). Isolation of recombination defective and UV sensitive mutants of Bacillus megaterium. J. Bacteriol. 163, 155–160. Espinosa, M., Del Solar, G., Rojo, F., and Alonso, J. C. (1995). Plasmid rolling circle replication and its control. FEMS Microbiol. Lett. 130, 111–120. Filutowicz, M., Dellis, S., Levchenko, I., Urh, M., Wu, F., and York, D. (1994). Regulation of replication of an iteron-containing DNA molecule. In “Progress in Nucleic Acid Research and Molecular Biology,” Vol. 48, pp. 239 –273. Academic Press, New York. Fliss, E. R., and Setlow, P. (1985). Genes for Bacillus megaterium small, acid-soluble spore proteins: Nucleotide sequence of two genes and their expression during sporulation. Gene 35, 151–157. Freundlich, M., Ramani, N., Mathew, E., Sirko, A., and Tsui, P. (1992). The role of integration host factor in gene expression in Escherichia coli. Mol. Microbiol. 6, 2557– 2563. Furth, M. E., and Wickner, S. H. (1983). Lambda DNA Replication. In “Lambda II” (R. W. Hendrix, J. W. Roberts, F. W. Stahl, and R. A. Weisberg, Eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Goodrich, J. A., Schwartz, M. S., and McClure, W. R. (1990). Searching for and predicting the activity of sites for DNA binding proteins: Compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF). Nucleic Acids Res. 18, 4993–5000. Grove, A., Figueiredo, M. L., Galeone, A., Mayol, L., and Geiduschek, E. P. (1997). Twin hydroxymethyluracil-A base pair steps define the binding site for the DNAbinding protein TF1. J. Biol. Chem. 272, 13084 –13087. Helinski, D. R., Toukdarian, A. E., and Novick, R. P. (1995). Replication control and other stable maintenance mechanisms of plasmids. In “Escherichia coli and Salmonella typhimurium” (F. C. Neidhardt et al., Eds.), 2nd ed. pp. 2295–2324. Am. Soc. for Microbiol., Washington, DC. Hill, C., Miller, L. A., and Klaenhammer, T. R. (1991). The bacteriophage resistance plasmid forms high-molecularweight multimers in lactococci. Plasmid 25, 105–112. Itaya, M., Kondo, K., and Tanaka, T. (1989). A neomycin resistance gene cassette selectable in a single copy state in the Bacillus subtilis chromosome. Nucleic Acids Res. 17, 4410. Janniere, L., Bruand, C., and Ehrlich, S. D. (1990). Structurally stable Bacillus subtilis cloning vectors. Gene 87, 53– 61. Janniere, L., Gruss, A., and Ehrlich, S. D. (1993). Plasmids. In “Bacillus subtilis and Other Gram-Positive Bacteria” (A. L. Sonenshein, J. A. Hoch, and L. Losick, Eds.), pp. 625– 644. Am. Soc. for Microbiol., Washington, DC. Kahn, S. A. (1997). Rolling circle replication of bacterial plasmids. Microbiol. Mol. Biol. Rev. 61, 442– 455. Keiselburg, M. K., Weickert, M., and Vary, P. S. (1984).
Analysis of resident and transformant plasmids in Bacillus megaterium. Biotechnology 2, 254 –259. Kiewiet, R., Bron, S., Jonge, K. D., Venema, G., and Seegers, J. F. M. L. (1993). Theta replication of the lactococcal plasmid pWV02. Mol. Microbiol. 10, 319 –327. Lovett, P. S., and Keggins, C. M. (1979). Bacillus subtilis as a host for molecular cloning. Methods Enzymol. 68, 342– 357. Meijer, W. J. J., Deboer, A. L., van Tongeren, S., Venema, G., and Bron, S. (1995). Characterization of the replication region of the Bacillus subtilis plasmid pLS20: A novel type of replicon. Nucleic Acids Res. 23, 3214 – 3223. Moriya, S., Fukuoka, T., Ogasawara, N., and Yoshikawa, H. (1988). Regulation of initiation of the chromosomal replication by DnaA-boxes in the origin region of the Bacillus subtilis chromosome. EMBO J. 2911–2917. Mountain, A. (Ed.) (1989). Gene expression systems for Bacillus subtilis. In “Bacillus” (C. R. Harwood, Ed.), pp. 73–113. Plenum, New York. Novick, R. (1989). Staphylococcal plasmids and their replication. Annu. Rev. Microbiol. 43, 537–565. Nugent, M. E. (1989). Plasmid replication and stability. In “Bacillus” (C. R. Harwood, Ed.), pp. 155–168. Plenum, New York. Pansegrau, W., Lanka, E., Barth, P. T., Figurski, D. H., Guiney, D. G., Haas, D., Helinski, D. R., Schwab, H., Stanisich, V. A., and Thomas, C. M. (1994). Complete nucleotide sequence of Birmingham IncPa plasmids: Compilation and comparative analysis. J. Mol. Biol. 239, 623– 663. Perego, M., Spiegelman, G. B., and Hoch, J. A. (1988). Structure of the gene for the transition state regulator, abrB: Regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis. Mol. Microbiol. 2, 689 – 699. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Molecular Cloning: A Laboratory Manual,” 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY. Seegers, J. F. M. K., Bron, S., Franke, C. M., Venema, G., and Kiewiet, R. (1994). The majority of lactococcal plasmids carry a highly related replicon. Microbiology 140, 1291–1300. Seiki, M., Ogasawara, N., and Yoshikawa, H. (1981). Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. Mol. Gen. Genet. 183, 227–233. Stevenson, D. M., Lach, D., and Vary, P. S. (1993). A gene required for germination in Bacillus megaterium is plasmid-borne. In “DNA Transfer and Gene Expression in Microorganisms” (E. Balla and G. Berencsie, Eds.), pp. 197–208. Intercept, Budapest. Sussman, M. D., Vary, P. S., Hartman, C., and Setlow, P. (1988). Integration and mapping of Bacillus megaterium genes which code for small, acid-soluble spore proteins and their protease. J. Bacteriol. 170, 4942– 4945. Swinfield, T.-J., Janniere, L., Ehrlich, S. D., and Minton, N. P. (1991). Characterization of a region of the Entero-
B. megaterium PLASMID REPLICON coccus faecalis plasmid pAMb1 which enhances the segregational stability of pAMb1-derived cloning vectors in Bacillus subtilis. Plasmid 26, 209 –221. Vary, P. S. (1992). Development of genetic engineering in Bacillus megaterium: An example of the versatility and potential of industrially important bacilli. In “Biology of Bacilli: Applications to Industry” (R. Doi and M. McGloughlin, Eds.), pp. 251–310. Butterworths–Heinemann, Boston. Vary, P. S. (1994). Prime time for Bacillus megaterium. Microbiology 140, 1001–1013. Von Tersch, M. A., and Carlton, B. C. (1983). Bacteriocin from Bacillus megaterium ATCC19213: Comparative studies with megacin A-216. J. Bacteriol. 155, 866 – 871.
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Yoshikawa, H., and Wake, R. G. (1993). Initiation and termination of chromosome replication. In “Bacillus subtilis and other Gram-Positive Bacteria” (A. L. Sonenshein, J. A. Hoch, and R. Losick, Eds.), pp. 507–528. Am. Soc. for Microbiol., Washington, DC. Zaghloul, T., Kawamura, F., and Doi, R. (1985). Translational coupling in Bacillus subtilis of a heterologous Bacillus subtilis–Escherichia coli gene fusion. J. Bacteriol. 164, 550 –555. Zhang, Y., Praszkier, J., Hodgson, A., and Pittard, A. J. (1994). Molecular analysis and characterization of a broad-host-range plasmid, pEP2. J. Bacteriol. 176, 5718 –5728. Communicated by S. A. Khan
40, 175–189 (1998) PL981359
ARTICLE NO.
Characterization of a Theta Plasmid Replicon with Homology to All Four Large Plasmids of Bacillus megaterium QM B1551 David M. Stevenson,1 Muthusamy Kunnimalaiyaan, Kerstin Mu¨ller,2 and Patricia S. Vary3 Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115 Received August 12, 1997; revised April 2, 1998 A replicon from one of an array of seven indigenous compatible plasmids of Bacillus megaterium QM B1551 has been cloned and sequenced. The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551. The cloned 2374-bp HindIII fragment was sequenced and contained two upstream palindromes and a large (.419-aminoacid) open reading frame (ORF) truncated at the 39 end. Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF. The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication. The ORF product was shown to act in trans. A small region with similarity to the B. subtilis chromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins. Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream. The predicted ORF protein of .48 kDa was basic and rich in glutamate 1 glutamine (16%). There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF. The data suggest that this is a theta replicon with an expressed rep gene required for replication. The replicon contains its iterons within the gene and has no homology to reported replicons. It is the first characterization of a B. megaterium replicon. © 1998 Academic Press Key Words: plasmid; theta replication; sporulation; Bacillus megaterium; Bacillus subtilis; Bacillus thuringiensis.
a bacteriocin, called a megacin (Keiselburg et al., 1984), and a gene required for spore germination on all single germinants (Stevenson et al., 1993). Most of the research on plasmid replication has been done in Escherichia coli [see recent reviews (Helinski et al., 1995; Kahn, 1997)], but progress has been made in characterizing some of the gram-positive plasmid replicons including those from Streptococcus, Staphylococcus, and Bacillus (Janniere et al., 1993; Novick, 1989). Unlike E. coli and most gramnegative bacteria, several small plasmids in the gram-positive bacteria replicate by a rolling circle mechanism (RCM)4 [although two small RCM plasmids have been reported in gram-
Bacillus megaterium is a gram-positive sporeforming bacterium used in many industrial applications and as a cloning host for expression of intact foreign proteins [see reviews by Vary (1992, 1994)]. Most B. megaterium strains isolated from soil as well as those used widely in industry contain numerous plasmids. We have previously reported that strain QM B1551 carries more than 11% of its cellular DNA as an array of seven indigenous plasmids (renamed pBM100 –pBM700) of sizes 5.5, 8.8, 20, 47, 71, 108, and 165 kb, respectively (Keiselburg et al., 1984), but their function in the cell remains elusive. Phenotypes found to be associated with these plasmids include the production of 1 Current address: Department of Biological Sciences, Dartmouth College, Hanover, NH 03755. 2 Current address: Institut for Klinische Mikrobiologie und Immunologie, Wasserturmstrasse, 391054 Erlangen, Germany. 3 To whom correspondence should be addressed. Fax: (815) 753–1753. E-mail: [email protected].
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Abbreviations used: RCM, rolling circle mechanism; Em, erythromycin; Cm, chloramphenicol; Nm, neomycin; Ap, ampicillin; PCR, polymerase chain reaction; IPTG, isopropyl-b-D-thiogalactoside; ORF, open reading frame; IHF, integration host factor; Ter, termination sequence; aa, amino acids. 0147-619X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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negative Helicobacter (del Solar et al., 1993)]. A survey of 40 gram-positive bacterial RCM plasmids showed the average size to be 4 kb, with the largest 8.8 kb. Theta plasmids, on the other hand, were found to range in size from 3.8 to 95 kb (Espinosa et al., 1995; Janniere et al., 1993; Kiewiet et al., 1993). Little is known about the large plasmids of gram-positive bacteria, and descriptions of their theta replicons are new [see reviews (Janniere et al., 1993; Novick, 1989)]. Most of these theta plasmids are from Streptococcus, Lactococcus, and Enterococcus. Of the very few Bacillus replicons described, most are from plasmids of B. thuringiensis carrying the cry insecticidal genes. Theta plasmids have been classified into classes A–D depending on the presence of a Rep protein, iterons, DnaA boxes, and dependence on Pol I proteins (Bruand et al., 1993; Helinski et al., 1995; Janniere et al., 1993; Novick, 1989). The gram-positive bacterial plasmid pAMb1 family (including pIP501 and pSM19035) defined class D, which has a dispensable oriA structure and dependence on Pol I (Bruand et al., 1993; Janniere et al., 1993). A possible class E replicon with no Rep protein gene or requirement for Pol I, but an oriA region, has been described by characterizing the 3.1-kb replication region from a 55-kb B. subtilis plasmid pLS20 (Meijer et al., 1995). Our aim in studying the seven compatible cryptic plasmids of B. megaterium is to gain a clearer understanding of their function and their maintenance. Plasmids provide a nonlethal system to study plasmid and host replication including the proteins involved, the regulation of replication, partitioning, and stability. In addition, most of the cloning vectors used in Bacillus have been from other genera and replicate by a RCM. These vectors are unstable in B. subtilis, an important cloning host (Janniere et al., 1993). Only three theta replicons—pHT1030 from B. thuringiensis, pIP501 and pAMb1 from Enterococcus faecalis— have so far been used for vector construction (Arantes and Lereclus, 1991; Behnke et al., 1981; Janniere et al., 1990). Characterization of replicons from optimally adapted indigenous plasmids of Bacillus, especially those with theta
replicons, should lead to the construction of more stable vectors for this genus. The array of seven compatible plasmids of strain QM B1551 provides a unique system. By studying it in detail, it is possible that the role and possible interaction of the plasmids within the cell can be better understood. As a first step, we report here the isolation of a replicon that was found by screening a HindIII plasmid DNA library from B. megaterium QM B1551. Further characterization demonstrated that it is most probably a theta replicon and exhibits some distinctive characteristics. The Sequence Accession number in GenBank is AFO15255. MATERIALS AND METHODS Bacterial Strains, Plasmids, and Growth Conditions Strains and plasmids used in this study are listed in Table 1. B. megaterium was grown in SNB or MC broth or plates with appropriate selection at 30°C as described previously (English and Vary, 1986). Antibiotics and concentrations used for Bacillus were erythromycin (Em) 5 mg/ml, chloramphenicol (Cm) 5 mg/ml, and neomycin (Nm) 5 mg/ml for plates and 1 mg/ml for broth. Sporulation was assayed microscopically and/or by changes in colony morphology after 70 h incubation at 30°C in SNB. E. coli and B. subtilis were routinely grown in LB medium (Sambrook et al., 1989) with appropriate selection [100 mg/ml ampicillin (Ap) for E. coli.]. An E. coli–Bacillus shuttle vector pHT315 (Arantes and Lereclus, 1991) was used for gene library construction. It maintains about 15 copies in B. subtilis and has both B. thuringiensis and E. coli replicons. It carries an ApR gene for E. coli and an EmR gene for Bacillus. Plasmid pBEST501(Itaya et al., 1989) was derived from pGEM4 and has a NmR cassette that expresses in Bacillus, but lacks an origin capable of replicating in Bacillus. Plasmid pYZ11 is a compatible derivative of the RCM 5.5-kb plasmid (pBM100) from B. megaterium QM B1551. Plasmid pGEM7Zf(1) (Promega) was specifically designed for the Erase-a-Base system. Plasmid pGEMT-easy (Promega) is
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B. megaterium PLASMID REPLICON TABLE 1 Strains and Plasmids Used in This Study Strain/plasmid Strains B. megaterium QM B1551 PV361 PV622 PV623 B. subtilis 168 1A1 1A43 1A226 E. coli DH5a
Plasmids pHT315 pBEST501 pJM103 pGEM7Zf(1) pGEM-T easy pYZ11 pDS100 pDS110 pDS571 pDS611 pDS6163 pDS61100 pDS61133 p133JM pKM55 a b
Genotype or description
Source or Reference
Wild-type (7p1)a Plasmidless (7p2) QM B1551 derivative (gerP) PV361/pDS100 PV361/pDS110
J. C. Vary, UIC Sussman et al., (1988) This study This study
trpC2 recA1 trpC2 hisH2 pheA1 polA5 trpC2
BGSCb BGSC BGSC
F9 endA1 hsdR17 (r-m-) supE44 thi-1 recA1 gyrA96 relA1 D(lacZYA-argF) U169 deoR (f80)dlac D(lacZ M15)
Promega
AmpR, EmR shuttle vector (Bacillus, E. coli) B. thuringiensis ori1030, pUC19 lacZ, 6.51 kb AmpR in pGEM4, NmR from pUB110 Bacillus integrative vector, 4.15 kb AmpR in pUC19, CmR from pC194, integrative vector, 3.72 kb AmpR f1ori lacZ::PrT7, PrSP6, 3.0 kb AmpR f1ori lacZ::PrT7, PrSP6, 3.02 kb linear with T overhang pBM100 TetR pUC19 AmpR, 10.3 kb 2.4-kb Clone I in pHT315 2.4-kb Clone I in pBEST501 2.4-kb Clone I in pGEM7Zf(1) D1 in pGEM7Zf(1) D63 in pGEM7Zf(1) D100 in pGEM7Zf(1) D133 in pGEM7Zf(1) Clone I D133 in pJM103 93-bp iteron PCR fragment in pYZ11
Arantes and Lereclus (1991) Itaya et al. (1989) Perego et al. (1988) Promega Promega Y. Zhou, unpublished This study This study This study This study This study This study This study This study This study
Contains all seven indigenous plasmids. Bacillus Genetic Stock Center, Ohio State University.
pGEM5Zf(1) linearized with EcoRV with added T nucleotides for PCR cloning. DNA Isolation and Plasmid Library Construction Small-scale plasmid DNA isolations were prepared using alkaline lysis procedures for Bacillus (Zaghloul et al., 1985) and E. coli. (Sambrook et al., 1989). Large-scale Bacillus plasmid DNA isolations were prepared by the method of Lovett and Keggins (1979) following
cell growth to OD660 5 0.75 in MC broth. DNA was additionally purified by CsCl– ethidium bromide gradient centrifugation (Sambrook et al., 1989). A QM B1551 plasmid library was constructed by digesting QM B1551 plasmid DNA with HindIII and ligating the resulting fragments into pHT315 digested with HindIII and dephosphorylated. The ligation mixture was transformed into E. coli DH5a and plated on LB(1) Ap 1 IPTG 1 X-gal. More than 1300 white transformants were individually picked
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and incubated for 3 h as separate minicultures in 250 ml LB broth 1 Ap. Glycerol was added to 15%, and the minicultures were stored at 270°C. Transformation and Plasmid Stability B. megaterium was transformed by protoplast fusion (Von Tersch and Carlton, 1983) and regenerated in RHAF medium as described (English and Vary, 1986). B. subtilis was transformed by the method of Cutting and Van der Horn (1990) and E. coli by the method of Sambrook et al. (1989). To test for plasmid stability, cells were grown at 30°C in SNB broth with appropriate antibiotic to an OD660 5 0.5, then diluted 1:100 into SNB broth without antibiotic and also plated on SNB agar. The subculture was grown for 6 h, again subcultured, and plated, continuing at 6-h intervals for 72 h. All dilution plates were replicated after overnight incubation to SNB 6 antibiotic to determine the presence of plasmid. Sequencing the 2.4-kb Fragment Nested deletions of the 2.4-kb HindIII fragment cloned into pGEM7Zf(1) and transformed into strain DH5a were generated with the Erase-a-Base kit (Promega) following the manufacturer’s directions. DNA was sequenced with [a-32P]dATP (1 mCi/reaction) using the USB Taq cycle sequencing kit (United States Biochemical). Primers were both synthesized oligonucleotides and universal primers. DNA and amino acid sequences were analyzed with the computer program PCGENE (Intelligenetics, Inc., A. Bairock, University of Geneva, Geneva, Switzerland). Computer searches were performed using the BLAST programs (Altschul et al., 1990) provided by GenBank at the National Center for Biotechnology Information, Bethesda, Maryland. BLASTN was used to search for nucleotide similarity and BLASTX for similarity to proteins. The amino acid sequence of the ORF was also searched against protein databases using BLASTP.
Southern Hybridizations and PCR The Polarplex chemiluminescent blotting kit (New England Biolabs) was employed for Southern hybridizations following the manufacturer’s suggestions. DNA was blotted onto nylon membrane, crosslinked using UV Stratalinker (Stratagene, Inc.), and then hybridized at 68°C. Chemiluminescent blots were exposed to Fuji X-ray film for 2 to 30 min, depending on signal strength. A fragment containing the tandem direct repeats was generated by PCR using sense and antisense primers at nucleotides 1568–1585 and 1643–1660. PCR was carried out for 30 cycles of 95°C for 1 min, 45°C for 2 min, and 72°C for 1 min. The 93-bp product was cloned into the pGEM-T easy vector (Promega) according to the manufacturer’s directions and confirmed by sequencing. The insert (iterons) were then recloned into a stable vector at the EcoRI site of pYZ11. RESULTS A HindIII library of QM B1551 plasmid DNA was constructed in shuttle plasmid pHT315 and transformed into DH5a as described under Materials and Methods. Plasmid DNA was isolated from pooled E. coli clones and transformed into B. megaterium PV361. A 2.4-kb fragment was detected as a clone (pDS100) that dramatically decreased sporulation in B. megaterium. The recombinant colonies containing the clone lysed on plates within 2–3 days. To determine which of the seven plasmids was the source of the fragment, the entire 2.4-kb fragment was used as a probe against QM B1551 plasmid DNA and hybridized under stringent conditions. As can be seen in Fig. 1, hybridization was observed with all four large plasmids of QM B1551 (lane b). There was no homology with chromosomal DNA since no hybridization occurred with total DNA from plasmidless strain PV361 (lane e). Sequencing of the 2.4-kb Fragment Plasmid pDS571 was constructed by cloning the 2.4-kb HindIII fragment into pGEM7Zf(1), and a series of nested deletions were made to
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FIG. 1. Southern hybridization of QM B1551 plasmid and PV361 total DNA using the HindIII 2.4-kb fragment as probe. (1) Agarose gel electrophoresis of DNA; (2) Southern blot of the gel probed with the 2.4-kb fragment. (A) The 2.4-kb HindIII fragment from pDS100; (B) QM B1551 CsCl–EtBr plasmid preparation. (C) l HindIII digest, (D) QM B1551 plasmids digested with HindIII; (E) PV361HindIII-digested total DNA (plasmidless strain).
sequence the 2.4-kb fragment as described under Materials and Methods. The complete DNA sequence is shown in Fig. 2. The 2374-bp fragment contained a large ORF, which encompassed more than half the clone (1257 bp), that could code for an incomplete basic polypeptide of 419 amino acids with a predicted molecular weight of .48 kDa. Within the ORF sequence were four tandem 12-bp direct repeats, three of which are identical (starting at bp 1592), the fourth differing by only two bases. Upstream from the ORF were two palindromic regions. The palindrome starting at position 531 was potentially the most thermodynamically stable, with a projected DG at 25°C of 238.8 kcal. It contained a 27-nucleotide inverted repeat with a two-nucleotide loop. Part of this region (552–584) showed 82% (26/29 bp) similarity to a portion of the Type II membrane binding region within the B. subtilis chromosomal
origin, oriC, as shown on the sequence (Fig. 2) and aligned in Fig. 5B. The second potential stem –loop structure, starting at position 687, was a small 9-bp G 1 C-rich inverted repeat with a 6-bp A 1 T-rich loop and a deduced DG of 222.4 kcal. The region surrounding the palindromes was A 1 T rich and contained 13 locations with runs of A . 5 and five runs of T $ 5. There was a possible s A promotor with a perfect consensus 210 at 836–841 (TATAAT) and a 235 at 809– 118 (TTATTGAAT) (consensus 5 TTATTGAAA). Several possible DnaA boxes were also observed and are marked. Interestingly, at least four putative integration host factor (IHF) boxes and a sequence with some homology to the termination sequences (Ter) of the B. subtilis chromosomal origin were found (Figs. 2 and 5). Searches of the database produced no significantly similar proteins, motifs, or nucleotide sequences. How-
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FIG. 2. DNA sequence of the 2374-bp fragment. Repeats (both direct and inverted) are indicated by thin arrows. Relevant restriction sites and possible s A promoter are indicated and asterisks mark a putative Shine–Dalgarno (SD) sequence. A sequence homologous to a region of oriC of the B. subtilis chromosome is indicated with a thick shaded line above the sequence. Possible IHF boxes are indicated with a single underline. Putative DnaA boxes are indicated with thicker arrows above the sequence, and differ by 1–2 bp from the consensus TTATCCACA. A sequence homologous to B. subtilis chromosomal terminators is marked by a double underline. Labeled small arrows mark the start and direction of DNA retained for deletions.
ever, the ORF was found to be somewhat similar to several plant gliadin proteins, apparently because of the large numbers of glutamine codons (16% glutamate 1 glutamine).
Replication by the 2.4-kb Fragment The presence of tandem direct repeats and homology with oriC of B. subtilis led to the hypoth-
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FIG. 3. Agarose gel electrophoresis of pDS110 transformants showing accumulation of highly polymerized plasmid DNA and loss of largest plasmid. (A) QM B1551/pDS110; (B) QM B1551 (wt); (C) PV361/pDS110; (D) PV361/pDS110 PstI digest; (E) l HindIII linear DNA standard.
esis that the fragment contained an origin of replication. To test this, the 2.4-kb HindIII fragment was cloned into pBEST501 containing only an E. coli replicon, to construct pDS110, which was then transformed into both QM B1551 and PV361. Greater than 3200 transformants were obtained in plasmidless PV361, showing that the fragment, indeed, contained a functional origin of replication. However, transformation of QM B1551 with an equal amount of pDS110 DNA produced no transformants. Using 20 times more DNA yielded only 18 transformants. Plasmid DNA from two of the transformants was isolated. The CsCl– ethidium bromide-purified plasmid profile of one of these is shown in Fig. 3, lane A. The QM B1551/pDS110 transformant had lost at least the 165-kb plasmid. Comparison with QM B1551 plasmids (lane B) showed that it retained the 5.5- and 8.8-kb plasmids, but had highermolecular-weight bands in addition to probably the 20-, 47-, and 71-kb plasmids. Because of the
highly polymerized DNA present, it is difficult to assign the bands specifically. When PV361 was transformed with pDS110 (lane C), the DNA was found to exist in highly polymerized forms. Digestion of the DNA from a PV361 transformant by PstI resolved all the forms into one linear 6.6-kb band (lane D). These data suggest that the cloned fragment may be incompatible with the 165-kb plasmid (a band present at 108 kb molecular weight could be the 108-kb plasmid or highly polymerized DNA) and also explained the difficulty in transforming this strain. Deletion Analysis to Define the Minimal Replicon and Sporulation Effect To find the region of the fragment responsible for replication and the observed inhibition of sporulation, nested deletions as well as restriction enzyme deletions were made and subcloned into integrative vectors pJM103 or
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FIG. 4. Deletion analysis. Lines represent the DNA present in the deletion. The diagram is drawn to scale. The restriction sites were derived from the sequence and have been verified by digestions. Sporulation and replication were assayed as described under Materials and Methods.
pBEST501 (for replication) or pHT315 (for sporulation assays). All constructions were transformed into PV361. The results are shown in Fig. 4 along with relevant restriction sites. Sporulation was assessed by comparing the degree of colony clearing on plates after 5 days as compared with the Spo1 control (PV361/pHT315) with microscopic verification. It is apparent from the deletion analysis that neither the region upstream of the ORF alone (deletions 114 and 53) nor the region containing most of the ORF (deletion 133) supported replication. However, deletions 63 and 100 delineated the upstream region required for replication. Deletion 63, which replicated, retained 536 bp upstream to the ORF, the putative promoter, and most, but not all, of the upstream palindrome (see Fig. 2). Deletion 100, with 291 bp upstream to ORF, retained the putative 210, but not the 235 promoter region, and did not replicate. Interestingly, deletions 1 and 63, which lacked an additional 161 nucleotides at the carboxyl end (XbaI site), were still capable of replicating. No deletions other than 1 and 63 supported replication, indicating that at least most of the ORF and the region upstream (between 63 and 100) were required for replication. Sporulation inhibition roughly correlated with replication.
Plasmid Stability Cells containing the 2.4-kb fragment in pBEST501 were subcultured at 6-h intervals with appropriate controls, maintaining the cultures vegetatively through 38 generations without selective pressure. The plasmid carrying the 2.4-kb fragment (with the incomplete ORF) in pBEST501 was unstable. About 18% of the viable cells had lost the plasmid within 10 generations, and 98% had lost the plasmid within 38 generations. Cultures with a control plasmid lost less than 5% after 38 generations. Host Range and Requirement for Pol I or RecA To test whether the B. megaterium plasmid replicon was functional in B. subtilis and whether it required Pol I (like several theta plasmids) or RecA (required for single-stranded DNA conversion in some RCM plasmids), pDS100 was transformed into B. subtilis wildtype, polA, and recA mutant strains. Similar numbers of transformants were observed with each of the three strains and a control plasmid (data not shown), demonstrating that the B. megaterium replicon could function in B. subtilis and that the replicon did not require DNA polymerase I or RecA for replication.
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B. megaterium PLASMID REPLICON TABLE 2 Incompatibility of Direct Repeats No. transformants/mg DNA
Donor plasmid
Recipient strain
Both donor and recipient selection
Donor selection
160 350
90 —
A. Transformation pKM55 (repeats) pKM55
a
PV361/pDS110 PV361
B. Stability of transformed plasmids Percentage of plasmid retained Donor
Recipient
Both
5 100
66 100
4 100
PV361/pDS110/pKM55 PV361/pDS110/pHT315
a pKM55: 93-bp direct repeat PCR fragment in pYZ11(RCM,TcR); pDS110: 2.4-kb clone I fragment in pBEST501 (NmR); pHT315: EmR B. thuringiensis replicon.
Complementation of the orf Product If a diffusible product is produced by the orf it should complement a defective orf in trans. Clone p133JM, which does not replicate in PV361 because of a deletion in the orf (D133) at the 59 end (Figs. 2 and 4), was transformed into PV361 containing a functional orf (pDS110). The resultant transformants were selected on donor antibiotic plates and replicated to plates selecting for both plasmids. In three experiments, an average of 28 transformants/mg DNA were recovered (340 in pHT315 control) selecting for the donor plasmid. All these contained both plasmids. Presence of both plasmids was verified by testing several colonies for both plasmids, restricting the isolated plasmid DNA with HindIII.
was transformed into PV361 containing either pDS110 or pHT315 as a control. The transformants were selected for the donor plasmid (TcR), incubated overnight, and then replica plated, as shown in Table 2A. A few transformants were then tested for incompatibility by streaking on SNB, inoculating into SNB broth and incubating 6 h, then diluting and plating on SNB plates, all without selection. After overnight growth, colonies were replica plated to SNB and SNB plus donor, recipient, and donor 1 recipient selection (Table 2B). In the experiment shown, 996 colonies were tested. As can be seen, the cloned direct repeats could not be sustained in the same cell and the number of cells carrying pDS110 was also reduced. DISCUSSION
Demonstration of Incompatibility of the Direct Repeats To test incompatibility, a fragment containing the tandem direct repeats was generated by PCR as described under Materials and Methods. The 93-bp insert was cloned first into pGEM-T easy vector, then subcloned into the EcoRI site of pYZ11 to construct pKM55. Plasmid pKM55
A 2374-bp fragment from a HindIII library of plasmid DNA of B. megaterium QM B1551 has been shown to function as a replicon. The hybridization of the replicon to all four, large compatible plasmids suggests that they are homologous. This homology and the lack of a selectable phenotype precluded the assignment of the replicon to its plasmid. However, trans-
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formation into QM B1551 caused the loss of the 165-kb plasmid and perhaps the 108-kb plasmid, suggesting the possibility that one of these is the source of the replicon. The replication driven by the cloned origin is probably theta replication for several reasons. First, it was derived from a large plasmid. Janniere et al. (1993) reported that no RCM plasmid in gram-positive bacteria has been found greater than 8.8 kb in size, although recently pNG2, a 14.4-kb RCM plasmid from Corynebacterium diphtheriae, has been reported (Zhang et al., 1994). The homologous B. megaterium plasmids are much larger. Second, regions of the sequence are similar to other theta replicons, including a sequence similar to the chromosomal origin of B. subtilis (a theta replicon), a region of tandem direct repeats, a typical oriA region, possible DnaA boxes, and a sequence similar to a chromosomal (theta) Ter sequence. Third, no DNA or amino acid similarity was found to known RCM plasmids or their Rep proteins, in contrast to most grampositive RCM plasmids. Fourth, the direct repeats function as an incompatibility locus typical of theta plasmids. Fifth, there is similarity to a group of large, homologous, thetareplicating Lactococcus plasmids (Baum and Gilbert, 1992; Seegers et al., 1994). These plasmids have very homologous replicons with iterons, upstream palindromes, and large (.400 aa) ORFs coding for Rep proteins that bind to the iterons. It has been reported that the iterons of these compatible plasmids differ from one another by two bases (Seegers et al., 1994). Sixth, there is a region that Filutowicz and co-workers (1994) have observed in gram-negative theta plasmids immediately downstream from the iterons with the consensus CCACAGGNNNAA. The 2.4-kb origin sequence immediately downstream from the direct repeats (1636 –1651) was CCAgAacGNNNnnAA. Whether this is significant or coincidental cannot be determined since the function of the region is not known for any plasmid. Seventh, no requirement for RecA was observed, which is observed for some RCM plasmids. While these observations are not direct
proof of theta replication, they are consistent with known theta-replicating plasmids. The minimal replicon size is between 1385 and 1675 bp. Deletion 63 containing 578 bp upstream to the ORF replicated, while a further deletion of 300 bp closer to the ORF (deletion 100), which lacked the 235 sequence of a possible sigma A promoter, did not replicate (Fig. 4). Deletion clones 63 and 100 defined the minimal replicon. It is significant that deletion clone 100 (Fig. 4), which lacked part of the putative promoter, could not replicate. The 235 to 210 spacing is one nucleotide further than the optimal spacing, but this has been found in B. subtilis not to be as important as the base composition (Mountain, 1989). In front of the ORF is a good ribosomal binding site that agrees closely with the consensus ribosomal binding site of B. megaterium (Fliss and Setlow, 1985), further suggesting that the gene needs to be translated for replication to occur. The ability of the 2.4-kb clone to act in trans, replicating a 59 deleted ORF, demonstrated that the ORF is expressed and capable of replicating another plasmid. It also strongly suggests it codes for a Rep protein. The size of the ORF is similar to that reported for most theta Rep proteins (Janniere et al., 1993; Helinski, 1995). The carboxyl end does not seem to be essential for replication since a further deletion of 53 amino acids from the carboxyl end was still functional. We have not been able to confirm the location of the promoter as yet. However, the complementation tests showed that a product from the ORF does work in trans and therefore is expressed. Similar difficulty in determining a start site for the Rep protein of R6K has been encountered (Filutowicz et al., 1994). Unusual features of this replicon are the position of the tandem direct repeats inside the ORF, the small number and size of direct repeats compared with many other gram-positive and -negative bacterial replicons, and the lack of similarity of the ORF to any other prokaryotic proteins. Janniere et al. (1993) surveyed several replicons from gram-positive hosts with various tandem direct repeats, almost all of which flank the Rep protein gene and are 17–22 bp. When the 2.4-kb fragment was transformed
B. megaterium PLASMID REPLICON
into wild-type QM B1551, it was incompatible with at least the 165-kb plasmid (Fig. 3). It was then necessary to demonstrate that the direct repeats, the site of binding for a Rep protein in many theta replicons, were the site of incompatibility. Indeed, when a 93-bp fragment containing the direct repeats was introduced into a cell already harboring the whole replicon (pDS110), incompatibility was observed. These direct repeats, therefore, should be the equivalent of the iterons described previously for many theta plasmids (Helinski et al., 1995; Janniere et al., 1993; Seegers et al., 1994). There are a few examples of iterons internal to the Rep protein ORF. The internal iteron arrangement is similar to the lambda phage origin, where four tandem 19-bp direct repeats are within the O gene, a DnaA homologue (Furth and Wickner, 1983). Internal interons have recently been reported in B. thuringiensis homologous plasmids p43, p44, and p60, which have two direct repeats and two sets of 19-bp inverted repeats within the RepA protein gene (Baum and Gilbert, 1992). The large lactococcal plasmids (Seegers et al., 1994) have homologous replicons, but their eleven 22-bp direct repeats are upstream from the rep gene. No DNA or amino acid similarity of the B. megaterium replicon to any of the above replicons was found. The sequence of the replicon (Fig. 2) had other interesting features. Several potential DnaA boxes were near the direct repeats, similar to theta replicons of gram-negative bacteria (Filutowicz et al., 1994). All the DnaA boxes have one or two mismatches from the consensus TTATCCACA, which is common (Kiewiet et al., 1993). It has been reported that when the chromosomal origin, oriC, from B. subtilis was cloned, it caused instability (Moriya et al., 1988; Seiki et al., 1981; Yoshikawa and Wake, 1993), attributable to the DnaA boxes (Moriya et al., 1988). When the number of boxes was decreased or the sequences were mutagenized, transformation was possible. However, the transformants were often slow-growing, probably because of interference with chromosome replication. Interference with chromosomal DNA replication by the B. megaterium plasmid origin might explain the inhibition of sporula-
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tion, since completion of a round of DNA replication is required in the initiation of B. subtilis sporulation (Mandelstam and Higgs, 1974). In addition, four possible sites were found and are shown in Fig. 5A aligned with the binding site consensus for IHF from gram-negative bacteria (Freundlich et al., 1992). Goodrich and co-workers (1990) statistically analyzed 27 E. coli IHF functional sequences and reported that there was considerable deviation in binding sites adjacent to and within the consensus sequence. But in the sequence AATCAAnnAnTTA, the capitalized bases are found in more than 80% of the sites and the boldface positions occur 95–100% of the time. As shown in Fig. 5A, all those positions are conserved in the bp 1522 site, and all but one of the conserved sites are present in the bp 1339, 1846, and 2332 sites. The possible IHF site starting at bp 1522 is a good candidate for a functional site since it is upstream from the direct repeats and near two putative DnaA boxes. Binding studies must be done to test whether the sites are functional. Interestingly, three of the possible IHF binding sites had a DNA box directly downstream within 9 –12 bases (Fig. 5A), consistent with a model in which DnaA and IHF bind and bend DNA to promote Rep protein binding at the iterons (Filutowicz et al., 1994). TF1 from B. subtilis phage SP01 has homology to bacterial HU proteins and the E. coli IHF (Andera et al., 1994; Grove et al., 1997) and has been extensively studied, but to our knowledge, there is no report of an equivalent Bacillus protein. The B. megaterium origin sequence also contained a region with some homology to the oriC (Fig. 5B) and Ter regions of the B. subtilis chromosome (Fig. 5C, G. Wake, personal communication). In the replicon, TerBm is upstream from the ORF between the two palindromes. Regions homologous to Ter sequences of the B. subtilis chromosome have been reported on the Bacillus theta plasmid pLS20 replicon (Meijer et al., 1995). As can be seen, the homology of the TerBm is weaker and must be tested for function before any conclusions can be made. The cloned 2.4-kb fragment caused the formation of multimeric DNA, as do some RCM plasmids (Janniere et al., 1993; Nugent, 1989). Al-
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FIG. 5. Possible homologous IHF, oriC, and Ter I sequences in the 2.4-kb fragment (A) Alignment of putative IHF boxes found in the origin sequence with the consensus sequence. aFreundlich et al. (1992). bGoodrich et al. (1990). (B) Alignment of oriC of the B. subtilis chromosome with a palindromic region of the plasmid replicon. (C) Alignment of a possible termination (Ter) sequence from the 2.4-kb fragment with the Ter sequences of the B. subtilis chromosome and pLS20. cR. G. Wake (personal communication). dMeijer et al. (1995).
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B. megaterium PLASMID REPLICON
though the bands as shown in Fig. 3 (lanes A and C) were faint and somewhat diffuse, the upper bands increased sequentially approximately 6–7 kb, suggesting that they represented multimeric forms of pDS110, confirmed by restriction digestion (lane D). Subclones of theta plasmid pAMb1 from E. faecalis accumulate highly polymerized DNA unless a 2.3-kb region directly downstream from the RepE protein is present (Swinfield et al., 1991). This region includes a gene with homology to the recombinases of INV–RES invertase class and resolvase of Tn917 and pIP404, and is required for stability (Janniere et al., 1993). Lack of such a downstream region in the B. megaterium 2.4-kb fragment could be a possible reason for the instability observed as well as the highly polymerized DNA. Highly polymerized DNA formation has also been observed in another large (49-kb), theta-replicating Lactococcus plasmid, pRT2030 (Hill et al., 1991). Attempts to clone a larger fragment containing the complete ORF and adjacent elements have been complicated by the homology of the origin clone with the four other large plasmids. Of eight hybridizing clones from another gene library, none had the sequence of this clone (Stevenson, unpublished data). Since theta-replicating plasmids have been classified into classes A–D by whether they contain a typical oriA structure and/or have a functional Rep protein, or require DNA polymerase I (Filutowicz et al., 1994; Pansegrau et al., 1994), the B. megaterium replicon was transformed into B. subtilis mutants to test both its host range and dependence on Pol I or RecA (needed by some RCM plasmids) and better define its replicon. Plasmid pDS110 successfully replicated in B. subtilis 168, as well as polA and recA mutants. This suggests that the replicon requires neither DNA polymerase I, nor RecA (another test suggesting it is not a RCM plasmid), and can replicate in a related species. Structurally, it has the typical oriA of several gram-negative bacterial plasmids, that is, tandem direct repeats, a GC-rich area (position 1672–1696) followed by an AT-rich region (82% A 1 T at position 1696 –1744). However, it differs from these in location of oriA within a large ORF. Computer searches have shown that the origin has no obvious similarity with any
known Rep proteins in class A, nor those of the Class E pLS20. In summary, the replicon described above has homology with three other plasmids from the same cell, is unstable in its cloned, minimal form, and posesses direct repeats within the Rep gene that cause incompatibility with its replicon. The product of the ORF is probably a Rep protein, which can act in trans. It is one of few theta plasmid replicons described in Bacillus and also has the capability to replicate in B. subtilis. The B. megaterium replicon may, therefore, define a new class of plasmid replicons. ACKNOWLEDGMENTS This work was supported in part by Grant 1R15 GM49440-01 from the National Institutes of Health (P.S.V.) and NATO Grant CRG.940770 (P.S.V.). The authors thank Dr. R. Tewari and Yansheng Zhou for critical reading of the manuscript and Gary Baisa for assisting in some of the plasmid constructions.
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