Genetic Manipulation of Bacillus Methanolicus, a Gram-Positive, Thermotolerant Methylotroph DAVID CUE,1† HONG LAM,1 RICHARD L

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1997, p. 1406–1420 Vol. 63, No. 4 0099-2240/97/$04.00ϩ0 Copyright ᭧ 1997, American Society for Microbiology

Genetic Manipulation of Bacillus methanolicus, a Gram-Positive, Thermotolerant Methylotroph DAVID CUE,1† HONG LAM,1 RICHARD L. DILLINGHAM,2 RICHARD S. HANSON,2 1,3 AND MICHAEL C. FLICKINGER * Biological Process Technology Institute1 and Department of Biochemistry,3 University of Minnesota, St. Paul, Minnesota 55108, and Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 554552

Received 17 September 1996/Accepted 29 January 1997

We report the ﬁrst genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efﬁciency from 7- to 11-fold. Two low-copy-number Escherichia coli-B. methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 carries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus.

Methylotrophic bacteria are capable of using reduced one- olicus, genetic studies of this organism have not been possible carbon compounds as carbon and energy sources. These or- because of the lack of an effective gene delivery system. ganisms are believed to possess great potential for use in the In this report, we describe the genetic transformation of B. fermentation industry for the production of single-cell protein, methanolicus protoplasts with integrational and multicopy polysaccharides, amino acids, and vitamins (8, 17, 18, 35). plasmid vectors. We also describe the isolation and character- Methanol is attractive as a fermentation substrate due to its ization of bifunctional plasmids that are stably maintained in low cost, availability, and water solubility (8, 13, 17, 35). both Escherichia coli and B. methanolicus. Bacillus methanolicus (1, 29) is a gram-positive, thermotolerant, facultative methylotroph that offers several advantages MATERIALS AND METHODS over other methylotrophs for industrial fermentations. The optimal growth temperature of B. methanolicus (50 to 53ЊC) Bacterial strains and plasmids. The bacterial strains, plasmids, and bacterio- ␣ significantly reduces fermentor cooling costs. The organism phage used in this work are listed in Table 1. The E. coli strains DH5 and DM1 served as host strains for the routine cloning and maintenance of plasmids. E. coli also possesses a NAD-linked methanol dehydrogenase that DH5␣ MCR was the host strain used for the construction of plasmid libraries of allows the bacterium to produce higher yields of ATP per mole B. methanolicus MGA3 and bacteriophage RD-1 DNA. B. methanolicus NOA2- of methanol oxidized than is possible for gram-negative methy- 13A5-2 (strain 13A5-2) was used for plasmid transformation experiments. lotrophs. Additionally, auxotrophic mutants of B. methanolicus The E. coli-Bacillus subtilis shuttle plasmids pEN1 (6) and pHP13 (11) have been described. pDQ499 was constructed by cloning a 1.3-kb Neor gene from can be readily isolated, whereas auxotrophs of gram-negative pBEST501 (14) and a 340-bp B. methanolicus lysC promoter-bearing fragment methylotrophs have proven difficult to isolate (17, 35). into pUC18; pDQ499 cannot be maintained in B. methanolicus for lack of a Classical mutagenesis and selection strategies have been em- functional origin of DNA replication. pDQ503 is pDQ499 carrying a 1-kb frag- ployed to isolate B. methanolicus mutants that overproduce ment of the B. methanolicus lysC gene (5). The plasmid pAA8671 (30) was the source of lysC DNA fragments. Plasmids pDQ507 and pDQ508 were constructed L-lysine (13, 29). Additionally, B. methanolicus genes encoding by cloning 3- and 4-kb HindIII fragments, respectively, from endogenous B. lysine biosynthetic enzymes have been cloned and sequenced methanolicus plasmids into pDQ499. (22, 30). While these studies have yielded important informa- Growth media and reagents for protoplast transformations. B. methanolicus Њ tion regarding the regulation of lysine synthesis in B. methan- was grown at 50 C on an orbital shaker (Labline) at 330 rpm in either MV medium (29) supplemented with 0.15 mM methionine and threonine, tryptone soytone broth (TSB; 1.5% tryptone, 0.5% soytone, 86 mM NaCl [pH 7.0]), or MYTM (MV medium plus threonine, methionine, and 0.1% yeast extract). E. coli strains were grown in Luria broth (27) at 37ЊC on an orbital shaker at 300 * Corresponding author. Mailing address: Biological Process Tech- rpm. Unless stated otherwise, solid media contained 1.5% agar (Difco). nology Institute, 240 Gortner Laboratory, 1479 Gortner Ave., St. Paul, Neomycin, chloramphenicol, and erythromycin were added to B. methanolicus media, when appropriate, at final concentrations of 5, 10, and 1 ␮g/ml, respec- MN 55108-6106. Phone: (612) 624-9259. Fax: (612) 625-1700. E-mail: tively. Ampicillin, neomycin, and chloramphenicol were added to E. coli media to mfl[email protected]. 100, 25, and 30 ␮g/ml, respectively. † Present address: Department of Microbiology, University of Min- For transformation of B. methanolicus protoplasts, cells were grown in SOB nesota, Minneapolis, MN 55455. (27) containing 0.25 M sucrose (pH 6.5). Regeneration medium used for the

1406 VOL. 63, 1997 GENETIC MANIPULATION OF BACILLUS METHANOLICUS 1407

TABLE 1. Bacterial strains, plasmids, and bacteriophage DNA isolations and Southern analysis. Plasmid DNA was isolated from E. coli and B. methanolicus by the alkaline lysis method (5, 27). Chromosomal DNA was Strain, plasmid, Source or isolated from B. methanolicus as described previously (5). Relevant characteristic(s) or phage reference Bacteriophage DNA was purified from concentrated lysates as follows. DNase I and RNase A were added to the lysates at concentrations of 25 and 250 ␮g/ml, E. coli respectively, and incubated for 30 min at room temperature. EDTA (25 mM), DH5␣ recA1 hsdR17 Gibco BRL sodium dodecyl sulfate (0.5%) and proteinase K (50 mg/ml) were then added, DM1 dam-13::Tn9 dcm mcrB hsdR Gibco BRL and the mixture was incubated at 56ЊC for 1 h. The proteinase-treated lysates DH5␣-MCR mcrA ⌬(mrr-hsdRMS-mcrBC) recA1 Gibco BRL were then extracted three times with phenol-chloroform and twice with chloroform. DNA was recovered by ethanol precipitation and redissolved in Tris- EDTA (TE). Approximately 750 ␮g of bacteriophage DNA could be recovered B. methanolicus from 1 liter of infected cells. MGA3 Wild type 29 Ϫ Ϫ Supercoiled plasmids and restriction fragments were purified from agarose NOA2-13A5-2 Thr Met ; 2-aminoethyl-L-cysteine 13 gels by the method of Vogelstein and Gillespie (37). Southern analyses (32) were resistant performed by use of the Genius labeling-detection system (Boehringer Mann- heim) in accordance with the manufacturer’s recommendations. Plasmids Recombinant DNA techniques. DNA manipulations were performed by stan- pAA8671 pUC18Cm containing the B. 30 dard techniques (27) with enzymes and reagents purchased from Gibco-BRL and methanolicus lysC-PstI fragment Boehringer Mannheim. DNA methylation in vitro was performed with dam methylase and S-adenosyl-L-methionine (New England Biolabs). Plasmid meth- pBEST501 Neor cassette vector 14 r ylation was verified by restriction of modified plasmids with Sau3AI, MboI, and pEN1 E. coli-B. subtilis shuttle plasmid (Cm )6 BclI. DNA sequencing was performed by Sequetech Corporation, Mountain r pHP13 E. coli-B. subtilis shuttle plasmid (Cm 11 View, Calif. Ermr) Nucleotide sequence accession number. The GenBank and EMBL accession pDQ499 Apr Neor 5 number of the primary nucleotide sequence of pDQ507 (see Fig. 11) is U81371. pDQ503 E. coli-B. methanolicus integrative 5 Transformation of B. methanolicus protoplasts. B. methanolicus was grown at Њ 8 shuttle plasmid (Apr Neor) 50 C in SOB-sucrose medium to an OD600 of 0.5 to 0.6 (approximately 10 ϫ Њ pDQ507 E. coli-B. methanolicus shuttle plasmid This study CFU/ml). Cells were harvested by centrifugation at 1,900 g for 15 min at 23 C. The cell pellets were suspended in 1/10 volume of SMMCB containing 1 ␮gof (Apr Neor) lysozyme per ml. Cells were incubated at 42ЊC, with aeration, for 30 min. Pro- pDQ508 E. coli-B. methanolicus shuttle plasmid This study toplast formation was monitored by phase-contrast microscopy. Protoplasts were r r (Ap Neo ) harvested by centrifugation at 1,500 ϫ g for 15 min. The pelleted protoplasts pDQ531 pHP13 containing lysC-PstI This study were resuspended in 1/10 volume of SMMCB and recentrifuged. The washed pDQ539 E. coli-B. methanolicus cloning vector This study protoplasts were then resuspended in 1/50 of the original volume of SMMCB. pDQ541 E. coli-B. methanolicus cloning vector This study One-hundred-microliter portions of the protoplast suspensions were trans- pDQ543 E. coli-B. methanolicus cloning vector This study ferred to 1.5-ml microcentrifuge tubes. Twenty microliters of plasmid DNA in pUC18 Cloning vector 39 SMMC was gently mixed with the protoplasts. One milliliter of 40% PEG solution was added to the protoplast-DNA mixtures, and the suspensions were mixed and allowed to stand at room temperature for 3 min. One-half milliliter of Bacteriophage Virulent B. methanolicus bacteriophage This study SMMCB was then added to each tube, and the protoplasts were harvested by RD-1 centrifugation at 2,000 ϫ g for 5 min. The pelleted protoplasts were washed once with 1.5 ml of SMMCB and then suspended in 0.5 ml of SOB-sucrose or (for transformations with pEN1 and pHP13) in 0.5 ml of SOB-sucrose containing 0.125 ␮g of chloramphenicol per ml. Cultures were then incubated, with aeration, at 50ЊC for 2 h and then plated on regeneration media containing the plating of protoplasts contained 0.75% tryptone, 0.25% soytone, 0.1% yeast appropriate antibiotic. For dilution of transformed cultures prior to plating, dilutions were performed at 45ЊC in a suspension of protoplasts that had been extract, 43 mM NaCl, 10 mM MgCl2, 0.25 M sucrose, 0.01% bovine serum albumin, 0.75% agar, and 1% gelatin at pH 6.5. SMMC (4) contained 0.5 M subjected to the transformation in the absence of DNA. Regeneration plates were incubated at 50ЊC for 6 days. To minimize dehydration of selection plates, sucrose, 20 mM disodium maleate, 10 mM MgCl2, and 10 mM CaCl2 (pH 6.5). SMMCB was SMMC in 1ϫ SOB. The polyethylene glycol (PEG) solution con- plastic bags were placed over stacks of four to six inverted plates. tained 40% (wt/vol) PEG (molecular weight, 8,000; Sigma) in SMMC. Transfection of protoplasts with bacteriophage RD-1 DNA was performed Bacteriophage methods. For determining titers of lysates of bacteriophage similarly. Detection of transfection was accomplished by mixing transfected pro- RD-1, bacterial plating cultures were prepared by diluting (1:10) a 12- to 16-h toplasts with 0.2 ml of log-phase B. methanolicus 13A5-2 cells. A 2.5-ml volume culture of strain 13A5-2 into TSB. Cultures were grown at 50ЊC to an optical of molten SOB-sucrose (pH 7.0) containing 0.6% agar was added to the protoplast-cell mixtures and poured onto plates containing SOB-sucrose (pH 7.0)– density at 500 nm (OD500) of 0.7 to 0.9. Two-tenths milliliter of the bacterial Њ cultures was transferred to borosilicate tubes (12 by 100 mm). RD-1 lysates were 0.8% agar plates. The plates were incubated overnight at 42 C. diluted in SM medium (27), and 0.1-ml portions of a diluted lysate were mixed Estimation of plasmid copy numbers. Genomic DNA was isolated from de- with the bacterial cells and incubated at 42ЊC for 15 min. A 2.5-ml volume of rivatives of strain 13A5-2 carrying each of the recombinant plasmids, i.e., molten (55ЊC) TSB containing 0.6% agar was then added to the tubes, and the pDQ507, pDQ508, and pHP13. The plasmids were digested with PstI and then contents were poured over the surface of tryptic soy agar (TSA) plates. The serially diluted in TE. The serially diluted plasmids (typically six different dilu- plates were incubated overnight at 42ЊC before plaques were counted. tions) were electrophoresed through 0.8% agarose, blotted, and probed with Large-scale lysates of bacteriophage RD-1 were prepared as follows. Twenty pAA8671. milliliters of an overnight culture of strain 13A5-2 was diluted into 500 ml of This plasmid probe hybridized to the 2.2-kb PstI fragment containing the B. Њ methanolicus chromosomal lysC gene and to the pUC18 portions of the three MYTM and grown at 50 C until the cultures reached an OD500 of 0.3. At this time, the culture was infected with 107 PFU of RD-1 and then incubated at 42ЊC recombinant plasmids. The relative intensities of the hybridizing bands (within Ӎ the linear range of detection) were determined by scanning densitometry. Hy- until cell lysis occurred ( 5 h). Ten milliliters of CHCl3 was added to the culture, and the culture was incubated at 42ЊC for 15 min. Cellular debris was removed bridizations between pAA8671 and pDQ531 and pAA8671 and pDQ552 were by two centrifugations at 8,000 ϫ g for 10 min at 5ЊC. also performed as controls for possible differential hybridization of the probe to Crystalline NaCl was added to the clarified lysate to a final concentration of 1 lysC and pUC18 sequences. M. The mixture was incubated on ice for 1 h and then centrifuged at 6,000 ϫ g for 30 min. Solid PEG 8000 (Sigma) was added to the resulting supernatant to a RESULTS final concentration of 10%, and the mixture was incubated on ice for 60 min. The bacteriophage were pelleted by centrifugation at 10,000 ϫ g for 90 min, sus- Transfection of B. methanolicus protoplasts with bacterio- Њ pended in 10 ml of SM medium, and stored overnight at 5 C. Debris was phage RD-1 DNA. Previous attempts by our laboratories to removed from the suspension by centrifugation at 2,000 ϫ g for 15 min. The resulting supernatant was centrifuged in a Beckman SW40 rotor at 22,000 rpm genetically transform B. methanolicus with foreign DNA by for3hat5ЊC. Bacteriophage was resuspended from the resulting pellet by electroporation had been unsuccessful. A number of factors overlaying the pellets with 3 ml of SM medium and allowing the tubes to stand could have contributed to those difficulties, including host re- Њ overnight at 5 C. The resuspended bacteriophage was transferred to 1.5-ml striction of transforming DNA, poor expression of selectable microcentrifuge tubes and stored at 5ЊC. RD-1 lysates prepared in this manner typically yielded 1013 PFU/ml. Concentrated lysates were stable at 5ЊC for at least markers, the inability of B. methanolicus to replicate plasmid 1 year. DNA, an inefficient homologous recombination system, and 1408 CUE ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 2. Transfection of B. methanolicus with TABLE 4. Transformation of B. methanolicus protoplasts bacteriophage RD-1 DNA with plasmid DNA

No. of No. of infectious Antibiotic No. of transformants/ RD-1 DNA Plasmid DNA Cells infectious centers/␮gof selection ␮gofDNA (␮g) centers DNA pEN1 Chloramphenicol 32 Protoplasts 0 0 0 pDQ503 Neomycin 24 0.3 372 1.24 ϫ 103 MGA3 plasmid library Neomycin 1.76 ϫ 103 0.6 624 1.04 ϫ 103 RD-1 plasmid library Neomycin 1.02 ϫ 103 1.2 791 6.60 ϫ 102 pHP13 Chloramphenicol 3.41 ϫ 104 5.0 Ͼ2 ϫ 103 Ͼ4.00 ϫ 102 pDQ507 Neomycin 1.78 ϫ 103 pDQ508 Neomycin 1.63 ϫ 105 Mock protoplasts 0 0 0 2.4 0 Ͻ0.5

in preparations of mock protoplasts remained relatively con- stant (Table 3). the failure to physically introduce sufficient amounts of DNA We therefore varied a number of parameters in an effort to into bacterial cells. We reasoned that we could circumvent find conditions that increased the efficiency of CFU recovery. most of these potential barriers to transformation by trans- The parameters included the composition of regeneration me- forming cells with bacteriophage DNA that had been isolated dia, incubation temperature during protoplast generation and from an infected B. methanolicus culture. Therefore, subse- recovery, the pH of growth media, protoplast generation buffer quent to these attempts, a bacteriophage capable of infecting and regeneration media, the incubation temperature during B. methanolicus cultures was sought and isolated. This phage PEG-mediated transformation, and culture dilution and plat- was designated RD-1. ing. Varying these conditions had only modest effects on pro- Bacteriophage DNA was isolated from a lysine-producing toplast recovery. These effects were not reproducible. A major culture of B. methanolicus 13A5-2 that had been infected with impediment to finding conditions that optimized CFU recov- RD-1. The purified RD-1 DNA was then used to transfect ery was that B. methanolicus protoplasts were extremely sen- protoplasts of 13A5-2 by a procedure similar to that developed sitive to dilution, thus making accurate quantification of CFU for transfection of Bacillus stearothermophilus (34) and B. sub- recovery difficult. The best conditions found for successful tilis (26) protoplasts. transformation of B. methanolicus (see Materials and Meth- Table 2 summarizes one of our RD-1 transfection experi- ods) typically result in recovery of only 1 to 2% of the original ments. The detection of infectious centers requires the addi- viable cell number (CFU). tion of RD-1 DNA, and the number of infectious centers Transformation of B. methanolicus protoplasts with plasmid produced increased when higher levels of DNA were used for DNA. We successfully transformed strain 13A5-2 with two transfection. Attempts to transfect cells treated identically but plasmids, pDQ503 and pEN1 (Table 4). Neither plasmid has a without lysozyme (sterile distilled water was substituted for recognition site for the B. methanolicus restriction endonucle- lysozyme) yielded no infectious centers. These results indi- ase BmeTI (5) and thus would not have been susceptible to cated that our protoplast transfection procedure resulted in BmeTI restriction upon introduction into bacterial cells. Selec- the transfer of RD-1 DNA into B. methanolicus, and thus this tion for pDQ503 transformants was accomplished by plating procedure should also be useful for the introduction of plasmid on regeneration agar containing 2.5 ␮g of neomycin per ml. A DNA into this thermotolerant methylotroph. concentration of chloramphenicol of 5 ␮g/ml was used to select A barrier to adapting the transfection procedure for the for pEN1 transformants. transformation of protoplasts with plasmid DNA was the inef- pDQ503 is a derivative of pUC18 that carries 1.3 kb of the ficient recovery of viable cells that had been exposed to the B. methanolicus lysC gene and the gene coding for kanamycin- transfection procedure (Table 3). The incubation of B. meth- neomycin nucleotidyltransferase. A single copy of this gene anolicus cells with lysozyme decreased the number of CFU by confers a high level of neomycin resistance (Neor) upon B. Ϫ 10 3. Subsequent handling steps (harvesting of protoplasts, subtilis (14). The E. coli-B. subtilis shuttle vector pEN1 carries PEG addition, and washing of protoplasts, etc.) further re- the replication origin of the thermotolerant Bacillus coagulans duced the number of CFU recovered. The apparent survival plasmid pBC1 and a gene encoding for chloramphenicol frequency of cells that had been subjected to the entire regi- acetyltransferase (cat). This plasmid has been successfully in- men was approximately 1/105. In contrast, the number of CFU troduced into a number of gram-positive organisms (6). Transformations performed with pDQ503 and pEN1 were very inefficient (Table 4), yielding only 24 and 32 transformants, respectively, per ␮g of plasmid DNA. Total (genomic) TABLE 3. Survival of B. methanolicus during protoplast transfection steps DNA was isolated from six of the pDQ503 transformants and the nontransformed parent strain, and Southern blotting was a Transfection step CFU/ml performed to verify the presence of pDQ503 in the Neor iso- Harvesting of cells...... 3.15 ϫ 107 lates. We did not anticipate replication of pDQ503 in B. meth- Suspension in 1/10 volume of SMMCB...... 5.88 ϫ 108 anolicus since the plasmid lacks an origin of DNA replication Lysozyme incubation, 60 min...... 2.90 ϫ 105 that is functional in Bacillus spp. However, since pDQ503 Mock lysozyme incubation, 60 min...... 1.88 ϫ 109 carries bacterial DNA, transformation could result from plas- Lysozyme incubation, 120 min...... 6.13 ϫ 105 mid integration into the B. methanolicus chromosome (Fig. 1). ϫ 9 Mock lysozyme incubation, 120 min...... 2.78 10 By using the pDQ503 DNA probe to hybridize to the same ϫ 3 Post-PEG treatment (protoplasts) ...... 7.85 10 PstI restriction fragments of DNAs isolated from each of the Post-PEG treatment (mock protoplasts)...... 1.08 ϫ 109 six Neor transformants, a band of 2.7 kb and a second band of a Colonies were counted after 48 h of incubation at 50ЊC. 2.2 kb (Fig. 2, lanes 10 and 18) were detected. Detection of the VOL. 63, 1997 GENETIC MANIPULATION OF BACILLUS METHANOLICUS 1409

FIG. 2. Southern analysis of pDQ503 transformants. Chromosomal DNA was isolated from strain 13A5-2 (lanes 4, 8, 12, and 16) and two 13A5-2::pDQ503 isolates (lanes 2, 3, 6, 7, 10, 11, 14, and 15). pDQ503 isolated from E. coli is also shown (lanes 1, 5, 9, and 13). The DNAs were digested with BsmI (lanes 1 to 4), BamHI (lanes 5 to 8), PstI (lanes 9 to 12), or HindIII (lanes 13 to 16). Southern hybridization was performed with pDQ503 as the probe. The sizes of the pDQ503 restriction fragments are 5.3 kb for BsmI, 4.3, 0.6, and 0.4 kb for BamHI, 2.7 and 2.6 kb for PstI, and 5.3 kb for HindIII.

FIG. 1. Integration of pDQ503 into lysC-PstI. (A) Partial restriction map of pDQ503. The integrative plasmid pDQ503 carries 340- and 989-bp fragments of lysC-PstI DNA (indicated as lysCЈ in the figure). The positions of restriction sites the pDQ503 transformants. Instead, two new fragments of 5.7 are numbered beginning with the HindIII site. (B) Partial restriction map of the 2.2-kb B. methanolicus PstI restriction fragment (lysC-PstI) that carries the lysC and 4.9 kb were detected. The latter was of the size predicted gene The positions of the restrictions sites (in kilobases) are based on the work for the BsmI restriction fragment comprised of lysC-PstI and of Schendel and Flickinger (30). (C) Restriction map of the lysC-PstI region of pDQ503 DNA. The former fragment would be comprised of pDQ503 transformants. lysC-PstI DNA is represented by the filled segments; pDQ503 and chromosomal DNA 3Ј to lysC-PstI. The 2.0-kb pDQ503 DNA is represented by the hatched segments. The point of the single crossover event that leads to integration of pDQ503 within lysC was arbitrarily BsmI fragment that was common to digests of strain 13A5-2 placed at bp 1800 of lysC-PstI. Abbreviations: B, BamHI; Bs, BsmI; H, HindIII; and strain 13A5-2::pDQ503 DNAs contained lysC-PstIatbp23 P, PstI. to 213 and chromosomal DNA 3Ј to lysC-PstI. Plasmid DNA was also isolated from strain 13A5-2 and six pDQ503 transformants. pDQ503 did not hybridize to plasmid DNA isolated from any of these strains. Plasmid DNA that 2.2-kb restriction fragment was due to hybridization of the hybridized to either pEN1 or pUC18 was readily isolated from probe with the PstI fragment that carries the lysC gene. Hy- pEN1 transformants (data not shown). Neither pEN1 nor bridization to this fragment is also observed for PstI-digested pUC18 hybridized to DNA from strain 13A5-2. DNA isolated from the nontransformed parent strain, 13A5-2 These results established successful transformation of B. (Fig. 2, lane 12). The larger fragment should be a doublet of methanolicus with the integrational plasmid pDQ503 and the 2.6- and 2.7-kb restriction fragments that result from PstI di- multicopy plasmid pEN1 but, unfortunately, at a low fre- gestion of pDQ503. quency. Additionally, these results indicated that the Neor and Further analysis by restriction digests was performed with Cmr genes could both serve as selectable markers at 50ЊC for DNA isolated from two pDQ503 transformants. Figure 2 the genetic manipulation of B. methanolicus. shows a Southern blot of genomic DNAs that had been digested with BsmI (lanes 2 to 4), HindIII (lanes 6 to 8), PstI (lanes 10 to 12), and BamHI (lanes 14 to 16) and then probed with pDQ503. The predicted and observed hybridizing frag- TABLE 5. Predicted restriction fragments resulting from digestion ments are listed in Table 5. of genomic DNA from B. methanolicus 13A5-2 and The results of the Southern analyses confirmed that the Neor 13A5-2::pDQ503 DNA isolates carried PDQ503 integrated within the lysC gene. For Size(s) of fragment(s) (kb) for strain: example, pDQ503 and lysC-PstI each possess a single BsmI Restriction 13A5-2 13A5-2::pDQ503 restriction site. Cleavage of pDQ503 with BsmI (Fig. 2, lane 1) enzyme yields a single 5.3-kb restriction fragment. BsmI cleavage of Predicted Observed Predicted Observed strain 13A5-2 DNA yields two detectable restriction fragments (lane 4). One of the strain 13A5-2 fragments is comprised of BsmI Ͼ2.0, Ͼ0.2 5.0, 2.0 4.9, Ͼ2.2, Ͼ0.2 4.7, 5.7, 2.0 lysC-PstI at bp 23 to 213 and chromosomal DNA located 5Ј to BamHI 0.8, Ͼ1.1, Ͼ0.3 0.7, 9.3, 6.8 4.3, 0.8, 0.6, 0.4, 4.4, 0.7, 9.3, Ͼ Ͼ lysC. The second fragment is comprised of lysC-PstIatbp214 1.1, 0.3 6.8 to 2223 and chromosomal DNA located 3Ј to this segment. PstI 2.2 2.1 2.7, 2.6, 2.2 2.7, 1.9 HindIII 0.8, 0.4, Ͼ1.0 0.7, 2.7 3.7, 0.8, 0.4, Ͼ2.7 3.7, 4.4 The 5.0-kb fragment is absent in BsmI digests of DNA from 1410 CUE ET AL. APPL.ENVIRON.MICROBIOL.

Construction of E. coli-B. methanolicus shuttle vectors. Two previously described E. coli-B. subtilis shuttle vectors, pEN1 and pHP13, were evaluated for their potential to serve as B. methanolicus-B. subtilis shuttle vectors. In addition, B. methanolicus shuttle vectors were constructed by cloning either B. methanolicus or RD-1 DNA fragments into pDQ499. This plasmid is essentially pUC18 carrying the same Neor marker as that of pDQ503. pDQ499 is unable to transform B. methanolicus since it lacks a functional Bacillus replication origin and signiﬁcant homology to the bacterial chromosome. Three E. coli-B. methanolicus shuttle plasmids, pDQ506, pDQ507, and pDQ508, were constructed by cloning endogenous B. methanolicus DNA fragments into pDQ499. These plasmids were isolated as a result of an attempt to clone a replication origin from bacteriophage RD-1. Bacteriophage DNA was isolated from an RD-1-infected culture of B. methanolicus 13A5-2, digested with HindIII, and ligated into HindIII-digested pDQ499. The ligated DNAs were used to transform E. coli DH5␣ to ampicillin and neomycin resistance. E. coli transformants were scraped from the surfaces of agar plates and used to inoculate Luria broth containing antibiotics. Plas- mid DNA was isolated from the pooled E. coli clones and used to transform strain 13A5-2 to neomycin resistance, resulting in approximately 1,000 Neor transformants. Six of these 13A5-2 transformants were inoculated into TSB containing 5 ␮g of neomycin per ml. Plasmid DNA was isolated from these cultures after overnight incubation and used for the transformation of E. coli DH5␣. Plasmid DNA was then isolated from the E. coli transformants, and the restriction patterns were analyzed. Four of the six B. methanolicus-transforming plasmids that were recovered were found to carry the same 4.0-kb HindIII fragment cloned into pDQ499. A partial restriction map of one plasmid, pDQ508, is shown in Fig. 3. One of the recovered plasmids, pDQ507 (Fig. 3), was found to carry a 3.1-kb HindIII fragment. A third plasmid, pDQ506, contained a 3.5-kb HindIII insert. Southern blotting was performed with either pDQ506, pDQ507, or pDQ508 as a probe to determine if the cloned HindIII fragments originated from RD-1 DNA. Surprisingly, we were unable to detect hybridization of any of the plasmids to bacteriophage RD-1 DNA. Instead, we found that the three plasmids all carried distinct HindIII fragments of B. methanolicus DNA (Fig. 4 and 5). This indicates that our preparation of bacteriophage DNA was contaminated with a small amount of host DNA that was serendipitously cloned into pDQ499. The HindIII fragments carried on these plasmids were found to originate from different sources since no cross-hybridization between the cloned inserts was detected. Maintenance of pDQ508 and pDQ507 as plasmids in B. meth- FIG. 3. Partial restriction maps of plasmids pDQ499, pDQ507, and pDQ508. pDQ499 is a pUC18 derivative that carries the Neor gene from pBEST501 (14). anolicus. To verify that pDQ508 was capable of replication as pDQ499 cannot be maintained in B. methanolicus for lack of a functional DNA a plasmid in B. methanolicus, pDQ508 isolated from DH5␣ replication origin or signiﬁcant homology to the bacterial chromosome. pDQ507 (pDQ508) was used to transform B. methanolicus. Plasmid and pDQ508 contain 3.1- and 4.0-kb, HindIII restriction fragments, respectively, DNA isolated from a new transformant and the original of B. methanolicus DNA. The cloned HindIII fragments (labeled as pBM1 ori and pmr-1) allow maintenance of pDQ507 and pDQ508 as plasmids in B. meth- 13A5-2(pDQ508) isolate was analyzed by Southern blotting, anolicus. Abbreviations: B, BamHI; Bm, BmeTI; C, ClaI; E, EcoRI; Ec, EcoRV; with pDQ508 isolated from E. coli as the probe (Fig. 4). The H, HindIII; N, NciI; P, PstI; X, XbaI. same restriction patterns were observed for plasmid DNAs isolated from E. coli and both B. methanolicus transformants, indicating that the plasmid was structurally stable in both bacterial species. slower-migrating band in lane 2 may be the open circular form Plasmid pDQ508 was found to hybridize to both plasmid and of the plasmid or a plasmid dimer. genomic DNA isolated from strain 13A5-2. Figure 4 shows the DNA from pBM1 was not detected in plasmid DNA isolated hybridization of pDQ508 to plasmid DNA isolated from strain from pDQ508 transformants of strain 13A5-2 (Fig. 4, lane 3). 13A5-2 (lane 2). These hybridizing bands likely represent su- Instead, the probe hybridized to three bands of 13A5-2 percoiled forms of the endogenous plasmid from which (pDQ508) plasmid DNA. The two largest bands (16.5 and 8.4 pDQ508 was derived. We estimated the size of the endogenous kb) are the sizes predicted for dimeric and monomeric forms, plasmid (hereafter referred to as pBM1) to be 17 kb. The respectively, of pDQ508. Hybridization to plasmid bands of the VOL. 63, 1997 GENETIC MANIPULATION OF BACILLUS METHANOLICUS 1411

and the two B. methanolicus transformants were then analyzed by Southern blotting, with pDQ507 as the probe. Plasmids isolated from the strain 13A5-2 transformants were indistin- guishable, indicating that the plasmid is structurally stable in B. methanolicus. Figure 5 shows the hybridization of pDQ507 to genomic (lane 2) and plasmid (lane 3) DNA isolated from nontransformed strain 13A5-2. pDQ507 did hybridize to an amorphous smear of undigested genomic DNA isolated from strain 13A5- 2 (lane 2). These results suggest that pDQ507 was derived either from a very large endogenous plasmid or from chromosomal DNA. Since we have yet to conclusively establish whether the B. methanolicus DNA fragment carried on pDQ507 is of chromosomal or extrachromosomal origin, we will refer to the endogenous genetic element as pmr-1, i.e., putative methanolicus replicon. Plasmid DNA that hybridized with pDQ507 could be readily isolated from strain 13A5-2(pDQ507) transformants (Fig. 5, lanes 4, 8, and 12). Restriction of these plasmid preparations with HindIII (lane 8) produced two restriction fragments that hybridized with pDQ507. The larger (4.3-kb) fragment (Fig. 5, lane 8) is the vector (pDQ499) portion of pDQ507. The smaller (3.1-kb) fragment (lane 8) is the B. methanolicus DNA fragment that was cloned into pDQ499 to construct pDQ507. FIG. 4. Southern analysis of pDQ508 transformants. Plasmid DNA was isolated from E. coli DH5␣(pDQ508) (lanes 1 and 6), strain 13A5-2 (lanes 2 and 7), This fragment is also apparent in HindIII-digested genomic strain 13A5-2(pDQ508) (lanes 3 and 8), and two Neos derivatives of 13A5-2 DNA from strain 13A5-2 (lane 6). The 4.3-kb fragment is not (pDQ508) (lanes 4, 5, 9, and 10). Undigested (lanes 1 to 5) and HindIII-digested present in digests of strain 13A5-2 DNA. (lanes 6 to 10) plasmids were blotted and probed with pDQ508. The open arrow PstI digestion of plasmid DNA from 13A5-2(pDQ507) (Fig. adjacent to lane 6 indicates the 4.3-kb pDQ499 portion of pDQ508. The ﬁlled ␣ arrow indicates the 4.0-kb pBM1 HindIII fragment that was cloned into pDQ499. 5, lane 12) and from DH5 (pDQ507) (lane 13) produced The numbers adjacent to the supercoiled DNA ladder (Gibco-BRL) in lane 11 7.4-kb restriction fragments that hybridized to the DNA probe. indicate the size of each marker in kilobases.

same apparent size was evident in preparations of the plasmid from E. coli (Fig. 4, lane 1). A 5.5-kb DNA band was present in undigested (Fig. 4, lane 3) and HindIII-digested (Fig. 4, lane 8) plasmid DNA isolated from 13A5-2(pDQ508). The 5.5-kb band is likely comprised of single-stranded pDQ508 DNA, which suggests rolling-circle replication of this plasmid in B. methanolicus (see below). HindIII cleavage of pDQ508 isolated from E. coli (Fig. 4, lane 6) or B. methanolicus (lane 8) produced two restriction fragments that hybridized to pDQ508. One of the HindIII fragments (lane 6) is the size predicted (4.3 kb) for HindIII-restricted pDQ499. This fragment is absent from digests of plasmid DNA isolated from the untransformed parent strain (lane 7). The 4.0-kb HindIII fragment is present in digests of strain 13A5-2 DNA as well. This 4.0-kb fragment is the HindIII restriction fragment that was cloned from pBM1 into pDQ499. Undigested pDQ508 from B. methanolicus comigrated with pDQ508 isolated from E. coli (Fig. 4, lanes 1 and 3). Addition- ally, analysis of B. methanolicus pDQ508 transformants and three Neos derivatives of 13A5-2(pDQ508) (Fig. 4, lanes 4, 5, 9, and 10) demonstrated that pBM1 is absent from pDQ508 transformants. These results support the contention that pDQ508 is maintained as an autonomously replicating plasmid in B. methanolicus. Experiments similar to those described above for pDQ508 were performed to establish the origin of the B. methanolicus FIG. 5. Southern analysis of pDQ507 transformants. Genomic DNA (lanes 2, 6, and 10) and plasmid DNA were isolated from the nontransformed parent DNA fragment carried by pDQ507 and to verify that pDQ507 strain 13A5-2. Plasmid DNA was isolated from 13A5-2(pDQ507) (lanes 4, 8, and was also maintained as a plasmid in B. methanolicus. Plasmid 12) and from DH5␣(pDQ507) (lanes 5, 9, and 13); uncut (lanes 2 to 5), pDQ507 was isolated from E. coli and used to transform strain HindIII-digested (lanes 6 to 9), and PstI-restricted (lanes 10 to 13) DNAs were 13A5-2. Plasmid DNA was then isolated from a new pDQ507 blotted and probed with pDQ507. The open arrow adjacent to lane 8 indicates the 4.3-kb (pDQ499) portion of pDQ507. The ﬁlled arrow indicates the 3.1-kb transformant and the original 13A5-2(pDQ507) isolate. The HindIII fragment that was cloned from pmr-1. The numbers adjacent to the restriction patterns of the plasmid DNAs isolated from E. coli supercoiled DNA ladder in lane 1 indicate the size of each marker in kilobases. 1412 CUE ET AL. APPL.ENVIRON.MICROBIOL.

The 7.4-kb fragment is absent from digests of strain 13A5-2 genomic DNA (lane 10), but a 4.2-kb PstI fragment was observed. Since pDQ507 possesses a single PstI restriction site, a 7.4-kb fragment should result from linearization of the plasmid. We interpret these results as indicating that PDQ507 is maintained as an extrachromosomal circular molecule in B. methanolicus. We found while working with pDQ507 that preparations of plasmid DNA obtained from E. coli were frequently contaminated with a variant form of pDQ507 (i.e., pDQ507a) that possessed a 0.3-kb HindIII fragment in addition to the 4.3- and 3.1-kb fragments. We have not observed the presence of the 0.3-kb fragment in B. methanolicus transformants. The variant plasmid appeared to carry a duplication of a pUC18 sequence, since pUC18 readily hybridizes to the 0.3-kb fragment. We were able to eliminate the problem of pDQ507 instability in E. coli by constructing subcloned derivatives of pDQ507 (described below). These derivatives appear to be superior to pDQ507 in being equally stable in E. coli and B. methanolicus. Structural and segregational stability of E. coli-B. methanolicus shuttle plasmids. We evaluated the segregational and structural stabilities of the four shuttle plasmids (pEN1, PDQ507, pDQ508, and pHP13) as follows. Transformed derivatives of B. methanolicus 13A5-2 were grown at 50ЊCinTSB without antibiotics. Portions of each culture were removed after approximately 10, 30, 40, 60, and 70 generations of growth and plated onto TSA and TSA containing antibiotics (5 ␮g of neomycin per ml for pDQ507 and pDQ508 transformants and 5 ␮g of chloramphenicol per ml for pEN1 and pHP13 transformants). The TSA plates were incubated for 18 to24hat50ЊC, and the numbers of colonies formed on the two types of media were determined (Fig. 6A). FIG. 6. Stability of B. methanolicus plasmids. (A) To determine the segrega- To verify the accuracy of the direct plating colony counts, tional stability of pEN1 (F), pHP13 (ç), pDQ507 (■), and pDQ508 (å), the colonies were picked from the 18- to 24-h-old TSA plates plasmids were introduced into B. methanolicus 13A5-2 and the transformed strains were grown in TSB lacking antibiotics. Portions of the cultures were lacking antibiotics and inoculated onto duplicate plates of TSA removed after approximately 10, 30, 40, 60, and 70 generations of growth and and TSA containing antibiotics. The plates were incubated for plated on TSA and on TSA containing the appropriate antibiotic. The percent- 18 to 24 h and then scored for the number of antibiotic- age of CFU that remained resistant to the antibiotics is reported. (B) Structural resistant and antibiotic-sensitive colonies. Approximately 100 stability of B. methanolicus plasmids. Plasmid DNAs were isolated from the cultures used in inoculation in the segregational stability experiments summa- colonies of each plating of each strain were assayed. rized for panel A above. The following plasmids isolated from the inoculum Plasmids pDQ507 and pHP13 were found to be very stable cultures are shown: pEN1 (lane 1), pDQ507 (lane 4), and pDQ508 (lane 7). in strain 13A5-2. Essentially 100% of the CFU retained the Strains 13A5-2(pDQ507) and 13A5-2(pDQ508) were grown in the absence of plasmids after growth in the absence of antibiotic selection for antibiotic selection for 66 and 63 generations, respectively, and then subcultured into TSB containing neomycin. Plasmid DNA was isolated from the neomycin- 66 and 63 generations, respectively. Plasmid pDQ508 was grown cultures. These plasmid DNAs were also run (pDQ507 [lane 5] and somewhat less stable than either pDQ507 or pHP13. Approx- pDQ508 [lane 8]). Strain 13A5-2(pEN1) was grown in the absence of chloram- imately 15% of the CFU in the culture lost the plasmid during phenicol for 35 generations and plated onto chloramphenicol-containing media. the ﬁrst 37 generations of growth. For reasons that we do not ACmrcolony that arose from the plating was inoculated into TSB containing chloramphenicol. Plasmid DNA that was isolated from this culture is also shown understand, loss of pDQ508 ceased after this point. Eighty-ﬁve (lane 3). Plasmid DNAs were restricted with PstI, blotted, and probed with percent of the CFU retained the plasmid after 55 and 63 pUC18. generations of growth. Plasmid pEN1 is very unstable in B. methanolicus. Only 4% of the CFU in the culture used as the inoculum (Fig. 6A) were resistant to chloramphenicol despite the fact that the inoculum selection for 66 and 63 generations, respectively, subcultured had been grown in TSB containing 5 ␮g of chloramphenicol into TSB containing neomycin, and incubated overnight. Plas- per ml. The plasmid was rapidly segregated out of the popu- mid DNAs that were isolated from the neomycin-grown cul- lation in the absence of antibiotic selection, with only 0.0001% tures are shown in lanes 5 and 8 in Fig. 6B. Strain 13A5- of the CFU being Cmr after 35 generations of growth. No Cmr 2(pEN1) was grown in the absence of chloramphenicol for 35 CFU were recovered after that point. generations and plated on chloramphenicol-containing media. The cultures used for the segregational stability experiments ACmrcolony that arose from the plating was inoculated into were also used to determine the structural stability of each TSB containing chloramphenicol. Plasmid DNA that was iso- of the shuttle plasmids after many generations of bacterial lated from this culture is shown in Fig. 6B, lane 2. Plasmid growth. For these experiments, plasmid DNAs were isolated DNAs were restricted with PstI, blotted, and probed with from the cultures used to inoculate the segregational stability pUC18. We detected no structural alterations of pEN1, experiments summarized above. Plasmids isolated from the pDQ507, or pDQ508 during extended growth of B. methanoli- inoculum cultures are shown in lanes 1, 4, and 7 of the blot cus transformants carrying the recombinant plasmids. Plasmid shown in Fig. 6B. Strains 13A5-2(pDQ507) and 13A5-2 pHP13 was also found to be structurally stable in strain 13A5-2 (pDQ508) were grown at 50ЊC in the absence of antibiotic for at least 63 generations of growth (data not shown). VOL. 63, 1997 GENETIC MANIPULATION OF BACILLUS METHANOLICUS 1413

Replication of pDQ507 and pDQ508 in B. methanolicus. Southern blot analysis (Fig. 6B) revealed that plasmid DNA isolated from B. methanolicus pEN1 (lanes 1 and 2) and pDQ508 (lanes 7 and 8) transformants contained fast-migrating plasmid forms that were not present in preparations of the same plasmids isolated from E. coli (lanes 3 and 9). Each of the plasmids shown in Fig. 6B had a single PstI restriction site. Cleavage of any of the plasmids with PstI should have yielded a single restriction fragment that hybridized to pUC18. This was the case for plasmid DNAs that were isolated from E. coli and for pDQ507 isolated from B. methanolicus but not for pEN1, pHP13, or pDQ508 isolated from B. methanolicus. Two of the plasmids that transformed B. methanolicus, pEN1 and pHP13, are plasmids known to replicate by a rolling- circle mechanism in Bacillus species, and as a result, single- stranded DNA molecules frequently accumulate in cells. It seemed likely that the fast-migrating plasmid forms that were isolated from B. methanolicus pEN1, pHP13, and pDQ508 transformants were single-stranded forms of the recombinant plasmids. To test this interpretation, plasmid DNA was isolated from B. methanolicus and E. coli transformants, electrophoresed through agarose gels, transferred to a membrane after denaturation (Fig. 7A) or without denaturation (Fig. 7B), and probed with pUC18. The probe was found to hybridize to plasmid DNA from all sources when the probed DNAs were denatured prior to blotting. In the absence of denaturation, however, hybridization to plasmid DNA from E. coli (Fig. 7B, lanes 2, 5, and 8) was barely detectable. Figure 7B clearly demonstrates the hybridization of pUC18 to plasmid DNA isolated from strain 13A5-2 transformed with pHP13 (lanes 3 and 4) and pDQ508 (lanes 6 and 7). The positions of the hybridizing bands in Fig. 7B corresponded with the positions of the fast-migrating plasmid forms observed in Fig. 7A. These results indicate that single-stranded pDQ508 DNA accumulates in B. methanolicus transformants, suggesting that pDQ508, like pEN1 and pHP13, is replicated by a rolling-circle mechanism. Distinct, fast-migrating bands have not been observed in plasmid preparations from pDQ507 transformants (Fig. 5 to 7), nor have we obtained convincing evidence for the accum- ulation of single-stranded pDQ507 DNA in strain 13A5-2 (pDQ507). We have, however, frequently detected hybridization of pDQ507 to a smear of DNA in preparations of 13A5-2 (pDQ507) DNA. The smeared DNA is present in genomic as well as plasmid DNA preparations from this strain (see Fig. 5 FIG. 7. Detection of single-stranded plasmid DNA in B. methanolicus transformants. Plasmid DNAs were isolated by the alkaline lysis method, electropho- and 7 for examples). The smeared DNA may result from the resed on agarose gels, and transferred to membranes after denaturation (A) or degradation of pDQ507 or pmr-1 DNA in vivo. This possible ex- without denaturation (B) and then probed with pUC18. Plasmid DNA was planation, however, seems at odds with the stability of pDQ507 isolated from strains DH5␣(pHP13) (lane 2), 13A5-2(pHP13) (lanes 3 and 4), in vivo. The absence of descrete bands of single-stranded DH5␣(pDQ508) (lane 5), 13A5-2(pDQ508) (lanes 6 and 7), DH5␣(pDQ507) (lane 8), and 13A5-2(pDQ507) (lanes 9 and 10). Plasmid DNA digested with PstI pDQ507 DNA does suggest that pDQ507 is replicated by a (lanes 2, 3, 5, 6, 8, and 9) and undigested plasmid (lanes 4, 7, and 10) were used, theta mode of DNA replication in B. methanolicus. Further and a supercoiled DNA ladder is included (lane 1). work is required to identify the progenitor of pDQ507 and to gain a fuller understanding of the mechanism of plasmid maintenance. The plasmid copy number of 13A5-2(pDQ508), 13A5-2 formants would lack the endogenous B. methanolicus replicon (pDQ507), and pHP13 was determined by Southern blotting from which the recombinant plasmid was derived, loss of the with pAA8671 as the probe. The relative intensities of these endogenous replicon being selected for by transformation. blots indicated that 3 to 5 copies of each of these plasmids are To test for incompatibility between pDQ507 and pmr-1 and maintained in B. methanolicus. between pDQ508 and pBM1, neomycin-sensitive (Neos) deriv- Analysis of plasmid-cured derivatives of 13A5-2(pDQ507) atives of 13A5-2(pDQ507) and 13A5-2(pDQ508) were iso- and 13A5-2(pDQ508). Typically, the efﬁciency of plasmid trans- lated. Total DNA was isolated from the Neos isolates, digested formation is low when a recombinant plasmid, such as pDQ507 with HindIII, and blotted and probed with pDQ507 (Fig. 8) or or pDQ508, is used for transformation of a bacterial strain pDQ508 (Fig. 4). As controls, DNAs from strain 13A5-2, carrying a resident plasmid with the same DNA replication 13A5-2(pDQ507), and 13A5-2(pDQ508) were also analyzed. origin (16). We anticipated that pDQ507 and pDQ508 trans- We found that pDQ508 did not hybridize to plasmid DNA 1414 CUE ET AL. APPL.ENVIRON.MICROBIOL.

In contrast, we found that pDQ507 transformants maintain the endogenous replicon from which pDQ507 was derived (Fig. 8). As anticipated, pDQ507 hybridized to two HindIII fragments of 13A5-2(pDQ507) DNA (lane 2), a 4.3-kb fragment (the pDQ499 portion of pDQ507), and a 3.1-kb fragment. The 3.1-kb fragment represents the cloned pmr-1 fragment. This fragment is also present in HindIII-digested 13A5-2 DNA (Fig. 8, lane 1). The 4.3-kb fragment was absent from digests of DNA isolated from the Neos isolates (lanes 3 to 5). Unexpectedly, the 3.1-kb fragment was present in these same digests. Earlier results (Fig. 5, 7, and 8) supported the interpretation that pDQ507 is maintained as a low-copy-number free plasmid in B. methanolicus. First, pDQ507 is readily isolated from B. methanolicus transformants by an alkaline lysis preparation procedure. Second, the hybridization proﬁles of plasmid DNA isolated from E. coli and B. methanolicus are virtually identical for HindIII-cut, PstI-cut, or uncut plasmid. These results would be very unlikely to have been obtained if pDQ507 lacked a replication origin and was maintained in B. methanolicus by integration into pmr-1. Mapping of the putative replication origin of pDQ507. To locate the region of pDQ507 that is required for maintenance of the recombinant plasmid in B. methanolicus, subcloned derivatives of the plasmid were constructed and evaluated for FIG. 8. Analysis of plasmid-cured derivatives of 13A5-2(pDQ507). Genomic their ability to transform strain 13A5-2 (Fig. 9). All of these DNA was isolated from 13A5-2 (lane 1), 13A5-2(pDQ507) (lane 2), and three Neos derivatives of 13A5-2(pDQ507) (lanes 3 to 5). The DNAs were digested with subclones were neomycin-resistant pUC18 derivatives. To dif- HindIII, and a Southern hybridization was performed with pDQ507 as the probe. ferentiate plasmids that were maintained extrachromosomally HindIII-cut pDQ507 isolated from E. coli is also shown (lane 6). The open and ﬁlled from plasmids that had integrated within pmr-1, genomic and arrows (lane 6) indicate the same restriction fragments as the arrows in Fig. 5 do. plasmid DNA from the various transformants were analyzed. Southern analysis of these DNA preparations (see Fig. 10 for example) revealed that pDQ550 (Fig. 9) was the smallest isolated from the three Neos derivatives of strain 13A5-2 pDQ507 derivative that was maintained extrachromosomally (pDQ508) tested (see Fig. 4, lanes 4, 5, 9, and 10 for examples). in B. methanolicus. pBM1 was not detected in pDQ508 transformants or any of the Four plasmids (pHL2, pHL3, pDQ551, and pDQ554) that Neos derivatives of 13A5-2(pDQ508). These results indicate had smaller DNA inserts than pDQ550 were not maintained that the introduction of pDQ508 into strain 13A5-2 results in extrachromosomally in strain 13A5-2. Plasmid DNA that hy- the loss of pBM1. bridized with pUC18 could not be isolated from 13A5-2 trans-

FIG. 9. Deletion analysis of the origin of replication of pDQ507. A partial restriction map of the 3.1-kb HindIII fragment of pDQ507 is shown. The leftmost HindIII site is arbitrarily designated as bp 1. The rightmost HindIII site is designated as bp 3100. The approximate locations of other restriction sites are indicated at the top of the ﬁgure. The locations of the 46-bp inverted repeat (IR) and putative ORF1 and ORF2 are indicated. The sequenced portion of pDQ507 is indicated by shading. Horizontal lines below the restriction map indicate the segments of pDQ507 that were subcloned to construct the individual plasmids that are listed to the right of each line. The column on the far right indicates whether a particular plasmid was free replicating (R) or integrative (I) in B. methanolicus or was not tested (NT). Restriction site abbreviations: Bc, BclI; C, ClaI; D, DraI; E, EcoRI; Ec, EcoRV; H, HindIII; X, XbaI. T4, endpoint of a T4 DNA polymerase-generated deletion. VOL. 63, 1997 GENETIC MANIPULATION OF BACILLUS METHANOLICUS 1415

imperfect 46-bp inverted repeat sequence. A search of the GenBank and EMBL databases did not produce any DNA sequences with significant homology to this portion of pDQ507. Since the 90-bp segment does not contain any open reading frames (ORFs) of significant size, we propose that this region functions as a DNA replication origin in B. methanolicus. The second region required for plasmid maintenance is a 1,457-bp sequence containing one intact (ORF1) (Fig. 11) and one truncated (ORF2) ORF. This sequence is 65.9% homologous to the dpp operon of B. subtilis (Fig. 12) which encodes components of a dipeptide transport operon. The predicted translation products of ORF1 and ORF2 have 86 and 84% similarity with the proteins encoded by the B. subtilis dppA and dppB genes, respectively (Fig. 13). ORF1 is apparently required for extrachromosomal maintenance of the recombinant plasmids. Plasmid pDQ550, which contains the inverted repeat region and an intact ORF1, is maintained as a plasmid in B. methanolicus. Deletion of two-thirds of the C-terminal coding sequence of ORF1 from pDQ550 resulted in a plasmid, pDQ554, that is maintained as an integrated DNA molecule (Fig. 9). Thus, it is possible that ORF1 encodes a plasmid replication protein. Effects of restriction on plasmid transformation efficiency. We have previously shown that B. methanolicus 13A5-2 possesses a major restriction endonuclease, BmeTI, that recognizes the sequence 5ЈTGATCA3Ј. We have also shown that BmeTI sites within the bacterial chromosome are methylated at the adenine within the recognition sequence (5). Since the E. coli dam methylase would introduce this same modification into BmeTI sites (24), we determined the effect of dam methylation of plasmid DNA on the transformation efficiency of B. methanolicus. Two E. coli-B. methanolicus shuttle plasmids, pDQ507 and pDQ508, each have a single BmeTI site. To assay the effect of dam methylation on transformation efficiency, pDQ507 and pDQ508 were isolated from the Damϩ Dcmϩ E. coli strain DH5␣ and from the DamϪ DcmϪ strain DM1. One-tenth to 1 ␮g of each plasmid was used to transform B. methanolicus cells, selecting for Neor. The results of these experiments (Table 6) show that pDQ508 isolated from DH5␣ transformed B. methanolicus 11 times more efficiently than plasmid isolated from DM1. Meth- ylation of pDQ507 increased the efficiency of transformation by approximately fivefold. These results suggest that dam methylation of plasmid DNA has only a modest effect on the efficiency of B. methanolicus transformation, at least for small plasmids possessing a single BmeTI site. FIG. 10. Analysis of plasmid DNA isolated from strain 13A5-2 transfor- In Bacillus thuringiensis (19), it has been reported that DNA mants. (A) Ethidium bromide-stained gel of BamHI-digested plasmid DNAs isolated from strain 13A5-2 transformed with pDQ507 (lane 3), pDQ550 (lane methylation by the E. coli dcm methylase can negatively affect 4), pDQ551 (lane 5), pDQ554 (lane 6), or pHL3 (lane 7). Plasmid DNA isolated the efficiency of transformation. It was possible that the results from strain 13A5-2 (lane 8) and DNA size standards (Gibco BRL) (lane 1) are of our transformation experiments were misleading in that also shown. (B) Southern analysis of the gel shown in panel A. Labeled pUC18 while dam-methylated DNA would be protected from BmeTI was used as the probe. restriction, plasmid DNA isolated from DH5␣ would also be dcm methylated and thus possibly susceptible to restriction by a methylation-dependent restriction system. formed with these plasmids (Fig. 10). Southern analysis of To investigate this possibility, we used dam methylase to genomic DNA isolated from the same transformants did indi- modify pDQ507 and pDQ508 isolated from E. coli DM1. The cate the presence of the recombinant plasmids integrated in vitro methylated DNA transformed B. methanolicus with an within B. methanolicus DNA (data not shown). Stable trans- efficiency comparable to that of transformation with plasmid formation with these plasmids apparently requires integration isolated from DH5␣ (Table 6), suggesting that dcm methyl- of the recombinant plasmid within pmr-1. ation of pDQ507 and pDQ508 has no significant effect on the The results of the deletion analysis indicate that two regions efficiency of B. methanolicus transformation. of pDQ507 are required for extrachromosomal maintenance. BmeTI is the only restriction endonuclease identified in B. The first region is a 90-bp sequence between the EcoRI and methanolicus. Despite this, it is possible that the presence of an XbaI sites of the plasmid (Fig. 11). This segment contains an uncharacterized restriction system may also limit the efficiency 1416 CUE ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 11. Partial nucleotide sequence and predicted ORFs of pDQ507. Restriction enzyme sites relevant to plasmid construction are indicated above the sequence. The predicted amino acid sequences of ORF1 and ORF2 are shown below the nucleotide sequence. Putative ribosome binding sites are underlined. The 46-bp inverted repeat is double underlined. The numbers at the left indicate the nucleotide positions with respect to the HindIII site. of B. methanolicus transformation. To test this possibility, plas- B. methanolicus with efficiencies that were comparable to those mids pDQ508 and pHP13 were isolated from transformed performed with the same plasmids isolated from B. methanoli- derivatives of 13A5-2 and DH5␣; the number of transformants cus. While these results do not exclude the possibility that B. for each of the four plasmid preparations was determined. methanolicus possesses multiple restriction systems, they did Since strain 13A5-2 carries several endogenous plasmids, it indicate that (i) any uncharacterized B. methanolicus restric- was difficult to accurately determine the amount of recombi- tion system would likely recognize an infrequently occurring nant plasmid isolated from this strain. To circumvent this prob- DNA sequence and (ii) the restriction of transforming DNA by lem, plasmid DNAs from B. methanolicus and E. coli were B. methanolicus did not pose a significant barrier to transfor- electrophoresed through 0.8% agarose and stained with mation. ethidium bromide and the plasmid bands of interest were ex- Construction of B. methanolicus cloning vectors. Neither cised from the gels. The DNAs were purified from the gel pDQ507 nor pDQ508 is highly suitable for use as a cloning slices, quantified, and used for transformation of B. methan- vector. Both have few unique restriction sites, and plasmids olicus (Table 6). carrying cloned inserts could not be easily detected. We there- The results in Table 6 were obtained from four duplicate fore constructed derivatives of pDQ507 and pDQ508 that are transformations performed with two different preparations of better suited for use as cloning vectors. These vectors, shown in each plasmid. Plasmid DNAs isolated from DH5␣ transformed Fig. 12, each contain the 1,581-bp EcoO109-to-DraI fragment VOL. 63, 1997 GENETIC MANIPULATION OF BACILLUS METHANOLICUS 1417

FIG. 12. Alignment of nucleotide sequences of pDQ507 (upper sequence) and the B. subtilis dpp operon (lower sequence; obtained with the Genetics Computer Group GAP program [9]; gap weight, 5.0; length weight, 0.3). The GenBank accession number for the dpp sequence is X56678. from pUC18 (35). This fragment contains the replication ori- 26) and B. stearothermophilus protoplasts (34, 41), our trans- gin and the multiple-cloning site of pUC18. These plasmids formation efficiencies are comparable to those reported for also carry the 1.3-kb Neor gene from pBEST501. numerous gram-positive bacteria (6, 18). Techniques which are Plasmid pDQ541 carries the same pBM1 fragment as does generally more efficient and less tedious than protoplast trans- pDQ508. Plasmid pDQ539 carries the 3.1-kb pmr-1 fragment formation, such as electroporation (12) or biolistic transforma- subcloned from pDQ507. Plasmid pDQ543 (not shown) carries tion (28), could be optimized for B. methanolicus transforma- the same pmr-1 fragment as pDQ550. Each of the three clon- tion by use of the vectors described here. ing vectors has been successfully introduced into B. methanoli- We have previously characterized the B. methanolicus BmeTI cus 13A5-2. restriction-modification (r-m) system (5). The BmeTI restriction endonuclease recognizes the 6-bp DNA sequence 5ЈTGA Ј DISCUSSION TCA3 . The cognate methyltransferase (mBmeTI) modifies this sequence to 5ЈTGm6ATCA3Ј. Since the E. coli dam meth- In this report, we describe the development of a gene trans- ylase would introduce the same modification as mBmeTI into fer system for the thermotolerant, gram-positive methylotroph BmeTI sites (24), it seemed likely that dam methylated plasmid B. methanolicus. A reliable protoplast transformation proce- DNA would transform B. methanolicus with a higher efficiency dure that permits the genetic transformation of B. methanoli- than nonmethylated DNA. cus with both integrative and low-copy-number plasmid vectors The transformation of B. methanolicus with pDQ508 DNA ϩ ϩ was developed. isolated from E. coli DH5␣ (Dam Dcm ) was 11 times While the efficiency of transformation of B. methanolicus greater than that with pDQ508 isolated from the DamϪ DcmϪ protoplasts (up to 105 transformants/␮g of DNA) is less than E. coli strain DM1. Incubation of nonmethylated DNA with the efficiencies reported for the transformation of B. subtilis (4, dam methylase and S-adenosylmethionine in vitro increased 1418 CUE ET AL. APPL.ENVIRON.MICROBIOL.

tional r-m systems. We can conclude, though, that if B. methanolicus does encode an additional r-m system, the system likely recognizes an infrequently occurring target sequence. We attempted to construct an E. coli-B. methanolicus shuttle plasmid by cloning the replication origin of bacteriophage RD-1 into the E. coli plasmid pDQ499. Autonomously replicating E. coli plasmids that carry the replication origins of bacteriophages ␭ (21) and P1 (33) have been described. How- ever, when a plasmid library of RD-1 DNA was constructed in E. coli and the pooled plasmids were used to transform B. methanolicus (selecting for Neor), three different plasmids were isolated. All three plasmids could be isolated from B. methanolicus as circular extrachromosomal DNAs. More surprisingly, none of the recovered plasmids hybridized to RD-1 DNA. Instead, the plasmids were each found to carry distinct cloned fragments of B. methanolicus DNA, indicating that the RD-1 bacteriophage DNA preparation was contaminated with a minute amount of host DNA that was serendipitously cloned into pDQ499. Host DNA may have been DNA that escaped nuclease digestion during the isolation of bacteriophage particles or packaged within phage particles. Two of the three plasmids that were isolated in this experi- ment were further characterized to assess their potential useful- ness as B. methanolicus cloning vectors. The shuttle plasmid pDQ508 was determined to carry the replication origin of a moderate-size (Ӎ17-kb) endogenous B. methanolicus plasmid, pBM1. This endogenous plasmid is absent from pDQ508 transformants. This is likely due to incompatibility between pDQ508 and pBM1. We have shown that pDQ508 is segregationally and structurally stable in B. methanolicus for more than 63 generations of growth in the absence of antibiotic selection. The shuttle plasmid can be readily introduced into B. methanolicus, yield- FIG. 13. Sequence alignment of ORF1 (A) and ORF2 (B) with DppA and ing nearly 106 transformants/␮g of DNA. We have detected the DppB, respectively. The alignments were generated with the GAP program (9) presence of single-stranded plasmid DNA in pDQ508 trans- by using a gap weight of 3.0 and a length weight of 0.1. Identical residues are indicated by lines; similar residues are indicated by dots. formants, providing strong evidence that the plasmid replicates by a rolling-circle mechanism (15). The properties of pDQ508 are similar to those of pHP13. The latter plasmid carries the replication origin of the cryptic B. subtilis plasmid pTA1060. B. the transformation efficiency nearly 10-fold. Similar results methanolicus can be transformed with pHP13 (104 transfor- were obtained for transformation experiments performed with mants/␮g of DNA), which is stably maintained at a low copy methylated and nonmethylated pDQ507. Thus, dam methyl- number in B. methanolicus 13A5-2. The plasmid is known to ation does increase the efficiency of plasmid DNA transformation of B. methanolicus. The effect of dam methylation is pre- sumably due to inhibition of DNA restriction by BmeTI, although further experiments are required to substantiate this TABLE 6. Effect of plasmid DNA methylation on assumption. We find no evidence that dcm methylation affects transformation efficiency the efficiency of B. methanolicus transformation as has been No. of No. of trans- Antibiotic Plasmid BmeTI Source of plasmid formants/␮g reported for B. thuringiensis (19). selection The modest enhancement of transformation by dam meth- sites of DNAa ylation perhaps should not be surprising since both pDQ507 pDQ508 1 E. coli (Damϩ) Neomycin 1.63 ϫ 105 and pDQ508 possess a single BmeTI recognition site (data not 1 E. coli (DamϪ DcmϪ) Neomycin 1.44 ϫ 104 shown). Haima et al. (11) demonstrated that small plasmids 1 E. coli (DamϪ DcmϪ)b Neomycin 8.07 ϫ 104 are relatively insensitive to restriction by B. subtilis Marburg but that sensitivity to restriction increases dramatically (up to pDQ507 1 E. coli (Damϩ) Neomycin 1.78 ϫ 103 Ϫ Ϫ 3 orders of magnitude) with increased plasmid size. Thus, the 1 E. coli (Dam Dcm ) Neomycin 3.70 ϫ 102 Ϫ Ϫ b 3 ability to protect transforming DNA from BmeTI restriction 1 E. coli (Dam Dcm ) Neomycin 3.44 ϫ 10 could prove to be crucial for successfully introducing larger ϩ E. coli c ϫ 3 B. methanoli- pDQ508 1 (Dam ) Neomycin 4.32 10 plasmids containing cloned DNA fragments into 1 B. methanolicusc Neomycin 7.86 ϫ 103 cus. We found that for pHP13 and pDQ508, plasmids isolated pHP13 1 E. coli (Damϩ)c Chloramphenicol 1.08 ϫ 104 from E. coli and B. methanolicus transformed B. methanolicus 1 B. methanolicusc Chloramphenicol 9.96 ϫ 103 with comparable efficiencies. These results suggest that BmeTI a Numbers represent the averages of at least two transformation experiments. may be the only restriction endonuclease possessed by B. meth- Ϫ b Plasmid DNA was isolated from a Dam E. coli strain (DM1) and then anolicus. We cannot dismiss the possibility, however, due to the methylated in vitro with dam methylase. small sizes of the shuttle plasmids, of the existence of addi- c Experiments were performed with plasmid DNA purified from agarose gels. VOL. 63, 1997 GENETIC MANIPULATION OF BACILLUS METHANOLICUS 1419 replicate by a rolling-circle mechanism (11). Although pDQ508 and pHP13 appear to be nearly equivalent plasmids, we have yet to directly compare the abilities of the two plasmids to stably maintain cloned genes in B. methanolicus. A second bifunctional plasmid containing B. methanolicus DNA is pDQ507. This plasmid is also stably maintained in B. methanolicus and transforms the bacterium with reasonable efficiency. We initially became interested in characterizing this plasmid since the early indications were that pDQ507 was derived from a large endogenous B. methanolicus plasmid. If this is correct, pDQ507 could make an ideal vector for moving large cloned DNA fragments into B. methanolicus. Several lines of evidence suggest that pDQ507 is maintained as an extrachromosomal circular molecule in B. methanolicus. First, pDQ507 can be readily isolated from B. methanolicus transformants by an alkaline lysis plasmid isolation procedure. Second, pDQ507 hybridizes to the same PstI, HindIII, and BamHI restriction fragments of plasmid DNA isolated from either E. coli or B. methanolicus (Fig. 5 and 7B). Also, hybridization of pDQ507 to undigested plasmid DNA indicates the presence of similarly sized plasmid molecules in E. coli and B. methanolicus, although the bulk of the B. methanolicus plasmid migrates as high-molecular-weight DNA. While these results could be taken as evidence that pDQ507 is an autonomously replicating plasmid, other results argue that this may not necessarily be the case. We have been unable to unambiguously demonstrate the hybridization of pDQ507 to plasmid DNA isolated from nontransformed strains of B. methanolicus. The plasmid does, however, hybridize to B. methanolicus genomic DNA. So while it is clear that pDQ507 contains a cloned fragment of B. methanolicus DNA, the origin is un- FIG. 14. Restriction maps of pDQ539 and pDQ541. Plasmid constructions are described in the text. The restriction sites indicated in the figure represent certain and hence we have named this sequence pmr-1 (puta- unique restriction sites within the polylinker of pUC18. Abbreviations: B, tive methanolicus replicon) to indicate the endogenous genetic BamHI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SmaI. element from which pDQ507 was derived. Since pmr-1 and pDQ507 are apparently present in the same host cells, pDQ507 may be dependent upon pmr-1 for replication. It is also conceivable that pDQ507 is not replicated as a be maintained in the same cell for many generations before plasmid; rather, pDQ507 could exist integrated into the host one of the replicons is excluded (36). genome, the extrachromosomal circular forms of the plasmids Our accumulated results are most consistent with pDQ507 being generated by replicative excision of the integrated plas- carrying a B. methanolicus DNA fragment of chromosomal mid. origin. Numerous autonomously replicating plasmids that At this point, we also cannot dismiss the possibility that the carry replicons derived from chromosomal sequences have progenitor of pDQ507 is a very large circular or linear plasmid. been described. The cloned replicons have in some cases Labeled pDQ507 does hybridize to undigested genomic DNA proven to be chromosomal replication origins (23, 40) or pseu- from B. methanolicus. Typically, the majority of the DNA pres- do-oriC sequences (40). In other instances, the cloned repli- ent in our preparation of genomic DNA migrates as 50- to cons were derived from defective bacteriophage (2, 7) or plas- 60-kb DNA fragments, the relatively small size of the frag- mid (10) replication origins. Autonomous replicons derived ments due to shearing of chromosomal DNA during the iso- from functional plasmids capable of site-specific integration lation and preparation procedure. A very large plasmid would into host chromosomes have also been described (25, 38). be sensitive to shearing as well and thus would comigrate with Deletion analysis and DNA sequencing of pDQ507 indi- chromosomal DNA on agarose gels. We have successfully iso- cated that two regions of the plasmid are required for extra- lated an approximately 50-kb endogenous B. methanolicus chromosomal maintenance. One region is a 90-bp sequence plasmid that hybridizes to pDQ506. Thus, if pDQ507 was de- that contains a 46-bp imperfect inverted repeat (Fig. 11). It is rived from an endogenous plasmid, the endogenous plasmid is likely that this sequence functions as a plasmid replication likely to be greater than 50 kb in size. origin in B. methanolicus. Deletion of this sequence from We had anticipated that we could cure B. methanolicus of pDQ550 to construct pHL2 resulted in the loss of extrachro- pBM1 and pmr-1 by transformation of the bacterium with, mosomal maintenance. This region of pDQ550 appears un- respectively, pDQ508 and pDQ507. This was true for pDQ508 likely to code for a DNA replication protein since no ORF of transformants since we have found no evidence for the pres- significant size was found within this fragment. No plasmid ence of pBM1 in transformed cells. In contrast, pmr-1 is ap- replication origin sequences with significant homology to this parently present in pDQ507 transformants as well as in Neos sequence were found in either the GenBank or EMBL data- derivatives of strain 13A5-2(pDQ507). These results suggest base. that pmr-1 and pDQ507 are compatible, but it is also conceiv- A second region is within a 1,457-bp sequence that is 65.9% able that either pmr-1 or pDQ507 may have acquired point homologous to the sequence of the dpp operon of B. subtilis mutations that permit maintenance of the two elements in the (Fig. 13), which encodes components of a dipeptide transport same host cell. Also, it is possible for incompatible replicons to operon (20, 31). The homologous region of pDQ550 contains 1420 CUE ET AL. APPL.ENVIRON.MICROBIOL.

one complete ORF (ORF1) and one truncated ORF (ORF2). 12. Hanahan, D., J. Jessee, and F. R. Bloom. 1991. Plasmid transformation of The sequenced portion of ORF2 indicates that the putative B. Escherichia coli and other bacteria. Methods Enzymol. 204:63–113. methanolicus 13. Hanson, R. S., R. Dillingham, P. Olson, G. H. Lee, D. Cue, F. J. Schendel, peptide has 84.0% similarity and 65.2% identity C. Bremmon, and M. C. Flickinger. 1996. Production of L-lysine and some to the protein encoded by dppB (Fig. 14B). DppB is a compo- other amino acids by mutants of B. methanolicus, p. 227–234. In M. E. nent of a membrane-associated complex that transports pep- Lidstrom and F. R. Tabita (ed.), Microbial growth on C1 compounds. Kluwer tides across the cytoplasmic membranes (20). It seems likely Academic Publishers, Dordrecht, The Netherlands. 14. Itaya, M., K. Kondo, and T. Tanaka. 1989. A neomycin resistance gene that the ORF2 product performs a similar function in B. meth- cassette selectable in a single copy state in the Bacillus subtilis chromosome. anolicus. Because of this, as well as the fact that pDQ550 Nucleic Acids Res. 17:4410. appears to carry only the amino-terminal coding portion of 15. Jannie`re, L., A. Gruss, and S. D. Ehrlich. 1993. Plasmids, p. 625–644. In ORF2, it seems unlikely that ORF2 encodes a protein that is A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. required for plasmid replication. ASM Press, Washington, D.C. The putative product of ORF1 is a 30.2-kDa protein that is 16. Kittel, B. L., and D. R. Helinski. 1993. Plasmid incompatibility and replica- 86.1% similar and 75.5% identical to the product of the B. tion control, p. 223–242. In D. B. Clewell (ed.), Bacterial conjugation. Ple- subtilis dppA gene (Fig. 14A). While the products of ORF1 and num Press, New York, N.Y. 17. Leak, D. J. 1992. Biotechnological and applied aspects of methane and dppA are obviously related peptides, neither protein has a se- methanol utilizers, p. 245–280. In J. C. Murrell and H. Dalton (ed.), Methane quence with significant similarity to protein sequences present and methanol utilizers. Plenum Press, New York, N.Y. in the GenBank and EMBL libraries (20, 31). The function of 18. Lidstrom, M. E., and D. I. Stirling. 1990. Methylotrophs: genetics and DppA in B. subtilis is unknown. commercial applications. Annu. Rev. Microbiol. 44:27–58. Ј 19. Macaluso, A., and A.-M. Mettus. 1991. Efficient transformation of Bacillus Deletion of ORF2 and the 3 516 bp of ORF1 from pDQ550 thuringiensis requires nonmethylated plasmid DNA. J. Bacteriol. 173:1353–1356. resulted in a plasmid (pDQ554) that is not maintained extra- 20. Mathiopoulos, C., J. P. Mueller, F. J. Slack, C. G. Murphy, S. Patankar, G. chromosomally in B. methanolicus (Fig. 9). While these results Bukusoglu, and A. L. Sonenshein. 1991. A Bacillus subtilis dipeptide trans- suggest that ORF1 may encode a protein necessary for repli- port system expressed during sporulation. Mol. Microbiol. 5:1903–1913. ␭ cation of pDQ507 derivatives, additional investigations are re- 21. Matsubara, K., and A. Kaiser. 1969. dv: an autonomously replicating DNA fragment. Cold Spring Harbor Symp. Quant. Biol. 33:769. quired to determine if this is true. 22. Mills, D. A., and M. C. Flickinger. 1993. Cloning and sequence analysis of Further work in progress should enable us to more precisely the meso-diaminopimelate decarboxylase gene from Bacillus methanolicus identify the minimal replicons of pDQ507 and pDQ508 and to MGA3 and comparison to other decarboxylase genes. Appl. Environ. Mi- determine whether the plasmids encode essential replication crobiol. 59:2927–2937. 23. Moriya, S., T. Atlung, F. G. Hansen, H. Yoshikawa, and N. Ogasawara. 1992. proteins. This information should provide insight into the Cloning of an autonomously replicating sequence (ars) from the Bacillus mechanism(s) of plasmid maintenance in B. methanolicus. subtilis chromosome. Mol. Microbiol. 6:309–315. 24. Nelson, M., E. Raschke, and M. McClelland. 1993. Effect of site-specific methylation on restriction endonucleases and DNA modification methyl- ACKNOWLEDGMENTS transferases. Nucleic Acids Res. 21:3139–3154. 25. Omer, C. A., and S. N. Cohen. 1989. SPL1: a paradigm for plasmids that We thank Diego Matteuzzi, University of Bologna, Italy, for pro- site-specifically integrate in actinomycetes, p. 289–296. In D. E. Berg and viding pEN1. Technical support for portions of this work was provided M. M. Howe (ed.), Mobile DNA. ASM Press, Washington, D.C. by Nobuharu Tsujimoto. 26. Perkins, J. B., and D. H. Dean. 1983. 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