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

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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/$04.0010 Copyright q 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 thermo- tolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transfor- mation 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. methan- olicus 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, thermotol- erant, facultative methylotroph that offers several advantages MATERIALS AND METHODS over other methylotrophs for industrial fermentations. The optimal growth temperature of B. methanolicus (50 to 538C) Bacterial strains and plasmids. The bacterial strains, plasmids, and bacterio- a 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 DH5a 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 8 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 378C 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 mg/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 mg/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 mg/ml, E. coli respectively, and incubated for 30 min at room temperature. EDTA (25 mM), DH5a 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 568C for 1 h. The proteinase-treated lysates DH5a-MCR mcrA D(mrr-hsdRMS-mcrBC) recA1 Gibco BRL were then extracted three times with phenol-chloroform and twice with chloro- form. DNA was recovered by ethanol precipitation and redissolved in Tris- EDTA (TE). Approximately 750 mg of bacteriophage DNA could be recovered B. methanolicus from 1 liter of infected cells. MGA3 Wild type 29 2 2 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 8 8 shuttle plasmid (Apr Neor) 50 C in SOB-sucrose medium to an OD600 of 0.5 to 0.6 (approximately 10 3 8 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 mgof (Apr Neor) lysozyme per ml. Cells were incubated at 428C, 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 3 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.
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