Journal of Bacteriology, March 2004, p . 1229-1238, Vol . 186, No . 5

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

Trygve Brautaset,^1* Øyvind M . Jakobsen,^1,2 Michael C . Flickinger,³ Svein Valla,¹ and Trond E . Ellingsen²

Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim,¹ SINTEF Applied Chemistry, SINTEF, N-7043 Trondheim, Norway,² BioTechnology Institute, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St . Paul, Minnesota 55108³

Received 8 October 2003/ Accepted 20 November 2003

 
Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of50°C . With the exception of mannitol, no sugars have beenreported to support rapid growth of this organism, which isclassified as a restrictive methylotroph . Here we describe theDNA sequence and characterization of a 19,167-bp circular plasmid,designated pBM19, isolated from B . methanolicus MGA3 . Sequenceanalysis of pBM19 demonstrated the presence of the methanoldehydrogenase gene, mdh, which is crucial for methanol consumptionin this bacterium . In addition, five genes [pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase] with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19 . A shuttle vector, pTB1.9, harboringthe pBM19 minimal replicon [repB and ori] was constructed and used to transform MGA3 . Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not ableto grow on methanol . A pTB1.9 derivative harboring the completemdh gene could not restore growth on methanol when it was introducedinto the pBM19-cured strain, suggesting that additional pBM19genes are required for consumption of this carbon source . Screeningof 13 thermotolerant B . methanolicus wild-type strains showedthat they all harbor plasmids similar to pBM19, and this isthe first report describing plasmid-linked methylotrophy inany microorganism . Our findings should have an effect on futuregenetic manipulations of this organism, and they contributeto a new understanding of the biology of methylotrophs.

 
The methylotrophs constitute a diverse group of microorganismsthat can utilize reduced one-carbon [C₁] compounds, such as methanol, as sole carbon sources for growth [2, 3] . The abundance,purity, and low price of methanol compared to sugars make methylotrophsinteresting candidate organisms for production of amino acids,vitamins, cytochromes, coenzymes, single-cell proteins, andrecombinant proteins [14, 21] . The key intermediate for biologicalC₁ fixation is formaldehyde, which may be assimilated via alternative biochemical routes . Bacteria that fix formaldehyde by the ribulose monophosphate [RuMP] pathway belong to three groups: gram-negative obligate methylotrophs, gram-positive facultative methylotrophs,and thermotolerant Bacillus species [14] . The RuMP pathway [Fig.1] has two unique enzymes, 3-hexulose-6-phosphate synthase [HPS]and 6-phospho-3-hexuloisomerase [PHI], which catalyze the two-stepfixation phase of formaldehyde with ribulose-5-phosphate [Ru-5-P],which yields fructose 6-phosphate [F-6-P] . RuMP pathway fixationoperons, including the hps and phi genes, have been cloned fromrepresentatives of all three bacterial groups and characterized,and these operons have certain similarities in both gene organizationand regulation [25, 31, 39] . In addition to the fixation phase,a complete RuMP pathway includes the cleavage and rearrangementphases, which are thought to share enzymes with the pentosephosphate and glycolytic or Entner-Doudoroff pathways [Fig. 1] . There are several RuMP pathways, and thermotolerant bacillihave been reported to use the fructose-1,6-bisphosphate aldolase[FBPA]-transaldolase [TA] variant [4, 16] . In this pathway theF-6-P generated by the action of HPS and PHI is phosphorylatedby 6-phosphofructokinase [PFK] before FBPA cleaves the resultingfructose 1,6-bisphosphate to form the triose phosphates glyceraldehyde3-phosphate and dihydroxyacetone phosphate . Dihydroxyacetonephosphate enters the central pathway for synthesis of cell constituents,while glyceraldehyde 3-phosphate enters the final phase of theRuMP cycle, in which the formaldehyde acceptor Ru-5-P is regenerated.Important enzymes for the regeneration phase are transketolase[TKT], ribose-5-phosphate isomerase, TA, and Ru-5-P 3-epimerase[RPE] . Another variant of the RuMP pathway, the FBPA-sedoheptulose-1,7-bisphosphatase [SBPase] variant, is found in certain facultative methylotrophic Bacillus species [16], in which the rearrangement phase includesSBPase instead of TA activity [Fig. 1].

 
Large plasmids, typically ranging in size from 50 to 200 kb,are commonly present in methylotrophic bacteria, including some methanotrophs [14, 22], yet our knowledge concerning the biologicalsignificance of any of these replicons in methylotrophs is limited.Methylotrophy does not correlate well with traditional methodsof bacterial classification [2, 21], and it is tempting to speculatethat methylotrophy might be plasmid linked . Hybridization experimentswith several plasmids isolated from a range of methylotrophsrevealed no homology, which argues against an episomal locationof genes involved in methanol metabolism [14] . To date, thereis no evidence for a direct role of plasmid-borne genes in theC₁ metabolism of any methylotrophic organism.

A number of thermotolerant and methylotrophic gram-positive Bacillus strains have been isolated from different locations,and based on physiological and 16S rRNA sequence analyses theseorganisms are collectively classified as Bacillus methanolicus[4, 33] . B . methanolicus uses an NAD[P]-dependent methanol dehydrogenase[MDH] to oxidize methanol to formaldehyde, and in addition toentering the RuMP pathway, a linear branch for dissimilationof formaldehyde to CO₂ was recently demonstrated [29] . It waspreviously shown that this bacterium can secrete 55 g of glutamateper liter at 50°C by using methanol as a carbon source infed-batch fermentation [11], and a homoserine dehydrogenasemutant [13A52-8A66] that secreted up to 35 g of L-lysine perliter at 50°C was described [18] . In a previous study [13] it was found that the B . methanolicus NOA2 mutant 13A52 possesses a plasmid with an estimated size of 17 kb, and a plasmid ofa similar size was later identified [N . Tsujimoto, H . Yasueda,and S . Sugimoto, 24 October 2000, Japanese patent applicationJP2000295988] in B . methanolicus PB1 [= NCIMB 13113] . No sequence information is available for any of these DNA molecules, andthe biological significance of them remains unknown . In thisreport we describe the DNA sequence and characterization ofa 19,167-bp circular plasmid, designated pBM19, isolated fromB . methanolicus MGA3 . Remarkably, both mdh and five putativeRuMP pathway genes were identified in this plasmid, and we foundthat pBM19 is essential for growth of this bacterium on methanol.

 
Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study are listedin Table 1 . Recombinant Escherichia coli cells were grown at 37°C in Luria-Bertani medium [32] supplemented with ampicillin[100 µg/ml] . For preparation of protoplasts B . methanolicuscells were grown at 50°C in SOB medium [which contains tryptoneand yeast extract] [Difco] supplemented with 0.25 M sucrose,and the resulting recombinant cells were grown in the same mediumsupplemented with neomycin [25 µg/ml] . For preparation of crude extracts B . methanolicus strains were grown in SOB containing 0.25 M sucrose, and methanol was added at a concentration of 150 mM 1 h prior to harvest to induce MDH expression . For all other purposes B . methanolicus cells were grown at 50°Cin methanol minimal vitamin medium [MVcMY medium] containing1 mM MgSO₄, high-salt buffer, vitamins, 0.025% yeast extract[Difco], 200 mM methanol, and trace metals essentially as describedpreviously [11] . Mannitol medium was MVcMY medium without methanolsupplemented with D-mannitol [10 g/liter; Sigma] as the sole carbon source . Bacterial growth was monitored by measuring the optical density at 600 nm.

 
DNA manipulations. All recombinant E . coli procedures [plasmid preparation, restrictionanalysis, ligation, and transformation] were performed as describedby Sambrook et al . [32] . Transformation of B . methanolicus MGA3strains was performed by using the protoplast method developedfor B . methanolicus NOA2 mutant 13A52, essentially as describedby Cue et al.[13] . Plasmid and total DNA were prepared fromB . methanolicus by using Qiagen Midi Prep and Dneasy tissuekits [Qiagen Gmbh, Hilden, Germany], respectively, accordingto the manufacturer's instructions . DNA fragments used for probes[Table 2] were isolated from agarose gels by using a Qiaex kit[Qiagen Gmbh], labeled by using a digoxigenin [DIG] kit fromBoehringer Mannheim, and used for Southern hybridization analysisaccording to the manufacturer's instructions . DNA sequencingwas performed by the Advanced Genetic Analysis Center [Universityof Minnesota, St . Paul], and the sequence data were analyzedby using the online programs Pfam [http://www.sanger.ac.uk/Software/Pfam/], BlastP [http://www.ncbi.nlm.nih.gov/BLAST/], Fasta [http://www.ebi.ac.uk/fasta33/], and Multialin [http://prodes.toulouse.inra.fr/multalin/multalin.html].

 
Preparation of crude cell extracts and enzyme assays. Crude extracts were prepared essentially as described by Arfmanet al . [5] . Late-exponential-phase cell cultures were harvestedby centrifugation [Sorvall GSA rotor; 4.500 rpm, 10 min, 4°C]and washed twice in 50 mM potassium phosphate buffer [pH 7.5]containing 5 mM MgSO₄. E . coli cells were disrupted by sonication[Branson Sonifer 250] as described previously [11] . B . methanolicus cells were incubated at 37°C for 60 min in the presenceof lysozyme [5 mg/ml] and 25 U of mutanolysin [10 U/ml; Sigma]before sonication . Cell debris was removed by centrifugation[13,000 x g, 30 min, 4°C], and the supernatants were collectedas crude extracts . NAD[P]-dependent MDH activity was measuredin the reverse direction by the formaldehyde reductase assayby monitoring the decrease in absorbance at 340 nm [ShimadzuUV-160A] due to formation of NAD⁺ at 50°C [5] . All experimentswere performed in duplicate . Polyacrylamide gels [15%, wt/vol]were stained with Coomassie brilliant blue.

Construction of pBM19-based shuttle vectors pTB1.9, pTB1.9mdh, and pTB1.9mdhL. The 5,220-bp BamHI/PstI fragment of pBM19 was cloned into thecorresponding sites of pUC19 . The resulting plasmid was digestedwith AflIII, and the 4.2-kb fragment including the pUC19 vectorbackbone, as well as the repB-ori region of pBM19, was religated.The resulting construct was linearized with BamHI/SacI, andthe cohesive SacI end was blunted with T4 DNA polymerase toobtain fragment 1 . The 1.6-kb BamHI/PstI fragment of plasmidpDQ508 [encoding the Neo^r gene] was isolated, and the cohesivePstI end was blunted as described above to obtain fragment 2.Fragments 1 and 2 were ligated to obtain plasmid pTB1.9 [seeFig . 5] . A DNA fragment including the mdh gene and its 237-bpupstream sequences was PCR amplified from pBM19 by using primersmdh-F and mdh-R [Table 3], and the resulting 1,580-bp PCR product was end digested with XbaI/SacI and cloned into the correspondingsites of plasmid pJB658 . From the resulting construct the 1,575-bpXbaI/BamHI insert was cloned into the corresponding sites ofpTB1.9, yielding vector pTB1.9mdh [Table 1] . The 3,289-bp HindIIIfragment of pBM19 was cloned into pGEM-3zf, yielding plasmidpTB3.3H . The 2,115-bp SacI/PstI fragment was isolated from thisconstruct and used to replace the corresponding 1,192-bp SacI/PstI fragment of pTB1.9mdh, which yielded the MDH expression vector pTB1.9mdhL . All constructs were verified by DNA sequencing.

 
PCR analysis of B . methanolicus strains for pBM19 DNA. For analysis of MGA3 strains for chromosomal copies of pBM19genes, total DNA were isolated and used as templates for PCRby using the following oligonucleotide primer pairs: repB-Fplus repB-R, pfk-F plus pfk-R, rpe-F plus rpe-R, tkt/fba-F plustkt/fba-R, glpX-F plus glpX-R, and mdh-F plus mdh-R [Table 3].These primer pairs correspond to the amplified regions of therepB, pfk, rpe, tkt and fba, glpX, and mdh genes of pBM19, respectively.To screen B . methanolicus wild-type strains for pBM19-like plasmids,both plasmid and total DNA were isolated and used as templatesfor PCR performed with primers tba15 and tba42 [Table 3] . Inall cases the 100-µl PCR mixture contained 0.1 to 0.5µg of DNA, 50 pmol of the forward primer, 50 pmol of thereverse primer, 350 µmol of each deoxynucleoside triphosphate,1x PCR buffer [GIBCO], and 2 U of the Taq DNA polymerase fromthe same system . The PCR was performed with a Perkin-Elmer GeneAmpPCR system 2400 by using the following program: one cycle ofdenaturation 94°C for 3 min, 30 cycles of denaturation at94°C for 60 s, annealing at 55°C for 60 s, and synthesisat 68°C for 2 min, and one cycle of synthesis at 68°Cfor 7 min . The DNA fragments obtained were verified by DNA gelelectrophoresis and partial DNA sequencing.

PCR cloning of the MGA3 RuMP pathway fixation operon. Based on the previously published DNA sequence of the RuMP pathwayfixation operon of Bacillus brevis S1 [39], the PCR primer pairsrmp-F plus rmp-R4, rmp-F5 plus rmp-R3, and rmp-F4 plus rmp-R5 were designed [Table 3] . By using these primers, three DNA fragmentswere PCR amplified from MGA3 total DNA and individually clonedinto pLITMUS28 or pGEM-11zf, which yielded plasmids pRMP1, pRMP2,and pRMP3, respectively [Table 1] . Both strands of the clonedinserts of these plasmids were sequenced.

Estimation of plasmid copy number. One DNA probe of chromosomal origin [rmp-P] and three DNA probesof pBM19 origin [repB-P, fba/tkt-P, and pfk-P2] were designed[Table 2] . Coupled amplification and DIG labeling of these probeswere performed by using a PCR-DIG probe synthesis kit [RocheMolecular Biochemicals, Mannheim, Germany] according to themanufacturer's instructions . The DNA concentrations of the resultingprobes were analyzed by gel electrophoresis and standardized.Total DNA was isolated from MGA3 cells grown in MVcMY mediumat 50°C to the late exponential phase and was digested withSacI . Various dilutions of digested DNA were separated by gelelectrophoresis and used for three independent two-probe Southernhybridizations with one chromosome-derived probe and one pBM19-derivedprobe . The hybridization bands obtained with both probes werescanned, and three-dimensional graphs of the intensity profilesof each band were generated by digital image analysis . Eachgraph was integrated, which gave the corresponding intensityvolume . The pBM19 copy number was calculated by comparing theintensity volumes of chromosomal and plasmid bands at different dilutions.

Nucleotide sequence accession numbers. The DNA sequences of plasmid pBM19 and the RuMP pathway fixationoperon reported in this paper have been deposited in the GenBanknucleotide sequence database under accession numbers AY386314and AY386313, respectively.

 
DNA sequencing of the B . methanolicus MGA3 plasmid pBM19. By analyzing B . methanolicus MGA3 DNA we isolated a plasmid with an estimated size of 19 kb, designated pBM19 . Overlapping fragments representing the entire pBM19 DNA were cloned in E.coli, and both strands of the cloned inserts were sequencedby the primer extension method . PCR analysis was used when appropriateto verify the overlap between assumed neighboring inserts . Usingthis strategy, we determined the complete pBM19 DNA sequenceconsisting of 19,167 bp [Fig . 2] . The overall G+C content was36.7%, and computer-assisted analysis of the DNA sequences ledto identification of the putative genes shown in Fig . 2 andTable 4 . Interestingly, the pBM19 plasmid is composed of two regions encoding genes arranged in opposite orientations, suggesting that the present form of this plasmid might have been generated by fusion between separate DNA molecules.

 
[i] Key gene for methanol oxidation, methanol dehydrogenase, is encoded by pBM19. Remarkably, the mdh gene, including its ribosome binding sitecoding region and proposed promoter region, which exhibited96.8% overall identity to the active mdh gene in B . methanolicusC1 [= NCIMB 13114] [15], was localized on pBM19 . The deducedprimary sequences of the mdh gene products [381 and 382 aminoacids [aa]] of B . methanolicus MGA3 and C1 are 97.9% identical.MDH, which belongs to the family III group of NAD[P]-dependentalcohol dehydrogenases [6], catalyzes the oxidation of methanolto formaldehyde [Fig. 1] and thus plays a key role in methylotrophic growth of B . methanolicus.

[ii] Genes with deduced roles in methanol assimilation via the RuMP pathway. Five putative genes with assigned roles in methanol assimilationvia the RuMP pathway [glpX, fba, tkt, pfk, and rpe] were identifiedon pBM19 [Fig. 1 and Table 4] . Except for rpe, these genes andmdh are arranged in the same orientation and occupy one continuousregion of the pBM19 plasmid [Fig . 2] . The fba and tkt codingsequences are separated by 12 nucleotides, suggesting that theymay be translationally coupled . The remaining three genes aremost probably transcribed from individual promoters.

The deduced glpX gene product is a 321-aa protein that exhibits the highest overall level of identity [55%] to the Bacillus halodurans class II fructose-1,6-bisphosphatase [FBPase] protein encoded by glpX [accession number BAB07502.1] . Bacterial FBPaseare bifunctional enzymes with both FBPase and SBPase activities.The class II variants typically have high SBPase-to-FBPase ratios[34] and play a role in one variant of the rearrangement phaseof the RuMP pathway.

The deduced fba gene product is a 285-aa protein which is 79% identical to the B . halodurans class II FBPA protein [accession number NP_244653] . Such aldolases are typically found in manybacterial autotrophs, including the chemoautotroph Xanthobacterflavus, in which its expression is induced during growth onmethanol by the ribulose bisphosphate pathway [35] . A numberof catalytically important residues in the E . coli class IIFBPA protein have been identified [28, 40], and sequence comparisonsconfirmed that these residues are conserved in the deduced B.methanolicus fba gene product.

The tkt gene encodes a 667-aa deduced protein whose primary sequence is 75% identical to the sequence of the TKT proteinof B . halodurans [accession number Q9KAD7] . TKT activity isneeded in the RuMP pathway rearrangement phase [Fig . 1], andin X . flavus TKT activity is induced sixfold upon growth onmethanol [35] . A number of residues found to be critical forcatalytic activity in the Saccharomyces cerevisiae TKT protein[24, 27, 37] are conserved in the pBM19-encoded TKT protein.

The deduced pfk gene product is a 322-aa protein that exhibits 62% overall identity with the PFK protein of B . halodurans [accessionnumber Q9K843] . In addition, it is 51% identical to the extensively characterized ATP-dependent PFK enzyme of E . coli [7] . The activesite motif TIDND, as well as the catalytically important residuesR72, D103, R162, and R252, are 100% conserved in these two proteins,indicating that the putative pfk gene of pBM19 encodes an ATP-dependentPFK protein presumably involved in the RuMP pathway cleavagephase in B . methanolicus [Fig . 1] . Interestingly, in the methylotrophicbacterium Amycolatopsis methanolica the ATP-dependent PFK proteinis specifically induced upon growth on methanol [1].

The rpe gene on pBM19 is separated from the other putative metabolic genes [Fig . 2] . The deduced 214-aa rpe gene product is 85% identicalto the RPE enzyme of Bacillus anthracis [accession number NP_846240.1].RPE catalyzes the interconversion of R-5-P and xylulose-5-phosphateand thus plays a role in the rearrangement phase of the RuMPpathway [Fig . 1] . Certain motifs and residues important forRPE activity have been reported [12], and these features, includingthe active site motif DGG, are conserved in the deduced B . methanolicusrpe gene product.

[iii] Genetic elements for plasmid replication and maintenance. The repB gene encodes a 412-aa putative protein, and the 200-aa N-terminal sequence of this gene product is 36% identical to the replication initiator protein RepB [accession number CAA71788]encoded by the Pseudomonas alcaligenes plasmid pECB2 . Immediatelyupstream of the repB gene is a distinct region with numerousdirect repeats, which may represent the pBM19 origin of replication[ori] . Another gene [parA] with a proposed function relatedto plasmid replication and maintenance is located 344 bp downstreamof repB . The primary sequence of the deduced parA gene product[256 aa] exhibits 37% overall identity to the chromosome partitionprotein ParA [accession number NP_624291] from Thermoanaerobactertengcongensis . ParA is an ATPase involved in active partitioningof bacterial chromosomes and plasmids during cell division [8].Together, repB, parA, and ori probably constitute genetic elements for pBM19 replication and segregational stability.

[iv] Mobile element-related genes. The deduced tnpI gene product is a 284-aa polypeptide exhibiting51% overall identity to a site-specific recombinase [TnpI] encodedby the Bacillus thuringiensis plasmid pGI2 [23] . TnpI belongs to the phage integrase family of resolvases, and these proteins mediate transposition processes by catalyzing the site-specific recombination of the cointegrated replicon, yielding the final transposition product . Two more genes [orf5 and orf6] with proposedfunctions related to the tnpI function are located downstreamof the rpe gene [Fig . 2] . The deduced gene products of orf5and orf6 are 37 and 79% identical to transposase proteins ofHelicobacter pylori and thermophilic bacterium PS3 [26], respectively. Four more open reading frames [orf1 to orf4] were identified, and the deduced gene products exhibited no significant similarity with proteins in the databases.

Cloning and sequencing of a chromosomal RuMP pathway fixation operon encoding HPS and PHI from MGA3. Recently, the RuMP pathway fixation operons including HPS [hps]and PHI [phi] genes from B . subtilis [38] and thermotolerantB . brevis S1 [39] were described . These genes are critical forthe fixation phase of the RuMP pathway [Fig . 1], and based onthe previously published DNA sequences we designed PCR primersfor amplification of the corresponding operon from B . methanolicusMGA3 . Three PCR fragments of the expected lengths were obtainedand cloned to obtain plasmids pRMP1, pRMP2, and pRMP3 [Table1] . The inserts of these plasmids were sequenced and were foundto represent a putative RuMP pathway fixation operon, as shownin Fig . 3 . Two genes, hps and phi, were identified, and theoverall level of identity between the MGA3 and B . brevis S1operons at the DNA level was 96%, suggesting that the clonedoperon represented the active RuMP pathway fixation genes ofB . methanolicus MGA3 . The high level of DNA sequence identitybetween these two operons is in agreement with previous reportswhich suggested that B . brevis S1 should be classified as aB . methanolicus strain [4].

 
Analysis of B . methanolicus MGA3 for chromosomal copies of pBM19 genes. After mdh and five putative RuMP pathway genes were found tobe located on plasmid pBM19, it was of interest to investigate whether chromosomal copies of these genes are present in B. methanolicus MGA3 . In Methylomonas aminofaciens a copy of the three-member RuMP pathway gene cluster was discovered [31], and two copies of the cbb operon are present in some autotrophs; one of these copies is located on the chromosome, and one is plasmid borne [10, 17] . Therefore, six different DNA probesrepresenting individual pBM19 genes [mdh-P, rpe-P, tkt-P, glpX-P,pfk-P1, and fba-P] [Table 2] were prepared . Both plasmid DNAand total DNA isolated from MGA3 were digested with restrictionenzymes having no [SalI], one [EcoRI], and three [BamHI] recognitionsites within the pBM19 plasmid, separated by gel electrophoresis,and used for Southern hybridization experiments . The resultsof these experiments are shown in Fig. 4 . The signal patternsobtained for plasmid DNA and total DNA were similar in all cases,indicating that none of these genes exists as a chromosomalcopy in B . methanolicus MGA3 . However, we cannot rule out thepossibility that isogenes with different sequences that do nothybridize to the pBM19-derived DNA probes are present in thisorganism.

 
Curing of pBM19 by using the shuttle vector pTB1.9. A shuttle vector, pTB1.9, harboring a 1.9-kb DNA fragment ofpBM19 covering the putative ori-repB region, was constructed[Fig. 5] . By using the protoplast method previously developed for B . methanolicus NOA2 mutant 13A52 [13], pTB1.9 was transformedinto MGA3 . Gel electrophoresis of plasmid DNA isolated fromMGA3[pTB1.9] confirmed the presence of pTB1.9, whereas pBM19was not detected . To analyze whether pBM19 was lost or chromosomallyintegrated, total DNA from MGA3[pTB1.9] was isolated and analyzedin a series of Southern hybridization experiments by using sixdifferent pBM19-derived DNA probes [mdh-P, rpe-P, fba-P, glpX-P,tkt-P, and pfk-P] [Table 2], as described above . No hybridizationsignals were obtained with any of the probes tested, suggestingthat the recombinant strain was cured of pBM19 [Fig . 4] . Next,a series of PCRs for amplification of regions representing sixpBM19 genes [mdh, rpe, tkt, fba, pfk, and glpX] were performedby using MGA3[pTB1.9] total DNA as the template . No bands appearedwhen we analyzed the resulting PCR products by gel electrophoresis,which is in agreement with the Southern hybridization results[see above] . As a control, total DNA from wild-type strain MGA3was used as a template in similar experiments, and in this caseall the desired DNA fragments were amplified [data not shown].Three additional MGA3[pTB1.9] transformants were analyzed ina similar manner, and in all cases the results obtained werethe same . Thus, we concluded that MGA3[pTB1.9] was cured ofpBM19, probably due to plasmid incompatibility.

MGA3 strains cured of pBM19 cannot grow on methanol but can grow on mannitol. The pBM19-cured strain MGA3[pTB1.9] was used to investigate the effect of pBM19 on the ability to utilize methanol . Mid-log-phase cultures of recombinant and wild-type MGA3 cells grown at 50°C in SOB medium containing 0.25% sucrose were diluted 100-foldin 50°C prewarmed MVcMY medium for continued growth . Whereasthe wild-type grew well under these conditions, the recombinantstrain was unable to grow, supporting the hypothesis that B.methanolicus is dependent on pBM19 for methanol utilization.We next compared these two strains in similar experiments inwhich methanol was replaced with mannitol as the sole carbonsource . This sugar was presumably taken up by the cells as F-6-P[Fig . 1], similar to what occurs in other Bacillus species [36]. Both strains grew well on this sugar, suggesting that pBM19genes are not critical for mannitol consumption in B . methanolicus.To rule out the possibility that there were any unwarrantedeffects caused by the presence of vector pTB1.9, we cured therecombinant strain of this plasmid . MGA3[pTB1.9] cells werecultivated at 50°C in SOB medium containing 0.25% sucrosefor approximately 80 generations and plated on solid SOB mediumcontaining 0.25% sucrose without antibiotic selection . Usingreplica plating, we identified neomycin-sensitive colonies,and one strain, designated MGA3C-A6, was isolated and characterized.Analysis by gel electrophoresis confirmed that pTB1.9 was notpresent in MGA3C-A6 . As expected, this strain did not grow onmethanol, whereas it grew well on mannitol . To completely excludethe possibility that the apparently cured strain was a contaminant,total DNA of MGA3C-A6 was isolated and used as template forPCR amplification of the RuMP pathway fixation operon [Fig. 3] by using PCR primers rmp-F and rmp-R3 [Table 3] . One strongband of the expected size appeared upon analysis of the PCRproduct by gel electrophoresis, and partial DNA sequencing ofthe purified fragment confirmed that it represented the expectedregion [data not shown].

Introduction of the mdh gene is not sufficient to restore methanol growth of the pBM19-cured strain MGA3C-A6. Besides mdh, it was unclear whether other pBM19 genes are involvedin methanol assimilation in B . methanolicus . To investigatethis, the mdh gene was introduced into MGA3C-A6 to test whetherthis gene is sufficient to restore the ability of this mutantstrain to utilize methanol . The mdh gene and 237 bp of upstreamsequence covering the deduced promoter region [15] was PCR amplified from pBM19 and cloned into the shuttle plasmid pTB1.9 to obtain plasmid pTB1.9mdh [Table 1] . Surprisingly, neither enzyme assaysnor sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts prepared from E . coli DH5 harboring pTB1.9mdhrevealed any MDH protein . We hypothesized that the endogenousmdh promoter region in pTB1.9mdh is not complete, and the analogousvector pTB1.9mdhL was constructed . This plasmid harbored themdh gene and 1,125 bp of upstream sequence, including the entireintergenic region between mdh and orf3 [Fig . 2] . When pTB1.9mdhLwas used, MDH activity was expressed in E . coli, similar tofindings reported previously [15] . Sodium dodecyl sulfate-polyacrylamidegel electrophoresis of crude extracts prepared from E . colicells harboring this plasmid produced a strong band at a positioncorresponding to the predicted MDH mass that was not presentin crude extracts prepared from cells harboring pTB1.9mdh [datanot shown] . Plasmid pTB1.9mdhL was therefore used to transform strain MGA3C-A6, and the resulting recombinant strain was tested for growth on methanol . The strain could still not grow on this carbon source . As reported by other workers [15], MDH activitywas virtually absent from B . methanolicus cells grown in complexmedia without methanol . This was also found to be the case forMGA3 strains [data not shown] . Therefore, as a genetic control, plasmid pTB1.9mdhL was isolated from recombinant MGA3C-A6 and transformed into E . coli, and the resulting recombinant strain was shown to express a high level of MDH activity . We believethat these results indicate that pBM19 genes besides mdh are required for methanol consumption under the conditions tested.

Estimation of the pBM19 copy number in B . methanolicus MGA3. Genes encoded on plasmids are often present at elevated doses compared to the doses of chromosomal genes, and this could potentially have biological significance . After identification of putative RuMP pathway genes having both chromosomal and plasmid origins,it was therefore of interest to determine the pBM19 copy numberin MGA3 . Two-probe Southern hybridization experiments, in whichone probe had a chromosomal origin and one probe had a pBM19origin, were used to estimate the pBM19 copy number in B . methanolicusMGA3 grown on methanol . Care was taken to design all probesso that their lengths were similar [0.78 to 0.99 kb] and theirG+C contents were similar [40.6 to 41.4%], and the concentrationsof the probes were standardized . Moreover, by using SacI-digestedtotal DNA we managed to ensure that the sizes of target DNAfragments for both the chromosome- and plasmid-directed probeswere similar . A total of three independent Southern experimentswere performed by using chromosomal probe rmp-P together withpBM19-derived probes repB-P, pfk-P2, and tkt/fba-P . The signalintensities in all experiments were similar [Fig . 6] . The intensityvolume of each band was calculated by digital image analysis.Plots of both chromosomal and plasmid intensity volumes versusDNA concentration showed that there was a good correlation [datanot shown] . The dilution ratio that gave the same intensityvolumes for chromosomal and plasmid bands could then be calculated.By using this method, the pBM19 copy number in MGA3 was estimatedto be 10 to 16 copies per chromosome.

 
Screening of B . methanolicus strains for pBM19-like plasmids. We had access to 11 different thermotolerant and methylotrophicB . methanolicus wild-type strains [designated DFS2, HEN9, TSL32, CFS, RCP, SC6, NIWA, BVD, DGS, JCP, and N2] [Table 1] isolated at the University of Minnesota . Previous preliminary characterizations of these strains at the University of Minnesota indicated that they exhibit considerable physiological variation [Rick Dillingham, unpublished results] . Thus, it was of interest to see whether pBM19-like plasmids are present in these wild-type strains.In addition, B . methanolicus PB1 [= NCIMB 13113], isolated elsewhere, as well as the previously characterized strain NOA2 [33], wereincluded in this analysis . Restriction analysis of plasmid DNA isolated from all the strains indicated that they all harbor pBM19-like plasmids . However, the restriction patterns obtainedwere not identical . Therefore, each plasmid was used as a templatefor PCR analysis with primers tba42 and tba15 [see Materialsand Methods] . These two primers amplify a 2,796-bp DNA fragmentincluding the 3'-terminal and 5'-terminal parts of the mdh andfba structural genes, respectively, as well as the entire glpX structural gene, of pBM19 . Gel electrophoresis analysis of thePCR products demonstrated that there was a single strong bandat the same predicted size in all cases [data not shown] . PartialDNA sequencing showed that the PCR fragments were similar, demonstratingthat pBM19-like plasmids or parts of such plasmids are presentin all of the B . methanolicus strains tested . These resultsindicate that plasmid-dependent methylotrophy is not restrictedto strain MGA3 and is widespread in nature.

 
Until recently it was assumed that HPS and PHI activities are restricted to methylotrophic organisms, and the presence ofthese activities was regarded as indicative of such organisms[16] . However, the identification of such enzymes in nonmethylotrophic bacteria suggests that the RuMP pathway is common in prokaryotes[30, 38] and presumably is used for detoxification of formaldehyde.Three genes, ywjI, fbaA, and ywjH, encoding class II FBPase,class II FBPA, and TA, respectively, are clustered in the B.subtilis chromosome [accession numbers Z99104 to Z99124] . Inthe obligate methylotroph M . aminofaciens 77a [31] and the facultativemethylotroph Mycobacterium gastri MB19 [25] the gene for TA was found in the same cluster as the genes for hexulose phosphate synthase and hexulose phosphate isomerase.

The remarkable finding that mdh and five putative RuMP pathway genes [pfk, rpe, tke, glpX, and fba] are present on a B . methanolicusplasmid provides crucial information regarding the genetic basisfor methanol metabolism in this organism . Chromosomal copieswere not detected for any of these genes in B . methanolicusMGA3, and the complete inability to grow on methanol of pBM19-curedstrain MGA3C-A6 confirms that this plasmid is essential formethanol consumption under the conditions tested . In B . methanolicusC1 [= NCIMB 13114] it has been unequivocally demonstrated thatmethanol oxidation is catalyzed by the NAD[P]-dependent MDHencoded by mdh [4, 15] . Despite the extensive biochemical characterization of this protein [15, 19, 20], the mdh gene has never been reported to originate on a plasmid . The failure to complement growthof MGA3C-A6 on methanol by introducing the MDH expression plasmid pTB1.9mdhL implies that additional pBM19 genes are requiredfor methanol consumption . The ability of MGA3C-A6 to grow rapidlyon mannitol suggests that B . methanolicus has isoenzymes ofboth PFK and FBPA to metabolize F-6-P [Fig . 1], and it also implies that MGA3C-A6 and MGA3 are similar in other respects.

The gene encoding the MDH activator protein ACT was not foundon pBM19, yet expression of this protein and expression of MDHin B . methanolicus have been reported to be regulated coordinately[19, 20] . This suggests that methanol oxidation in B . methanolicusmay be governed by the concerted action of both chromosomallyand plasmid-borne genes . The latter notion is supported by ourfinding that the formaldehyde fixation genes hps and phi arepresent on the B . methanolicus chromosome . Also not presenton pBM19 are genes for the RuMP pathway enzymes TA and ribose-5-phosphateisomerase [Fig . 1] . Although low levels of activity of bothproteins have been detected in crude extracts of this bacterium[4], it is not known whether the proteins are crucial for methanolconsumption . This fact, together with the presence of the glpXgene on pBM19, suggests that the SBPase variant, and not theTA variant, is the relevant RuMP pathway in this organism [Fig.1] . The advantage of possessing certain RuMP pathway genes ona multicopy plasmid is unknown . However, based on the presentresults it is tempting to speculate that methylotrophy may bea transferable metabolic property in nature.

Previous reports have shown that B . methanolicus is sensitive to rapid changes in methanol concentrations, presumably dueto toxic intracellular formaldehyde accumulation [29] . Although HPS synthesis has been reported to be induced by formaldehyde, there is noncoordinate expression of this enzyme and MDH inB . methanolicus, and cells may have high MDH levels and lowHPS levels [5] during growth on methanol . Based on the present findings it is tempting to speculate that this feature is partially caused by the multiple copies of the mdh gene compared to the chromosomal hps gene [and the phi gene] . It is possible that engineering of a pBM19 derivative that includes these two genes may result in improved formaldehyde tolerance, as well as higher methanol assimilation rates if the derivative is introducedinto B . methanolicus strains.

 
This work was supported by a grant from the Research Councilof Norway.

We are grateful to Rick Dillingham for isolation of B . methanolicus strains, and we thank Trine Aakvik for helping with the cloning and sequencing of the RuMP pathway fixation operon . Also, wethank Sergey B . Zotchev for carefully reading the manuscriptand Arne Strøm and Kjell Josefsen for encouraging discussionsduring the course of this work.

 
* Corresponding author . Mailing address: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway . Phone: 47 73 59 86 89 . Fax: 47 73 59 12 83 . E-mail: trygve.brautaset@biotech.ntnu.no .

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