Journal of Bacteriology, March 2004, p . 1565-1570, Vol . 186, No . 5

Characterization of Two Methanopterin Biosynthesis Mutants of Methylobacterium extorquens AM1 by Use of a Tetrahydromethanopterin Bioassay

Madeline E . Rasche,^* Stephanie A . Havemann, and Mariana Rosenzvaig

Microbiology and Cell Science Department, University of Florida, Gainesville, Florida 32611-0700

Received 7 July 2003/ Accepted 12 November 2003

 
An enzymatic assay was developed to measure tetrahydromethanopterin [H₄MPT] levels in wild-type and mutant cells of Methylobacterium extorquens AM1 . H₄MPT was detectable in wild-type cells but not in strains with a mutation of either the orf4 or the dmrA gene, suggesting a role for these two genes in H₄MPT biosynthesis.The protein encoded by orf4 catalyzed the reaction of ribofuranosylaminobenzene5'-phosphate synthase, the first committed step of H₄MPT biosynthesis.These results provide the first biochemical evidence for H₄MPT biosynthesis genes in bacteria.

 
Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium capable of growth on succinate and one-carbon [C₁] compounds . Growth on C₁ compounds requires several clusters of genes found on the chromosomal DNA [5, 6], and a number ofthese genes code for enzymes which have archaeal homologs thatdepend on tetrahydromethanopterin [H₄MPT] or structurally relatedcoenzymes [6, 7, 24, 25] . Previously, these coenzymes had beenfound only in methanogenic or hyperthermophilic sulfur-dependentarchaea [9, 19, 22, 29, 32].

M . extorquens cells contain a form of H₄MPT called dephospho-H₄MPT[7] . Although it has been assumed that this bacterium producesdephospho-H₄MPT biosynthetic enzymes, these proteins have notyet been identified, and their evolutionary relationship toarchaeal enzymes is unknown . In archaea, the genes encodingonly 4 of the 18 putative H₄MPT biosynthesis enzymes have beenidentified [14, 15, 28, 33, 34] . One of these enzymes, ribofuranosylaminobenzene 5'-phosphate [RFAP] synthase, catalyzes the first committed step of H₄MPT biosynthesis [26, 28] . In M . extorquens, a geneencoding an RFAP synthase homolog [orf4, also called mptG] hasbeen found clustered among several genes encoding H₄MPT-dependentenzymes [6, 7] . The orf4 gene product is 29% identical to RFAP synthase from Archaeoglobus fulgidus [28] . The protein encodedby a second putative H₄MPT biosynthesis gene [dmrA] shows homologyto bacterial dihydrofolate reductases and has been proposedby Marx et al . [21] to encode dihydromethanopterin reductase,which would catalyze the final step of H₄MPT biosynthesis . ThedmrA mutant cannot grow on C₁ compounds and exhibits a methanol-and formaldehyde-sensitive phenotype characteristic of mutantsdeficient in H₄MPT-dependent metabolism.

To test the hypotheses that orf4 and dmrA encode H₄MPT biosynthesisenzymes, we have developed an enzymatic assay to measure H₄MPTlevels in M . extorquens mutants . The assay is based on the NAD⁺-reducingactivity of methylene-H₄MPT dehydrogenase B [MtdB] [16] [Fig.1] . Here, we provide the initial biochemical evidence for twoH₄MPT biosynthetic genes in M . extorquens and demonstrate thatthe protein encoded by orf4 has RFAP synthase activity.

 
Methanosarcina thermophila cells were grown anaerobically on acetate as previously described [28] . M . extorquens AM1 wild-typeand mutant strains were generously provided by the laboratoryof Mary Lidstrom . It has previously been shown that the orf4,dmrA, and fae mutants are unable to grow on methanol and thatcomplementation of each mutant with the corresponding plasmid-bornegene restores the wild-type phenotype, indicating that the mutantphenotype is not due to a polar effect [7, 21, 31] . Wild-typeM . extorquens cells were grown at 30°C on modified minimalmedium at pH 7.0 with 20 mM succinate or 0.5% [vol/vol] methanolas previously described [1] except that the concentration ofCaCl₂ · 2H₂O was 2.5 mg per liter . M . extorquens AM1is naturally resistant to rifamycin, which was routinely addedto wild-type and mutant cultures at 50 µg per ml to prevent contamination by other microorganisms . Cultures of the orf4, dmrA, and fae mutants were grown on succinate, rifamycin, and kanamycin [50 µg per ml] . When the cultures reached anoptical density at 600 nm [OD₆₀₀] of 0.6, either 10 ml of 1M succinate [pH 7.0] or 5 ml of 100% methanol was added . Atan OD₆₀₀ between 0.8 and 1.0, the cells were harvested by centrifugationand washed with 50 mM TES [tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid] [pH 7.0; Fisher Scientific, Suwanee, Ga.], 10 mM MgCl₂, and either 10 mM succinate [for cells grown on succinate] or1% methanol . Cells were stored in liquid N₂.

For high-level expression of MtdB, the mtdB gene [16] was amplifiedby the PCR [27] for cloning into the NdeI and BamHI sites ofpET28b [Novagen, Inc., Madison, Wis.] . This vector introducesan N-terminal six-histidine [His₆] tag . The template was plasmidpALS8 [7], and the primers were 5'-GGACGTCCATATGGCCCGCTCGATCCTGCACAand 5'-GAAGGATCCTCATCCGGCGATCTCGAC . After amplification withPfu polymerase [Stratagene, La Jolla, Calif.], the PCR productwas purified with a PCR purification kit [QIAGEN, Valencia,Calif.], cut with NdeI and BamHI [New England Biolabs, Beverly, Mass.], and ligated [T4 DNA ligase; New England Biolabs] intopET28b cut with the same enzymes . The DNA was used to transform electrocompetent Escherichia coli DH1 . The sequence of the insert was verified by dideoxy sequencing [27], and the plasmid wastransformed into E . coli BL21[DE3]:RIL cells [Stratagene] . Theexpression cell line was called SW11.

For overproduction of His₆-MtdB, SW11 cells were grown in Luria-Bertanimedium with kanamycin [50 µg per ml] at 37°C . When cells reached an OD₆₀₀ of 0.8, expression was induced with isopropylthiogalactoside[IPTG; Inalco Pharmaceuticals, San Luis Obispo, Calif.] at 1mM . Cells were harvested after 3 h, washed with 50 mM MOPS [morpholinepropanesulfonicacid] [pH 7.0] and 10 mM MgCl₂, suspended in the same buffer[2 ml of buffer per g of cells], disrupted by French pressurecell lysis at 20,000 lb/in², and centrifuged at 27,000 x g for 60 min . The supernatant [cell extract] was stored in 400-µl portions at -80°C . Because H₄MPT is oxygen sensitive, His₆-MtdB was partially purified in an anaerobic chamber by using Ni-nitriloacetic acid [NTA] spin columns [QIAGEN] . The protein was eluted with 250 mM imidazole [pH 8.0] according to the manufacturer's instructions.

For determination of H₄MPT concentrations, M . extorquens cells[10 to 16 g] were thawed in an anaerobic chamber [Coy Products, Inc., Grass Lake, Mich.] containing 2% H₂ and 98% N₂ . Breakagebuffer [50 mM TES [pH 7.0], 10 mM MgCl₂, 20 mM 2-mercaptoethanol]with DNase I [Sigma Chemical Co., St . Louis, Mo.] was addedat a ratio of 1 ml of buffer per g of cells . Cells were disruptedanaerobically by two passages through a French pressure celland centrifuged for 2 h at 27,000 x g [4°C] . The supernatantwas filtered through a 0.45-µm-pore-size filter [Millipore,Bedford, Mass.] . Proteins were removed by using a Centricon-3filtration device [Millipore] in the absence of O₂ . The filtrate[filtered cell extract] was stored anaerobically in a glassvial covered with foil to protect H₄MPT from light inactivation.

H₄MPT was partially purified from filtered M . extorquens cellextracts by using ion-exchange and hydrophobic-interaction chromatographyin an anaerobic chamber [10] . To filtered cell extract [12 to16 ml], an equal volume of buffer A [50 mM MOPS [pH 6.8], 1%[vol/vol] 2-mercaptoethanol] was added . The mixture was loadedonto a 1-ml column of DEAE-Sephadex A25-125 [Sigma] . AlthoughH₄MPT did not bind to the column, some contaminants bound tothe column and were removed . H₄MPT was concentrated on a 0.5-mlSerdolit Pad I column [Serva, Heidelberg, Germany] equilibratedwith buffer B [1.4% [vol/vol] formic acid [pH 3], 10 mM 2-mercaptoethanol.]The column was washed with 2 ml of buffer B, followed by a methanolgradient of 1 ml each of 15, 25, and 50% [vol/vol] in bufferB . The pH of each fraction was adjusted to 7 . Formaldehyde [2µl of a 37% [vol/vol] solution] was added to 800 µlof the fractions, and the mixtures were incubated at room temperaturefor 10 min . After the solutions were transferred to a 3-ml glasscuvette, 1.1 ml of assay buffer [120 mM KH₂PO₄ [pH 6.8], 3 mMformaldehyde] and 20 µl of Ni-NTA-purified His₆-MtdB wereadded . The absorbance at 340 nm [A₃₄₀] was monitored for 25s, and the reaction was initiated with 100 µl of 2 mMNAD⁺ . The amount of NADH produced was estimated by using anextinction coefficient at 340 nm of 6.22 per mM NADH per cm [8].

To prepare samples containing tetrahydrosarcinapterin [H₄SPT] from Methanosarcina thermophila TM1, cells [5 g] were sealed in a stoppered serum vial and purged with H₂ gas for 5 min. H₂ treatment was required for the enzymatic reduction of the oxidized forms of sarcinapterin to H₄SPT . Anaerobic acetate buffer [10 ml of 30 mM sodium acetate [pH 4.0], 200 mM 2-mercaptoethanol] was added, and the cells were autoclaved for 15 min . The autoclaved cell extract was centrifuged anaerobically at 13,000 x g for20 min to remove precipitated proteins . The supernatant containingH₄SPT was stored in anaerobic vials at -80°C . For the measurementof H₄SPT, the assay mixture contained 1.8 ml of assay buffer[120 mM KH₂PO₄ [pH 6.8], 3 mM formaldehyde], 20 µl ofNi-NTA-purified His₆-MtdB, and 100 µl of heat-treatedcell extract . The reaction was initiated with 100 µl of2 mM NAD⁺.

PCR was used to amplify the orf4 gene from plasmid pALS8 [7]. The primers [5'-GATCCATATGAGACCGTGGCCCGAGGTCCCG and 5'-CATGGGATCCCTAAACTTCCGCAACCGAG;Genosys] introduced a 5' NdeI site and a 3' BamHI site for cloninginto pET15b [Novagen], which provides an N-terminal His₆ tag.The plasmid [pCL1] was transformed into chemically competentDH1 cells, and the sequence of the insert was verified . Theplasmid was transformed into BL21[DE3] cells [Novagen] containingthe pG-Tf2 plasmid for expression of a chaperone to assist inprotein folding [HSP Research Institute, Hayashibara BiochemicalLaboratories, Inc., Okayama, Japan] [23] . Expression of theHis₆-orf4 gene was induced as previously described for the RFAPsynthase gene from Methanothermobacter thermautotrophicus [2] except that ampicillin [125 µg per ml] was used insteadof kanamycin.

RFAP synthase activity was measured as previously described[28] except that the reaction mixtures were incubated for 16h at 30°C in 50 mM TES [pH 7.0] . Protein concentrationswere measured by using the Bradford assay [Bio-Rad] [3] withbovine serum albumin as the standard . Proteins were separatedby sodium dodecyl sulfate-polyacrylamide gel electrophoresisand stained with Coomassie brilliant blue R-250 [Bio-Rad] [12]. Phosphoribosylpyrophosphate [PRPP] was obtained from Sigma.All other chemicals were obtained from Fisher Scientific.

 
To facilitate the discovery of H₄MPT biosynthetic genes, an enzymatic assay was developed to enable the rapid screeningof mutants deficient in H₄MPT production . In this assay, formaldehyde is added to protein-free cell extracts to chemically convertH₄MPT to methylene-H₄MPT [Fig . 1] . The oxidation of methylene-H₄MPTis coupled to the reduction of NAD⁺ via MtdB from M . extorquens,producing an increase in A₃₄₀ . MtdB is highly specific for H₄MPTand does not react with tetrahydrofolate [16] . Thus, the enzymecan be used to distinguish between H₄MPT and tetrahydrofolatein bacterial cells . The production of a histidine-tagged versionof the enzyme [His₆-MtdB] allowed for the rapid purificationof large quantities of the enzyme by nickel affinity chromatography.

Because M . extorquens cells contain low concentrations of H₄MPT relative to those of methanogens [7, 13], the assay conditionswere first optimized by using extracts of the methanogen Methanosarcinathermophila . This organism produces H₄SPT, an H₄MPT analog [20]. When H₂-reduced Methanosarcina thermophila extracts were heatedto remove proteins and combined with formaldehyde, NAD⁺, andHis₆-MtdB, an increase in A₃₄₀, corresponding to the productionof NADH, was observed [Fig. 2, line 1] . No increase in A₃₄₀ was observed if any of the reaction components [formaldehyde,heated methanogen cell extract, His₆-MtdB, and NAD⁺] were omitted [Fig . 2, lines 2 to 5] . These results demonstrate that methylene-H₄SPTis a substrate for His₆-MtdB and that His₆-MtdB can be usedto detect H₄MPT analogs in cell extracts.

 
The MtdB assay was then used to measure H₄MPT levels in wild-type M . extorquens extracts . Initial attempts to measure H₄MPT levelsin M . extorquens extracts were unsuccessful due to the highbackground A₃₄₀ . To decrease the absorbance due to contaminatingmolecules, H₄MPT was partially purified by DEAE-Sephadex andhydrophobic-interaction chromatography . By this procedure, H₄MPTwas detected at a concentration of 44 µM in wild-typeM . extorquens cells grown on methanol [Fig . 3, line 1] . Whencells were grown on succinate, the H₄MPT concentration was abouthalf the level found in methanol-grown cells [Fig . 3, line 2]. This result was expected based on the report that H₄MPT-dependent cyclohydrolase activity in M . extorquens is lower during growth on succinate than during growth on methanol [30] . This findingmay indicate that the H₄MPT-dependent pathway is inducible duringgrowth on methanol.

 
The orf4 and dmrA genes of M . extorquens have previously beenproposed to encode bacterial H₄MPT biosynthetic enzymes [21,28] . To test these hypotheses, the enzymatic assay was usedto determine whether the orf4 and dmrA deletion mutants grownon succinate were capable of producing H₄MPT . When the orf4mutant was tested by using the His₆-MtdB assay, no increasein A₃₄₀ was detected [Fig . 3, line 4], indicating the absenceof H₄MPT in orf4 mutant extracts . Similarly, no H₄MPT was detectedin extracts of the dmrA mutant [Fig . 3, line 5] . This resultis consistent with roles for orf4 and dmrA as H₄MPT biosyntheticgenes.

As an additional control, we measured the level of H₄MPT in a mutant for a gene that is not involved in H₄MPT biosynthesis. The fae gene codes for the formaldehyde-activating enzyme [31], which catalyzes the reaction between formaldehyde and H₄MPT to produce methylene-H₄MPT . This enzyme is not required for H₄MPT biosynthesis . As predicted, H₄MPT was detected in extractsof the fae mutant [Fig . 3, line 3] at about two-thirds the levelfound in wild-type cells grown on succinate . We suspect thatthis difference may be due to the inefficiency of the fae mutantin converting formaldehyde and H₄MPT to methylene-H₄MPT, thesubstrate for His₆-MtdB . In support of this hypothesis, we foundthat the complete nonenzymatic conversion of formaldehyde andH₄MPT to methylene-H₄MPT required 10 min in wild-type cell extracts but 2 h in fae mutant extracts, suggesting that a smaller proportion of the H₄MPT in fae mutants was originally present as methylene-H₄MPT.

 
To provide biochemical evidence that the orf4 gene codes for RFAP synthase, we measured the RFAP synthase activity of M. extorquens wild-type and orf4 mutant cells . However, because of the low activity of the enzyme in M . extorquens cell extracts, it was necessary to incubate the assay solutions for an extended time period [16 h] to obtain reliable results . Extracts of wild-type M . extorquens cells contained a low level of RFAP synthase activity [0.49 nmol of RFAP produced in 16 h with 4 mg of protein] [Table 1] . This value is about 100 times lower than the specific activityof RFAP synthase in methanogen cells [28] . RFAP synthase activitywas not observed when the substrate PRPP was omitted from theassay . Furthermore, the RFAP synthase activity of M . extorquenscells was inhibited by a known RFAP synthase inhibitor, p-methylaminobenzoicacid, under conditions that inhibit RFAP synthase from methanogens[26] . In contrast, no RFAP synthase activity was detectablein extracts of the orf4 mutant [Table 1].

 
Attempts to purify RFAP synthase from M . extorquens cells were unsuccessful because of enzyme instability . Therefore, the orf4 gene was cloned into the pET15b vector for expression in E. coli . Initial attempts to express orf4 at 37°C with or without a His₆ tag resulted in large amounts of insoluble protein . Both the soluble and the insoluble fractions from the cells lacked RFAP synthase activity [data not shown] . A similar difficultywas previously encountered in expressing RFAP synthase from Methanothermobacter thermautotrophicus [2] . This problem wasovercome by coexpressing the RFAP synthase gene with a plasmid-encodedchaperone at 20°C . Under these same conditions, a smallproportion of the His₆-Orf4 protein was produced as solubleRFAP synthase . Over a period of 16 h, cell extract [1.5 mg of protein] produced 4.3 nmol of RFAP [Table 1] . The His₆-Orf4 protein was partially purified [23-fold] by nickel affinity chromatography; however, this procedure did not result in pure protein because of the low level of enzyme produced in the soluble form . RFAP synthase activity was undetectable in extracts ofcells containing the pET15b vector without orf4 . Taken together, these results demonstrate that M . extorquens cells contain RFAP synthase activity and that orf4 functions in H₄MPT biosynthesisas a bacterial RFAP synthase gene.

 
M . extorquens contains several clusters of genes required for C₁ metabolism, including genes that encode homologs of archaeal H₄MPT-dependent and methanofuran-dependent enzymes [6, 7] . Thefunctions of many of the C₁ metabolism genes are unknown, butsome have been proposed to play roles in H₄MPT and methanofuranbiosynthesis [6] . In this work, the production of a His₆-taggedform of MtdB enabled us to develop an enzymatic assay to measureH₄MPT levels in cell extracts and assign H₄MPT biosynthetic functions to two of the uncharacterized C₁ gene products . The orf4 mutant lacked RFAP synthase activity, while the recombinant His₆-Orf4 protein catalyzed the RFAP synthase reaction [Table 1] . This is the first biochemical evidence for an RFAP synthasegene outside the archaea . The proposed role of dmrA as a dihydromethanopterinreductase [21] is supported by the inability of the dmrA mutantto produce H₄MPT [Fig . 3] and by additional evidence obtainedin our laboratory that the DmrA protein catalyzes the NAD[P]H-dependent reduction of H₂MPT to H₄MPT [M . A . Caccamo, C . S . Malone, andM . E . Rasche, Abstr . 103rd Gen . Meet . Am . Soc . Microbiol., abstr. K-065, 2003] . The His₆-MtdB assay described here will be used to identify additional genes of the H₄MPT biosynthesis pathway in methylotrophic bacteria.

The distribution of H₄MPT-dependent pathways among bacteria and archaea is becoming clearer in light of the many prokaryotic genomes being sequenced . H₄MPT-dependent enzymes have been foundin autotrophic Xanthobacter strains, in methanotrophs, and inmethylotrophic bacteria that use the serine pathway or the ribulosemonophosphate [RuMP] pathway to assimilate formaldehyde [30]. Genome sequencing indicates that the aerobic hyperthermophilic archaeon Aeropyrum pernix and other diverse microorganisms contain RFAP synthase homologs [4, 11, 17, 18, 28] . These organismsmay contain previously unidentified forms of H₄MPT . At leastsix derivatives of H₄MPT have been characterized by structuralanalyses [7, 19, 20, 32], and the MtdB enzyme used in this workreacts with at least three of these analogs [H₄MPT from Methanothermobactermarburgensis [16], H₄SPT from Methanosarcina thermophila [Fig. 2], and dephospho-H₄MPT from M . extorquens [Fig . 3]] . Thus,the enzymatic assay for H₄MPT may offer a convenient methodfor detecting previously uncharacterized forms of H₄MPT as wellas for identifying the remaining H₄MPT biosynthetic genes ofbacteria and archaea.

 
We are grateful to Mary Lidstrom, Ludmila Chistoserdova, and Christopher Marx for their generosity in sharing the plasmidpALS8 and the M . extorquens wild-type, orf4, dmrA, and fae mutantstrains . We thank Jack Shelton for sequencing the mtdB and orf4genes and Vicki Kopf and Chi Bissett for their research contributions.

This work was supported by National Science Foundation grant numbers MCB-9876212 and MCB-9815924 and the Florida Agricultural Experiment Station.

 
* Corresponding author . Mailing address: Microbiology and Cell Science Department, University of Florida, P.O . Box 110700, Gainesville, FL 32611-0700 . Phone: [352] 392-1192 . Fax: [352] 392-5922 . E-mail: mrasche@ufl.edu .

Florida Agricultural Experiment Station journal series no . R-09891.

Present address: Chemistry Department, University of Florida, Gainesville, Florida.

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What Is Rhizobia?, What Is Biofilm?, What Is Growth Medium?, What Is Functional Genomics?, What Is Botulism?, e, Microorganism, a, Bacteriology, r, Microbiology, c, Microorganisms, o, Bacteria, n, Rhizobacter, s, Listeriosis, r, Bacillus, o, Yeasts, c, Escherichia coli, r, Wastewater treatment, a, Escherichia coli, r, Fermentations, a, Fermentations, i, Klebsiella, r, Clostridia, n, Vibriosis, s, Antibiotics, a, Prokaryotes, n, Neisseria, a, Staphylococcus, n, Bacteriological, s, Enterococci, e, Fermentations, n, Escherichia coli, n, Rhizobacter




 

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