Expression Vectors for Methanococcus maripaludis: Overexpression of Acetohydroxyacid Synthase and ß-Galactosidase

Corresponding author: William B. Whitman, Department of Microbiology, University of Georgia, Athens, GA 30602-2605., whitman{at}arches.uga.edu (E-mail)

Communicating editor: A. KLEIN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

A series of integrative and shuttle expression vectors was developed for use in Methanococcus maripaludis. The integrative expression vectors contained the Methanococcus voltae histone promoter and multiple cloning sites designed for efficient cloning of DNA. Upon transformation, they can be used to overexpress specific homologous genes in M. maripaludis. When tested with ilvBN, which encodes the large and small subunits of acetohydroxyacid synthase, transformants possessed specific activity 13-fold higher than that of the wild type. An expression shuttle vector, based on the cryptic plasmid pURB500 and the components of the integrative vector, was also developed for the expression of heterologous genes in M. maripaludis. The ß-galactosidase gene from Escherichia coli was expressed to ~1% of the total cellular protein using this vector. During this work, the genes for the acetohydroxyacid synthase (ilvBN) and phosphoenolpyruvate synthase (ppsA) were sequenced from a M. maripaludis genomic library.

METHANOGENS are important catalysts in the global carbon cycle. These strictly anaerobic archaea are the source of most of the earth's atmospheric methane, a potent greenhouse gas, and they are responsible for processing 1–2% of the carbon fixed every year during photosynthesis (REEBURGH et al. 1993 ). Given the enormity of this biogeochemical process, it is not surprising that methanogens are found in many different types of environments, ranging from psychrophilic to hyperthermophilic conditions (for a review see BOONE et al. 1993 ). In this regard, they are the most cosmopolitan archaea; they are abundant in extreme environments, such as deep-sea hydrothermal vents, as well as temperate environments, such as the gastrointestinal tracts of man and animals.

Methanococcus maripaludis strain JJ is typical of other hydrogenotrophic methanococci. It was isolated from the sediment of a salt water marsh and grows by the conversion of carbon dioxide and hydrogen gas or formate into methane (JONES et al. 1983A ). Amino acids and acetate are assimilated, but they are not significantly metabolized, indicating that methanogenesis is the sole or major energy source (WHITMAN et al. 1987 ). An important characteristic of M. maripaludis is its relatively rapid doubling time of 2.3 hr at the optimal temperatures of 35–39° (JONES et al. 1983A ). Thus, it is one of the fastest growing mesophilic methanogens.

M. maripaludis has also proven amenable to genetic analysis (TUMBULA and WHITMAN 1999 ). Integrative and shuttle vectors have been developed for Methanococcus species (GERNHARDT et al. 1990 ; COHEN-KUPIEC et al. 1997 ; TUMBULA et al. 1997 ). These vectors are efficiently transformed into M. maripaludis by a polyethylene glycol-based method (TUMBULA et al. 1994 ). Like other methanogens, M. maripaludis contains many oxygen-sensitive enzymes with complex prosthetic groups. These enzymes are not frequently expressed in an active form in Escherichia coli. Thus, a genetic system in M. maripaludis might be suitable for the expression of genes encoding these types of enzymes. We report here the development of expression shuttle and integrative vectors for M. maripaludis that use the M. voltae histone promoter (P_hmvA).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Bacterial strains, media, and growth conditions:
M. maripaludis JJ was obtained from W. J. Jones. The plasmids used in this study are listed in Table 1. M. maripaludis was grown at 37° on 275 kPa of H₂:CO₂ (80:20) in McN (mineral medium), McC (complex medium minus the vitamin solution), or McNA (McN plus 10 mM sodium acetate; WHITMAN et al. 1986 ). The sodium bicarbonate in the medium was reduced from 5 g liter^-1 to 2 g liter^-1 during growth at 100 kPa in 1-liter Wheaton bottles. Transformation of M. maripaludis with plasmid DNA was described previously (TUMBULA et al. 1994 ). For selection of puromycin-resistant methanococci, a stock solution of 500 µg ml^-1 puromycin dihydrochloride (Sigma, St. Louis) in distilled water was filter sterilized and added to the medium at a final concentration of 2.5 µg ml^-1. Strictly anaerobic techniques were used for medium preparation and cultivation.

The E. coli strains were grown at 37° on low-salt Luria-Bertani (LB) medium with the NaCl at 50% of the regular concentration (MANIATIS et al. 1982 ). Antibiotic concentrations were 60 µg ml^-1 for ampicillin and 50 µg ml^-1 for kanamycin in both liquid and solid LB medium. ß-Galactosidase was used as a blue/white screen on LB agar plates with isopropylthio-ß-D-galactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) according to the method of SAMBROOK et al. 1989 . Plasmids were transformed into E. coli strains by electroporation with a Gene Pulser (Bio-Rad, Richmond, CA) using the settings of 200 , 2.5 kV, and 25 µF with cuvettes (0.2 cm gap width).

Growth rates of E. coli and M. maripaludis were measured at 600 nm with a spectrophotometer (Spectronic 20).

Plasmid purification from M. maripaludis and E. coli:
Plasmid isolation from M. maripaludis was begun by centrifuging a 5-ml culture at 16,000 x g at 4° followed by resuspension of the cells in 100 µl of methanococcal medium. After resuspension, the Wizard miniprep kit (Promega, Madison, WI) minus the resuspension step was used for preparation of the plasmid DNA for transformation into E. coli. pURB500 was isolated from M. maripaludis C5 as described previously (TUMBULA et al. 1994 ). Plasmids from E. coli, grown with selective conditions, were isolated with the Wizard miniprep kit.

Construction of M. maripaludis genomic library:
M. maripaludis genomic DNA was isolated using a modified procedure from SAITO and MIURA 1963 . M. maripaludis was grown in 150 ml of McN to A₆₀₀ = 0.8. The cells were harvested by centrifugation at 7700 x g at 4° for 15 min in a JA-21 rotor (Beckman, Fullerton, CA) and washed once in 28 ml of sterile 0.4 M NaCl. The cell pellet was then resuspended in 3 ml of 0.15 M NaCl/0.1 M EDTA (pH 8.0). The cell suspension was frozen by plunging the centrifuge tube into an ethanol/dry ice bath. The frozen suspension was slowly thawed at 18°, at which time the cells lysed. The cell extract was then transferred to a Corex tube, and 5 ml of 50% phenol saturated with 0.15 M NaCl/0.1 M EDTA (pH 8.0) and 50% chloroform:isoamyl alcohol (24:1 v/v) were added. The solution was gently mixed by inversion before centrifugation at 8200 x g for 10 min. The red aqueous phase was extracted with another 5 ml of phenol/chloroform/isoamyl alcohol. Two volumes of cold 100% ethanol were then added to the aqueous phase, and the suspension was gently inverted before centrifugation at 4° for 15 min at 8200 x g. The supernatant was decanted, and the pellet was dried at room temperature. The pellet was resuspended in 100 µl of 15 mM NaCl/1.5 mM trisodium citrate (SSC) and stored at 4°. Additional SSC was added every 24 hr until the DNA was resuspended.

To make a genomic library for M. maripaludis, the genomic DNA (12 µg in 100 µl) was partially digested with 0.01 units of Sau3AI at 37° for 5 min. The reaction was terminated by the addition of phenol/chloroform/isoamyl alcohol, pH 8.0 (Ameresco). The resulting linear DNA fragments ranged from 1 to 6 kbp, as measured by gel electrophoresis. The DNA was precipitated upon the addition of one-half volume of 7.5 M ammonium acetate and two volumes of cold 100% ethanol followed by centrifugation. After the supernatant was decanted, the pellet was resuspended in 10 µl of distilled water. Five micrograms of digested DNA (5 µl) was ligated with 2 µl of Zap Express (Stratagene, La Jolla, CA) that was predigested with BamHI in a 10-µl reaction. After the ligation, the mixture was packaged and amplified once in E. coli XLOLR (Stratagene) before storage at -80° according to the company's directions.

Isolation and sequencing of the M. maripaludis ilvBN:
The M. maripaludis genomic library was screened with a polymerase chain reaction (PCR) product of the acetohydroxyacid synthase (AHAS) large subunit gene (ilvB) from M. aeolicus using pTLB30 as the template. The PCR contained the primers 5'-GAACGGAGCAGAGGC-3' and 5'-ACCAACCATACCAAGGGC-3', 5 units Taq DNA polymerase, 1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65 mM dTTP, and 0.35 mM digoxigenin-deoxyuridine-5'-triphosphate (DIG-dUTP) from Boehringer Mannheim (Indianapolis, IN). The annealing temperature was 48°, and the extension time was 2 min at 72°. This DIG-dUTP-labeled PCR product was used to screen plaque lifts from the library according to Boehringer Mannheim protocols. The hybridization temperature was 63°. The isolated phagemid was then converted to the plasmid form (pWLG1). SacI was used to subclone a 3.8-kbp fragment of pWLG1. This fragment was ligated into a SacI-digested pUC18, yielding pWLG3. The remaining portion of pWLG1 was religated together to form pWLG2. These three plasmids were sequenced by primer walking utilizing API sequencers at the Molecular Genetics Facility, University of Georgia. All oligonucleotides were made by Integrated DNA Technologies (Coralville, IA). Sequence analysis was performed with the GCG software package (Genetics Computer Group, Madison, WI). FRAMES was used to identify open reading frames (ORFs) using ATG, GTG, or TTG as the start codons. GAP was used to calculate the percentage identities between the methanococcal proteins. PILEUP was used to identify the truncated regions for the phosphoenopyruvate synthase gene (ppsA) and the AHAS small subunit gene (ilvN).

Plasmid construction:
The integrative vectors developed in this study were based on pMEB.2 (Figure 1). To remove the -complementation fragment of lacZ, pMEB.2 was digested with NdeI and SacI, followed by mung bean nuclease treatment and blunt-end ligation (Figure 1). This deletion allowed a unique NdeI site to be introduced into the multiple cloning site (MCS). The MCS was added after digestion of the plasmid with EcoRV and EcoRI and dephosphorylation with CIAP (calf intestine alkaline phosphatase). The phosphorylated oligonucleotides, 5'-AATTCAAGCATCATATGAAGCATACGCGTCTTAAGAGATCTCATGAT-3' and 5'-ATCATGAGATCTCTTAAGACGCGTATGCTTCATATGATGCTTG-3', were annealed and ligated into the vector forming pWLG11. The EcoRI fragment of Mipuid, which contained the M. voltae histone promoter (P_hmvA) and ribosome binding site and the E. coli ß-glucuronidase (uidA), was then cloned into the EcoRI site of pWLG11. Removal of uidA by NdeI digestion formed the M. maripaludis integrative expression vector pWLG13. To form pWLG13+5'-ilvB, the first 641 bp of the M. maripaludis ilvB were cloned into pWLG13. First, the 5' end of ilvB was amplified by PCR using the primers 5'-AAAAAAACATATGAAAGGAGCAGAGGCTATGATG-3' and 5'-AAAAAAAGATCTCCGCCTGCAATAATAACAGGTCTT-3'. These primers contained NdeI and BglII sites at the 5' and 3' ends, respectively. The PCR contained 2.5 units of Pfu DNA polymerase (Stratagene), and it was performed at an annealing temperature of 55° for 1 min and an extension time of 2 min at 72°. The PCR product was drop dialyzed against distilled water with VSWP membranes (Millipore, Bedford, MA) before digestion with NdeI and BglII for 4 hr. The enzymes in the reactions were removed using the Wizard clean-up kit (Promega).

View larger version (25K): In this window In a new window Download PPT slide   Figure 1. Construction of the integrative vectors pWLG13, pWLG14, and pWLG18. Only the unique or otherwise useful restriction sites are shown. pMEB.2 was digested with NdeI and SacI to remove lacZ' before a MCS was added to form pWLG11. pWLG14 was constructed by the addition of PhmvA between the EcoRI and BglII sites of pWLG11. lacZ, modified by PCR to contain NsiI and BglII restriction sites, was ligated into pWLG14 to form pWLG18. pWLG12A was developed from pWLG11 by the addition of PhmvA and uidA from Mipuid. The uidA was released from pWLG12A by a NdeI digestion to form pWLG13. The MCS was designed so that multiple restriction enzymes can be utilized in a single buffer. Using either NdeI or NsiI plus a second restriction enzyme allows directed cloning into the vectors. Addition of a third endonuclease with a site between the first endonuclease sites can lower the background associated with the religation of a single digestion. PhmvA can also be replaced using EcoRI (or ClaI in pWLG14) and NsiI or NdeI.

After the digestion of pWLG13 with NdeI and BglII, the PCR product was ligated in to form pWLG13+5'-ilvB. The sequence of the PCR product was confirmed by sequencing with the primers that flanked the insertion site, pMEB.2seq1, 5'-AGGCACCCCAGGCTTTACAC-3' (5' end), and pMEB.2seq2, 5'-GCGTTTTTTATTACCTACTA-3' (3' end).

To replace the NdeI site near the histone promoter of pWLG13 with a NsiI site, the promoter was PCR amplified using the primers 5'-ATCTGCGAATTCAGCTGATCGATCAAAATATAACATAAATAACATAGGTTTAA-3' (P_hmvANsiI1) and 5'-GAAGATCTTCAGCGCTAAACATGCATTTCACCTATTAGTTATCTATAAAATTATA-3' (P_hmvANsiI2). The first 12 nucleotides of P_hmvANsiI1 contained an EcoRI site and 5' extension for EcoRI digestion. Restriction sites for PvuII and ClaI were also added immediately upstream of the M. voltae histone promoter. P_hmvANsiI2 possessed a BglII site plus a two-nucleotide extension that allowed endonuclease digestion. The PCR annealing temperature was 43°, and the extension time was 1 min at 72°. The PCR product was digested with EcoRI and BglII for directed ligation into pWLG11, which had been digested with the same enzymes and treated with CIAP. The product, pWLG14, contained the NsiI site as part of the start codon downstream of the histone promoter.

The E. coli lacZ gene was engineered by PCR for ligation into pWLG14. The PCR contained pTer7 as the template and the primers 5'-CCAATGCATGACCATGATTACGGATTCACTGG-3' and 5'-GAAGATCTTTCCTTACGCGAAATACGGGCAG-3' to introduce flanking NsiI and BglII sites. It was performed with 2.5 units of Taq DNA polymerase and an annealing temperature of 52.3° and an extension time of 3.75 min at 72°. The lacZ product was cloned into pGEM-T, yielding pGEM-T+lacZ. This vector was digested with NsiI and BglII to release the lacZ fragment. After treatment of pWLG14 with NsiI and BglII followed by CIAP, the lacZ fragment was ligated in to yield pWLG18. Upon electroporation into E. coli XL1-Blue MRF' (Stratagene), pWLG18 was identified by ß-galactosidase production on plates that contained X-gal and IPTG.

pWLG30, the expression shuttle vector, was constructed by ligation of a partial EcoRI digestion of pURB500 with a complete EcoRI digestion of pWLG14 (Figure 2). The ligation mixture was then transformed into M. maripaludis. The transformants were then inoculated into McC/puromycin broth to select for plasmids that contained both a methanococcal origin of replication from pURB500 and the pac cassette from pWLG14. Plasmids were then prepared from the broth culture of the transformants and electroporated into E. coli XL1-Blue MRF'. The location and orientation of pWLG14 within pURB500 was determined by digestion with SacII and EcoO109I and was confirmed by sequencing. To form pWLG30 + lacZ, pWLG30 was digested with NsiI, BglII, and XbaI. BglII lowered the background of the plasmids that were digested by only NsiI or XbaI. pWLG18 was digested with NsiI and XbaI, and the NsiI-XbaI fragment containing lacZ was gel purified. After ligation and transformation into E. coli XL1-Blue MRF', pWLG30 + lacZ was found by screening for blue colonies on LB + ampicillin + X-gal plates. The sequencing primers designed for confirming cloned DNA in pWLG30 or pWLG30 + lacZ were 5'-ACTCTCCAGAATACATAAAA-3' (pWLG30seq1) and pMEB.2seq2.

View larger version (23K): In this window In a new window Download PPT slide   Figure 2. Construction of methanococcal expression shuttle vectors pWLG30 and pWLG30 + lacZ. pWLG30 was constructed by ligation of a partial EcoRI digestion of the cryptic methanococcal plasmid pURB500 into the EcoRI site of pWLG14. lacZ from pWLG18 (Figure 1) was then added between the unique NsiI and BglII sites of pWLG30. The low level of lacZ expression of pWLG30 + lacZ forms a blue/white screen for clones in E. coli. The unique ClaI site in pWLG14 and pWLG30 allows the PhmvA to be replaced with other promoters in future studies.

Assays of AHAS activity:
The wild-type strain JJ1 or a strain transformed with pWLG13 + 5'-ilvB was grown in 100 ml of McNA in modified Wheaton bottles at 100 kPa of H₂:CO₂ to A₆₀₀ = 0.60–0.75. The cells were harvested by centrifugation under anaerobic conditions (SHIEH et al. 1988 ). The pellets were resuspended in 100 µl of dilution solution per 100 ml of medium. The dilution solution, which was sparged with N₂ for 1 hr, contained 1 mM cysteine hydrochloride, 1 mM DL-dithiothreitol, 1 mg bovine pancreatic DNase I (Boehringer Mannheim), 7.5 ml NaCl solution (293 g liter^-1), 50 ml distilled water, and 50 ml general salts solution (WHITMAN et al. 1986 ). The cells were placed in an aluminum seal vial on ice and flushed with hydrogen gas for 2 min before freezing at -20° for 2 hr. Upon thawing, the cells had lysed, and the cell extract was dialyzed in 12,000–14,000 molecular weight cut off (MWCO) tubing overnight at 4°. The dialysis buffer was the basic purification buffer that had been sparged with N₂ and autoclaved (XING and WHITMAN 1994 ). The AHAS assays were performed at 37° as described previously (PARK et al. 1995 ). For protein quantification by the bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL), the dialyzed extract was first diluted 1:50 in 0.1 M sodium hydroxide, boiled for 10 min, and cooled on ice.

Assays of ß-galactosidase activity:
The ß-galactosidase assay was performed with 2-nitrophenyl-ß-D-galactopyranoside (ONPG) at a temperature of 37° (MILLER 1992 ). E. coli XL1-Blue MRF' containing pWLG30+lacZ was grown overnight in low salt LB broth medium/ampicillin without IPTG. These cells were inoculated into fresh LB broth/ampicillin with and without 1 mM IPTG and grown to OD₆₀₀ = 0.64–0.70 before being assayed. M. maripaludis was grown on McC medium/puromycin unless stated otherwise. M. maripaludis assays used the same conditions as E. coli.

SDS-PAGE:
This procedure was performed according to the method of LAEMMLI 1970 for a 10% polyacrylamide gel.

Nucleotide sequence accession numbers:
The GenBank accession nos. for the M. maripaludis AHAS gene (ilvBN) and ppsA are AF118061 and AF118060, respectively. The accession nos. for pWLG11, pWLG13, pWLG14, and pWLG30 are AF134196, AF134197, AF134198, and AF134199, respectively.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Cloning of the ilvBN genes of M. maripaludis:
The two subunits of AHAS, which catalyze an early step in branched-chain amino acid biosynthesis, are encoded by the ilvBN genes in M. aeolicus (BOWEN et al. 1997 ). Screening of the M. maripaludis genomic library identified one plaque that hybridized with the M. aeolicus ilvB probe. Upon conversion of the phagemid to the plasmid form, the plasmid pWLG1 was found to contain ~7 kbp of M. maripaludis genomic DNA. A total of 4838 bp was sequenced, and three ORFs with homology to known genes were identified (Figure 3A). Two ORFs encoded the large and small AHAS subunits (E.C. 4.1.3.18), ilvB and ilvN, respectively. The deduced amino acid sequence of the large subunit (IlvB), which contained 587 amino acids, had 68.1, 72.9, and 31.4% sequence identity to the M. aeolicus, M. jannaschii (MJ0227), and M. jannaschii (MJ0663) IlvB homologs, respectively. The low amino acid sequence similarity to MJ0663 was consistent with the assignment of this ORF to another function (BOWEN et al. 1997 ). Fourteen nucleotides separated ilvB from ilvN. Upon comparison to the M. aeolicus gene, ilvN appeared to be truncated at the 3' end to form a deletion equivalent to eight amino acids. This conclusion was supported by the presence of a Sau3A I site and the beginning of the pBK-CMV vector before a stop codon in the ilvN sequence. The deduced amino acid sequence of the small subunit (IlvN) from M. maripaludis was 71 and 73% identical to the M. aeolicus and M. jannaschii IlvN homologs, respectively. The ilvBN genes from M. aeolicus, which were separated by 172 bp, and M. maripaludis were linked (BOWEN et al. 1997 ). In contrast, the M. jannaschii AHAS genes were unlinked (BULT et al. 1996 ).

View larger version (34K): In this window In a new window Download PPT slide   Figure 3. (A) Sequenced region of pWLG1 containing the M. maripaludis genes encoding AHAS (ilvBN) and phosphoenolpyruvate synthase (ppsA). The junction between ppsA and ilvB indicating the position of the Sau3AI site likely to be involved in formation of a chimera is shown. The arrow corresponds to the position of the putative intein splice junction in the M. jannaschii ppsA. (B) Deduced amino acid sequence of PpsA from M. maripaludis and M. jannaschii near the intein splice junction. Arrow represents putative intein splice junction.

Enzyme activity corresponding to a phosphoenolpyruvate synthase (E.C. 2.7.9.2; pyruvate, water dikinase) has been described in the cellular extracts of M. maripaludis (SHIEH and WHITMAN 1987 ), and ppsA appeared immediately upstream of ilvB. When compared to the M. jannaschii, Pyrococcus furiosus, and Staphylothermus marinus homologs, the M. maripaludis PpsA was between 29 and 38 amino acids shorter at the N terminus. In addition, an initiation codon was not observed at the 5' end of the cloned ORF, but a Sau3AI site was present, suggesting that the cloned ppsA had been truncated and that the cloned DNA in pWLG1 was chimeric (Figure 3A). Alternatively, it is also possible that the ppsA cloned was a pseudogene naturally present in the genome. In contrast, ilvB did not appear to be truncated because the 5' end of the gene encoded an amino acid sequence nearly identical to the N-terminal sequence of the M. aeolicus AHAS (XING and WHITMAN 1994 ). At the amino acid level the M. maripaludis PpsA was 72% identical to the M. jannaschii PpsA (MJ0542). The M. jannaschii ppsA contained an internal insertion that translated into a putative intein of 412 amino acids (BULT et al. 1996 ). At the intein/extein junction, a mechanistically important cysteinyl residue was found in the first position of the second extein (BULT et al. 1996 ; CHONG et al. 1996 ; Figure 3B). The deduced amino acid sequence of the M. maripaludis PpsA was nearly identical to the M. jannaschii homolog near the intein/extein junction, and the mechanistically important cystienyl residue was conserved. The putative intein, however, was absent.

Overexpression of ilvBN in M. maripaludis:
AHAS is normally expressed at the low levels typical of biosynthetic genes within the methanococci (XING and WHITMAN 1987 ). Because this enzyme is both unstable and O₂ labile, it would be much easier to characterize biochemically if it could be overexpressed. To overexpress this gene, pWLG13+5'-ilvB was constructed. In this plasmid, the start codon of ilvB overlapped with the NdeI site 4 bp downstream of the ribosome-binding site and promoter (P_hmvA) for the M. voltae histone (Figure 1). This plasmid also contained 641 bp of the 5' end of ilvB to allow for efficient homologous recombination into the genomic copy of ilvB. After transformation into M. maripaludis, integration of the pWLG13+5'-ilvB into the genome would place a truncated ilvB gene under control of the native promoter and place the intact ilvBN operon under the control of P_hmvA. To test this hypothesis, the specific activity of AHAS in extracts of a pWLG13+5'-ilvB transformant was determined (Table 2). Compared to extracts of wild-type cells, AHAS specific activity was 13-fold higher in the transformant. The anticipated specific activity was calculated from the expression of the Methanothermus fervidus histone. In this organism, the number of histone molecules was 1–2 x 10⁴ molecules per genome, and the genome possessed two genes (STROUP and REEVE 1992 ). The level of expression per gene would then be expected to be ~0.5–1 x 10⁴ molecules. On the basis of the specific activity and molecular weight of the M. aeolicus AHAS (XING and WHITMAN 1994 ), the same level of expression of the M. maripaludis ilvBN operon would yield a specific activity of 0.08–0.17 units (mg of protein)^-1 or very close to the observed value. This result also demonstrated that the M. voltae promoter was active in M. maripaludis.

To facilitate cloning into the integration vectors, pWLG18 was constructed with lacZ downstream of the P_hmvA to provide an opportunity for blue/white screening of clones in E. coli (Figure 1). Because lacZ contains an NdeI site, pWLG18 was constructed with an NsiI site immediately downstream of the P_hmvA, overlapping the start codon. The availability of a vector with an NsiI site also provides an alternative method for cloning genes that possess NdeI sites near their 5' end and that would not be suitable for cloning in pWLG13.

Construction of the expression shuttle vector:
To express heterologous genes in M. maripaludis, an expression shuttle vector was constructed from pWLG14 and pURB500 (Figure 2). pURB500 is a cryptic plasmid from M. maripaludis C5 and contains a methanococcal origin of replication, an NdeI site, and three EcoRI sites (TUMBULA et al. 1997 ). The NdeI site precluded use of this same site near the P_hmvA. Therefore, pWLG14 was chosen because it contained an NsiI site downstream of P_hmvA. The expression vector was then constructed by ligation of a partial EcoRI digestion of pURB500 into pWLG14 and transformation of M. maripaludis to select for the methanococcal origin of replication and puromycin resistance. Plasmids isolated from two independent transformants contained the same SacII and EcoO109I endorestriction maps, indicating that pWLG14 was inserted in the same EcoRI site of pURB500, as utilized in pDLT44 (Figure 2, TUMBULA et al. 1997 ). The expression shuttle vector pWLG30 was then further modified by adding lacZ to allow for blue/white screening in E. coli (Figure 2).

E. coli XL1-Blue MRF' containing pWLG30 + lacZ produced blue colonies in the presence of X-gal. The production of ß-galactosidase was independent of the addition of IPTG, indicating that expression was not from the lac promoter. Enzymatic assays of E. coli in LB broth plus ampicillin detected ß-galactosidase activity slightly above background levels. Weak expression with P_hmvA in E. coli was also observed by BENEKE et al. 1995 using Mipuid. Like pDLT44, pWLG30 transformed M. maripaludis with a high efficiency even though the plasmid yields were low from E. coli. pWLG30, isolated from M. maripaludis, was checked for rearrangements after transformation back into E. coli. No rearrangements were observed.

Expression of lacZ in M. maripaludis and E. coli:
The expression shuttle vector pWLG30 was tested for in vivo expression of the ß-galactosidase gene (lacZ) from E. coli. M. maripaludis does not contain ß-galactosidase, and activity was not detectable in wild-type cells (COHEN-KUPIEC et al. 1997 ). However, ß-galactosidase was readily detectable in cell-free extracts of a transformant bearing pWLG30 + lacZ (Table 3). A protein band corresponding to the expected size of ß-galactosidase was found by SDS-PAGE in the transformants of pWLG30 + lacZ, but not in cells transformed with pWLG30 alone (Figure 4).

View larger version (104K): In this window In a new window Download PPT slide   Figure 4. Overexpression of ß-galactosidase in M. maripaludis by the expression shuttle vector pWLG30. SDS-PAGE of total protein from M. maripaludis stained with Coomassie blue. Bio-Rad low-molecular-weight standards (lane A): rabbit muscle phosphorylase b (97.4 kD), bovine serum albumin (66.2 kD), hen egg white ovalbumin (45.0 kD), and bovine carbonic anhydrase (31.0 kD). The transformants were grown in McC medium/puromycin to an absorbance of 0.5, and the wild type was grown to an absorbance of 0.8 in McC medium. Lane B, wild-type cells; lane C, cells transformed with pWLG30; lane D, cells transformed with pWLG30+lacZ; arrow is a protein with the expected Mr of ß-galactosidase (116.3 kD).

The specific activity of ß-galactosidase varied with the growth phase of the culture. During growth on H₂:CO₂, exponential growth was usually observed only at low cell densities, i.e., A₆₀₀ < 0.4 (JONES et al. 1983B ). At higher cell densities, the rate of H₂ transfer from the gas to liquid phases probably limited growth, and arithmetic growth was observed (Figure 5A). In cultures of the transformant, the specific activity of ß-galactosidase increased threefold from exponential to stationary phase. During the stationary phase, where absorbance (600 nm) = 1, the ß-galactosidase specific activity was 7.5 µmol min^-1 (mg of protein)^-1. Purified ß-galactosidase has a specific activity of 600 µmol min^-1 (mg of protein)^-1 (Boehringer Mannheim). Thus, the ß-galactosidase appeared to represent ~1% of the cellular protein of the transformant. Whether or not the increase in specific activity resulted from changes in the level of expression from P_hmvA or in the copy number of the plasmid was not determined.

View larger version (19K): In this window In a new window Download PPT slide   Figure 5. Expression of ß-galactosidase in a transformant of pWLG30 + lacZ. Specific activity was in micromoles of X-gal transformed min-1 (mg of protein)-1. (A) Effect of growth phase on the specific activity. Cells, 4% v/v, were inoculated into McC/puromycin medium. (B) Stability of pWLG30 + lacZ in M. maripaludis upon serial transfers of the culture. M. maripaludis was grown to an absorbance (600 nm) of 0.62–0.85 in McN (), 0.40–0.58 in McC (), and 0.44–0.66 in McC/puromycin (•). The inoculum was 4%. Every 24 hr, the culture was transferred to fresh medium, and the specific activity was determined after 14 hr of growth. The average number of generations in the three media after transfers 1, 2, 3, and 4 were 5, 10, 14.5, and 23, respectively. Data points were the average of duplicate assays. For McN, McC, and McC/puromycin media, correlation coefficients of the specific activity with the number of transfers were 0.99, 0.98, and 0.74, respectively. Critical values for significance at P = 0.05 and 0.01 were 0.88 and 0.96, respectively.

The stability of pWLG30 in methanococci is important for the production of large amounts of recombinant protein. In one experiment, pWLG30 + lacZ was transferred every 3 wk in McC+puromycin medium for 3 mon. Upon electroporation into E. coli, all the >500 colonies examined on LB-amp + X-gal-containing medium were blue, indicating that the lacZ had been maintained. Restriction endonuclease mapping of the plasmid from one of the clones failed to detect any differences with the original plasmid. To further address this issue, the levels of ß-galactosidase were tested after multiple transfers in media without puromycin (Figure 5B). In both mineral (McN) and rich (McC) media, the levels of ß-galactosidase activity rapidly dropped after four serial transfers in media without puromycin. Although the initial specific activity in mineral medium was lower than in complex medium, the relative rate at which activity was lost was ~20–25% per transfer in both media. If the loss in ß-galactosidase activity was an indication of loss of the plasmid, the availability of the amino acids and other components of the rich medium appeared to have little affect on this process. In contrast, in the presence of puromycin, the level of ß-galactosidase activity was maintained for at least four transfers, and the apparent decline was not statistically significant (Figure 5B).

Summary:
To our knowledge, this report is the first description of an expression shuttle vector for the methanogenic archaea. Because these strict anaerobes contain many oxygen-sensitive enzymes and unusual coenzymes, they are good candidates for an expression system for enzymes that are not expressed in an active form in E. coli. Among the methanogens, the methanococci may be especially useful in this regard. Their rapid growth facilitates genetic manipulations. They can be cultured on a large scale on formate, which substantially reduces the hazards and expense of cultivation with H₂:CO₂. Thus, even though the levels of expression are only ~1% of the total cellular protein, it is possible to obtain large amounts of protein.

In addition to expressing homologous enzymes for biochemical studies, the integrative expression vectors may be useful for manipulating the physiology of M. maripaludis. For instance, a spontaneous mutation in Methanosarcina barkeri overexpressed the genes encoding pyruvate oxidoreductase and allowed the mutant to grow on pyruvate as an electron donor (BOCK and SCHONHEIT 1995 ). With the integrative expression vector, it would be possible to recreate such a mutant in M. maripaludis to determine if it also allows heterotrophic growth on pyruvate.

	ACKNOWLEDGMENTS

The authors are grateful to Elliot Altman for helpful discussions on pTer7. We are also grateful to Albrecht Klein for the plasmids pMEB.2 and Mipuid. This work was supported by Department of Energy grant DE-FG02-97ER20269 and National Science Foundation award DBI-9413236 to Warren L. Gardner.

Manuscript received March 23, 1999; Accepted for publication May 5, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

BENEKE, S., H. BESTGEN, and A. KLEIN, 1995  Use of the Escherichia coli uidA gene as a reporter in Methanococcus voltae for the analysis of the regulatory function of the intergenic region between the operons encoding selenium-free hydrogenases. Mol. Gen. Genet. 248:225-228[Medline].

BOCK, A.-K. and P. SCHÖNHEIT, 1995  Growth of Methanosarcina barkeri (Fusaro) under nonmethanogenic conditions by the fermentation of pyruvate to acetate: ATP synthesis via the mechanism of substrate level phosphorylation. J. Bacteriol. 177:2002-2007[Abstract/Free Full Text].

BOONE, D. R., W. B. WHITMAN and P. ROUVIÈRE, 1993 Diversity and taxonomy of methanogens, pp. 35–80 in Methanogenesis: Ecology, Physiology, Biochemistry, and Genetics, edited by J. G. Ferry. Chapman & Hall, New York.

BOWEN, T. L., J. UNION, D. L. TUMBULA, and W. B. WHITMAN, 1997  Cloning and phylogenetic analysis of the genes encoding acetohydroxyacid synthase from the archaeon Methanococcus aeolicus.. Gene 188:77-84[Medline].

BULT, C. J., O. WHITE, G. J. OLSEN, L. ZHOU, and R. D. FLEISCHMANN et al., 1996  Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.. Science 273:1017-1140.

CHONG, S., Y. SHAO, H. PAULUS, J. BENNER, and F. B. PERLER et al., 1996  Protein splicing involving the Saccharomyces cerevisiae VMA intein. J. Biol. Chem. 271:22159-22168[Abstract/Free Full Text].

COHEN-KUPIEC, R., C. BLANK, and J. A. LEIGH, 1997  Transcriptional regulation in Archaea: in vivo demonstration of a repressor binding site in a methanogen. Proc. Natl. Acad. Sci. USA 94:1316-1320[Abstract/Free Full Text].

GERNHARDT, P., O. POSSOT, M. FOGLINO, L. SIBOLD, and A. KLEIN, 1990  Construction of an integration vector for use in the archaebacterium Methanococcus voltae and expression of a eubacterial resistance gene. Mol. Gen. Genet. 221:273-279[Medline].

JONES, W. J., M. J. B. PAYNTER, and R. GUPTA, 1983a  Characterization of Methanococcus maripaludis sp. nov., a new methanogen isolated from salt marsh sediment. Arch. Microbiol. 135:91-97.

JONES, J. W., W. B. WHITMAN, R. D. FIELDS, and R. S. WOLFE, 1983b  Growth and plating efficiency of methanococci on agar media. Appl. Environ. Microbiol. 46:220-226[Abstract/Free Full Text].

LAEMMLI, U. K., 1970  Cleavage of structural protein during the assembly of the head of the bacteriophage T4. Nature 227:680-685[Medline].

MANIATIS, T., E. F. FRITSCH and J. SAMBROOK, 1982 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

MILLER, J. H., 1992 A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

PARK, S. H., R. XING, and W. B. WHITMAN, 1995  Nonenzymatic acetolactate oxidation to diacetyl by flavin, nicotinamide and quinone coenzymes. Biochim. Biophys. Acta. 1245:366-370[Medline].

REEBURGH, W. S., S. C. WHALEN and M. J. ALPERIN, 1993 The role of methylotrophy in the global methane budget, pp. 1–14 in Microbial Growth on C₁ Compounds, edited by J. C. MURRELL and D. P. KELLY. Intercept Ltd., Andover, MA.

SAITO, H. and K.-I. MIURA, 1963  Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta. 72:619-629[Medline].

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Cloning: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SHIEH, J. S. and W. B. WHITMAN, 1987  Pathway of acetate assimilation in autotrophic and heterotrophic methanococci. J. Bacteriol. 169:5327-5329[Abstract/Free Full Text].

SHIEH, J., M. MESBAH, and W. B. WHITMAN, 1988  Pseudoauxotrophy of Methanococcus voltae for acetate, leucine, and isoleucine. J. Bacteriol. 170:4091-4096[Abstract/Free Full Text].

STROUP, D. and J. N. REEVE, 1992  Histone HMf from the hyperthermophilic archaeon Methanothermus fervidus binds to DNA in vitro using physiological conditions. FEMS Microbiol. Lett. 91:271-276.

TUMBULA, D. L. and W. B. WHITMAN, 1999  Genetics of Methanococcus: possibilities for functional genomics in archaea. Mol. Microbiol. in press.

TUMBULA, D. L., R. A. MAKULA, and W. B. WHITMAN, 1994  Transformation of Methanococcus maripaludis and identification of a PstI-like restriction system. FEMS Microbiol. Lett. 121:309-314.

TUMBULA, D. L., T. L. BOWEN, and W. B. WHITMAN, 1997  Characterization of pURB500 from the archaeon Methanococcus maripaludis and construction of a shuttle vector. J. Bacteriol. 179:2976-2986[Abstract/Free Full Text].

WHITMAN, W. B., J. SHIEH, S. SOHN, D. S. CARAS, and U. PREMACHANDRAN, 1986  Isolation and characterization of 22 mesophilic methanococci. Sys. Appl. Microbiol. 7:235-240.

WHITMAN, W. B., S. SOHN, S. KUK, and R. XING, 1987  Role of amino acids and vitamins in nutrition of mesophilic Methanococcus spp. Appl. Environ. Microbiol. 53:2373-2378[Abstract/Free Full Text].

XING, R. and W. B. WHITMAN, 1987  Sulfometuron methyl-sensitive and -resistant acetolactate synthases of the archaebacteria Methanococcus spp. J. Bacteriol. 169:4486-4492[Abstract/Free Full Text].

XING, R. and W. B. WHITMAN, 1994  Purification and characterization of the oxygen-sensitive acetohydroxy acid synthase from the archaebacterium Methanococcus aeolicus.. J. Bacteriol. 176:1207-1213[Abstract/Free Full Text].

This article has been cited by other articles:

				R. Matsumi, K. Manabe, T. Fukui, H. Atomi, and T. Imanaka Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance J. Bacteriol., April 1, 2007; 189(7): 2683 - 2691. [Abstract] [Full Text] [PDF]

				S.-V. Albers, M. Jonuscheit, S. Dinkelaker, T. Urich, A. Kletzin, R. Tampe, A. J. M. Driessen, and C. Schleper Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus Appl. Envir. Microbiol., January 1, 2006; 72(1): 102 - 111. [Abstract] [Full Text] [PDF]

				T. J. Lie, G. E. Wood, and J. A. Leigh Regulation of nif Expression in Methanococcus maripaludis: ROLES OF THE EURYARCHAEAL REPRESSOR NrpR, 2-OXOGLUTARATE, AND TWO OPERATORS J. Biol. Chem., February 18, 2005; 280(7): 5236 - 5241. [Abstract] [Full Text] [PDF]

				B. C. Moore and J. A. Leigh Markerless Mutagenesis in Methanococcus maripaludis Demonstrates Roles for Alanine Dehydrogenase, Alanine Racemase, and Alanine Permease J. Bacteriol., February 1, 2005; 187(3): 972 - 979. [Abstract] [Full Text] [PDF]

				E. L. Hendrickson, R. Kaul, Y. Zhou, D. Bovee, P. Chapman, J. Chung, E. Conway de Macario, J. A. Dodsworth, W. Gillett, D. E. Graham, et al. Complete Genome Sequence of the Genetically Tractable Hydrogenotrophic Methanogen Methanococcus maripaludis J. Bacteriol., October 15, 2004; 186(20): 6956 - 6969. [Abstract] [Full Text] [PDF]

				K. Pfluger, S. Baumann, G. Gottschalk, W. Lin, H. Santos, and V. Muller Lysine-2,3-Aminomutase and {beta}-Lysine Acetyltransferase Genes of Methanogenic Archaea Are Salt Induced and Are Essential for the Biosynthesis of N{varepsilon}-Acetyl-{beta}-Lysine and Growth at High Salinity Appl. Envir. Microbiol., October 1, 2003; 69(10): 6047 - 6055. [Abstract] [Full Text] [PDF]

				G. E. Wood, A. K. Haydock, and J. A. Leigh Function and Regulation of the Formate Dehydrogenase Genes of the Methanogenic Archaeon Methanococcus maripaludis J. Bacteriol., April 15, 2003; 185(8): 2548 - 2554. [Abstract] [Full Text] [PDF]

				W. B. Whitman, F. Pfeifer, P. Blum, and A. Klein What Archaea Have to Tell Biologists Genetics, August 1, 1999; 152(4): 1245 - 1248. [Full Text]

				W. Kim and W. B. Whitman Isolation of Acetate Auxotrophs of the Methane-Producing Archaeon Methanococcus maripaludis by Random Insertional Mutagenesis Genetics, August 1, 1999; 152(4): 1429 - 1437. [Abstract] [Full Text]