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Departments of Microbiology,1 Chemical Engineering, University of Washington, Seattle, Washington 98195-17502
Received 26 August 2002/ Accepted 16 October 2002
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-proteobacterium and is a facultative methylotroph capable of growth on C1, C2, C3, and C4 compounds (4, 14). Methylotrophic metabolism in M. extorquens AM1 begins with the oxidation of C1 substrates in the periplasm, producing formaldehyde. In the cytoplasm, formaldehyde condenses with either tetrahydrofolate (H4F) or tetrahydromethanopterin (H4MPT) to form the methylene derivatives of each C1 carrier (3). Methylene-H4F can either be assimilated into cell material via the serine cycle or oxidized to formate and then CO2, whereas methylene-H4MPT is solely oxidized to formate (19) and then CO2. Eighty-six genes involved in C1 metabolism in M. extorquens AM1 have been identified elsewhere (4, 14). These include genes involved in methanol oxidation, methylamine oxidation, formaldehyde oxidation, formate oxidation, and the serine cycle, most of which are organized in a small number of large gene clusters. Since the known methylotrophy gene clusters have now been analyzed, a search for any remaining methylotrophy genes requires more global approaches. Past attempts to identify methylotrophy genes using transposon mutagenesis have been problematic and found only previously known genes (13, 25). Recently, however, mutagenesis with mini-Tn5 identified novel genes involved in chloromethane utilization in the closely related strain Methylobacterium chloromethanicum CM4 (23). We have used another mini-Tn5 derivative, IsphoA/hah-Tc (2, 6, 15), to perform a transposon mutagenesis screen in M. extorquens AM1 to search for previously unknown methylotrophy genes.
Transposon mutagenesis and mutant screen.
Biparental matings were performed to introduce pCM639 (6) from Escherichia coli SM10 
pir (17) into wild-type M. extorquens AM1 (18) on nutrient agar (Difco, Detroit, Mich.) overnight at 30°C. Dilutions of the biparental mating mixtures were then plated onto minimal salt medium (1) containing succinate (15 mM) for growth with rifamycin (50 µg/ml) and tetracycline (10 µg/ml) for selection. Analogous experiments using the related transposons TnphoA and mini-Tn5 Tc resulted in a significantly lower rate of transposition (C. J. Marx and M. E. Lidstrom, unpublished data). Fresh cultures of ISphoA/hah-Tc-containing mutants were then screened on plates containing methanol (125 mM) for growth defects. From the 24,000 transposon insertion colonies obtained on succinate, 55 methanol-defective mutant strains were isolated.
Phenotypic analysis. To further classify the metabolic defect in each strain, the methanol-defective mutants were tested for growth defects on methylamine (35 mM), formate (35 mM), or ethylamine (30 mM). Growth phenotypes were assessed after 3 to 5 days of growth based on colony size relative to wild-type M. extorquens AM1 and placed into three categories: wild type (++), intermediate (+), and trace to none (-). Defects in methanol oxidation will show a growth defect only on methanol, while defects in the serine cycle and H4F or H4MPT pathway will show a growth defect on all C1 compounds. A portion of C1 and C2 metabolism overlaps in the conversion of acetyl-coenzyme A to glyoxylate, so defects in this part of metabolism will show growth defects on all C1 and C2 substrates (11, 12). Strains were also tested on medium containing both succinate and methanol (at 125 mM) to identify methanol-sensitive mutants, a phenotype specifically associated with defects in H4MPT-dependent formaldehyde oxidation (8, 24). Of the 55 methanol-defective mutants, 22 were only defective on methanol, 11 were C1 defective, 19 were C1 and C2 defective, and three were C1 defective and methanol sensitive.
Identification of the transposon insertion sites. A semirandom two-step PCR procedure to amplify the junction at the site of transposon insertion into the chromosome was performed essentially as described elsewhere (2, 5, 15). Modifications included using boiled colony preparations or chromosomal DNA preparations as template, use of primer CEKG 2B in the first amplification, and supplementation of the reaction mixtures with either 0.1 mg of bovine serum albumin/ml or 5% dimethyl sulfoxide. PCR products were purified using QIAquick PCR purification columns (Qiagen, Hilden, Germany) and sequenced (University of Washington Biochemistry Department DNA Sequencing Facility) using the following primer: 5'-AAACGGGAAAGGTTCCGTCCA-3'. Sequence analyses were performed using the ERGO website (www.ergo.integratedgenomics.com/ERGO/CGI; Integrated Genomics, Chicago, Ill.) and the analysis tools available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).
The 55 independent ISphoA/hah-Tc mutant strains with a methanol growth defect contained insertions into 31 different genes, 12 of which were represented more than once. In those cases in which multiple insertions were identified in the same gene, the insertions were located in different sites and the mutants exhibited a generally consistent mutant phenotype. Of the 86 previously characterized genes involved in methylotrophy, only 49 give clear growth defects on methanol. The others are either involved in the oxidation of methylamine (mau genes), are genes involved in redundant functions for which single mutants do not generate a growth phenotype, or carry out functions in both heterotrophic and methylotrophic metabolism so that null mutants cannot be isolated. The transposon mutagenesis identified 21 known methylotrophy genes (data not shown), which is about half of the methylotrophy genes for which methanol-defective mutants would be expected, indicating that the mutagenesis was not saturating. In addition to the 21 characterized genes, however, this screen also identified 10 previously uncharacterized methylotrophy genes (Table 1).
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Seven of the C1-defective mutants contained insertions into two previously uncharacterized genes that are unlinked to known methylotrophy genes (Table 1). Four independent mutants were obtained with insertions into an ORF with significant similarity to cbbR (LysR-type transcriptional regulator of autotrophy). The predicted amino acid sequence of the M. extorquens AM1 CbbR has 44% identity and 61% similarity to the CbbR in Xanthobacter flavus (GenBank accession no. P25545). Three mutants were also found with insertions into an ORF that is predicted to encode formate-tetrahydrofolate ligase (FtfL). The M. extorquens AM1 FtfL has a predicted amino acid sequence with 65% identity and 78% similarity to the FtfL of M. loti (GenBank accession no. BAB49812).
Eight C1- and C2-defective mutants were isolated with insertions into six genes previously uncharacterized in M. extorquens AM1 and unlinked to previously known C1 genes. Two C1- and C2-defective mutants were found to contain insertions into genes apparently involved in molybdenum cofactor biosynthesis. Mutant S06-42 contains an insertion into an ORF with significant similarity to moaA (molybdenum cofactor biosynthesis, protein A), which encodes a protein with 73% identity and 83% similarity to the MoaA of Brucella melitensis (GenBank accession no. AAL52200). Mutant S165-21 contains an insertion into an ORF with significant similarity to mobA (molybdopterin-guanine dinucleotide biosynthesis protein A), which has a predicted amino acid sequence with 56% identity and 64% similarity to the MobA from Rhodobacter sphaeroides (GenBank accession no. CAA70754). Additionally, four C1- and C2-defective mutants were obtained in genes of uncertain function, here designated mea genes (methyl and ethyl assimilation defective). This designation has been used previously for genes required for C1 and C2 growth (22). Mutant S06-17 contains an insertion into an ORF, here designated meaC, which encodes a conserved hypothetical protein with 67% identity and 80% similarity to a MaoC family protein of Caulobacter crescentus (GenBank accession no. AAK25618). Three mutants were isolated with an insertion into an ORF, here designated meaD, encoding another conserved hypothetical protein. The predicted amino acid sequence of meaD has 71% identity and 82% similarity to an ortholog in B. melitensis (GenBank accession no. AAL51247) and 43% identity and 56% similarity to the PduO protein of Salmonella enterica serovar Typhimurium LT2 (GenBank accession no. AAD39014). Mutants S109-30 and S151-59 contain insertions into ORFs, here designated meaE and meaF, which encode small hypothetical proteins with no significant similarity to other known proteins.
Finally, one C1-defective and methanol-sensitive mutant strain (S64-67) was identified in a previously uncharacterized ORF unlinked to all known methylotrophy genes. Previously, this phenotype had only been observed for M. extorquens AM1 mutants defective for the H4MPT-dependent enzymes fae (encodes formaldehyde-activating enzyme) (24) and mtdB (encodes methylene-H4MPT dehydrogenase) (8) and is believed to be caused by the inability of these mutant strains to detoxify formaldehyde. This ORF, here designated dmrA (putative dihydromethanopterin reductase, see below), encodes a protein (GenBank accession number AY093431) that has 26% identity and 44% similarity to the dihydrofolate reductase (DHFR) of Lactobacillus casei (GenBank accession no. P00381) (Fig. 1). In order to better understand functions required for formaldehyde oxidation, this mutant was chosen for further study.
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dmrA::kan mutant strain and phenotypic analysis.
In order to confirm that the ISphoA/hah-Tc insertion into the gene designated dmrA is responsible for the methanol-sensitive mutant phenotype of the S64-67 strain, a directed dmrA::kan mutant was generated. The chromosomal regions directly upstream and downstream of dmrA were amplified by PCR, and the resulting products were cloned into pCR2.1 (Invitrogen, Carlsbad, Calif.) to generate pCM207 and pCM208, respectively. The 0.6-kb BglII-NotI fragment from pCM207 was cloned between the BglII and NotI sites of the new broad-host-range allelic exchange vector pCM184 (16) to create pCM211. The 0.6-kb SacII-SacI fragment from pCM208 was then inserted between the corresponding sites of pCM211 to generate pCM212, which was then transferred into wild-type M. extorquens AM1 by conjugation using the helper strain E. coli S17-1 (20). Kanamycin-resistant transconjugants were screened for tetracycline sensitivity, indicating a double-crossover event resulting in a null mutant genotype. PCR analysis confirmed the generation of a dmrA::kan strain, designated CM212K.1.
The dmrA::kan strain, CM212K.1, was indistinguishable from the transposon mutant S64-67 in all phenotypic characterizations performed (Fig. 2, data not shown). The MIC of methanol for the dmrA mutants S64-67 and CM212K.1 was determined by comparing the rate and extent of colony formation of wild-type M. extorquens AM1. Methanol at final concentrations ranging from 125 mM to 50 nM was added to the plates containing succinate immediately prior to the addition of the molten agar. Because an undetermined fraction of the methanol will volatilize, the reported MIC of methanol is a maximum value. The MIC of methanol for S64-67 and CM212K.1 was 1 µM during growth on succinate plates. The MIC for the wild type is above 125 mM, and the MIC for mutants defective in fae, which encodes an enzyme catalyzing the first step of the H4MPT-dependent formaldehyde oxidation pathway, is 50 µM methanol (24). This suggests that the insertion into dmrA results in a severe defect in formaldehyde oxidation.
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Complementation analysis. As a final test that the C1-defective and methanol-senstive phenotype is due to loss of dmrA function, a plasmid expressing dmrA from lacZp was constructed for complementation analysis. The coding region of dmrA was amplified by PCR and cloned into pCR2.1 (Invitrogen) to produce pCM185. The 0.4-kb HindIII-BglII fragment of pCM185 was cloned between the HindIII and BamHI sites of pCM62 to produce pCM186. Introduction of pCM186 into either S64-67 or CM212K.1 using the helper plasmid pRK2073 (7) resulted in wild-type growth on methanol or succinate plus methanol. These results confirm that the loss of dmrA is responsible for the methanol-sensitive mutant phenotype observed in both S64-67 and CM212K.1.
Multiple DHFR homologs present in M. extorquens AM1.
If dmrA encoded DHFR, interruption by mutation should be lethal due to the central role of DHFR in biosynthetic reactions involving C1 units. However, the genome sequence contains three ORFs with identity to DHFR. One of these ORFs, here designated dfrA, has a predicted amino acid sequence significantly more similar to other DHFRs than are the other two (Fig. 1). The DfrA sequence (GenBank accession no. AY093432) has 50% identity and 63% similarity to the DHFR present in the closely related -proteobacterium M. loti (GenBank accession no. BAB48827). Additionally, dfrA is located directly downstream of an apparent thymidylate synthase, a gene organization that is conserved across a wide variety of bacteria. These sequence analyses suggest that dfrA encodes the general DHFR activity.
Antibiotics such as trimethoprim and methotrexate inhibit bacteria through the inhibition of DHFR activity. Resistance to trimethoprim is most often accomplished by expression of an alternate antibiotic-resistant DHFR (21). Because Methylobacterium strains generally exhibit resistance to this class of antibiotics (9), we determined whether dmrA was involved. Wild-type M. extorquens AM1, S64-67, and CM212K.1 were grown on plates containing up to 400 µg of trimethoprim/ml. No differences in growth were observed, indicating that the dmrA gene product is not required for trimethoprim resistance. The third ORF with identity to DHFR, here designated dfrB, has a predicted amino acid sequence (GenBank accession number AY093433) with 25% identity and 47% similarity to the trimethoprim-resistant DHFR found on the E. coli plasmid pCJ001 (10) (Fig. 1). Mutational and biochemical analyses will be required to assess the role of dfrA and dfrB in M. extorquens AM1.
Suggested role for DmrA in methylotrophy.
The dmrA mutants have a C1-defective and methanol-sensitive phenotype that has been associated with defects in H4MPT-dependent formaldehyde oxidation (8, 24). Given the similarity of the predicted DmrA protein sequence to that of DHFR, we suggest that dmrA encodes dihydromethanopterin reductase. If so, this would represent a novel enzymatic activity analogous to DHFR but functioning with H4MPT rather than with H4F:
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If this is the function of DmrA, it would explain the severe methanol sensitivity of the mutant, since the entire H4MPT pathway would be inoperable with no H4MPT in the cell. Consistent with this hypothesis, an apparent dmrA ortholog was found in the genome sequence of Methylococcus capsulatus Bath (www.jgi.doe.gov/JGI_microbial/html/index.html), a methanotrophic bacterium that contains the H4MPT pathway, but not in any other available genome sequences (L. Chistoserdova, C. J. Marx, and M. E. Lidstrom, unpublished data). Furthermore, it has recently been determined that M. extorquens AM1 dmrA mutants fail to synthesize H4MPT (S. Wyles and M. Rasche, personal communication). Detailed biochemical characterization will be required to test our hypothesis that dmrA encodes a dihydromethanopterin reductase.
Conclusions. The results presented here represent the first successful application of transposon mutagenesis to the study of M. extorquens AM1 and bring the total number of known methylotrophy genes in this organism to 96. In addition to the putative dihydromethanopterin reductase, nine other novel methylotrophy genes have been identified and are presently under investigation in our laboratory. Since only about half of the known methylotrophy genes were identified in the mutagenesis screen in each of the four mutant categories, it is expected that several more methylotrophy genes are as yet undiscovered. New approaches involving expression microarrays and proteomics may uncover new methylotrophy genes, and these experiments are presently in progress. It is clear, however, that the ability to grow on C1 compounds requires a substantial genetic investment by the organism, involving many dozens of genes.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the NIH (GM58933).
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FOOTNOTES |
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), and the dmrA mutants are S64-67 (
) and CM212K.1 (
).