1 Department of Microbiology, University of Washington, Seattle, WA 98195, USA
2 Department of Chemical Engineering, University of Washington, Seattle, WA 98195, USA
Correspondence
Mary E. Lidstrom
lidstrom{at}u.washington.edu
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ABSTRACT |
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Present address: 2215 Biomedical Physical Sciences, Michigan State University, East Lansing, MI 98824-4320, USA.
The GenBank accession numbers for the vector sequences reported in this article are AY307999 (pCM168) and AY308000 (pCM172).
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INTRODUCTION |
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One problem of central metabolism in methylotrophy that requires a new genetic tool involves formaldehyde utilization. In M. extorquens AM1, the formaldehyde produced from the primary oxidation of C1 substrates condenses with either tetrahydrofolate (H4F) or tetrahydromethanopterin (H4MPT) to form the respective methylene derivatives (Fig. 1; reviewed by Vorholt, 2002
). The reaction with H4MPT, a folate analogue long thought unique to methanogenic archaea (Chistoserdova et al., 1998
), is catalysed by the formaldehyde-activating enzyme, Fae, or can occur spontaneously (Vorholt et al., 2000
). Methylene-H4MPT is converted to methenyl-H4MPT and then formyl-H4MPT through the action of the NAD(P)-dependent methylene-H4MPT dehydrogenases MtdA (Vorholt et al., 1998
) and MtdB (Hagemeier et al., 2000
), and methenyl-H4MPT cyclohydrolase, Mch (Pomper et al., 1999
). The C1 unit is then hydrolysed by the formyltransferasehydrolase complex, Fhc (Pomper & Vorholt, 2001
), to produce formate and free H4MPT (Pomper et al., 2002
). Mutants defective for the H4MPT pathway fail to grow on C1 substrates and are sensitive to the presence of compounds that lead to the production of formaldehyde (Hagemeier et al., 2000
; Marx et al., 2003
b; Vorholt et al., 2000
), leading to the suggestion that the H4MPT-linked pathway serves as the primary formaldehyde oxidation and detoxification pathway in M. extorquens AM1.
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The enzymes of the H4F pathway are found at high specific activities during heterotrophic growth and are generally present at three- to fourfold higher levels during growth on C1 compounds (Chistoserdova & Lidstrom, 1994b; Marison & Attwood, 1982
; Pomper et al., 1999
; Vorholt et al., 1998
). This finding led to the original suggestion that the H4F pathway functions as the primary formaldehyde oxidation route during methylotrophy (Marison & Attwood, 1982
). The discovery of the H4MPT pathway in the methylotrophic bacteria and archaea (Chistoserdova et al., 1998
) and the elucidation of its critical role in formaldehyde oxidation (Chistoserdova et al., 1998
; Hagemeier et al., 2000
; Marx et al., 2003
b; Vorholt et al., 2000
) have brought this suggestion into question. It has also been suggested that the H4F pathway potentially could function in the reductive direction to produce methylene-H4F from formate during methylotrophic growth (Pomper et al., 2002
; Vorholt, 2002
). Recently, a C1-defective mutant with a transposon insertion into an ftfL homologue has been obtained (Marx et al., 2003
b). Unfortunately, a complete understanding of the role of the H4F pathway has been complicated by the inability to obtain null mutants of mtdA or fch even during growth on succinate (Chistoserdova & Lidstrom, 1994a
, b
; Vorholt et al., 1998
), suggesting a role for these gene products in heterotrophic metabolism. Although mutants with a reduced activity of MtdA or Fch were obtained and these strains were found to be defective for growth on C1 compounds, the presence of significant activities of the two gene products in these mutants makes the role of the H4F pathway in methylotrophy and heterotrophy uncertain. It is possible that the requirement for this pathway during heterotrophic growth is due to the need for formyl-H4F for biosynthesis. However, this does not explain the lack of growth on methanol, as formyl-H4F can be synthesized from formate via the FtfL reaction during methylotrophy (Fig. 1
). In addition, MtdA differs from a standard methylene-H4F dehydrogenase, in that it also has significant activity with methylene-H4MPT (Fig. 1
; Vorholt et al., 1998
). Therefore, it was not possible to rule out a role for MtdA in the H4MPT pathway during heterotrophic and/or methylotrophic growth.
To define the role of the H4F pathway in heterotrophy and methylotrophy, an approach was taken that required a new genetic tool, an insertional expression system that allows expression of genes from a stable, unmarked chromosomal locus. This system has been utilized to express folD, which encodes a bifunctional NADP-dependent methylene-H4F dehydrogenase/methenyl-H4F cyclohydrolase that does not have activity with H4MPT derivatives, from Methylobacterium chloromethanicum CM4T in an unmarked M. extorquens AM1 strain. This allowed the generation of null mutants lacking mtdA and/or fch. Additionally, we found that null mutants of mtdA and/or fch could be generated in the wild-type by supplementing the medium with formate. These approaches have demonstrated that the apparent essentiality of mtdA and fch during growth on succinate is due to the need for formyl-H4F and have clearly demonstrated the requirement for MtdA and Fch during methylotrophy.
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METHODS |
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The E. coli rrnB terminator (trrnB) from pCM130 (Marx & Lidstrom, 2001) and the T7 terminator (tT7) from pET-3a (Novagen) were amplified by PCR and cloned into pCR2.1 (Invitrogen) to generate pCM119 and pCM120, respectively. The 0·5 kb BamHIHincII fragment from pCM119 containing trrnB was ligated into the same sites of pMTL23 to generate pCM123. The 0·4 kb NruIXhoI fragment from pCM120 was inserted into the same sites of pCM123 to generate pCM124. A terminator-flanked cassette bearing PmxaF was generated by inserting the 0·3 kb NruIHindIII fragment from pCM80 (Marx & Lidstrom, 2001
) between the HincII and HindIII sites of pCM124 to generate pCM126.
The insertional vector backbone pCM167 contains unique BglII and StuI sites between which terminator-flanked cassettes were inserted as BamHINruI fragments. The insertional cloning vector pCM168 contains the 0·9 kb fragment from pCM124, whereas the insertional expression vector pCM172 contains the 1·2 kb pCM126 fragment. Additionally, a construct was made to generate a katA : : kan strain that allows the identification of those recombinants with a complete allelic exchange at the katA locus. The 3·4 kb EcoRISphI fragment from pLC1128.Km (Chistoserdova & Lidstrom, 1996) was blunted and cloned into the SmaI site of pAYC61 (Chistoserdov et al., 1994
) to generate pCM82.
Construction of plasmids to test the utility of the insertional systems.
To test the efficiency of transcription termination afforded by trrnB or tT7 in M. extorquens AM1, PmxaF present in the 0·4 kb BamHIEcoRI fragment from pCM27 (Marx & Lidstrom, 2001) was introduced between the same sites upstream of the reporter gene xylE in pCM76 (Marx & Lidstrom, 2001
) to generate pCM77. The 0·6 kb BamHISphI fragment from pCM119 and the 0·4 kb BamHISphI fragment from pCM120 were then ligated into the same sites of pCM77 between PmxaF and xylE to generate pCM121 and pCM122, respectively. The expression level afforded by pCM172 was examined by inserting the 0·8 kb HindIIINsiI fragment from pCM21 with gfp (green fluorescent protein) into the same sites of pCM172 to generate pCM174.
Construction of plasmids containing folD.
As a functional test of the insertional expression vector, folD and purU from M. chloromethanicum CM4T (Vannelli et al., 1999) were cloned and introduced into pCM172. The coding regions of purU and folD were amplified from a chromosomal DNA preparation of M. chloromethanicum CM4T by PCR and cloned into pCR2.1 (Invitrogen) to produce pCM201 and pCM202, respectively. Both constructs were sequenced to confirm no errors had been introduced. The 0·9 kb XbaIKpnI fragment from pCM201 was cloned into the same sites of pCM80 (Marx & Lidstrom, 2001
) to generate pCM203; subsequently, the 1·0 kb KpnISacI fragment from pCM202 was introduced between the same sites of pCM203 to generate pCM205. The 1·9 kb XbaINsiI fragment from pCM205 containing purUfolD was then inserted into the same sites of pCM172 to generate pCM206. A construct for expression of folD alone was made by self-ligating the 9·3 kb blunted pCM206 XbaIAsp718I fragment to produce pCM219. The purU constructs were not used in this study.
Construction of donor plasmids to generate mutants defective for mtdA and/or fch.
M. extorquens AM1 deletion mutants lacking mtdA and/or fch were generated using the allelic exchange vector pCM184 (Marx & Lidstrom, 2002). Approximately 0·5 kb regions upstream and downstream of each of these genes were amplified by PCR. The resulting mtdA flanks were introduced into pCR2.1 (Invitrogen) to generate pCM272 and pCM273; the fch flanks are contained in pCM276 and pCM277. The construct to generate
mtdA : : kan mutants was made by introducing the 0·5 kb SacIIAgeI fragment from pCM273 between the corresponding sites of pCM184 to produce pCM274; subsequently, the 0·5 kb BglIINdeI fragment from pCM272 was ligated into the same sites of pCM274 to produce pCM275. The construct to generate
fch : : kan mutants was made by introducing the 0·6 kb ApaISacI fragment from pCM277 into the same sites of pCM184 to produce pCM278; subsequently, the 0·5 kb EcoRINdeI fragment from pCM276 was ligated into the same sites of pCM278 to produce pCM279. Finally, a construct to make
mtdAfch : : kan mutants was generated by introducing the 0·5 kb BglIINdeI fragment from pCM272 into the same sites of pCM278 to produce pCM280.
Generation of mutant strains.
Strains carrying insertion vectors were generated by electroporating the appropriate constructs into the katA : : kan strain CM82.1 as described previously (Toyama et al., 1998). Tetracycline-resistant transformants were then screened for kanamycin sensitivity. Unmarked (tetracycline-sensitive) insertion strains were generated using the cre-expressing plasmid pCM158 as described previously (Marx & Lidstrom, 2002
). Mutants were generated in the various strain backgrounds by introducing the appropriate donor constructs by conjugation from E. coli S17-1 (Simon et al., 1983
) as described previously (Chistoserdov et al., 1994
). All deletion mutants and insertion strains were confirmed by diagnostic PCR analysis. Plasmids were introduced into the appropriate strains via triparental matings using the helper plasmid pRK2073 (Figurski & Helinski, 1979
).
Phenotypic analyses of mutant strains.
To compare the growth of wild-type M. extorquens AM1 with mutants in liquid medium, cultures were grown to mid-exponential phase, centrifuged and then resuspended in fresh medium containing the carbon source described. To test for sensitivity to methanol, methanol was added to one set of succinate flasks to the reported final concentration. Mutant phenotypes were also assessed on solid medium by comparing the relative rate of colony formation. All phenotypic analyses were performed at least twice.
Enzymic assays.
NADP-dependent methylene-H4F dehydrogenase (Chistoserdova & Lidstrom, 1994b), methenyl-H4F cyclohydrolase (Pomper et al., 1999
), formyl-H4F hydrolase (Nagy et al., 1995
) and catechol 2,3-dioxygenase (Kataeva & Golovleva, 1990
) activities were assayed as described with extracts prepared from cell material that was harvested from exponential-phase cultures. The H4MPT-dependent activity of MtdA was not assayed. FolD activity was determined in a strain lacking MtdA (CM219-275K.1) or by the difference between the folD-expressing strain and the wild-type. Activities are reported in mU [nmol min-1 (mg protein)-1] unless otherwise noted. Between culture variability in enzyme activities was less than 20 %. Total protein content of the extracts was determined spectrophotometrically (Kalb & Bernlohr, 1977
; Whitaker & Granum, 1980
) using a Beckmann DU 640B spectrophotometer. GFP expression was assayed in whole cells by measuring the relative fluorescence per OD600 unit using a Shimadzu RF-5301 PC spectrofluorophotometer with excitation and emission wavelengths of 410 and 509 nm, respectively.
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RESULTS |
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The expression level afforded by the insertional expression vector was determined by comparing GFP fluorescence in CM174.1 to wild-type M. extorquens AM1 carrying pCM88 (Marx & Lidstrom, 2001), in which gfp is transcribed by PmxaF of the plasmid expression vector pCM80 (Marx & Lidstrom, 2001
). Succinate-grown wild-type with pCM88 had a fluorescence/OD600 of 320 or 130 for methanol- and succinate-grown cells, respectively, compared to a relative fluorescence/OD600 of 680 versus 240 for CM174.1 grown under the same conditions. Thus, for the case of GFP, the insertional expression vector provided twofold higher expression than the plasmid system but exhibited the same regulation pattern (2·6-fold induction on methanol) as that obtained with pCM88 (2·4-fold induction). Additionally, the termination efficiency of trrnB and tT7 in M. extorquens AM1 was examined by inserting each of the terminators between PmxaF and xylE (which encodes catechol 2,3-dioxygenase). The XylE activities of cells containing the parental plasmid, pCM77 (PmxaFxylE), were 800 and 190 mU in extracts prepared from methanol- and succinate-grown cultures, respectively. These values dropped to 5 and 2 mU for pCM121 (PmxaFtrrnBxylE) and 290 and 95 mU for pCM122 (PmxaFtT7xylE). Therefore, the E. coli trrnB terminator provided a 99 % reduction in activity, compared to only a 5064 % reduction by tT7. Collectively, these data indicate that the insertional expression vector pCM172 provides significant expression from a chromosomal locus that is largely transcriptionally isolated from the surrounding genes.
M. extorquens AM1 mutants lacking mtdA and/or fch can be generated in a strain expressing folD from M. chloromethanicum CM4T
To better understand the role of the M. extorquens AM1 H4F pathway in methylotrophy and the apparent essentiality of mtdA and fch during heterotrophic growth, mutants defective for these H4F pathway activities were generated in strains expressing an analogous but non-orthologous enzyme from the related methylotroph M. chloromethanicum CM4T. The folD gene, which encodes a bifunctional NADP+-dependent methylene-H4F dehydrogenase/methenyl-H4F cyclohydrolase from M. chloromethanicum CM4T (Studer et al., 2002; Vannelli et al., 1999
), was cloned and introduced into the insertional expression vector pCM172. This construct was introduced into CM82.1 and tetracycline-resistant, kanamycin-sensitive transformants were isolated and confirmed to contain the folD chromosomal insertion, transcribed by PmxaF. Enzymic assays confirmed that FolD was expressed in an active form, with 81 and 39 mU of NADP-dependent methylene-H4F dehydrogenase activity in extracts of cells grown on methanol and succinate, respectively. The folD-expressing strain CM219T.1 was unmarked using a cre-expression vector (Marx & Lidstrom, 2002
) to generate the antibiotic-resistance-free strain CM219.1 [katA : : (loxPtrrnBPmxaFfolDtT7)] for further experiments.
Constructs based on the allelic-exchange vector pCM184 (Marx & Lidstrom, 2002) were generated to delete mtdA, fch or both, and these were introduced into both wild-type M. extorquens AM1 and the folD-expressing strain CM219.1. As had been reported previously, null mutants were not obtained in the wild-type on succinate medium (Chistoserdova & Lidstrom, 1994b
; Pomper et al., 1999
), but were readily obtained in CM219.1. The resulting strains CM219-275K.1 [katA : : (loxPtrrnBPmxaFfolDtT7),
mtdA : : kan], CM219-279K.1 [katA : : (loxPtrrnBPmxaFfolDtT7),
fch : : kan], CM219-280K.1 [katA : : (loxPtrrnBPmxaFfolDtT7),
mtdAfch : : kan] and CM219.1 grew like the wild-type in medium containing succinate (Fig. 3
). The addition of methanol to the medium did not inhibit growth (Fig. 3
), unlike the phenotype of mutants defective for the H4MPT pathway (Hagemeier et al., 2000
; Marx et al., 2003
b; Vorholt et al., 2000
). The folD-expressing strain CM219.1 grew more slowly on methanol than the wild-type, however, and the mtdA and fch mutants generated in this strain failed to grow at all on methanol (Fig. 3
). Similar results were obtained on solid medium; additionally, CM219-275K.1, CM219-279K.1 and CM219-280K.1 failed to grow on methylamine, formate or oxalate.
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DISCUSSION |
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The availability of the insertional expression vector pCM172 enabled experiments aimed at determining the role of the H4F pathway in methylotrophy in M. extorquens AM1. Using this system to generate a FolD-expressing insertion strain, we showed that null mutants in mtdA and/or fch could be obtained on succinate in a background containing folD. These results suggested that the role of these genes during growth on succinate is to generate formyl-H4F for biosynthetic purposes and showed that the H4MPT-dependent activity of MtdA is not required during heterotrophic growth. This hypothesis was confirmed by the demonstration that the requirement for mtdA and fch could be alleviated by the FtfL reaction, provided that formate or other compounds generating formate were supplied in the medium.
Both sets of null mutants in mtdA and fch failed to grow on C1 compounds, confirming a specific role in methylotrophy for the products of these genes. However, it was surprising that the mutants expressing FolD failed to grow on C1 compounds. FolD carries out the same reactions as MtdA/Fch together, except that MtdA has significant activity with methylene-H4MPT (Vorholt et al., 1998). However, if the methylene-H4MPT dehydrogenase activity of MtdA was important during methylotrophic growth, the Fch mutant should still be complemented by FolD. Fch does not show detectable activity with methenyl-H4MPT, and a different enzyme (Mch) carries out this reaction in M. extorquens AM1 (Pomper et al., 1999
). FolD from M. chloromethanicum CM4T is required for growth on chloromethane, a C1 substrate that is not oxidized to formaldehyde, but rather is catabolized through C1-H4F pathway intermediates by MetF (methylene-H4F reductase), FolD and PurU (formyl-H4F hydrolase) (Studer et al., 2002
; Vannelli et al., 1999
). It has been suggested (Pomper et al., 2002
; Vorholt, 2002
) that the H4F pathway of M. extorquens AM1 (MtdA, Fch and FtfL) may function in the assimilatory direction during growth on methanol to supply methylene-H4F for the serine cycle from some fraction of the formate that is produced from formaldehyde by the H4MPT pathway. Therefore, it is possible that the net fluxes through these two H4F pathways are in opposite directions. This may be reflected, for example, in different affinities for substrates and/or the effect and identity of potential effector molecules that may modulate flow through C1-H4F intermediates. This hypothesis is supported by the growth inhibition in methanol medium observed in the wild-type expressing FolD, which could be explained by a futile cycle involving the methylene-H4F/formyl-H4F interconversions. Alternatively, it is possible that the in vivo activity of FolD is not sufficient for growth on C1 compounds, as the in vitro FolD activity in methanol-grown cells was about one-third the in vitro activity of MtdA in the wild-type (Vorholt et al., 1998
). Regardless of which explanation is correct, the results we present here clearly demonstrate that MtdA and Fch are necessary for growth on C1 compounds.
The insensitivity to methanol during growth on succinate of mtdA and/or fch mutants provides additional support for the hypothesis that the H4F pathway does not contribute significantly to formaldehyde oxidation to formate, and ultimately CO2. So far, all mutants in the H4MPT pathway are sensitive to methanol and other formaldehyde-producing substrates (Hagemeier et al., 2000; Marx et al., 2003
b; Vorholt et al., 2000
), underscoring the important role of this pathway in formaldehyde oxidation. In the case of mtdA, in particular, the lack of sensitivity to formaldehyde-producing substrates suggests that MtdB activity alone is sufficient for the detoxification of formaldehyde. This confirms the previous suggestion (Hagemeier et al., 2000
) that MtdB is the primary methylene-H4MPT dehydrogenase in vivo.
With the exception of the requirement for supplementation with formate to grow on succinate, or a compound that can be converted into formate (methanol, methylamine or oxalate), the growth of the mtdA and/or fch mutants is consistent with that reported for ftfL mutants (Marx et al., 2003b). Therefore, there is a consistent phenotype associated with a defective H4F pathway: no growth on C1 compounds including formate, but lack of inhibition by methanol during growth on succinate. The inability to grow on formate or oxalate confirms that the H4F pathway is required to convert formate into methylene-H4F for assimilation of these substrates (Large et al., 1961
). Finally, the insensitivity to methanol during growth on succinate by mutants defective for the H4F pathway contrasts with the distinct growth inhibition observed for H4MPT pathway mutants. This difference in mutant phenotype suggests that these two C1-transfer pathways may play distinct roles. Experiments designed to directly test the direction of carbon flow through the H4F pathway are in progress.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Chambers, S. P., Prior, S. E., Barstow, D. A. & Minton, N. P. (1988). The pMTL nic- cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68, 139149.[CrossRef][Medline]
Chistoserdov, A. Y., Chistoserdova, L. V., McIntire, W. S. & Lidstrom, M. E. (1994). Genetic organization of the mau gene cluster in Methylobacterium extorquens AM1: complete nucleotide sequence and generation and characteristics of mau mutants. J Bacteriol 176, 40524065.[Abstract]
Chistoserdova, L., Vorholt, J. A., Thauer, R. K. & Lidstrom, M. E. (1998). C1 transfer enzymes and coenzymes linking methylotrophic bacteria and methanogenic Archaea. Science 281, 99102.
Chistoserdova, L., Chen, S. W., Lapidus, A. & Lidstrom, M. E. (2003). Methylotrophy in Methylobacterium extorquens AM1 from a genomic point of view. J Bacteriol 185, 29802987.
Chistoserdova, L. V. & Lidstrom, M. E. (1994a). Genetics of the serine cycle in Methylobacterium extorquens AM1: identification, sequence, and mutation of three new genes involved in C1 assimilation, orf4, mtkA, and mtkB. J Bacteriol 176, 73987404.[Abstract]
Chistoserdova, L. V. & Lidstrom, M. E. (1994b). Genetics of the serine cycle in Methylobacterium extorquens AM1: identification of sgaA and mtdA and sequences of sgaA, hprA, and mtdA. J Bacteriol 176, 19571968.[Abstract]
Chistoserdova, L. V. & Lidstrom, M. E. (1996). Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1. Microbiology 142, 14591468.[Abstract]
Figurski, D. H. & Helinski, D. R. (1979). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 16481652.[Abstract]
Hagemeier, C. H., Chistoserdova, L., Lidstrom, M. E., Thauer, R. K. & Vorholt, J. A. (2000). Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1. Eur J Biochem 267, 37623769.
Kalb, V. F. & Bernlohr, R. W. (1977). A new spectrophotometric assay for protein in cell extracts. Anal Biochem 82, 362371.[Medline]
Kataeva, I. M. & Golovleva, L. A. (1990). Catechol 2,3-dioxygenases from Pseudomonas aeruginosa 2x. Methods Enzymol 188, 115121.[Medline]
Large, P. J., Peel, D. & Quayle, J. R. (1961). Microbial growth on C1 compounds. 2. Synthesis of cell constituents by methanol- and formate-grown Pseudomonas AM1, and methanol-grown Hyphomicrobium vulgare. Biochem J 81, 470479.[Medline]
Laukel, M., Chistoserdova, L., Lidstrom, M. E. & Vorholt, J. A. (2003). The tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1: purification and properties. Eur J Biochem 270, 325333.
Lidstrom, M. E. (2001). Aerobic methylotrophic prokaryotes. In The Prokaryotes, posting date 2 November, 2001 ( http://141.150.157.117:8080/prokPUB/chaprender/jsp/showchap.jsp?chapnum=300&initsec=01_00 ). Edited by M. Dworkin.
Marison, I. W. & Attwood, M. M. (1982). A possible alternative mechanism for the oxidation of formaldehyde to formate. J Gen Microbiol 128, 14411446.
Marx, C. J. & Lidstrom, M. E. (2001). Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology 147, 20652075.
Marx, C. J. & Lidstrom, M. E. (2002). Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. BioTechniques 33, 10621067.[Medline]
Marx, C. J., Laukel, M., Vorholt, J. A. & Lidstrom, M. E. (2003a). Purification of the formate-tetrahydrofolate ligase from Methylobacterium extorquens AM1 and demonstration of its requirement for methylotrophic growth. J Bacteriol 185, 71697175.
Marx, C. J., O'Brien, B. N., Breezee, J. & Lidstrom, M. E. (2003b). Novel methylotrophy genes of Methylobacterium extorquens AM1 identified by using transposon mutagenesis including a putative dihydromethanopterin reductase. J Bacteriol 185, 669673.
Nagy, P. L., Marolewski, A., Benkovic, S. J. & Zalkin, H. (1995). Formyltetrahydrofolate hydrolase: a regulatory enzyme that functions to balance pools of tetrahydrofolate and one-carbon tetrahydrofolate adducts in Escherichia coli. J Bacteriol 177, 12921298.[Abstract]
Nunn, D. N. & Lidstrom, M. E. (1986). Isolation and complementation analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structural gene of Methylobacterium sp. strain AM1. J Bacteriol 166, 581590.[Medline]
Palmeros, B., Wild, J., Szybalski, W., Le Borgne, S., Hernandez-Chavez, G., Gosset, G., Valle, F. & Bolivar, F. (2000). A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria. Gene 247, 255264.[CrossRef][Medline]
Pomper, B. K. & Vorholt, J. A. (2001). Characterization of the formyltransferase from Methylobacterium extorquens AM1. Eur J Biochem 268, 47694775.
Pomper, B. K., Vorholt, J. A., Chistoserdova, L., Lidstrom, M. E. & Thauer, R. K. (1999). A methenyl tetrahydromethanopterin cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in Methylobacterium extorquens AM1. Eur J Biochem 261, 475480.
Pomper, B. K., Saurel, O., Milon, A. & Vorholt, J. A. (2002). Generation of formate by the formyltransferase/hydrolase complex (Fhc) from Methylobacterium extorquens AM1. FEBS Lett 523, 133137.[CrossRef][Medline]
Purdy, D., O'Keeffe, T. A., Elmore, M., Herbert, M., McLeod, A., Bokori-Brown, M., Ostrowski, A. & Minton, N. P. (2002). Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier. Mol Microbiol 46, 439452.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Simon, R., Priefer, U. & Puhler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1, 784791.
Studer, A., McAnulla, C., Buchele, R., Leisinger, T. & Vuilleumier, S. (2002). Chloromethane-induced genes define a third C1 utilization pathway in Methylobacterium chloromethanicum CM4. J Bacteriol 184, 34763484.
Toyama, H., Anthony, C. & Lidstrom, M. E. (1998). Construction of insertion and deletion mxa mutants of Methylobacterium extorquens AM1 by electroporation. FEMS Microbiol Lett 166, 17.[CrossRef][Medline]
Vannelli, T., Messmer, M., Studer, A., Vuilleumier, S. & Leisinger, T. (1999). A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane. Proc Natl Acad Sci U S A 96, 46154620.
Vorholt, J. A. (2002). Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria. Arch Microbiol 178, 239249.[CrossRef][Medline]
Vorholt, J. A., Chistoserdova, L., Lidstrom, M. E. & Thauer, R. K. (1998). The NADP-dependent methylene tetrahydromethanopterin dehydrogenase in Methylobacterium extorquens AM1. J Bacteriol 180, 53515356.
Vorholt, J. A., Marx, C. J., Lidstrom, M. E. & Thauer, R. K. (2000). Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol. J Bacteriol 182, 66456650.
Whitaker, J. R. & Granum, P. E. (1980). An absolute method for protein determination based on the difference at 235 and 280 nm. Anal Biochem 109, 156159.[Medline]
Received 22 June 2003;
revised 8 September 2003;
accepted 9 September 2003.
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