Reconstruction of C3 and C4 metabolism in Methylobacterium extorquens AM1 using transposon mutagenesis

Stephen J. Van Dien1, Yoko Okubo1, Melinda T. Hough2, Natalia Korotkova1, Tricia Taitano2 and Mary E. Lidstrom1,2

1 Departments of Chemical Engineering, University of Washington, Seattle, WA 98195, USA
2 Departments of Microbiology, University of Washington, Seattle, WA 98195, USA

Correspondence
Mary E. Lidstrom
lidstrom{at}u.washington.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The growth of Methylobacterium extorquens AM1 on C1 compounds has been well-studied, but little is known about how this methylotroph grows on multicarbon compounds. A Tn5 transposon mutagenesis procedure was performed to identify genes involved in the growth of M. extorquens AM1 on succinate and pyruvate. Of the 15 000 insertion colonies screened, 71 mutants were found that grew on methanol but either grew slowly or were unable to grow on one or both of the multicarbon substrates. For each of these mutants, the chromosomal region adjacent to the insertion site was sequenced, and 55 different genes were identified and assigned putative functions. These genes fell into a number of predicted categories, including central carbon metabolism, carbohydrate metabolism, regulation, transport and non-essential housekeeping functions. This study focused on genes predicted to encode enzymes of central heterotrophic metabolism: 2-oxoglutarate dehydrogenase, pyruvate dehydrogenase and NADH : ubiquinone oxidoreductase. In each case, the mutants showed normal growth on methanol and impaired growth on pyruvate and succinate, consistent with a role specific to heterotrophic metabolism. For the first two cases, no detectable activity of the corresponding enzyme was found in the mutant, verifying the predictions. The results of this study were used to reconstruct multicarbon metabolism of M. extorquens AM1 during growth on methanol, succinate and pyruvate.


Abbreviations: TCA, tricarboxylic acid

The GenBank accession numbers for the M. extorquens AM1 pdhABCD, sucABC and fumA sequences reported in this paper are AF497851, AF497852 and AF497854, respectively.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Methylotrophic bacteria are organisms capable of growth using C1 compounds such as methanol as the only carbon and energy source and therefore, are of interest as biocatalysts for the conversion of methanol to useful products (Lidstrom, 1992). Since bacteria are not optimized to make products under large-scale commercial process conditions, metabolic engineering of methylotrophs will be required to develop strains with more-desirable process characteristics. Ideally, for the development of economically viable bioprocesses, cellular-wide resources should be redirected to synthesizing a specific product from methanol at high efficiency and yield. To achieve such a goal, it is necessary to understand and manipulate central metabolism. One of the most widely studied of the methanol-utilizing bacteria is the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. This organism is capable of growth on a variety of C2, C3 and C4 compounds as well as methanol and methylamine, assimilates methanol at the level of formaldehyde by the serine cycle and has served as the primary model system for the study of methylotrophic metabolism and methylotrophic enzymes (Chistoserdova, 1996; Lidstrom, 1992). Methylotrophy in M. extorquens AM1 is well-understood at the physiological and genetic levels and tools are available for genetic manipulations, making it an attractive system for the development of metabolic engineering techniques in methylotrophs. Eighty-six genes have been characterized, most of which are involved in the methanol assimilation and oxidation pathways unique to methylotrophic bacteria (Chistoserdova, 1996). In contrast, little is known about the growth of M. extorquens AM1 on C3 and C4 compounds, or of the metabolism of multicarbon compounds once they are produced from formaldehyde via the serine cycle. This unexplored region of heterotrophic central metabolism includes parts of the tricarboxylic acid (TCA) cycle, anapleurotic pathways, gluconeogenesis and the pentose-phosphate pathway. Past work has shown that TCA cycle enzymes are present at higher levels in cells grown on multicarbon substrates than in cells grown on C1 substrates (Salem et al., 1973; Taylor & Anthony, 1976). In addition, mutants in 2-oxoglutarate dehydrogenase (Taylor & Anthony, 1976) or pyruvate dehydrogenase (Bolbot & Anthony, 1980) were capable of growth on C1 compounds but not multicarbon compounds, indicating the specificity of these reactions for heterotrophy. However, some of the mutants were leaky and the site of the mutations was not characterized.

The recent availability of the M. extorquens AM1 genome sequence along with the advent of genetic tools for the construction of insertion mutants (Chistoserdov et al., 1994) and the overexpression of genes (Marx & Lidstrom, 2001) now enable the application of genomic approaches to the understanding of metabolic pathways in this organism. In a previous study, five genes predicted to be involved in growth on succinate or pyruvate encoding citrate synthase, succinate dehydrogenase, malic enzyme, phosphoenolpyruvate synthase and phosphoenolpyruvate carboxykinase were identified in the genome sequence. Mutants were generated and characterized, providing initial information on pathways of C3 and C4 metabolism (Van Dien & Lidstrom, 2002).

In this study, we apply a more-global approach to the metabolic reconstruction of central heterotrophic metabolism in M. extorquens AM1. It has recently been demonstrated in our laboratory that a mini-Tn5 derivative, ISphoA|hah-Tc (D'Argenio et al., 2001), could be used successfully in M. extorquens (Marx et al., 2003). We describe here the generation of a pool of random ISphoA|hah-Tc insertion mutants, and the screening of these mutants for defective growth on non-C1 substrates. Through identification and further characterization of the gene interruptions responsible for these growth phenotypes, a more-complete understanding of M. extorquens AM1 heterotrophic and methylotrophic central metabolism has been obtained.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Growth of M. extorquens AM1.
Cultures were grown aerobically at 30 °C either in liquid or on agar plates using mineral salts medium (Attwood & Harder, 1972) containing either 0·5 % methanol, 0·4 % succinate or 0·4 % pyruvate as the growth substrate. To generate growth curves, 30 ml of methanol-grown culture in exponential phase was harvested and resuspended in medium without carbon source to a final OD600 value of 6·0. One millilitre of this suspension was used to inoculate 30 ml cultures of different carbon sources, and the OD600 value of these cultures was measured as a function of time for approximately 30 h. Each growth experiment was performed twice with reproducible results, so only one curve is shown in each case. Doubling times, when given, represent means over the curves for both experiments.

Generation and screening of transposon mutants.
Transposon mutagenesis of M. extorquens AM1 was performed using the ISphoA|hah-Tc delivery plasmid pCM639 (D'Argenio et al., 2001; Marx et al., 2003). pCM639 was introduced into wild-type M. extorquens AM1 by biparental mating using Escherichia coli SM10 {lambda}pir (Miller & Mekalanos, 1988). Recombinants were selected on minimal salts medium agar plates containing methanol as the growth substrate, 10 µg tetracycline ml-1 and 50 µg rifamycin ml-1 for selection. Individual colonies were purified by streaking on fresh plates of the same composition.

After growth for 3 days on plates containing methanol, mutants were tested for C3 and C4 growth phenotype by streaking on minimal salts medium agar plates containing pyruvate or succinate as the carbon source and 10 µg tetracycline ml-1. Mutants were allowed to grow for 3 days, at which time those with a visible growth deficiency were selected. These strains were then retested on all three types of minimal plates to verify phenotype. After 3 days of growth, mutants were assigned a phenotype on each substrate based on their observed growth characteristics: normal (++), if growth appeared similar to wild-type on the screening medium; slow (+), if after 3 days it was possible to observe colony growth but it was less than wild-type; minus (-), if after 3 days there was almost no observable growth on the screening medium.

PCR amplification of interrupted chromosomal region.
The chromosomal region adjacent to the transposon insertion in each of the mutant strains was amplified using a semi-random, two-step PCR protocol (Chun et al., 1997; Marx et al., 2003). The products were purified using Qiaquick spin columns (Qiagen), and sequence analysis was performed by the University of Washington Sequencing Facility.

Prediction of interrupted gene function.
Identity searches were performed to locate the sequences in the M. extorquens AM1 partial genome sequence (Integrated Genomics; http://www.integratedgenomics.com/genomereleases.html#list6). Putative gene function was assigned by BLAST search of the corresponding translated sequence against the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST).

Generation of directed mutations.
Data from the M. extorquens AM1 genome project were used to design PCR primers specific for regions of the genome containing candidates for multicarbon-specific genes. Putative genes encoding the alpha subunit of the pyruvate dehydrogenase E1 component, the B subunit of NADH : ubiquinone oxidoreductase and fumarase were identified in the M. extorquens AM1 genome sequence and amplified by PCR using chromosomal DNA as a template. Products of the expected sizes were obtained and isolated, cloned directly into pCR2.1 (Invitrogen) and subcloned into pUC19 (Promega) as either EcoRI–EcoRI or XbaI–KpnI fragments. Unique blunt restriction sites located near the beginning of the gene were found and used for the insertion of a 1·4 kb HincII fragment from pUC4K (van der Oost et al., 1989) containing a kanamycin resistance (KmR) cassette. Orientation was chosen so that the KmR gene was transcribed in the same direction as the M. extorquens AM1 gene. The interrupted gene was subsequently removed and cloned into the suicide vector pAYC61 (Chistoserdov et al., 1994); the resulting plasmid was transformed into E. coli S17-1 (Simon et al., 1983). The resulting strains were used as donor strains in biparental matings with wild-type M. extorquens AM1, and KmR TcS progeny were obtained on minimal medium agar plates containing methanol as described previously (Chistoserdov et al., 1994). In all cases, the identity of the double-crossover mutants was confirmed by PCR using chromosomal DNA as a template and the gene-specific primers described above.

Overexpression of genes in M. extorquens AM1.
Genome sequence data obtained as described above were used to design PCR primers for the amplification of the wild-type gene. Products of the expected sizes were obtained and isolated, cloned directly into pCR2.1 and subcloned downstream of the PmxaF promoter in the M. extorquens AM1 expression vector pCM80 (Marx & Lidstrom, 2001) as either EcoRI–EcoRI or XbaI–KpnI fragments.

DNA manipulations.
Plasmid isolation, E. coli transformation, restriction enzyme digestion and ligation were carried out by standard protocols (Sambrook et al., 1989). The chromosomal DNA of M. extorquens AM1 was isolated by the procedure of Saito & Miura (1963). Biparental matings between E. coli and M. extorquens AM1 were performed as described previously (Chistoserdov et al., 1994).

Enzyme assays.
Enzyme activities were determined in wild-type or mutant M. extorquens AM1 crude extracts obtained by passing cells through a French pressure cell at 1·2x108 Pa, followed by centrifugation at 15 000 g. All measurements were done aerobically at room temperature in a total volume of 1 ml using published methods as follows: pyruvate dehydrogenase (Thissen et al., 1986) and glucose-6-phosphate isomerase (Schreyer & Bock, 1980). Fumarase activity was determined in the malate to fumarate direction using the technique of Flint (1994). However, due to interference from the crude cell extracts at 240 nm, the formation of fumarate was monitored by following the increase in absorbance at 300 nm. 2-Oxoglutarate dehydrogenase assay was performed by monitoring the release of 14CO2 from [1-14C]2-oxoglutarate in the presence of NAD+ and Coenzyme A (Green et al., 2000). For all assays, a milliunit (mU) of activity is defined as 1 nmol substrate reacted min-1.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Transposon mutagenesis using ISphoA|hah-Tc (D'Argenio et al., 2001) was performed on wild-type M. extorquens AM1 using the procedure outlined above to create a bank of random, tetracycline-resistant mutants capable of growth on methanol. Fifteen thousand insertion strains were screened for growth phenotype on pyruvate and succinate as representative C3 and C4 growth substrates, respectively. Since the M. extorquens AM1 genome contains approximately 7000 open reading frames, this theoretically represented slightly over two times coverage and thus was not intended to be saturating. Seventy-one mutants with impaired growth on one or both substrates were identified and subjected to further characterization (Table 1). The chromosomal region adjacent to the transposon insertion of mutant strains was amplified using a semi-random, two-step PCR protocol (Chun et al., 1997), and the resulting products were sequenced. The amplification was successful with 63 of the mutants and a total of 55 genes were identified, after accounting for duplications. Putative gene functions could be assigned to many genes based on BLAST searches of the translated sequence in all reading frames against the NCBI non-redundant sequence database (http://www.ncbi.nlm.nih.gov/BLAST). Fifteen genes either showed no significant identity with any genes of known function or could only be identified as ‘hypothetical proteins' (Table 1). Most of the remaining 40 genes are predicted to belong to one of several categories: those involved in central carbon metabolism, those involved in carbohydrate metabolism, putative regulators, putative transporters and non-essential housekeeping genes. A number of genes in these last three categories were identified only as belonging to broad groups of proteins. A few of these genes are of special interest, including the putative transcriptional regulators and a putative C4-dicarboxylate transport protein. These and the genes of unknown function represent a pool of functions yet to be identified in C3 and C4 metabolism and will be analysed in separate studies. The gene functions given in Table 1 are those of the protein with highest identity to the mutated M. extorquens AM1 gene, and are not necessarily the function of the M. extorquens AM1 gene itself. In each case, experimental evidence will be required to confirm function. However, in those cases in which the identity is high, it is likely that the functions are similar.


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Table 1. List of insertion mutants for which sequence data were obtained, growth phenotypes on methanol (MeOH), pyruvate (Pyr) and succinate (Succ) plates, and predicted gene function determined by BLAST search (see text)

‘No significant homology’ indicates that the insertion site could be located in the genome sequence, but the translated ORF has no homology with any protein in the NCBI database with E-value less than e-4. Mutants chosen for further study are shown in bold type. GenBank accession numbers are provided for the sequence used in the functional assignment. Numbers beginning with NP are predictions from other genome sequences; all others are sequences of functionally characterized products.

 
Since the aim of this study was the reconstruction of multicarbon metabolism in M. extorquens AM1, we focused our initial experimental verification studies on genes predicted to encode known metabolic functions that were of interest for filling in knowledge gaps and confirming model predictions (Van Dien & Lidstrom, 2002). These include 2-oxoglutarate dehydrogenase (M38-24), pyruvate dehydrogenase (M106-62) and NADH : ubiquinone oxidoreductase (M06-14), all of which are predicted to not be required for growth on C1 compounds (Taylor & Anthony, 1976; Bolbot & Anthony, 1980; Van Dien & Lidstrom, 2002), and glucose-6-phosphate isomerase (M02-75), which is predicted to be required for all growth conditions (Van Dien & Lidstrom, 2002). A mutant was also obtained in a gene predicted to encode NAD-dependent malic enzyme (M121B-13), which catalyses the reversible decarboxylation of malate to pyruvate. However, an insertion mutant in this gene having the same growth phenotype was already constructed by a directed approach and characterized in a previous study (Van Dien & Lidstrom, 2002), so this strain was not investigated further.

2-Oxoglutarate dehydrogenase
Strain M38-24 contains a transposon insertion in a gene predicted to encode the E1 component of 2-oxoglutarate dehydrogenase (EC 1.2.4.2), approximately 400 bp downstream of the 5' end of the gene. The identity of this gene is supported by the location of putative genes encoding E2 and dihydrolipoamide dehydrogenase components of the enzyme immediately downstream of the gene for the E1 subunit. These genes were named sucA, sucB and sucC. 2-Oxoglutarate dehydrogenase activity in the wild-type strain increased during growth on succinate as compared to methanol as shown previously (Taylor & Anthony, 1976), and the activity was non-detectable in the mutant strain (Table 2). This mutant does not grow appreciably on succinate or pyruvate either on agar plates or in broth culture, but has nearly a wild-type growth rate on methanol (Fig. 1).


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Table 2. 2-Oxoglutarate dehydrogenase activity in cell extracts of various strains

One milliunit of activity is defined as the reduction of 1 nmol NAD+ min-1. Where error ranges are given, each value represents the mean±SD of three measurements. ND, No activity detected in any of three independent measurements.

 


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Fig. 1. Representative growth curves of M38-24 (2-oxoglutarate dehydrogenase mutant) compared to those of wild-type M. extorquens AM1. {blacksquare}, Wild-type on methanol; {bullet}, wild-type on succinate; {blacktriangleup}, wild-type on pyruvate; {square}, M38-24 on methanol; {circ}, M38-24 on succinate; {triangleup}, M38-24 on pyruvate.

 
Pyruvate dehydrogenase
The transposon insertion in strain M106-62 is located approximately 300 bp upstream of the 3' terminus of a gene predicted to encode the E2 component of pyruvate dehydrogenase (EC 2.3.1.12). The gene is preceded by genes with identity to both subunits of the E1 component and is followed by a possible dihydrolipoamide dehydrogenase gene. This gene cluster was named pdhABCD. M106-62 grows slowly on succinate and pyruvate as compared to the wild-type strain, and the pyruvate dehydrogenase activity in this strain is approximately one-third that of the wild-type during methanol growth (Table 3). Furthermore, the activity in the wild-type strain increases more than two-fold during growth on pyruvate or succinate, as described previously (Salem et al., 1973). Finally, to demonstrate that no sharing of the E1 and E2 subunits occurs between the pyruvate and 2-oxoglutarate dehydrogenase complexes, assays for both dehydrogenases were performed on the strains mutant in each enzyme. 2-Oxoglutarate dehydrogenase activity in the pyruvate dehydrogenase mutant M106-62 was comparable to that of the wild-type (Table 2). Similarly, the 2-oxoglutarate dehydrogenase mutant M38-24 did not exhibit reduced pyruvate dehydrogenase activity (Table 3).


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Table 3. Pyruvate dehydrogenase activity in cell extracts of various strains

One milliunit of activity is defined as the reduction of 1 nmol NAD+ min-1. Each value represents the mean±SD of at least three measurements from multiple cultures. ND, No activity detected in any of three independent measurements.

 
In the transposon mutant screen we also identified a strain (M75-58) with an insertion in the putative dihydrolipoamide dehydrogenase gene upstream of the putative E2 subunit gene. This mutant had the same phenotype on agar plates as M106-62. In an attempt to generate a non-leaky pyruvate dehydrogenase mutant, an insertion mutant in the putative E1 subunit gene was generated by the directed approach described in Methods. This new strain, AM1-PDH1, does not grow appreciably on succinate or pyruvate on agar plates or in broth culture (Fig. 2) and has no detectable pyruvate dehydrogenase activity (Table 3).



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Fig. 2. Representative growth curves of AM1-PDH1 (pyruvate dehydrogenase subunit E1 mutant) compared to those of wild-type M. extorquens AM1. {blacksquare}, Wild-type on methanol; {bullet}, wild-type on succinate; {blacktriangleup}, wild-type on pyruvate; {square}, AM1-PDH1 on methanol; {circ}, AM1-PDH1 on succinate; {triangleup}, AM1-PDH1 on pyruvate.

 
NADH : ubiquinone oxidoreductase
Several transposon mutants were obtained with insertions in genes predicted to encode different subunits of the NADH : ubiquinone oxidoreductase (EC 1.6.5.3), all with a pyruvate-negative growth phenotype. This enzyme is highly conserved among bacterial species and consists of 14 subunits. In the M. extorquens AM1 genome sequence, putative genes for these 14 subunits are clustered on a single contig and show high identity to genes of other organisms. For example, the translated amino acid sequence of the M. extorquens AM1 subunit B gene is 83 % identical to that of Sinorhizobium meliloti and 79 % identical to that of Agrobacterium tumefaciens. Unfortunately, all of our presumed NADH : ubiquinone oxidoreductase mutant strains grew normally on pyruvate when revived from -80 °C freezer stocks, while retaining tetracycline resistance. This result suggests the mutants acquired a compensating mutation that not only allowed growth on pyruvate, but also exerted strong selective pressure during culturing on methanol. In order to confirm the preliminary phenotype assignments, it was necessary to regenerate a mutant by a directed approach. A strain containing an insertion mutation in the gene predicted to encode the B subunit of NADH : ubiquinone oxidoreductase was constructed as described in Methods. Subunit B was chosen because one of the strains obtained in the transposon mutagenesis screening process contains an insertion in this gene and there are no known paralogues of this gene in the M. extorquens AM1 genome sequence. In contrast, putative paralogues for several other subunits exist elsewhere on the chromosome. In agreement with the phenotype of the original transposon mutant M06-14, this putative NADH : ubiquinone oxidoreductase mutant (YO1) does not grow on pyruvate plates but grows normally on succinate plates.

To further study the growth phenotype of the mutant strain YO1, growth rates were measured in liquid mineral salts medium with each of the three substrates and compared with that of wild-type (Fig. 3). Although the pyruvate culture grew, with a doubling time of 9·9 h compared to 4·7 h with the wild-type, there was a significant lag upon transfer from methanol culture that did not occur in the wild-type. A less-severe lag also occurred with the succinate culture, and the final succinate doubling time was 5·4 h, compared to 4·2 h with the wild-type.



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Fig. 3. Representative growth curves of YO1 (NADH : ubiquinone oxidoreductase subunit B mutant) compared to those of wild-type M. extorquens AM1. {blacksquare}, Wild-type on methanol; {bullet}, wild-type on succinate; {blacktriangleup}, wild-type on pyruvate; {square}, YO1 on methanol; {circ}, YO1 on succinate; {triangleup}, YO1 on pyruvate.

 
Glucose-6-phosphate isomerase
An insertion (M02-75) was also obtained in a putative glucose-6-phosphate isomerase gene; this mutant grew slowly on both pyruvate and succinate on agar plates. The existence of this transposon mutant is surprising, since glucose-6-phosphate isomerase is expected to be a necessary enzyme for all carbon sources, given the essential role it performs in sugar metabolism. To assure correct prediction of gene function, activity assays were performed. Glucose-6-phosphate isomerase (EC 5.3.1.9) activity in extracts from M02-75 is 15·8±1·67 mU (mg protein)-1, as opposed to 408·5±3·06 mU (mg protein)-1 in extracts from wild-type M. extorquens AM1, both during growth on methanol, indicating that this gene does affect the predicted activity. It seems likely that the residual activity in the mutant is sufficient to allow normal growth on methanol, providing a possible explanation for why this mutant was obtained.

Directed mutagenesis of a putative fumarase gene
A few genes that would be expected to function in central metabolism during C3 and C4 growth were not identified by this random mutagenesis procedure. One such gene of interest is that encoding the TCA cycle enzyme fumarase (EC 4.2.1.2) since it functions both in the TCA cycle during heterotrophic growth and in the glyoxylate regeneration cycle during methylotrophic growth (Korotkova et al., 2002). Two putative fumarase genes were identified in the M. extorquens AM1 genome sequence. One of these genes, named fumA, was cloned behind the PmxaF promoter of pCM80 and the resulting plasmid mated into wild-type M. extorquens AM1. The resulting strain exhibited a fumarase activity of 2120±170 mU (mg protein)-1, as opposed to 140±39 mU (mg protein)-1 in the wild-type strain with no plasmid, thus confirming the predicted enzyme activity. A double-crossover mutant in this gene could not be obtained, suggesting that it is required for growth on all substrates tested. These results suggest that under these growth conditions the other putative fumarase gene either does not encode fumarase or is not expressed at a sufficient level to rescue the mutant.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To further extend our understanding of central metabolic pathways in M. extorquens AM1, we have undertaken a transposon mutagenesis screen to isolate mutants defective in growth on pyruvate and/or succinate. In addition, other genes predicted to be involved in growth on multicarbon compounds have been analysed by directed mutation.

With the results presented here in combination with those from a recent paper (Van Dien & Lidstrom, 2002), we now have a nearly complete genetic and biochemical characterization of the TCA cycle and anapleurotic pathways and can thus reconstruct this region of central metabolism. The growth phenotypes of mutants in the genes encoding the various steps of these pathways are summarized in Fig. 4. As predicted previously (Taylor & Anthony, 1976; Anthony, 1982), during growth on methanol a complete TCA cycle is not required, as evidenced by the wild-type growth rate of the 2-oxoglutarate dehydrogenase mutant M38-24. The enzymes leading to the biosynthetic precursor 2-oxoglutarate are necessary, as are those leading from succinate to malate because they form part of the essential pathway for the conversion of acetyl-CoA to glyoxylate (Korotkova et al., 2002). Pyruvate can be formed either from malate by the NAD-dependent malic enzyme or from phosphoenolpyruvate via pyruvate kinase, so null-mutants in either of these enzymes have no growth defect on methanol (Van Dien & Lidstrom, 2002). Likewise, acetyl-CoA is formed from the serine cycle (Anthony, 1982), so pyruvate dehydrogenase is not required during methylotrophic growth.



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Fig. 4. Summary of growth phenotypes for insertion mutants in genes encoding enzymes of the TCA cycle and anapleurotic pathways. (a) Methanol growth; (b) succinate growth; (c) pyruvate growth. Heavy solid line, mutant will not grow on substrate; heavy dashed line, mutant grows slowly; heavy dotted line, mutant grows like wild-type; light solid line, not determined. Enzymes are as follows: 1, citrate synthase (Van Dien & Lidstrom, 2002); 2, 2-oxoglutarate dehydrogenase (this work); 3, succinate dehydrogenase (Van Dien & Lidstrom, 2002); 4, fumarase (this work); 5, malate dehydrogenase (M. Chistoserdova, unpublished data); 6, malic enzyme (Van Dien & Lidstrom, 2002); 7, phosphoenolpyruvate carboxylase (Arps et al., 1993); 8, phosphoenolpyruvate carboxykinase (Van Dien & Lidstrom, 2002); 9, pyruvate kinase (Chistoserdova & Lidstrom, 1997); 10, phosphoenolpyruvate synthase (Van Dien & Lidstrom, 2002); 11, pyruvate dehydrogenase (this work). Also noted are enzymes for which mutants have not been obtained but for which enzyme activities have been detected in cell extracts (this work; M. Chistoserdova, unpublished data), (12) aconitase, (13) isocitrate dehydrogenase and (14) succinyl-CoA synthetase or succinyl-CoA hydrolase.

 
During growth on either succinate or pyruvate a functional TCA cycle is required, as indicated previously (Taylor & Anthony, 1976). The inability of the pyruvate dehydrogenase null-mutant AM1-PDH1 to grow on multicarbon compounds confirms that pyruvate dehydrogenase is the primary means of generating the acetyl-CoA required to drive the TCA cycle (Bolbot & Anthony, 1980). With succinate as the substrate, pyruvate must be formed either by malic enzyme or from oxaloacetate by a combination of phosphoenolpyruvate carboxykinase and pyruvate kinase. Based upon the relative activities of these enzymes, we have predicted malic enzyme to be the primary route for pyruvate synthesis (Van Dien & Lidstrom, 2002). Labelling studies have demonstrated that with pyruvate as the growth substrate, significant flux occurs through the TCA cycle (Salem et al., 1973). However, the anapleurotic enzymes are necessary to replenish TCA cycle intermediates that are lost to biosynthesis. This requirement can be fulfilled either by phosphoenolpyruvate synthase followed by phosphoenolpyruvate carboxylase or by malic enzyme.

The final genes of interest detected in this study are those predicted to encode the various subunits of the NADH : ubiquinone oxidoreductase. This enzyme forms an integral part of energy metabolism during heterotrophic growth and is thus important to understanding the energy and redox balance of the cell. The oxidation of NADH by this enzyme complex is the first step of oxidative phosphorylation, and is necessary for the conversion of reducing power, in the form of NADH, to energy in the form of ATP. A metabolic model of M. extorquens AM1 predicts that the entry of NADH into oxidative phosphorylation is important during growth on succinate and pyruvate, but not on methanol (Van Dien & Lidstrom, 2002). According to the model, methanol oxidation by methanol dehydrogenase (Lidstrom, 1992) produces sufficient reduced cytochrome, which enters the oxidative phosphorylation chain below NADH, so that NAD(P)H is more valuable to the cell for biosynthetic needs than for energy production. The growth phenotype data presented here agree with the prediction. An insertional mutant in a putative NADH : ubiquinone oxidoreductase gene grew normally on methanol and showed impaired growth on succinate and pyruvate. A similar heterotrophic phenotype of NADH : ubiquinone oxidoreductase mutants has been observed in Rhodobacter capsulatus, with impaired aerobic growth on malate and succinate (Dupuis et al., 1998).

The definition of the steps in C3 and C4 metabolism and their relationship to methylotrophy now provides a framework within which to assess growth on both multicarbon and single carbon compounds in M. extorquens, a necessary step for metabolic engineering of central metabolism. In addition, the pool of transposon insertion mutants with altered growth on C3 and/or C4 compounds is now available for further in-depth analysis of central heterotrophic metabolism in this bacterium.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the National Institutes of Health (GM58933). We thank D. D'Argenio, L. Gallagher and C. Manoil for providing the ISphoA|hah-Tc delivery strain.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Anthony, C. (1982). The Biochemistry of Methylotrophs. London: Associated Press.

Arps, P. J., Fulton, G. F., Minnich, E. C. & Lidstrom, M. E. (1993). Genetics of serine pathway enzymes in Methylobacterium extorquens AM1: phosphoenolpyruvate carboxylase and malyl coenzyme A lyase. J Bacteriol 175, 3776–3783.[Abstract]

Attwood, M. M. & Harder, W. (1972). A rapid and specific enrichment procedure for Hyphomicrobium spp. Antonie Van Leeuwenhoek 38, 369–377.[Medline]

Bolbot, J. A. & Anthony, C. (1980). The metabolism of 1,2-propanediol by the facultative methylotroph Pseudomonas AM1. J Gen Microbiol 120, 245–254.

Chistoserdov, A. Y., Chistoserdova, L. V., McIntire, W. S. & Lidstrom, M. E. (1994). The genetic organization of the mau gene cluster in Methylobacterium extorquens AM1: complete nucleotide sequence and generation and characteristics of mau mutants. J Bacteriol 176, 4052–4065.[Abstract]

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Received 20 August 2002; revised 1 October 2002; accepted 18 November 2002.