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
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ABSTRACT |
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The GenBank accession numbers for the M. extorquens AM1 pdhABCD, sucABC and fumA sequences reported in this paper are AF497851, AF497852 and AF497854, respectively.
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INTRODUCTION |
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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.
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METHODS |
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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
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 EcoRIEcoRI or XbaIKpnI 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 EcoRIEcoRI or XbaIKpnI 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.
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RESULTS |
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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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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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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.
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DISCUSSION |
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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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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.
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ACKNOWLEDGEMENTS |
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Received 20 August 2002;
revised 1 October 2002;
accepted 18 November 2002.