JOURNAL OF BACTERIOLOGY, Apr. 2010, p. 1813–1823
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 192, No. 7
Alternative Route for Glyoxylate Consumption during Growth on
Two-Carbon Compounds by Methylobacterium extorquens AM1?
Yoko Okubo,1Song Yang,1Ludmila Chistoserdova,1and Mary E. Lidstrom1,2*
Department of Chemical Engineering1and Department of Microbiology,2University of Washington, Seattle, Washington 98195-2180
Received 29 August 2009/Accepted 17 January 2010
Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and
multicarbon compounds. Mutants defective in a pathway involved in converting acetyl-coenzyme A (CoA) to
glyoxylate (the ethylmalonyl-CoA pathway) are unable to grow on both C1and C2compounds, showing that
both modes of growth have this pathway in common. However, growth on C2compounds via the ethylmalonyl-
CoA pathway should require glyoxylate consumption via malate synthase, but a mutant lacking malyl-CoA/
?-methylmalyl-CoA lyase activity (MclA1) that is assumed to be responsible for malate synthase activity still
grows on C2compounds. Since glyoxylate is toxic to this bacterium, it seemed likely that a system is in place
to keep it from accumulating. In this study, we have addressed this question and have shown by microarray
analysis, mutant analysis, metabolite measurements, and13C-labeling experiments that M. extorquens AM1
contains an additional malyl-CoA/?-methylmalyl-CoA lyase (MclA2) that appears to take part in glyoxylate
metabolism during growth on C2compounds. In addition, an alternative pathway appears to be responsible for
consuming part of the glyoxylate, converting it to glycine, methylene-H4F, and serine. Mutants lacking either
pathway have a partial defect for growth on ethylamine, while mutants lacking both pathways are unable to
grow appreciably on ethylamine. Our results suggest that the malate synthase reaction is a bottleneck for
growth on C2compounds by this bacterium, which is partially alleviated by this alternative route for glyoxylate
consumption. This strategy of multiple enzymes/pathways for the consumption of a toxic intermediate reflects
the metabolic versatility of this facultative methylotroph and is a model for other metabolic networks involving
high flux through toxic intermediates.
Methylobacterium extorquens AM1 grows on one-carbon (C1)
compounds using the serine cycle for assimilation (25). This
metabolism requires the conversion of acetyl-coenzyme A
(CoA) to glyoxylate, which occurs via a novel pathway in which
acetyl-CoA is converted to methylsuccinyl-CoA via aceto-
acetyl-CoA, ß-hydroxybutyryl-CoA, and ethylmalonyl-CoA
(30–33). Recently, the steps involved in the conversion of
methylsuccinyl-CoA to glyoxylate have been elucidated, and
the pathway has been termed the ethylmalonyl-CoA (EMC)
pathway (1, 19, 20, 40). Careful labeling measurements cou-
pled to measurements of intermediates has confirmed that,
during the growth of M. extorquens AM1 on methanol, meth-
ylsuccinyl-CoA is converted to glyoxylate and propionyl-CoA
via mesaconyl-CoA and ß-methylmalyl-CoA (40).
This finding has raised questions regarding how M. ex-
torquens AM1 grows on two-carbon (C2) compounds. The
pathway involved in the conversion of acetyl-CoA to glyoxylate
is known to operate during growth on both C1and C2com-
pounds, as mutants in genes involved in this conversion are
unable to grow on either C1or C2compounds, and in both
cases they are rescued by glyoxylate (11, 15–17, 44). If glyoxy-
late is produced as an end product of this pathway during C2
growth, then it must be converted to an intermediate of central
metabolism, which has been proposed to involve a malate
synthase activity (2, 14–17) (Fig. 1). In M. extorquens AM1, the
apparent malate synthase activity is carried out in two steps,
first by converting acetyl-CoA and glyoxylate to malyl-CoA by
malyl-CoA lyase and then by converting malyl-CoA to malate
by malyl-CoA hydrolase (Fig. 1) (14). However, a mutant
(PCT57) defective in malyl-CoA lyase (MclA1) (22), which
contains no detectable malate synthase activity during growth
on methanol, is able to grow on C2compounds (43).
Clearly, the finding that glyoxylate is generated as a direct
product of the EMC pathway presents a conundrum. Appar-
ently acetyl-CoA is converted to glyoxylate via this pathway,
but M. extorquens AM1 lacking malate synthase is able to grow
on C2compounds. Another apparent conundrum involving the
malyl-CoA lyase (mclA1) mutant is that the EMC pathway
requires an enzyme that carries out ß-methylmalyl-CoA cleav-
age, a reaction that homologs of MclA1 are known to carry out
(38). The MclA1 enzyme has been purified from M. extorquens
AM1 and shown to have activity with glyoxylate and propionyl-
CoA (27), which would produce ß-methylmalyl-CoA. These
results have led to the suggestion that MclA homologs actually
are malyl-CoA/ß-methylmalyl-CoA lyases (38). Since the
mclA1 mutant does not contain detectable malyl-CoA lyase
activity, and by inference has correspondingly low ß-methyl-
malyl-CoA lyase activity, it was not clear how M. extorquens
AM1 could convert acetyl-CoA to propionyl-CoA and glyoxy-
late via the EMC pathway in the mclA1 mutant.
The purpose of this study was to solve these conundrums
and determine how mutants of M. extorquens AM1 grow on C2
compounds in the absence of malyl-CoA/ß-methylmalyl-CoA
lyase or malate synthase activity. Our results show (i) that the
known homolog of MclA1 (MclA2) appears to be capable of
supporting both ß-methylmalyl-CoA cleavage and condensa-
tion between glyoxylate and acetyl-CoA in the mclA1 mutant,
* Corresponding author. Mailing address: Office of Research, Uni-
versity of Washington, Box 351202, Seattle, WA 98195. Phone: (206)
616-5282. Fax: (206) 616-5721. E-mail: firstname.lastname@example.org.
?Published ahead of print on 29 January 2010.
and (ii) that an alternative route for glyoxylate consumption
occurs in this bacterium, in which it is converted to interme-
diates of central metabolism via a part of the serine cycle
coupled with the glycine cleavage system.
MATERIALS AND METHODS
Cultivation. M. extorquens AM1 (39) was grown at 28°C in batch culture using
a mineral salts medium described previously (42). Ethylamine was used at 20
mM. Antibiotic concentrations (in ?g ml?1) were the following: tetracycline, 10;
kanamycin, 50; rifamycin 50.
Microarray analysis. Cultures for microarray analysis were grown to an optical
density at 600 nm (OD600) of 0.4 to 0.8, harvested, and used for RNA isolation.
RNA isolation, purification, and digestion with DNase I was carried out as
previously described (42). cDNA production and labeling, hybridization, and
array scanning were carried out by MOgene (St. Louis, MO), using the DNase
I-digested RNA and custom Agilent 60-mer microarrays. RNA from ethylamine-
grown cells was compared to RNA from succinate-grown cells, and four repli-
cates were carried out. Microarray data are available at the NCBI Gene Expres-
sion Omnibus (18) and are accessible through GEO Series accession number
The complete genome sequence of M. extorquens AM1 is available in GenBank
(50). Data were analyzed as previously described (42) using standard deviation
instead of P value.
Enzyme activities. Two hundred-milliliter cultures for enzyme activities were
grown to an OD600of 0.6 to 1.3, washed, and resuspended in 3 ml of 0.2 M
Tris-HCl (pH 8.0) buffer. Enzyme activities were determined in crude extracts
obtained by passing the cell suspension through a French pressure cell at 1.2 ?
108Pa, followed by centrifugation for 10 min at approximately 21,000 ? g,
ultracentrifugation for 1 h at 90,000 ? g, and the concentration of the soluble
fraction by an Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-3 membrane
(Millipore, MA). Measurements were done at 30°C in a total volume of 0.9 ml.
Malate synthase was measured in the direction of malate synthesis as described
Mutant generation. An insertion mutation was generated in gcvP by inserting
the kanamycin (Km) resistance gene using the pAYC61 suicide vector, essen-
tially as previously described (6). To generate mutants in mclA2 and sga, the
respective genes were replaced in the chromosome with the Km resistance gene
using pCM184 (36), and unmarked versions of these mutants were generated by
expressing cre recombinase as previously described (36). The double mclA1
mclA2, sga mclA1, and sga mclA2 mutants were generated by introducing the
mclA1 mutation into the unmarked mclA2 and sga mutant, using the previously
described donor construct (12) and by introducing the mclA2 mutation into the
unmarked sga mutant, respectively. All mutants were confirmed by diagnostic
Metabolite determinations. (i) Extracellular sample preparation. The samples
for the determination of extracellular metabolites were removed from each flask
when the cells had reached an OD600of 0.5 ? 0.15. Ten ml of the cell culture was
filtered using a Millipore membrane (0.22 ?m) and then lyophilized at approx-
imately ?45°C in a FreeZone 4.5-liter Benchtop Freeze Dry System lyophilizer
(Labconco, MO). One ml of double-distilled H2O (ddH2O) containing 150 ?l of
6N HCl was added to the dried culture medium and then was applied to a C18
solid-phase extraction (SPE) column (1 ml; Restek, Bellefonte, PA) to remove
the salts. The eluate from the SPE column was collected into a 2-ml glass vial and
dried in a vacuum centrifuge (CentrivVap Concentrator System; Labconco,
MO). The complete dried sample that was analyzed by gas chromatography
(GC) ? GC-time-of-flight mass spectrometry (TOFMS) was further derivatized
in two steps (26). First, keto groups were methoximated by adding 50 ?l
methoxyamine solution (25 mg ml?1methoxyamine hydrochloride in pyridine)
and incubation at 60°C for 30 min. In the second step, trimethylsiylation was
performed by adding 50 ?l trimethylsilyl (TMS) reagent (N,O-bis(trimethylsilyl)-
trifluoroacetamide-trimethylchlorosilane 99:1) and heating at 60°C for 60 min.
(ii) Intracellular sample preparation. Six ml of the cell culture at an OD of
0.5 ? 0.15 was carefully and rapidly pipetted into the center of 25 ml of the
quenching solution [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered
(70 mM, pH 6.8) aqueous 60% methanol (vol/vol) solution (?40°C)]. The
quenched biomass was precipitated in a refrigerated centrifuge (6 min, 10,000
rpm, ?20°C; Dupont Sorvall RC5B, Waltham, MA). The supernatant was re-
moved, and the cell pellets were resuspended in 5 ml of the same methanol
solution and again centrifuged for 6 min at 10,000 rpm.
The intracellular metabolites were extracted by using a previously reported
protocol (52). D6-salicylic acid was added as the internal standard to correct for
variation due to sample extraction and injection. One ml of boiling ethanol
solution (75% [vol/vol] ethanol-water) was added to a given cell pellet and
incubated at 100°C for 5 min. The extracted cell suspension was cooled on ice for
3 min, and the cell debris was removed by centrifugation at 5,000 rpm for 5 min.
The cell-free metabolite extract was centrifuged again at 14,000 rpm for 8 min.
The supernatant was transferred into a 2-ml glass vial and dried in a vacuum
FIG. 1. Enzymes and genes involved in the ethylmalonyol-CoA
pathway. The colors of gene names denote a change in gene expression
from microarray results comparing wild-type cells grown on ethyla-
mine to those grown on succinate: dark red, ?3-fold increase; light red,
1.5- to 3-fold increase; black, no significant change (1.49-fold increase
to 1.49-fold decrease); light green, 1.5- to 3-fold decrease; dark green,
?3-fold decrease. Parentheses denote a predicted function not con-
firmed by the mutant phenotype. See Table 1 for enzyme names.
1814OKUBO ET AL. J. BACTERIOL.
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