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.
centrifuge (CentriVap Concentrator System; Labconco, MO) to complete dry-
ness. The dried sample was derivatized further for GC ? GC-TOFMS analysis in
two steps as described above.
(iii) GC ? GC-TOFMS measurement. GC ? GC-TOFMS experiments were
performed using a LECO Pegasus III time-of-flight mass spectrometer with the
4D upgrade (LECO Corp., St. Joseph, MI) as described previously (52). Differ-
ences between samples were assessed by a laboratory-written software method-
ology, referred to as the signal ratio method (S-ratio method) (41).
(iv)13C labeling. The incubation of cells with uniformly labeled glyoxylate
(1,2-13C; 99%; Cambridge Isotope Laboratories, Andover, MA) was carried out
at 28°C in a 25-ml glass tube containing 9.5 ?l of 0.532 M labeled glyoxylate. Five
ml of an ethylamine-grown culture was rapidly pipetted into the tube and vor-
texed for about 1 s. The final labeled glyoxylate concentration was 1 mM. After
various incubation times, the quenching solution noted above was added, and
amino acids and organic acids were extracted as described above and further
analyzed by liquid chromatography (LC)-MS/MS.
(v) LC-MS/MS measurement. LC-MS/MS experiments were carried out on a
Waters (Milford, MA) LC-MS system consisting of a 1,525-? binary high-pres-
sure liquid chromatography (HPLC) pump with a 2777C autosampler coupled to
a Quattro Micro API triple quadrupole mass spectrometer (Micromass,
Manchester, United Kingdom). The mass spectrometer was operated in both the
positive and negative electrospray ionization modes and scanned using multiple
reaction monitoring (MRM) to determine mass (M) isotopomer distribution
patterns. The MRM pairs (parent3daughter ions) of12C and13C metabolites
are the following: glycine (M ? 0, 76330; M ? 1, 77330 and 77331; M ? 2,
78331), serine (M ? 0, 106360; M ? 1, 107360 and 107361; M ? 2, 108361
and 108362; M ? 3, 109362), and malate (M ? 0, 1333115; M ? 1, 1343116;
M ? 2, 1353117; M ? 3, 1363118; M ? 4, 1373119). LC solvents for the
pentafluorophenylpropyl-bonded silica column (Luna PFPP; 150 mm by 2 mm;
3 ?m; Phenomenex, Torrance, CA) were the following: mobile phase A consisted
of 0.1% formic acid in water, while mobile phase B was acetonitrile. The fol-
lowing linear gradient was used: 100% A for 8 min, 100 to 70% A for 7 min, 70
to 50% A for 1 min, 50% A for 5 min, 50 to 0% A for 1 min, 0% A for 4 min,
0 to 100% A for 1 min, and 100% A for 8 min. The total run time was 35 min at
0.20 ml/min. The mass isotopomer distributions were corrected for the natural
isotope contribution by using a matrix-based method (21, 53) and calculated as
the relative abundances of the different possible mass isotopomers of a metab-
Flux calculations. Fluxes through the EMC pathway and to glyoxylate re-
quired to support a specific doubling time were calculated by assuming cells are
47% carbon (dry weight) (23) and 50% protein (dry weight) (48), and that 40%
of the carbon flux through the EMC pathway ends up as glyoxylate, 60% as
propionyl-CoA based on the proportions shown in Fig. 1.
Microarray comparison of ethylamine- and succinate-grown
cells. To provide global information on gene expression during
growth on C2compounds, the microarray analysis of M. ex-
torquens AM1 was carried out as described previously (42),
comparing ethylamine-grown cells to succinate-grown cells
(Table 1, Fig. 1). All of the known mau genes required for
methylamine dehydrogenase, which carries out ethylamine ox-
idation during growth on ethylamine (6), were highly induced
(8- to 52-fold). The genes involved in the portion of the EMC
pathway for converting acetyl-CoA to ß-hydroxybutyryl-CoA
(phaA, phaB) showed little change. The genes involved in the
portion of the EMC pathway for converting ß-hydroxybutyryl-
CoA to glyoxylate and propionyl-CoA were induced 2.1- to
2.5-fold (croR, ccr, ecm, msd), except for mcd, encoding the
mesaconyl-CoA hydratase, for which expression was decreased
slightly (1.5-fold decrease). The genes for converting propio-
nyl-CoA to R-methylmalonyl-CoA (pccA, pccB) did not show a
significant change, while three of the genes for converting
R-methylmalonyl-CoA to succinyl-CoA were induced 1.6- to
2.4-fold (mcmA, mcmB, meaD). The genes for converting suc-
cinyl-CoA to malate (sdhA, sdhC, sdhD, fum) all showed either
no significant change or decreased expression compared to
those converting to succinate. In keeping with its apparent
nonessential role in C2growth, mclA1 (formerly termed mclA
) showed slightly decreased expression (1.5-fold decrease),
while a second Mcl homolog, termed mclA2, was induced 2.4-
fold. In addition, a cluster of genes (MexAM1_META1p1550
to MexAM1_META1p1553) predicted to be involved in nitro-
gen metabolism via glutamate synthase was highly induced (4.5 to
18-fold; data not shown), as might be expected for growth on an
amine. An additional gene of interest also was induced, predicted
showed a 3.3-fold increase. Two others that were induced were
predicted to be involved in acetaldehyde oxidation, MexAM1_
META1p3652 (14-fold increase), which had homology to al-
dehyde dehydrogenases, and MexAM1_META1p3924 (3-fold
increase), which had homology to alcohol oxidases (Table 1,
Fig. 1). These results are in keeping with previous results
showing that multiple acetaldehyde-oxidizing enzymes are
present in this bacterium (51) and are consistent with an eth-
ylamine utilization route via acetaldehyde, acetate, and then
acetyl-CoA (Fig. 1).
Phenotypes of mcl mutants. A possible reason for the ability
of the original malyl-CoA lyase mutant (PCT57) to grow on C2
compounds is that it contained multiple mutations, one of
which allowed C2growth, and/or that C2-grown cells contained
malate synthase activity, since activity in cells grown on C2
compounds was not reported (43). We retested the phenotype
of an mclA1 insertion mutant that had been generated previ-
ously in our laboratory (12). This insertion mutant had no
detectable malyl-CoA lyase activity in cells grown on succinate
or succinate induced with methanol, and it was unable to grow
on C1compounds (12). Although previous work on C2com-
pounds in M. extorquens AM1 generally has involved ethanol as
a substrate, in our hands, growth on both plates and in liquid
culture is more robust with ethylamine, and our work has
utilized that substrate. In either case, the doubling time is 10 to
12 h, as reported previously for ethanol (16). When mclA1
mutants were streaked on ethylamine plates, colonies arose
more slowly than they did with the wild type (Table 2). Growth
curves in liquid medium confirmed a partial growth defect and
showed that the mclA1 mutant had a doubling time of about
twice that of the wild type. No malate synthase activity was
detected in this mutant grown on succinate, succinate induced
with methanol, or ethylamine, although activity was present in
the wild type under these conditions at levels of 16 to 35 nmol
min?1mg protein?1, which is similar to values previously re-
ported (16, 46). In our hands, the detection limit of this assay
was 1 to 2 nmol min?1mg protein?1. As reported previously
for PCT57 (43), the mclA1 mutant was inhibited by glyoxylate
at a lower concentration than that for the wild type. On plates,
apparent second-site suppressor mutants grew up readily at the
top of the streak, and care was necessary to grow liquid cul-
tures that maintained the original phenotype. These apparent
second-site suppressor mutants still were impaired for growth
on C2compounds and grew on ethylamine with a doubling
time between that of the original mclA1 mutant and that of the
wild type. Ethylamine-grown cultures of this more rapidly
growing strain also did not contain detectable malate synthase
As noted above, the M. extorquens AM1 genome (50) contains
another gene with homology to mclA1, MexAM1_META1p4295,
VOL. 192, 2010C2METABOLISM IN M. EXTORQUENS1815
TABLE 1. Microarray gene expression results for selected genes involved in C1and C2metabolism, comparing
ethylamine-grown cells to succinate-grown cells
Gene category and no.a
Gene nameDescriptionFold changeb
Required for growthc
Methylamine dehydrogenase genes
MexAM1_META1p2769 mauF Essential for methylamine
small subunit maturation
small subunit maturation
Essential for methylamine
Essential for methylamine
Essential for methylamine
MexAM1_META1p2771mauE 32 (33%)
MexAM1_META1p2772 mauD 27 (39%)
MexAM1_META1p2773mauA 24 (31%)
MexAM1_META1p2776 mauG 9 (43%)
Ethylmalonyl-CoA pathway genes
Crotonase, R-specific enoyl-
mcd (meaC) Mesaconyl-CoA hydratase
mclA2 Malyl-CoA lyase homolog
See Table 2
See Table 2
Genes for converting propionyl-
CoA to succinyl-CoA
(MCM) alpha subunit
Essential for MCM reaction
Genes for converting succinyl CoA
?1.7 NMNM NM 50
?2.0 (36%) ?
NR NR33, 49
Continued on following page
1816OKUBO ET AL. J. BACTERIOL.
which will be referred to as mclA2. The protein products of
these homologs also have been denoted Mcl1a and Mcl1b,
respectively (38). The translated product of mclA2 classes in a
separate phylogenetic branch from the products of mclA1 and
the mcl in Rhodobacter sphaeroides that is proposed to function
in the EMC pathway in this bacterium during growth on ace-
tate (38). The role of MclA2 in C1or C2metabolism in M.
extorquens AM1 is not known, but the MclA2 protein was
induced in a proteomics study comparing methanol growth to
succinate growth (35). In the microarray analysis in this paper,
Gene category and no.a
Gene name DescriptionFold changeb
Required for growthc
9, 29 See Table 2
See Table 2
See Table 2
MexAM1_META1p1218pgm Phosphoglycerate mutase NCNM NMNM 50
1.7 NM NM NM50
MexAM1_META1p0620gcvPGlycine decarboxylase NCSee Table 2 NRSee Table 2
Aldehyde and acetate conversions
aGene numbers reflect their order on the chromosome.
bFold change in the ethylamine/succinate level; standard deviations were within 25% of the means, unless otherwise indicated in parentheses.
c?, partial growth defect; ??, on ethanol; NR, not reported; NC, no significant change (change between 1.3- and 0.77-fold); E, presumed essential (mutants not
obtained); NM, no mutant available; MeOH, methanol; MtNH2, methylamine; EtNH2, ethylamine.
TABLE 2. Growth characteristics of M. extorquens AM1 wild type and mutants on agar platesa
Without glyoxWith glyox Without glyoxWith glyox
Doubling time (h)
aGlyox, 2 mM glyoxylate. ????, colonies in 3 days; ??? , colonies in 4 to 5 days; ??, colonies in 6 to 8 days; ?, colonies in 10 to 20 days;1⁄2?, small colonies
in 10 to 14 days; ?, no growth above the level of a control with no substrate. Two replicates were used for doubling times; values agreed within ?10%. NG, no growth;
ND, not determined; ?, maximum OD600? 0.5. All growth curves showed a lag phase similar to that of the wild type and reached a final OD600similar to that of the
wild type. All mutants grew like the wild type on succinate in liquid culture.
VOL. 192, 2010C2METABOLISM IN M. EXTORQUENS 1817
mclA2 shows a 2.4-fold induction during growth on ethylamine,
which is consistent with a role in C2metabolism (Fig. 1, Table
1). However, no malate synthase activity was detected in the
mclA1 mutant. A deletion mutant was generated in mclA2, but
no detectable growth phenotype was observed on plates con-
taining succinate, methanol, methylamine, or ethylamine (Ta-
ble 2). In liquid culture, the doubling time was slightly longer
than that for the wild type (15 h). However, a double mclA1
mclA2 mutant was unable to grow on ethylamine, suggesting
that MclA2 is able, at least in part, to functionally replace
Other mutants with partial defects for growth on C2com-
pounds. It has been reported previously that mutants lacking
serine hydroxymethyltransferase (glyA) and phosphoserine
phosphatase (serB) activities both had defects in growth on C2
compounds (10, 28, 29). The phenotypes of these mutants
suggest that interconversions of glycine and serine play a role
in C2metabolism. A possible pathway for converting glyoxylate
to three-carbon (C3) and four-carbon (C4) compounds via gly-
cine and serine involving known enzymes and genes in M.
extorquens AM1 is outlined in Fig. 2 (Table 1 lists the enzyme
names). To further pursue this concept, a mutant was gener-
ated in a gene of the glycine cleavage system (gcvP), which
interconverts glycine and methylene-H4F plus CO2, generating
NADH. M. extorquens AM1 previously has been shown to
generate C1units from glycine or glyoxylate (29). The gcvP
mutant and existing mutants defective in serine-glyoxylate ami-
notransferase (sga) and hydroxypyruvate reductase (hpr) were
tested for growth on C1and C2compounds. The sga and hpr
mutants are unable to grow on methanol (9, 29), as these
enzymes play a central role in the serine cycle. The gcvP mu-
tant grew normally on methanol. Both the gcvP and sga mu-
tants were found to have a partial defect for growth on ethyl-
amine on plates similar to that observed for the mclA1 mutant.
Like the mclA1 mutant, the sga mutant was inhibited by glyoxy-
late at a lower concentration than that of the wild type, but the
gcvP mutant did not respond to glyoxylate supplementation
(Table 2). In liquid culture, the sga mutant showed a strong
growth rate defect on ethylamine (47-h doubling time), while
the gcvP mutant showed a small growth rate defect (16-h dou-
bling time) but did not grow past an OD600of 0.5, while the
others all grew to an OD600of 1.2 to 1.5, like the wild type. As
noted previously (8), the hpr mutant showed no defect for
growth on C2compounds. Interestingly, both types of double
mutants with defects in mclA genes (mclA1 and mclA2) com-
bined with a defect in sga revealed diminished growth on eth-
ylamine plates, with the mclA1 sga mutant demonstrating a
more dramatic defect compared to that of the mclA1 sga mu-
tant. This result once again points to the fact that MclA1 and
MclA2 must be fulfilling similar functions, with MclA1 being
more active, in agreement with the activity measurements (as
In the microarray analysis, several of the genes in this possible
pathway were overexpressed (1.5- to 2.2-fold) in ethylamine-
grown cells compared to the expression of succinate-grown cells,
including sga, gck (glycerate kinase), serA (phosphoglycerate
dehydrogenase), ppc (phosphoenolpyruvate [PEP] carboxylase),
and mdh (malate dehydrogenase) genes (Table 1, Fig. 2).
Metabolite analysis. The sga, mclA1, and gcvP mutants were
analyzed for metabolites, both in cell extracts (intracellular)
and in the supernatant, and were compared to the wild type to
assess possible metabolic imbalances occurring that might con-
tribute to their impaired growth on ethylamine. M. extorquens
AM1 has a number of possible routes for glyoxylate consump-
tion, including conversion to glycolate and oxalate (Fig. 3), so
glycolate and oxalate were measured, as well as a number of
other targeted metabolites. In addition, an S-ratio analysis (41)
was carried out to identify the major differences between the
cultures (Fig. 4). The patterns in the supernatant and intracel-
lular analyses were significantly different, showing that the
metabolites in the supernatant were not simply a result of cell
lysis. The most striking differences between the wild type and
mutants involved amino acids. Several amino acids, including
proline, alanine, leucine, valine, and threonine, were high in all
or most of the three mutants, either intracellularly, in the
supernatant, or both. In addition, intermediates of valine, iso-
leucine, or leucine synthesis (2-oxoisovaleric acid, 3-methyl-2-
oxopentanoic acid, 4-methyl-2-oxopentanoic acid), respec-
tively, were high in the supernatant mainly in the sga mutant
but also, to some extent, in the other mutants. The level of
glycine was very high in the gcvP mutant, both intracellularly
and in the supernatant, which may explain the lack of growth
to high cell densities, since glycine is known to be inhibitory to
this bacterium (28). In addition, the sga and gcvP mutants
showed slightly higher intracellular oxalic acid, while the sga
FIG. 2. Potential pathway for converting glyoxylate to 2-phospho-
glycerate via glycine and serine. The net reaction is shown at the
bottom. Gene name colors are the same as those describe for Fig. 1.
FIG. 3. Possible fates of glyoxylate in M. extorquens AM1. Paren-
theses denote a predicted function not confirmed by the mutant phe-
notype. See Table 1 for enzyme names.
1818OKUBO ET AL. J. BACTERIOL.
mutant showed higher mesaconic acid, the non-CoA derivative
of a CoA intermediate of the EMC pathway. Other than the
amino acids that also were elevated in the other mutants, the
mclA1 mutant showed relatively minor (2-fold or less) changes
in the tested metabolites. The increase in amino acid pools may
reflect higher ammonium transfer activity as a result of growth
on an amine in combination with the lower growth rate.
13C-labeling analysis. The results described above suggested
that some glyoxylate is converted to central metabolic inter-
mediates via glycine and serine. If the pathway shown in Fig. 2
operates to carry out this conversion, then it should be possible
to observe carbon flow from labeled glyoxylate into glycine and
serine.13C-labeled glyoxylate was used for these experiments,
and all isotopomers were measured for these three compounds
in samples taken at short time points (10 s to 5 min) after the
addition of the labeled substrate. Major possible labeling pat-
terns are shown in Fig. 5. Glyoxylate processed by the glycine/
serine (Sga/Gcv/GlyA) route would be expected to generate
doubly labeled serine with unlabeled methylene H4F, or triply
labeled serine as methylene H4F becomes labeled. Singly la-
beled serine could come from unlabeled glycine reacting with
labeled methylene H4F. The labeled serine then would gener-
ate the same label in malate via PEP as that shown in Fig. 3.
Glyoxylate fluxing through the malyl-CoA route would pro-
duce doubly labeled malate, if acetyl-CoA is not made from
glyoxylate, or quadruply labeled malate, in the unlikely sce-
nario that acetyl-CoA can be made from glyoxylate. Malate
that enters the tricarboxylic acid (TCA) cycle loses a label, and
serine synthesized from malate via oxaloacetate, PEP, and the
phosphoserine pathway would maintain the same label as that
in the malate.
The results are consistent with both pathways operating to
utilize glyoxylate (Fig. 6). More than 95% of the glycine pool
becomes doubly labeled within 10 s (Fig. 6A), demonstrating
the rapid conversion of glyoxylate to glycine under these con-
ditions. For serine, 75% or more of the pool remains unlabeled
FIG. 4. Comparison of intracellular and supernatant metabolites in wild-type and mutant strains grown on ethylamine. Results are ratios of
values from each mutant compared to wild-type values. Top, supernatant; bottom, intracellular. ?, significant difference (P ? 0.05) between
mutants and the wild type. Error bars show the range for three biological replicates, each with three technical replicates. 3Mt2oxoPen,
3-methyl-2-oxopentanoic acid; 4Mt2oxoPen, 4-methyl-2-oxopentanoic acid; MtSuc, methylsuccinic acid; Pyru, pyruvate.
VOL. 192, 2010C2METABOLISM IN M. EXTORQUENS1819
throughout the experiment (Fig. 6B), suggesting that about 3/4
of it is synthesized from the phosphoserine pathway without
incorporating label. However, by 90 s, about 25% of the pool
is labeled. The doubly labeled compound appears first, fol-
lowed by the triply labeled compound, which is consistent with
a flow of glyoxylate-derived carbon through the glycine/serine
pathway and also with the labeling of the methylene H4F pool
within 30 s. A small amount of singly labeled serine appears
later, which could be derived from the small unlabeled glycine
pool reacting with labeled methylene H4F. About one-third of
the malate pool is quickly generated as doubly labeled, with 55
to 65% of the pool remaining unlabeled. Only a very small
amount of the malate pool occurs in other labeled species
(singly and quadruply labeled). These results suggest that more
than half of the malate is being generated within the TCA cycle
without incorporating label, but that the remainder is gener-
ated via glyoxylate. If all of the serine were generated from
malate, the labeling pattern for serine would parallel that for
malate. However, it does not. The doubly labeled malate stays
high, but the doubly labeled serine drops and is replaced by
triply labeled serine, which is the pattern predicted for the
glycine/serine route. These results do not rule out a previously
unknown, novel pathway for the synthesis of serine, but they
are consistent with the proposal that a significant amount of
the labeled serine is generated from glycine.
Two other conversion pathways are known for glyoxylate
based on in vitro results, involving conversion to glycolate (3, 8,
17, 34) and oxalate (5). It was not possible to accurately de-
termine the labeling of glycolate due to the presence of con-
taminating glycolate in the [13C]glyoxylate preparation, but no
labeled glycolate was observed above the background amount.
It also was not possible to detect labeled oxalate, suggesting
that if flux to oxalate occurs, it was below our detection limit.
Since both glycolate and oxalate are detected in ethylamine-
grown cells (52), it is likely that some flux to these compounds
The prediction that malate synthase activity is required for
M. extorquens AM1 to grow on C2compounds via the EMC
pathway stands in contrast to the literature evidence that a
mutant lacking malate synthase activity still is able to grow on
C2compounds (43). Possible explanations for this contradic-
tion are the following: (i) the EMC pathway does not operate
during growth on C2compounds, (ii) glyoxylate or a product
FIG. 5. Predicted labeling patterns from [13C]glyoxylate. Unlabeled products are not shown.
FIG. 6. Time course of ratios of isotopomers of glycine (top),
serine (middle), and malate (bottom) after the incubation of ethyl-
amine-grown cells with [13C]glyoxylate in the presence of [12C]ethyl-
amine as a fraction of the total compound. M ? 0, unlabeled; M ? 1,
singly labeled; M ? 2, doubly labeled; M ? 3, triply labeled; M ? 4,
1820OKUBO ET AL. J. BACTERIOL.
made from glyoxylate is excreted, (iii) malate synthase activity
actually is present in the malyl-CoA lyase mutant grown on C2
compounds, or (iv) an alternative glyoxylate consumption
Explanation i clearly is not correct, since a large body of
evidence has shown that M. extorquens AM1 grows on C2
compounds using the same pathway that is involved in con-
verting acetyl-CoA to glyoxylate during growth on C1com-
pounds (2, 11, 15–17, 30–33, 43). Most compelling is the fact
that mutants in this pathway are defective for growth on both
C1and C2compounds and are rescued for growth on both C1
and C2compounds by the addition of glyoxylate (11, 15–17,
30–33, 44). The results presented here are consistent with the
operation of the EMC pathway during growth on ethylamine.
Most of the genes involved in the conversion of acetyl-CoA to
glyoxylate and propionyl-CoA by the EMC pathway showed
higher expression in cells grown on ethylamine than in cells
grown on succinate, similarly to the result found with metha-
nol-grown cells (42) (Fig. 1, Table 1).
Likewise, the utilization of propionyl-CoA during growth on
C2compounds has been suggested to follow a standard con-
version scheme via propionyl-CoA carboxylase and methyl-
malonyl-CoA mutase, succinate dehydrogenase, and fumarase
(Fig. 1) (30–33, 40), and mutants in these genes also show
defects in growth on both C1and C2compounds that are
rescued by the addition of glyoxylate or glycolate (7, 11, 30). Of
these genes, only those involved in generating active methyl-
malonyl-CoA mutase showed higher expression in cells grown
on ethylamine than succinate. However, the slow growth ob-
served on C2compounds does not require high enzyme levels
to support the flux required. It can be calculated that the flux
through this pathway required to support a 12-h doubling time
is approximately 15 nmol min?1mg protein?1, and these en-
zymes are present in succinate-grown cells at in vitro activities
greater than this (11, 33). These results all are consistent with
the previous mutant data showing that the EMC pathway must
operate for the growth of M. extorquens AM1 on C2com-
For explanation ii, the excretion of glyoxylate or related
compounds would drop the yield, but it could allow the cells to
grow on the propionyl-CoA generated from ß-methylmalyl–
CoA. About 40% of the total carbon flux to biomass goes to
glyoxylate via the EMC pathway, so the amount to be excreted
would be significant. M. extorquens AM1 is known to have in
vitro activities for interconverting glyoxylate and glycolate (3, 8,
17, 34) and interconverting glyoxylate and oxalyl-CoA, which
then can be converted to oxalate (5), although not all of the
genes involved in these activities have been identified. Mea-
surements of intracellular and extracellular glyoxylate, glyco-
late, and oxalate showed that these compounds all were de-
tectable in the wild type both intracellularly and extracellularly.
However, the mclA1 mutant did not show major differences in
the levels of these compounds compared to those of the wild
type, either intracellularly or extracellularly, suggesting that
the excretion or accumulation of glyoxylate or products de-
rived from glyoxylate does not explain the growth of the mclA1
mutant on C2compounds. It is notable that the intracellular
levels of glyoxylate in the mclA1 mutant were not significantly
different from those of the wild type, suggesting that an alter-
native glyoxylate consumption route exists. Although the me-
tabolite detection methods used here did not determine abso-
lute concentrations, based on previous analyses (52) it can be
estimated that the total amount of glyoxylate, glycolate, and
oxalate detected in the supernatant of the mclA1 mutant was at
least two orders of magnitude lower than expected if the
glyoxylate flux was directed to excretion.
Explanation iii, that the mclA1 mutant contains malate syn-
thase activity during growth on C2compounds, is at best only
a partial explanation as, based on Mcl activity measurements,
the low activity would not account for the utilization of all of
the glyoxylate generated via the EMC pathway and would not
support the 25-h doubling time of this mutant on ethylamine
(approximately 7.5 nmol min?1mg protein?1). However, we
did show that the growth of the mclA1 deletion mutant is only
partially impaired on ethylamine, suggesting that MclA2 per-
forms a similar function but at a reduced level. The existence
of this isoenzyme thus provides a partial solution to the co-
Explanation iv, the presence of an alternative glyoxylate
consumption pathway, was shown to provide the final solution
to the conundrum. This pathway involves sga, glyA, and gcv,
and it appears to operate not only in mutants defective in the
EMC pathway but also in the wild type during growth on C2
compounds. Evidence for this pathway was obtained by observ-
ing mutant phenotypes and was consistent with labeling exper-
iments involving both mutants and the wild type, as described
above. Further confirmation of the importance of this glycine/
serine pathway during growth on C2compounds was obtained
from the finding that both mclA1 sga and mclA2 sga double
mutants have (partially) impaired glyoxylate consumption
routes and show significant growth defects on ethylamine, un-
like each of the single mutants.
Once glyoxylate is converted to serine, it has multiple routes
for incorporation, including conversion to protein and to other
C2, C3, and C4compounds via the routes shown in Fig. 3.
Strains containing mutations in some steps of these pathways
are known to grow on C2compounds, including strains lacking
hpr (8) and gck (3, 13), suggesting either that serine is routed
into central metabolites via alternative pathways or that, in
vivo, other enzymes are present that carry out this function at
the low flux required to support the observed growth rate. A
possible pathway involving these steps can be drawn for con-
verting 2-glyoxylate to a malate (Fig. 2 and 3), using known
enzymes and genes in M. extorquens AM1, although it is ener-
getically expensive, requiring one NADH molecule and one
ATP molecule per malate molecule, and even more if an en-
ergy-requiring transamination reaction is involved. However,
glyoxylate is a reactive aldehyde and is inhibitory to M. ex-
torquens AM1 above 2 mM in the external medium. Accumu-
lation inside the cell is likely to be detrimental, and it is pos-
sible that the slow growth observed on C2compounds in the
wild type is due in part to the tradeoff between the detriments
of glyoxylate accumulation and the low yield involved in this
pathway. Our results show that even in the mutants analyzed
here, glyoxylate does not accumulate, pointing to a careful
control system to keep it from building up.
In summary, our results demonstrate that during growth on
ethylamine, M. extorquens AM1 uses an alternative route for
glyoxylate consumption via glycine and serine to complement
the expected malate synthase route. Neither pathway alone
VOL. 192, 2010C2METABOLISM IN M. EXTORQUENS1821
supports wild-type growth, but the combination allows this
bacterium to grow normally on C2compounds. This finding
suggests that the two-step malate synthase reaction in M. ex-
torquens AM1 creates a bottleneck for glyoxylate consumption,
which the cell has overcome by shunting glyoxylate through a
second pathway. Although the measured in vitro activity of
malate synthase (16 nmol min?1mg protein?1for ethylamine-
grown cells) should be just sufficient to support the growth rate
on C2compounds, our results show that the in vivo activities
must restrict flux through this route. The presence of alterna-
tive routes for the consumption of a toxic intermediate is a
logical metabolic strategy and demonstrates the versatility and
flexibility of the metabolic network in this facultative methyl-
otroph. In addition, it represents a possible model for other
metabolic networks involving high flux through a toxic inter-
This work was funded by a grant from NIGMS (GM58933).
We thank Marina Kalyuzhnaya and Elizabeth Skovran for the crit-
ical reading of the manuscript.
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