Post on 21-Mar-2022
transcript
Article
Selection Maintains Appar
ently DegenerateMetabolic Pathways due to Tradeoffs in UsingMethylamine for Carbon versus NitrogenHighlights
d Two degenerate methylamine oxidation modules have
distinct roles in methylotrophs
d Methylamine dehydrogenase enables rapid use of
methylamine as a growth substrate
d The N-methylglutamate pathway enables nitrogen
assimilation from methylamine
d Tradeoffs between ammonium toxicity and cellular
localization select for degeneracy
Nayak et al., 2016, Current Biology 26, 1416–1426June 6, 2016 ª 2016 Elsevier Ltd.http://dx.doi.org/10.1016/j.cub.2016.04.029
Authors
Dipti D. Nayak, Deepa Agashe,
Ming-Chun Lee, Christopher J. Marx
Correspondencecmarx@uidaho.edu
In Brief
Metabolic degeneracy is commonly
observed in microbial genomes;
however, an adaptive basis for this
phenomenon is rarely understood. Nayak
et al. uncover physiological tradeoffs
underlying the utilization of methylamine
as a growth substrate or as nitrogen
source that selects for two methylamine
oxidation pathways in methylotrophs.
Current Biology
Article
Selection Maintains Apparently DegenerateMetabolic Pathways due to Tradeoffsin Using Methylamine for Carbon versus NitrogenDipti D. Nayak,1,5 Deepa Agashe,1,6 Ming-Chun Lee,1,7 and Christopher J. Marx1,2,3,4,*1Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA2Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA3Institute for Bioinformatics and Evolutionary Studies, University of Idaho, Moscow, ID 83844, USA4Center for Modeling Complex Interactions, University of Idaho, Moscow, ID 83844, USA5Present address: The Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, IL 61801, USA6Present address: National Centre for Biological Sciences, Bangalore 560065, India7Present address: Department of Biochemistry, University of Hong Kong, Pokfulam, Hong Kong*Correspondence: cmarx@uidaho.edu
http://dx.doi.org/10.1016/j.cub.2016.04.029
SUMMARY
Microorganisms often encode multiple non-ortholo-gous metabolic modules that catalyze the same re-action. However, little experimental evidence actu-ally demonstrates a selective basis for metabolicdegeneracy. Many methylotrophs—microorganismsthat grow on reduced single-carbon compounds—like Methylobacterium extorquens AM1 encode tworoutes for methylamine oxidation: the periplasmicmethylamine dehydrogenase (MaDH) and the cyto-plasmic N-methylglutamate (NMG) pathway. InMethylobacterium extorquens AM1, MaDH is essen-tial for methylamine growth, but the NMG pathwayhas no known physiological role. Here, we use exper-imental evolution of two isolates lacking (or inca-pable of using) MaDH to uncover the physiologicalchallenges that need to be overcome in order touse the NMG pathway for growth on methylamineas a carbon and energy source. Physiological char-acterization of the evolved isolates revealed regula-tory rewiring to increase expression of the NMGpathway and novel mechanisms to mitigate cyto-plasmic ammonia buildup. These adaptations ledus to infer and validate environmental conditionsunder which the NMG pathway is advantageouscompared to MaDH. The highly expressedMaDH en-ables rapid growth on high concentrations of methyl-amine as the primary carbon and energy substrate,whereas the energetically expensive NMG pathwayplays a pivotal role during growth with methylamineas the sole nitrogen source, which we demonstrateis especially true under limiting concentrations(<1 mM). Tradeoffs between cellular localizationand ammonium toxicity lead to selection for thisapparent degeneracy as it is beneficial to facultativemethylotrophs that have to switch between using
1416 Current Biology 26, 1416–1426, June 6, 2016 ª 2016 Elsevier L
methylamine as a carbon and energy source or justa nitrogen source.
INTRODUCTION
Multiple modules that apparently perform the same biological
function are often encoded in a genome [1–4]. In some instances,
these modules are closely related paralogs (genetic redun-
dancy), but in others, they are structurally and evolutionarily
distinct entities (functional degeneracy) [5]. Metabolic degener-
acy—non-orthologous enzymes and pathways that catalyze
the same metabolic transformation through distinct biochemical
reactions—is especially prevalent in bacterial genomes [1–4, 6].
Despite the cost, degeneracy (or even redundancy) at a cellular
level is proposed to increase robustness to environmental pertur-
bations [7], mutations [8], and stochasticity in cellular processes
[9]. Consider one of the classic examples of metabolic degener-
acy, the two alternate routes for assimilation of ammonium ions
(NH4+) into glutamate in Escherichia coli. Biochemical tradeoffs
between the energetically efficient glutamate dehydrogenase
(GDH)-mediated route and the higher-affinity route using gluta-
mine synthetase and glutamate synthase (GS-GOGAT) have
been proposed to allow optimal nitrogen assimilation across a
wide range of growth conditions and NH4+ concentrations [10].
Although there is some evidence that GDH is specifically benefi-
cial under free energy (or carbon) limitation [11], neither a growth
defect nor an overt NH4+ assimilation phenotype has been
consistently observed for mutants lacking GDH [10]. In fact,
the recurring proposition that GDH is a completely dispensable
enzyme really questions whether we understand the putative
selective basis for this particularly well-studied example of
metabolic degeneracy [10, 12]. Some other studies [2–4] have
invoked compensating tradeoffs between degenerate pathways
to explain stable coexistence, but there is surprisingly little direct
experimental evidence that explains the prevalence and mainte-
nance ofmetabolic degeneracy in bacterial genomes over evolu-
tionary timescales.
Methylotrophs are a polyphyletic group of microorganisms
that grow on reduced single-carbon (C1) compounds as the
td.
A B
periplasm
cytoplasm
CH3NH3+
CH3NH3+ HCHO
NH4+, 2e-
MaDH
HCHOFAESpontaneous
HCOO-
CO2
Biomass H4MPT oxidation pathway
H4Freduction pathway
periplasm
cytoplasm
CH3NH3+
CH3NH3+ HCHO
HCOO-
ATP ADP
NH4+ NADH + H+
CO2
H4MPT oxidation pathway
Biomass
NMG pathway
Figure 1. MethylamineMetabolism inMeth-
ylobacterium extorquens Strains
(A) A schematic representation of carbon flow
during methylamine growth mediated by MaDH
(orange) in M. extorquens AM1. The founding
ancestor A1 lacks the entire mau gene cluster
encoding MaDH. The founding ancestor A2 lacks
FAE (formaldehyde-activating enzyme), which
catalyzes the condensation of formaldehyde with
the C1 carrier molecule tetrahydromethanopterin
(H4MPT) and is only essential for methylamine
growth mediated by MaDH.
(B) Methylamine growth mediated by the NMG
pathway (blue) in other M. extorquens strains. The
spontaneous reaction between formaldehyde and
H4MPT, which occurs at a slower rate, is sufficient
for methylamine growth mediated by the NMG
pathway.
See also Figure S1. MaDH, methylamine dehy-
drogenase; NMG, N-methylglutamate.
sole carbon and energy source [13, 14]. Methylamine (CH3NH2),
a degradation product of proteins, nitrogenous pesticides, and
osmolytes like glycine betaine (N,N,N-trimethylglycine), can
serve as either a growth substrate or a nitrogen source for meth-
ylotrophs [15]. Here, we uncover selective pressures that lead to
the maintenance of two apparently degenerate modules for
methylamine oxidation in sequenced methylotrophs.
Two biochemically distinct pathways for methylamine
oxidation have been characterized in methylotrophic bacteria
[14]. Methylamine dehydrogenase (MaDH), along with ancil-
lary proteins required for proper assembly and localization, is
encoded by the mau gene cluster [16, 17]. MaDH catalyzes
the oxidation of methylamine to formaldehyde in a single step
in the periplasm, transferring a pair of electrons to cytochrome
c via an amicyanin electron acceptor (Figure 1A) [16, 18]. Unlike
MaDH, the cytoplasmic N-methylglutamate (NMG) pathway
mediates the primary oxidation of methylamine in three enzy-
matic steps (Figures 1B and S1), produces an NADH, and
also uses an ATP per mol of methylamine oxidized [19]. Further-
more, the flow of oxidized C1 units into assimilatory pathways
also differs during methylamine growth mediated by MaDH
versus the NMG pathway. All formaldehyde produced by
MaDH flows through the tetrahydromethanopterin (H4MPT)-
dependent formaldehyde oxidation pathway to formate [13,
20], which is partially oxidized to CO2 via a panel of four formate
dehydrogenases [21, 22] and partially assimilated into biomass
via the successive action of the tetrahydrofolate (H4F)-depen-
dent pathway and the serine cycle [14, 23]. In contrast, growth
mediated by the NMG pathway has no requirement for the H4F
pathway; C1 units are either dissimilated by the H4MPT-depen-
dent formaldehyde oxidation pathway or enter the serine cycle
directly [15]. Curiously, many methylotrophs encode MaDH as
well as the NMG pathway [24]. In Methylobacterium extorquens
AM1 (referred to as AM1 hereafter), an extensively studied
facultative methylotroph belonging to the Alphaproteobacteria
[13], the NMG pathway was observed to be incapable of
rescuing the methylamine growth defect of mutants lacking
MaDH [17]. A paradox thus arises: why does AM1 encode the
NMG pathway if it does not appear to contribute to growth on
methylamine?
In this study, we use experimental evolution to uncover the
physiological challenges that need to be overcome in order to
use the NMG pathway for growth on methylamine as a carbon
and energy source and in the process discover the environ-
mental conditions under which the NMG pathway is advanta-
geous compared to MaDH. We began with two distinct strains
of AM1 that were incapable of growth on methylamine despite
an intact NMG pathway. After an extended period of time in
liquid medium with methylamine as the sole carbon and energy
source, one replicate for each genotype recovered slow growth.
Remarkably similar physiological hurdles were overcome in both
cases: an increase in the expression of the NMG pathway and
unique strategies to mitigate cytoplasmic ammonium buildup.
Inspired by these findings, we demonstrate that the presence
of the NMG pathway is a substantial advantage to cells in envi-
ronments where %1 mM methylamine is used as the sole nitro-
gen source. Thus, whereas MaDH is a highly expressed, poorly
regulated, and energetically favorable enzyme that enables rapid
growth of methylamine as a carbon source, the energetically
expensive NMG pathway enhances growth in environments
with limiting methylamine as the sole nitrogen source.
RESULTS
Fortuitous Evolution of NMG-Dependent Growth onMethylamineTwo strains of AM1, obtained from unrelated, independent
studies [25, 26], were previously observed to be incapable of
methylamine growth despite an intact NMG pathway. We
wanted to explore whether growth could be re-established by
experimental evolution on methylamine as the sole carbon and
energy source. The underlying rationale behind this experiment
was to use the beneficial mutations that enable methylamine
growth using the NMG pathway alone to help elucidate its phys-
iological role when present in a genome together with MaDH.
The first ancestral strain (referred to hereafter as ‘‘A1’’) was
isolated from the final time point from one of eight populations
of AM1 that were evolved by serial transfer for 1,500 generations
on a combination of methanol and succinate [25, 27]. Upon
sequencing the genome, besides other mutations not involved
Current Biology 26, 1416–1426, June 6, 2016 1417
in this study, we observed that A1 had lost genes encoding
MaDH due to homologous recombination between insertion se-
quences (ISs) flanking the 10-kb mau gene cluster (Table S1).
The second strain (referred to hereafter as ‘‘A2’’) contains an
in-frame, markerless deletion of fae [26] and was originally
used as a negative control for an evolution experiment involving
selection upon codons in synonymous variants of fae [28]. Form-
aldehyde-activating enzyme (FAE) catalyzes rapid condensa-
tion of formaldehyde and H4MPT, although this reaction is also
known to occur spontaneously in vivo at a slower rate [20, 29].
FAE is essential for rapid methylamine growth mediated by
MaDH [20]. Thus, even though A2 encodes a functionally active
MaDH, Dfae prevents methylamine growth due to the buildup of
toxic formaldehyde in the cell (Figure 1A) [20, 29]. Furthermore,
methylamine growth in other M. extorquens strains that only
contain the NMG pathway is at least 3-fold slower and can be
supported by the spontaneous reaction between formaldehyde
and H4MPT, which makes FAE dispensable (Figure 1B) [15].
A1 and A2 were each inoculated in minimal medium with
20 mM methylamine as the sole carbon and energy source for
2–4 weeks until a significant rise in optical density (DOD600 z0.05) was observed. After this initial rise in optical density, the
population initiated with A1 was plated to obtain single colonies
of evolved isolates, one of which we studied here (‘‘E1’’). On the
other hand, the population initiated with A2 was further propa-
gated by serial transfer for a further 150 generations and then
plated to obtain single colonies. An isolate from the final time
point of this evolution experiment was also studied further
(‘‘E2’’). E1 and E2 exhibited methylamine growth rates of
0.04 h�1 and 0.09 h�1 (average of three replicate cultures; Fig-
ure 2A), respectively. Despite this improvement in growth, the
growth rate of E1 and E2 was 79% (p < 0.0001; unpaired two-
sided Student’s t test comparing the mean growth rate of three
biological replicates each) and 55% (p < 0.0001) lower than
that observed for wild-type (WT). The yield (measured as
maximum OD600) of the two evolved isolates was 80% (p <
0.0001) and 15% (p = 0.0008) lower too (Figures 2A and 2B).
Mutations Re-establishing Growth Using the NMGPathwayThe genomes of E1 and E2 were sequenced to identify the
mutations that restored methylamine growth (Table S1). First,
we noted an IS-mediated recombination event in E2 that deleted
the mau gene cluster. Remarkably, this was exactly the same
event that had occurred during the evolution of A1 (Figure S2A).
Second, one mutation in each evolved isolate involved the NMG
pathway. E1 has a nonsense mutation (W173*) in Meta1_1544,
immediately upstream of genes encoding enzymes of the NMG
pathway. E2 has a tandem duplication of a 12-kb region of the
genome containing genes of the NMG pathway (Figure S2A).
In addition, E1 contains an IS insertion in the ykkC/yxkD RNA
element [30] located between two ABC transporters (Figure S2B)
and E2 contains a nonsense mutation (Q446*) in kefB, encoding
a glutathione-dependent K+/H+ antiporter [31] (Figure S2B).
Genetic Confirmation of NMG-Dependent MethylamineGrowthKey genes in dissimilatory pathways were deleted to establish
whether mutations in E1 and E2 rerouted primary oxidation
1418 Current Biology 26, 1416–1426, June 6, 2016
through the NMG pathway in the same manner as has been
established in other methylotrophs that encode only the
NMG pathway [15, 19]. First, we deleted NMG dehydrogenase
[15, 19] (encoded by mgdABCD), an enzyme that catalyzes the
last step of the NMG pathway (Figure S1), in WT, E1, and E2.
The DmgdABCDmutant of WT did not have a significant change
in growth rate (p = 0.11) but did have a 5.4% decrease in yield
(p = 0.05) on methylamine (Figures 2A and 2B). Neither the
DmgdABCD mutant of E1 nor a mutant strain of E2 lacking
both copies of the mgdABCD operon could grow on methyl-
amine. Next, in order to determine whether formaldehyde oxida-
tion by the H4MPT pathway was required for methylamine
growth [20, 32], the first dedicated gene for H4MPT biosynthesis
(mptG) [32] was deleted in E1 and E2. No methylamine growth
was observed in the DmptG mutant of either strain. These data
corroborate that methylamine growth mediated by the NMG
pathway involves the H4MPT-dependent formaldehyde oxida-
tion pathway [15].
Selective Coefficients of Evolved AllelesBy performing competition assays with strains containing one
or more evolved alleles against a fluorescently labeled WT
strain, we determined that both mutations in E1 were benefi-
cial in isolation (Figures 2C and 2D). Introducing the nonsense
mutation in Meta1_1544 (now termed nmgR; see below)
increased the fitness of A1 to 2.9% that of WT (p = 0.04),
whereas introducing the IS insertion in the ykkC/yxkD RNA
element increased the fitness of A1 to 13.3% that of the WT
(p = 0.0002). The fitness of E1 was indistinguishable from the
product of the fitness effect of each single mutant (W[E1] =
W[nmgRW173*] 3 W[IS insertion into ykkC/yxkD]; p = 0.6; Fig-
ures 2C and 2D).
In contrast to E1, adaptive mutations need to be obtained in a
particular order in E2 (Figures 2C and 2E). Re-introducing the
mau gene cluster on a plasmid (pAYC139) [17] in E2 resulted in
negative fitness indicating net death, likely due to formaldehyde
buildup (Figure 2E), suggesting that the Dmau mutation arose
first. In the Dmau background, a duplication of the genomic
region containing the NMG pathway increased methylamine
fitness by 9-fold (p < 0.0001), and when the Q446* nonsense
mutation in kefB was introduced, subsequently, the fitness
rose by another 16.3% (p = 0.003; Figure 2E). To test whether
this truncated allele behaves like a null mutation, the kefB locus
was deleted in E2. The methylamine fitness of the DkefB mutant
of E2 was significantly lower than E2 (8.5%; p = 0.002) yet was
significantly higher than a mutant strain of E2 containing the
ancestral kefBWT (7.1%; p = 0.04), indicating that kefBQ446* is
not a complete loss-of-function allele (Figure S3). Collectively,
these results confirm that the identified mutations are beneficial
and account for the improvement observed in the evolved
isolates.
NMGPathway Is Repressed duringMethylamine Growthin WTIn both E1 and E2, mutations changing the dosage of the NMG
pathwayorneighboringgeneswereacritical step toward restoring
methylamine growth. To quantify the change in expression of the
NMG pathway, the activity of NMG dehydrogenase (NMGDH)
was measured in crude cell extracts from methylamine-induced
A
0
0.05
0.1
0.15
0.2
0.25
WT A1 A2 E1 E2
Gro
wth
Rat
e (h
-1)
N.D.
N.D.
mgdABCD mutant
B
0
0.05
0.1
0.15
0.2
0.25
0.3
WT A1 A2 E1 E2
Yie
ld (
Max
imu
m O
D60
0)
Series2
N.D. N.D.
mgd ABCDmutant
C
0
0.2
0.4
0.6
0.8
1
1.2
WT A1 A2 E1 E2
Fit
nes
s
D
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
Fit
nes
s
A1
2
121 E1
nmgRW173*
IS insertion in ykkC/yxkD
E
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
Fit
nes
s
A2 1 1
21
2
3
3
E2
mau
kefB Q446*
12kb duplication
E2+
MaDH
Figure 2. Growth and Fitness of WT, Ancestral, and Evolved M. extorquens AM1 Isolates on Methylamine as the Sole Carbon and EnergySource
(A and B) Growth rate (h�1; A) and yield or maximum OD600 (B) on 20 mM methylamine for WT, the two ancestral genotypes (A1 and A2), and the two evolved
isolates (E1 and E2) with (gray) and without (blue) the NMG pathway.
(C) Competitive fitness of WT, the two ancestral strains (A1 and A2), and the two evolved isolates (E1 and E2) on 20 mM methylamine relative to a strain of
M. extorquens AM1 expressing the fluorescent protein mCherry.
(D) Competitive fitness of strains with different combinations of mutations from E1 on 20 mM methylamine.
(E) Competitive fitness of strains with different combinations of mutations from E2 on 20 mMmethylamine. The error bars represent the 95% confidence interval
(CI) of the mean of three independent fitness assays.
See also Figures S2 and S3. N.D., not detected.
cultures. The activity of NMGDH in A1 and A2 was indistinguish-
able from WT (p = 0.09 and p = 0.50; Figure 3A). The activity of
NMGDH in E1 and E2, however, was 5.2-fold (p < 0.0001) and
9.3-fold higher (p < 0.0001) than their respective ancestors (Fig-
ure 3A). Genomic proximity, along with phenotypic evidence that
a nonsense mutation in Mext_1544 increased NMGDH activity in
E1, indicated that the gene product plays a regulatory role influ-
encing expression of one or more enzymes of the NMG pathway.
Hence, Mext_1544 has been renamed NMG pathway regulator
(nmgR).
Cytoplasmic pH Homeostasis in E1 Achieved via UreaExcretionConsidering the stoichiometry of biomass, it became apparent
that, when used as a growth substrate, the 1:1 ratio of carbon:
nitrogen in methylamine greatly exceeds the cellular demand
for nitrogen (�5:1 carbon:nitrogen). This is further confounded
because 50% of the carbon from methylamine is respired as
CO2 [2, 23, 33]. Thus, even in the absence of extracellular
NH4+, only 10% of the NH4
+ generated during methylamine
growth will be assimilated and, to prevent the toxic effects of
Current Biology 26, 1416–1426, June 6, 2016 1419
0
10
20
30
40
50
60
70
80
90
WT A1 A2 E1 E2
NM
GD
HA
ctiv
ity
(nm
ol H
CH
O/m
g pr
otei
n/m
in)
B
0
20
40
60
80
100
120
140
A1 E1U
rea
in s
up
ern
atan
t (
M)
Succinate Succinate + MA
N. D. N. D. 0
0.05
0.1
0.15
0.2
0.25
0.3
5.5 6 6.5 7 7.5
Gro
wth
Rat
e (h
-1)
Extracellular pH
E1
E2
kefBAnc
E1nmgR
E2 kefB WT
A1 nmgRW173*
C
periplasm
cytoplasm
CH3NH3+
CH3NH3+ HCHO
HCOO-
NH4+
CO2
Biomass
Urea
Urea amidolyase
Urea efflux pump
A
D E
periplasm
cytoplasm
CH3NH3+
CH3NH3+ HCHO
HCOO-
NH4+
CO2
Biomass
H+
K+
H+
K+
H+
K+
KefBQ446*
FAE spontaneous
HCHO
NMG pathway NMG pathway
XΔMaDH
Figure 3. Physiological Adaptations in Evolved M. extorquens AM1 Isolates that Use the NMG Pathway for Growth on Methylamine as the
Sole Carbon and Energy Source
(A) Mean activity of the NMG dehydrogenase enzyme (nmol formaldehyde produced per mg protein per minute) in crude extracts from methylamine-induced
cultures of WT, the two ancestral strains (A1 and A2), and the two evolved isolates (E1 and E2).
(B) Mean concentration of urea in the supernatant for A1 and E1 during succinate growth (open) and after methylamine induction (filled).
(C) Growth rate of E1 (gray), E2 (black), A1 containing the evolved allele of nmgR (dashed gray), and E2 containing the ancestral allele of kefB (dashed black) in
minimal media with 3.5 mM succinate, buffered to pH values ranging from 5.5–7.5. The error bars represent the 95%CI of the mean of three biological replicates.
(D) Mutations in a putative repressor (nmgR) leading to overexpression of the NMG pathway and mitigation of NH4+ accumulation by urea excretion leads to
methylamine growth mediated by the NMG pathway in E1.
(E) Deletion of MaDH, a tandem duplication of the genes encoding the NMG pathway, and balancing cytoplasmic pH through a constitutive kefB mutant
(kefBQ446*) leads to methylamine growth mediated by the NMG pathway in E2.
See also Figure S4. MA, methylamine.
NH4+ buildup, the remainder must be released. Whereas the
periplasmic location of MaDHmay be an advantage for releasing
this excess NH4+, the cytoplasmic localization of the NMG
pathway might prove to be a growth-limiting constraint. There-
fore, we hypothesized that mitigating cytoplasmic NH4+ accu-
mulation, especially due to overexpression of the NMG pathway,
is likely to have been a target of other beneficial mutations that
restored methylamine growth in E1 and E2.
Similar to results in other organisms, prior studies in
M. extorquens have shown that IS insertions commonly change
1420 Current Biology 26, 1416–1426, June 6, 2016
expression of flanking genes [34, 35].We used qRT-PCR tomea-
sure the mRNA level for each of the ABC transporters flanking
the ykkC/yxkD RNA element in methylamine-induced cultures
of E1 and A1 (Figure S4A). The expression of the nitrate/sulfo-
nate/bicarbonate ABC efflux transporter in E1 was 58% higher
than in A1 (Meta1_4100 to Meta1_4102; p < 0.05), but there
was no significant change in expression of the glycine/betaine/
proline transporter (Meta1_4103 to Meta1_4105; 2% increase;
p = 0.86). The genomic proximity of the nitrate/sulfonate/
bicarbonate ABC efflux transporter to urea metabolism genes
A
0
0.05
0.1
0.15
0.2
0.25
WT
Gro
wth
Rat
e (h
-1)
WTnmgRW173
WTkefB Q446*
B
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
WTY
ield
(M
axim
um
OD
600)
WT
nmgRW173WT
kefB Q446*
Figure 4. Growth and Fitness of WT Strains
with Mutations that Enable Growth on NMG
Pathway
(A) Growth rate (h�1) and (B) yield or maximum
OD600 of WT, WT with the evolved allele of nmgR
from E1, and WT with the evolved allele of kefB
from E2. The error bars represent the 95% CI of
the mean of three biological replicates. See also
Figure S5.
(Figure S4A) further led to the hypothesis that it might serve as an
efflux pump for urea. No significant amount of urea (<20 mM) was
detected in the supernatant of A1 cultures during succinate
growth or after methylamine induction (Figure 3B). For E1, how-
ever, �100 mM urea was detected in the supernatant indepen-
dent of the growth conditions (OD600 of cultures varied by
<25%), suggesting that an IS insertion in the ykkC/yxkD RNA
element renders the neighboring nitrate/sulfonate/bicarbonate
ABC efflux transporter constitutively active (Figures 3B and 3D).
Truncated KefB Facilitates Growth of E2 in AlkalineMediaDue to the absence of any ABC transporter in the genomic prox-
imity of kefB, an alternate mechanism for suppressing the dele-
terious effects of NH4+ buildup is likely to have been adopted in
E2. Based on the location of the amber codon, the truncated
kefBQ446* allele encodes the H+/K+ exchanger domain without
the glutathione-dependent regulatory domain: when bound to
reduced glutathione, this domain represses the KefB H+/K+ anti-
porter activity to prevent acidification of the cytoplasm at the
expense of K+ ions (Figure S4B) [36]. KefBQ446* could thus func-
tion constitutively, and the constant proton inflow might buffer
the rise in cytoplasmic pH due to NH4+ accumulation. Extrapo-
lating this effect further, KefBQ446* might enable cells to maintain
pH homeostasis in the cytoplasm during growth in alkalinemedia
as well. When E2 and amutant strain of E2 containing the ances-
tral kefBWT allele were grown in minimal media buffered to
different pH values ranging from 5.5 to 7.5, the growth rate of
E2 was 2.3-fold greater (p < 0.0001) in medium buffered to
pH = 7.5 (Figure 3C) and indistinguishable at lower pH values.
Similarly, E1 also grew 37.2% (p < 0.0001) faster than a mutant
of A1 with the evolved nmgRW173* allele in media buffered to
pH = 7.5, but not at lower pH values (Figures 3C and 3E).
Selective Coefficient of Adaptive Mutations IsLower in WTPrevious work in M. extorquens has demonstrated that benefi-
cial mutations that arise in the genomic context of strains with
distinct metabolic pathways are often deleterious in WT
[35, 37]. Therefore, we hypothesized that, if the NMG pathway
plays a distinct role from MaDH in AM1, the mutations in E1
and E2 may not be beneficial when moved into WT. When the
nmgRW173* mutation from E1 was introduced in WT, no signifi-
Current
cant difference in growth rate (p = 0.65)
or yield (p = 0.79) on methylamine was
observed and there was a marginally sig-
nificant 2.9% decrease in methylamine
fitness (p = 0.07; Figures 4A, 4B, and
S5). In contrast, when the kefBQ446* mutation from E2 was intro-
duced in WT, the resulting strain was 8.5% more fit (p < 0.0001)
and grew 12.0% faster (p < 0.0001) but did not have a significant
change in yield (p = 0.20; Figures 4A, 4B, and S5). However, this
fitness increase was only half that observed when the kefBQ446*
mutation was introduced in the A2 genomic background. These
results are consistent with the hypothesis that traits critical for
using the NMG pathway are at least somewhat distinct from
those needed for MaDH-dependent growth.
NMG Pathway Enables Growth on Methylamine as aNitrogen SourceHaving uncovered that the NMGpathway is tightly regulated and
may have inherent rate-limiting constraints on methylamine
metabolism due to cytoplasmic NH4+ accumulation, we devel-
oped the hypothesis that the NMG pathway might play a role
in using methylamine as a nitrogen source, especially at low
concentrations when NH4+ will not accumulate. In order to test
this idea, we performed two complementary experiments. In
the first experiment, we sought to determine whether increased
expression of the NMG pathway was beneficial during growth
with methylamine as the sole nitrogen source for E1 and E2.
As a metric for use of methylamine as a nitrogen source, we
compared the ratio of growth rates on succinate as a carbon
source with methylamine versus ammonia as the sole nitrogen
source (ksuccinate, methylamine/ksuccinate, ammonia). For E1, this ratio
was 18% greater than A1 (p = 0.002), and for E2, this ratio was
110% (p < 0.0001) greater than A2 (Figures 5A and 5B; Table
S2). Next, we tested whether AM1 strains lacking the NMG
pathway suffered a growth defect in media with succinate as
the carbon source and a wide concentration gradient of methyl-
amine as the sole nitrogen source. We observed a 30%–50%
decrease in growth rate when the methylamine concentra-
tion dropped to 1 mM or below (Figure 5C). This translates to a
2-fold advantage to the WT genotype possessing both degen-
erate pathways over the one solely containingMaDH. In addition,
the yield of the DmgdABCD mutant was significantly lower
across the entire concentration range tested (Figure 5D).
DISCUSSION
In this study, we demonstrate that, even though the NMG
pathway and MaDH each catalyze the oxidation of methylamine
Biology 26, 1416–1426, June 6, 2016 1421
A
0
0.05
0.1
0.15
0.2
0.25
0.3
WT A1 A2 E1 E2
Gro
wth
Rat
e (h
-1)
B
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
WT A1 A2 E1 E2
Yie
ld (
Max
imu
m O
D60
0)
C
0
0.5
1
1.5
2
2.5
0 2 4 6 8 10 12
Gro
wth
Rat
e (h
-1)
ofW
T /
Gro
wth
Rat
e (h
-1)
of
mg
d m
uta
nt
Methylamine Concentration (mM)
D
0
0.25
0.5
0.75
1
1.25
1.5
0 2 4 6 8 10 12
Yie
ld (
Max
. OD
600)
ofW
T/
Yie
ld (
Max
. OD
600)
of
mg
dm
uta
nt
Methylamine Concentration (mM)
periplasmcytoplasm
CH3NH3+
CH3NH3+ HCHO
NH4+, 2e-
MaDH
HCHOFAE
HCOO-
CO2
Biomass
E
periplasm
cytoplasm
F
CH3NH3+
CH3NH3+ HCHO
HCOO-
ATP ADP
NH4+
NADH + H+
CO2
Biomass
NMG pathway
Alternate Carbon Source
e-
Figure 5. A Role for the NMG Pathway during Growth in Medium with Methylamine as the Sole Nitrogen Source
(A and B) The growth rate (h�1; open; A) and yield or maximum OD600 (filled; B) of WT, the two ancestral strains (A1 and A2), and the two evolved isolates (E1 and
E2) in nitrogen-free media with 3.5 mM succinate as the carbon source and 10 mM methylamine as the sole nitrogen source.
(legend continued on next page)
1422 Current Biology 26, 1416–1426, June 6, 2016
in AM1, the former enables faster growth and higher yields on
limiting concentrations of methylamine as a nitrogen source
whereas the latter leads to rapid growth on methylamine as the
primary carbon and energy substrate.
Decades of research that had established that MaDH was the
sole pathway for the primary oxidation of methylamine in AM1
was recently challenged when a study reported that enzymes
of the NMG pathway are also encoded and expressed in this
strain [38]. To uncover the role of the NMG pathway, two strains
of AM1, previously known to be incapable of methylamine
growth but containing an intact NMG pathway [25, 26], were
experimentally evolved on methylamine as the sole carbon and
energy source. The first strain (A1) lacks the mau gene cluster
encoding MaDH (Figure 1A), whereas the second strain (A2)
has a lesion in the first gene (fae) of the formaldehyde oxidation
module (Figure 1A) that prevents methylamine growth mediated
by MaDH due to the buildup of toxic formaldehyde from methyl-
amine oxidation. Remarkably, the methylamine fitness of A1 and
A2 was significantly lower than other M. extorquens strains that
encode only the NMG pathway for methylamine oxidation [24].
Genomic, genetic, and biochemical analysis of the evolved
isolates revealed two classes of mutations that overcame the
regulatory and physiological constraints preventing methyl-
amine growth mediated by the NMG pathway. The first class
of mutations led to a significant increase in the expression level
of the NMG pathway (Figures S2 and 3A). Low expression of
enzymes of the NMG pathway in crude extracts from methyl-
amine-induced WT cells (Figure 3A) indicated that this pathway
is typically repressed, even in the presence of high concen-
trations of methylamine. Contrary to some other examples of
degeneracy, a significant growth or fitness advantage was not
observed when MaDH and the NMG pathway were simulta-
neously expressed during methylamine growth in WT (Figures
4A and S5) [1, 4]. Hence, these two degenerate pathways,
despite catalyzing the same biochemical transformation, seem
to be playing distinct roles in AM1.
The second class of adaptive mutations resulted in physiolog-
ical innovations to buffer the buildup of NH4+ ions in the cyto-
plasm, especially due to overexpression of the NMG pathway
in AM1, which typically uses a periplasmic MaDH for methyl-
amine growth. It is well established that eukaryotes mitigate
NH4+ buildup by converting it to the less-toxic, less-basic, nitrog-
enous compound urea, which is subsequently excreted (Figures
3B and 3D) [39]. To the best of our knowledge, this is the first
study to demonstrate urea excretion as a physiological response
to ameliorate the accumulation of NH4+ in the cytosol of bacterial
cells (Figure 3B). The opposite response of importing urea and
using urease to release NH4+ in order to increase the pH of the
cytoplasm is critical to how Helicobacter pylori thrives in the
acidic environment of the human stomach [40]. Additionally,
our physiological analyses also suggest that the ykkC/yxkD
RNA element represses the expression of the flanking efflux
pump unless bound to an unknown ligand. Whether this efflux
(C and D)Mean ratio of the (C) growth rate (h�1) and (D) yield ormaximumOD600 of
or 10 mM methylamine as the sole nitrogen source. The error bars represent the
(E) A schematic representing the metabolic route followed by methylamine when
(F) A schematic representing the metabolic route followed by methylamine wh
presence of other, likely more abundant, carbon substrate(s).
pump is specific for urea or has a wider substrate breath for
urea-based compounds, including many natural antimicrobial
agents, remains to be determined [41]. In E2, the kefBQ446* mu-
tation significantly enhances growth in alkalinemedia (Figure 3C);
likely, by removing the regulatory domain of the KefB K+/H+ anti-
porter and rendering it constitutively active, the H+ influx in the
cytoplasm is increased (Figures 3C and 3E). Until very recently,
cytoplasmic acidification by K+/H+ antiporters like KefB and
KefC has been singularly linked to a stress response mechanism
to counter the toxic effects of electrophiles like methylgloxal
in E. coli [31]. The results of this work broaden the cellular
role of KefB to the maintenance of pH homeostasis as well.
Interestingly, the kefBQ446* mutation in E2 mirrors the use of
dedicated monovalent cation/proton antiporters to maintain
cytoplasmic pH homeostasis under alkaline conditions in al-
kali-tolerant bacteria [42]. Furthermore, it was recently reported
that beneficial kefB alleles arose during experimental evolution of
a poor-growing M. extorquens AM1 strain engineered to use a
glutathione-based formaldehyde oxidation module, suggesting
that KefB activity may be more generally relevant to methylo-
trophy [37].
In nature, facultative methylotrophs like AM1 likely sense the
concentration of methylamine in its environment and the need
for alternative carbon or nitrogen sources in order to configure
the metabolic network accordingly to use methylamine either
as a growth substrate and/or as a nitrogen source. As a result,
the use of methylamine as a growth substrate and/or as a
nitrogen source might not be as simple as a direct correlation
with concentrations of methylamine in the environment. In envi-
ronments where methylamine is abundant and likely serves as
the preferred carbon and energy source, the highly expressed,
energetically efficient MaDH should lead to rapid growth and
the excess NH4+ produced can be released readily from the peri-
plasm (Figure 5E). In environments with higher concentrations of
other carbon sources but with limiting concentrations of methyl-
amine as the sole/preferred nitrogen source, the NMG pathway
is likely to be recruited for nitrogen assimilation (Figure 5F). Our
discovery of nmgR in regulating expression of the NMG pathway
enzymes is a start to understanding how M. extorquens AM1
make this decision. The concentration threshold for a strain
containing both pathways to have an advantage is 1 mMmethyl-
amine andmirrors the range ofmethylamine concentrations typi-
cally observed in different environments, thereby suggesting the
concentration-dependent advantage of degeneracy observed
here may be of great relevance in nature [43].
These two functionally degenerate pathways for methylamine
oxidation coexist in one-third of sequenced methylotrophs with
at least one methylamine oxidation pathway [24]. Functionally
degenerate pathways specialized for unique ecological roles
would only coexist long-term in a genome if there were tradeoffs
associated with each pathway. The cytoplasmic localization
of the NMG pathway [19] might allow cells to capture a higher
fraction of NH4+ ions released from low concentrations of
WTand theDmgdABCDmutant inmedia with 3.5mMsuccinate and 0.5, 1.0, 5,
95% CI of the mean of three biological replicates.
M. extorquens AM1 uses methylamine as a growth substrate.
en M. extorquens AM1 uses methylamine as the sole nitrogen source in the
Current Biology 26, 1416–1426, June 6, 2016 1423
methylamine relative to MaDH, a periplasmic enzyme. On the
other hand,MaDH iswell situated tousemethylamineasagrowth
substrate because NH4+ excretion can occur without transport
costs from the periplasm [16]. Thus, despite catalyzing the
same biochemical reaction, the two modules play ecologically
distinct roles to enable methylotrophs to optimally utilize methyl-
amine either as a growth substrate or as a nitrogen source.
EXPERIMENTAL PROCEDURES
Chemicals and Media
All chemicals were purchased from Sigma-Aldrich. E. coli strains were grown
in Luria-Bertani broth at 37�C with the standard antibiotic concentrations.
Standard growth conditions for AM1 utilized a modified version of Hypho min-
imal medium described previously [44]. Nitrogen-free Hypho minimal media
was made with a nitrogen-free sulfate salts solution (7 g of MgSO4 , 7 H2O
in 1 l deionized water). All components were autoclaved separately before mix-
ing under sterile conditions. Filter-sterilized carbon sources were added just
prior to inoculation in liquid minimal media with a final concentration of
3.5 mM for sodium succinate and 20 mM for methylamine hydrochloride.
Experimental Evolution
Experimental Evolution I
M. extorquens AM1 was serially transferred for 1,500 generations in medium
with 1.75 mM disodium succinate and 7.5 mM methanol as described earlier
[25, 27]. An evolved isolate (CM1054; here, A1) that could not grow on methyl-
amine was subsequently grown on 20 mM methylamine hydrochloride as
described in Supplemental Experimental Procedures.
Experimental Evolution II
A single population of a Dfae Dcel strain ofMethylobacterium extorquens AM1
(CM2770; here, A2) was serially transferred in modified Hypho medium with
20 mM methylamine hydrochloride as described in Supplemental Experi-
mental Procedures.
Growth Rate Measurements
Cells were acclimated and grown in 48-well microtiter plates (CoStar-3548)
containing 640 ml of growthmedium, in a shaking incubator at 650 rpm (Liconic
USA LTX44 with custom fabricated cassettes), in a room that was constantly
maintained at 30�C and 80% humidity as described previously [45]. The spe-
cific growth rate of cultures was calculated from the log-linear growth phase
using an open source, custom-designed growth analysis software called
CurveFitter available at http://www.evolvedmicrobe.com/CurveFitter/.
Plasmids and Strains
Markerless chromosomal allele replacements or gene deletions were con-
ducted using the allelic exchange vector pCM433 [46]. Genomic regions
were amplified by PCR and ligated on the pCM433 backbone cut with NotI
using Gibson assembly [47]. Mutant strains ofM. extorquens AM1were gener-
ated through conjugation by a tri-parental mating technique described else-
where [46]. Mutants were confirmed by a diagnostic PCR and validated by
Sanger sequencing. All strains and plasmids used and generated for this study
are listed in Table S3.
Competitive Fitness Assays
Fitness measurements were estimated by competing each strain against
M. extorquens AM1Dcel-DkatA::Ptac-mCherry (CM3120) described elsewhere
[45] and counting the fraction of fluorescent to non-fluorescent cells using the
LSR Fortesssa (Becton Dickinson) as described in the Supplemental Experi-
mental Procedures section. The competitive fitness was calculated as
W = logðR1 � N=R0Þ=logðð1� R1Þ � N=ð1� R0ÞÞ, where R1 and R0 represent
the population fraction of the test strain before and after mixed growth and
N represents the fold increase in the population density.
NMG Dehydrogenase Assay
Methylamine-induced cultures were lysed using a FastPrep-24 (MP Bio)
and lysing matrix B (MP Bio). Lysates were centrifuged at 13,000 rpm for
5 min in a centrifuge maintained at 4�C, and total protein was quantified
1424 Current Biology 26, 1416–1426, June 6, 2016
using the Quick Start Bradford Assay (Bio-Rad) using BSA as a standard.
0.1 mg of protein was incubated in sodium phosphate buffer (pH = 7.6)
with 5 mM NMG and 0.5 mM NAD+ at room temperature for 40 min and at
58�C for 30 min after Nash reagent [48] was added. Formaldehyde produc-
tion was estimated by measuring the absorbance at 412 nm, and NMGDH
activity was measured as the nmol formaldehyde produced per mg protein
per minute.
Genome Sequencing
Cell pellets were lysed using a combination of lysozyme and heat shock at
90�C for 10 min. Lysed cells were treated with proteinase K and RNase A at
37�C, and genomic DNA was extracted using the phenol-chloroform extrac-
tion protocol. Single-end 50-bp reads for Illumina sequencing were prepared
using the TruSeq DNA sample prep Kit (Illumina) and sequenced using an Illu-
mina Hi-Seq at the Microarray and Genomics Core Facility, Huntsman Cancer
Institute. Reads were assembled and aligned to the M. extorquens AM1
genome [49] using Breseq version 0.21 [50], and mutations were verified by
PCR and Sanger sequencing.
qRT-PCR
RNA from methylamine-induced cultures was extracted using an RNeasy Kit
(QIAGEN) and quantified using a Nanodrop spectrophotometer ND-1000
(Thermo Fisher Scientific). One microgram RNAwas reverse transcribed using
SuperScript III (Life Technologies), and qPCR was performed using a CFX-96
qPCR machine (Bio-Rad) and EvaGreen qPCR mix (Biotium). Ribosomal pro-
tein RpsB was used as an internal standard, and the relative expression of
Meta1_4101 and Meta1_4103 was quantified using a protocol described else-
where [34, 51]. Three technical replicateswere performed for each of three bio-
logical replicates. A no-template control, a no-RT control, and a standard
curve with DNA concentrations from 1 ng to 10�6 ng was run for every primer
pair. Gene-specific primers designed for rpsB, Meta1_4101, and Meta1_4103
are listed in Table S4.
Urea Quantification Assay
Spent media from cultures was collected by centrifugation and filtration using
a sterile 0.2-mmmembrane filter (Millipore). Urea concentration was measured
using the Urea Assay Kit (Sigma-Aldrich) per the manufacturer’s instructions.
Absorbance was measured in 96-well, flat-bottom plates (CoStar-3595) using
the Tecan Safire2 plate reader (Tecan Group). Two technical replicates were
performed for each of three biological replicates.
ACCESSION NUMBERS
The accession number for the sequence data reported in this paper is Bio-
Project: PRJNA316889.
SUPPLEMENTAL INFORMATION
Supplemental Information includes five figures, four tables, and Supplemental
Experimental Procedures and can be found with this article online at http://dx.
doi.org/10.1016/j.cub.2016.04.029.
AUTHOR CONTRIBUTIONS
D.D.N. and C.J.M. designed experiments. D.D.N., D.A., and M.-C.L. per-
formed experiments. D.D.N. and C.J.M. analyzed data. D.D.N. and C.J.M.
wrote the manuscript.
ACKNOWLEDGMENTS
We would like to thank members of the C.J.M. lab for their feedback on the
manuscript. C.J.M. acknowledges financial support from the NIH (GM078209).
Received: December 29, 2015
Revised: February 17, 2016
Accepted: April 11, 2016
Published: May 19, 2016
REFERENCES
1. Marx, C.J., Miller, J.A., Chistoserdova, L., and Lidstrom, M.E. (2004).
Multiple formaldehyde oxidation/detoxification pathways in Burkholderia
fungorum LB400. J. Bacteriol. 186, 2173–2178.
2. Marx, C.J., Van Dien, S.J., and Lidstrom, M.E. (2005). Flux analysis un-
covers key role of functional redundancy in formaldehyde metabolism.
PLoS Biol. 3, e16.
3. Denef, V.J., Klappenbach, J.A., Patrauchan, M.A., Florizone, C.,
Rodrigues, J.L.M., Tsoi, T.V., Verstraete, W., Eltis, L.D., and Tiedje, J.M.
(2006). Genetic and genomic insights into the role of benzoate-catabolic
pathway redundancy in Burkholderia xenovorans LB400. Appl. Environ.
Microbiol. 72, 585–595.
4. Gerth, M.L., Ferla, M.P., and Rainey, P.B. (2012). The origin and ecological
significance of multiple branches for histidine utilization in Pseudomonas
aeruginosa PAO1. Environ. Microbiol. 14, 1929–1940.
5. Tononi, G., Sporns, O., and Edelman, G.M. (1999). Measures of degener-
acy and redundancy in biological networks. Proc. Natl. Acad. Sci. USA 96,
3257–3262.
6. Boucher, Y., Douady, C.J., Papke, R.T., Walsh, D.A., Boudreau, M.E.R.,
Nesbø, C.L., Case, R.J., and Doolittle, W.F. (2003). Lateral gene transfer
and the origins of prokaryotic groups. Annu. Rev. Genet. 37, 283–328.
7. Kafri, R., Springer, M., and Pilpel, Y. (2009). Genetic redundancy: new
tricks for old genes. Cell 136, 389–392.
8. Wang, Z., and Zhang, J. (2009). Abundant indispensable redundancies in
cellular metabolic networks. Genome Biol. Evol. 1, 23–33.
9. Stelling, J., Sauer, U., Szallasi, Z., Doyle, F.J., 3rd, and Doyle, J. (2004).
Robustness of cellular functions. Cell 118, 675–685.
10. van Heeswijk, W.C., Westerhoff, H.V., and Boogerd, F.C. (2013). Nitrogen
assimilation in Escherichia coli: putting molecular data into a systems
perspective. Microbiol. Mol. Biol. Rev. 77, 628–695.
11. Helling, R.B. (2002). Speed versus efficiency in microbial growth and the
role of parallel pathways. J. Bacteriol. 184, 1041–1045.
12. Brenchley, J.E., Prival, M.J., and Magasanik, B. (1973). Regulation of the
synthesis of enzymes responsible for glutamate formation in Klebsiella
aerogenes. J. Biol. Chem. 248, 6122–6128.
13. Chistoserdova, L., Chen, S.W., Lapidus, A., and Lidstrom, M.E. (2003).
Methylotrophy inMethylobacterium extorquensAM1 from a genomic point
of view. J. Bacteriol. 185, 2980–2987.
14. Chistoserdova, L. (2011). Modularity of methylotrophy, revisited. Environ.
Microbiol. 13, 2603–2622.
15. Nayak, D.D., and Marx, C.J. (2014). Methylamine utilization via the
N-methylglutamate pathway in Methylobacterium extorquens PA1 in-
volves a novel flow of carbon through C1 assimilation and dissimilation
pathways. J. Bacteriol. 196, 4130–4139.
16. McIntire, W.S., Wemmer, D.E., Chistoserdov, A., and Lidstrom, M.E.
(1991). A new cofactor in a prokaryotic enzyme: tryptophan tryptophylqui-
none as the redox prosthetic group in methylamine dehydrogenase.
Science 252, 817–824.
17. Chistoserdov, A.Y., Chistoserdova, L.V., McIntire, W.S., and Lidstrom,M.E.
(1994). Genetic organization of the mau gene cluster in Methylobacterium
extorquens AM1: complete nucleotide sequence and generation and
characteristics ofmaumutants. J. Bacteriol. 176, 4052–4065.
18. Chen, L., Durley, R.C., Mathews, F.S., andDavidson, V.L. (1994). Structure
of an electron transfer complex: methylamine dehydrogenase, amicyanin,
and cytochrome c551i. Science 264, 86–90.
19. Latypova, E., Yang, S., Wang, Y.S., Wang, T., Chavkin, T.A., Hackett, M.,
Schafer, H., and Kalyuzhnaya, M.G. (2010). Genetics of the glutamate-
mediated methylamine utilization pathway in the facultative methylo-
trophic beta-proteobacterium Methyloversatilis universalis FAM5. Mol.
Microbiol. 75, 426–439.
20. Marx, C.J., Chistoserdova, L., and Lidstrom, M.E. (2003). Formaldehyde-
detoxifying role of the tetrahydromethanopterin-linked pathway in
Methylobacterium extorquens AM1. J. Bacteriol. 185, 7160–7168.
21. Chistoserdova, L., Laukel, M., Portais, J.C., Vorholt, J.A., and Lidstrom,
M.E. (2004). Multiple formate dehydrogenase enzymes in the facultative
methylotroph Methylobacterium extorquens AM1 are dispensable for
growth on methanol. J. Bacteriol. 186, 22–28.
22. Chistoserdova, L., Crowther, G.J., Vorholt, J.A., Skovran, E., Portais,
J.-C., and Lidstrom, M.E. (2007). Identification of a fourth formate dehy-
drogenase in Methylobacterium extorquens AM1 and confirmation of the
essential role of formate oxidation in methylotrophy. J. Bacteriol. 189,
9076–9081.
23. Crowther, G.J., Kosaly, G., and Lidstrom, M.E. (2008). Formate as the
main branch point for methylotrophic metabolism in Methylobacterium
extorquens AM1. J. Bacteriol. 190, 5057–5062.
24. Nayak, D.D., and Marx, C.J. (2015). Experimental horizontal gene transfer
of methylamine dehydrogenase mimics prevalent exchange in nature and
overcomes the methylamine growth constraints posed by the sub-optimal
N-methylglutamate pathway. Microorganisms 3, 60–79.
25. Lee, M.C., and Marx, C.J. (2012). Repeated, selection-driven genome
reduction of accessory genes in experimental populations. PLoS Genet.
8, e1002651.
26. Agashe, D., Martinez-Gomez, N.C., Drummond, D.A., and Marx, C.J.
(2013). Good codons, bad transcript: large reductions in gene expression
and fitness arising from synonymousmutations in a key enzyme. Mol. Biol.
Evol. 30, 549–560.
27. Lee, M.C., Chou, H.H., and Marx, C.J. (2009). Asymmetric, bimodal trade-
offs during adaptation of Methylobacterium to distinct growth substrates.
Evolution 63, 2816–2830.
28. Agashe, D., Sane, M., Phalnikar, K., Diwan, G.D., Habibullah, A., Martinez-
Gomez, N.C., Sahasrabuddhe, V., Polachek, W., Wang, J., Chubiz, L.M.,
and Marx, C.J. (2016). Large-effect beneficial synonymous mutations
mediate rapid and parallel adaptation in a bacterium. Mol. Biol. Evol. 33,
1542–1553.
29. Vorholt, J.A., Marx, C.J., Lidstrom, M.E., and Thauer, R.K. (2000). Novel
formaldehyde-activating enzyme in Methylobacterium extorquens AM1
required for growth on methanol. J. Bacteriol. 182, 6645–6650.
30. Barrick, J.E., Corbino, K.A., Winkler, W.C., Nahvi, A., Mandal, M., Collins,
J., Lee, M., Roth, A., Sudarsan, N., Jona, I., et al. (2004). New RNA motifs
suggest an expanded scope for riboswitches in bacterial genetic control.
Proc. Natl. Acad. Sci. USA 101, 6421–6426.
31. Ferguson, G.P., Nikolaev, Y., McLaggan, D., Maclean, M., and Booth, I.R.
(1997). Survival during exposure to the electrophilic reagent N-ethylmalei-
mide in Escherichia coli: role of KefB and KefC potassium channels.
J. Bacteriol. 179, 1007–1012.
32. Rasche,M.E.,Havemann,S.A., andRosenzvaig,M. (2004).Characterization
of two methanopterin biosynthesis mutants of Methylobacterium extor-
quens AM1 by use of a tetrahydromethanopterin bioassay. J. Bacteriol.
186, 1565–1570.
33. Van Dien, S.J., and Lidstrom, M.E. (2002). Stoichiometric model for
evaluating the metabolic capabilities of the facultative methylotroph
Methylobacterium extorquens AM1, with application to reconstruction of
C(3) and C(4) metabolism. Biotechnol. Bioeng. 78, 296–312.
34. Chou, H.H., and Marx, C.J. (2012). Optimization of gene expression
through divergent mutational paths. Cell Rep. 1, 133–140.
35. Michener, J.K., CamargoNeves, A.A., Vuilleumier, S., Bringel, F., andMarx,
C.J. (2014). Effective use of a horizontally-transferred pathway for dichloro-
methane catabolism requires post-transfer refinement. eLife 3, 1–16.
36. Roosild, T.P., Castronovo, S., Miller, S., Li, C., Rasmussen, T., Bartlett, W.,
Gunasekera, B., Choe, S., and Booth, I.R. (2009). KTN (RCK) domains
regulate K+ channels and transporters by controlling the dimer-hinge
conformation. Structure 17, 893–903.
37. Carroll, S.M., Chubiz, L.M., Agashe, D., andMarx, C.J. (2015). Parallel and
divergent evolutionary solutions for the optimization of an engineered cen-
tral metabolism inMethylobacterium extorquens AM1. Microorganisms 3,
152–174.
Current Biology 26, 1416–1426, June 6, 2016 1425
38. Martinez-Gomez, N.C., Nguyen, S., and Lidstrom,M.E. (2013). Elucidation
of the role of the methylene-tetrahydromethanopterin dehydrogenase
MtdA in the tetrahydromethanopterin-dependent oxidation pathway in
Methylobacterium extorquens AM1. J. Bacteriol. 195, 2359–2367.
39. Singer, M.A. (2003). Do mammals, birds, reptiles and fish have similar ni-
trogen conserving systems? Comp. Biochem. Physiol. B Biochem. Mol.
Biol. 134, 543–558.
40. Weeks, D.L., Eskandari, S., Scott, D.R., and Sachs, G. (2000). A H+-gated
urea channel: the link between Helicobacter pylori urease and gastric
colonization. Science 287, 482–485.
41. Tegos, G.P., Masago, K., Aziz, F., Higginbotham, A., Stermitz, F.R., and
Hamblin, M.R. (2008). Inhibitors of bacterial multidrug efflux pumps poten-
tiate antimicrobial photoinactivation. Antimicrob. Agents Chemother. 52,
3202–3209.
42. Padan, E., Bibi, E., Ito, M., and Krulwich, T.A. (2005). Alkaline pH homeo-
stasis in bacteria: new insights. Biochim. Biophys. Acta 1717, 67–88.
43. Ge, X., Wexler, A.S., and Clegg, S.L. (2011). Atmospheric amines - Part I. A
review. Atmos. Environ. 45, 524–546.
44. Nayak, D.D., and Marx, C.J. (2014). Genetic and phenotypic comparison
of facultative methylotrophy between Methylobacterium extorquens
strains PA1 and AM1. PLoS ONE 9, e107887.
45. Delaney, N.F., Kaczmarek, M.E., Ward, L.M., Swanson, P.K., Lee, M.C.,
and Marx, C.J. (2013). Development of an optimized medium, strain and
1426 Current Biology 26, 1416–1426, June 6, 2016
high-throughput culturing methods for Methylobacterium extorquens.
PLoS ONE 8, e62957.
46. Marx, C.J. (2008). Development of a broad-host-range sacB-based vector
for unmarked allelic exchange. BMC Res. Notes 1, 1.
47. Gibson, D.G., Young, L., Chuang, R.-Y., Venter, J.C., Hutchison, C.A., 3rd,
and Smith, H.O. (2009). Enzymatic assembly of DNA molecules up to
several hundred kilobases. Nat. Methods 6, 343–345.
48. Nash, T. (1953). The colorimetric estimation of formaldehyde by means of
the Hantzsch reaction. Biochem. J. 55, 416–421.
49. Vuilleumier, S., Chistoserdova, L., Lee, M.C., Bringel, F., Lajus, A., Zhou,
Y., Gourion, B., Barbe, V., Chang, J., Cruveiller, S., et al. (2009).
Methylobacterium genome sequences: a reference blueprint to investi-
gate microbial metabolism of C1 compounds from natural and industrial
sources. PLoS ONE 4, e5584.
50. Barrick, J.E., Colburn, G., Deatherage, D.E., Traverse, C.C., Strand, M.D.,
Borges, J.J., Knoester, D.B., Reba, A., and Meyer, A.G. (2014). Identifying
structural variation in haploid microbial genomes from short-read rese-
quencing data using breseq. BMC Genomics 15, 1039.
51. Chou, H.H., Berthet, J., and Marx, C.J. (2009). Fast growth increases the
selective advantage of a mutation arising recurrently during evolution un-
der metal limitation. PLoS Genet. 5, e1000652.
Current Biology, Volume 26
Supplemental Information
Selection Maintains Apparently Degenerate
Metabolic Pathways due to Tradeoffs
in Using Methylamine for Carbon versus Nitrogen
Dipti D. Nayak, Deepa Agashe, Ming-Chun Lee, and Christopher J. Marx
Figure S1: Related to Figure 1. A schematic of the N-methylglutamate pathway. The topology of the N-methylglutamate pathway in M. extroquens species has been shown to be semi-linear: N-methylglutamate synthase is capable of synthesizing N-methylglutamate from γ-glutamylmethylamide (blue arrow) as well as methylamine (gray arrow) albeit only in the ∆gmas mutant. NMGDH: N-methylglutamate dehydrogenase, MGS: N-methylglutamate synthase, GMAS: γ-glutamylmethylamide synthetase, NMG – N-methylglutamate, GMA - γ-glutamylmethylamide
CH3NH2
NM
GS*
GM
AS Glutamate, ATP
ADP
(GMA)
HCHO
NM
GD
H
NH4+
H2O, NAD+
Glutamate, NADH
(NMG)
NM
GS
NH4+
Figure S2: Related to Figure 2. Mutations in E1 and E2. A schematic representation of the adaptive mutations in A) E1 and B) E2 that enable methylamine growth mediated by the N-methylglutamate pathway.
IS IS
mau gene cluster
IS
∆mau
kefB
Q446*
Truncated kefB
NMG pathway gene cluster
Duplication
1 2 3
1 2NMG pathway gene clusterG1617391A, W173*
nmgR
Truncated nmgR
ykkC-yxkD
ABC Transporter
IS
IS insertion in ykkC - yxkD
G/B/P TransporterIS
ABC Transporter G/B/P Transporter
A
B
Figure S3: Related to Figure 2. Fitness of kefB alleles the E2 genomic background. Competitive fitness of E2, a mutant of E2 with an in-frame deletion in the kefB CDS, and a mutant of E2 with the WT allele of kefB on 20 mM methylamine hydrochloride relative to CM3120 (M. extorquens AM1 ∆cel-∆katA::Ptac-mCherry). The error bars indicate the 95% CI of the mean fitness value of three replicate competition assays. Note: The y-axis does not start at 0 to highlight the fitness different between the three strains.
0.3
0.32
0.34
0.36
0.38
0.4
0.42
0.44
1 2 3
Fitn
ess
E2 E2 ∆kefB E2 kefBWT
Figure S4: Related to Figure 3. Mutations ameliorating cytoplasmic NH4+ accumulation in E1 and E2. A)
Genes flanking the ykkC/yxkD RNA element in M. extorquens AM1. In pink and blue are the two ABC transporters on either side of this RNA element. The ABC-type nitrate/sulfonate/bicarbonate efflux transporter (in pink) is commonly associated with the ykkC/yxkD RNA element. This efflux transporter is in close proximity to urea metabolism genes (in yellow) and other nitrogen metabolism genes as well (in gray).B) The amino acid sequence of kefB (Meta1_2712) showing the K+/H+ ion exchanger domain in green and the RCK (regulator of K+ conductance) domain in orange. The mutated residue in E2 is highlighted in blue.
ykkC-yxkD
ABC efflux transporter (Meta1_4100 to Meta1_4102)
Glycine/betaine/proline transporter(Meta1_4013 to Meta1_4015)
Creatininase
gmaS homolog
Urea amidolyase with urea carboxylase and allophanate hydrolase subunits
(Meta1_4095)
Putative regulator
A
MASATDHASFLPPVLTFLSAAVIGVPLVRLLGQSAVLGYLVAGVVIGPAGFSLIAEPETAASVAEIGVVLLLFIVGLELEISRLVSMRRDIFGLGAAQLALCSLVIAGAALAYGLTPAAASVVGIAIALSATAVALQLLEERGDLGSPYGGRSFAVLLFQDISVVAILALLPLLASAGATPKDGWLDEGLRSTGRAVVAVAGVVLVGRYGLNPFFRLLAAAGGREVMTAAALLVVLGTALVMEKAGLSMAMGAFLAGVMLAESNFRHQLEADIEPFRGMLLGLFFMSVGMSIDGGLLTNHWLALLAATMAAIAVKIALVAGLFRLFGSPWLDALRGAAVLAPAGEFAFVILPAAGDLRILASDVTRFCVALAALTMLVGPIAAKGLDALIARRPKEAEPPSDVGEAQESG
DTRVLVVGFGRFGQILAQVLLAEGINITVIDKDVEQ IRNATSFGFRIYYGDGTRLDVLRASGLAKADLICVCIDDAPAALKIVDIVHEEFPNVRTYVRAYDRTHAIELMNRDVDFQLRETVESALGFGRATLESLGLPAEAAARRVEDVRKRDVARLVLQQAGAMPDGSGWLRGTPELRPEPLTAPKSPSRALSAETRGLIEQQPHESAARALEPEDAEPNA*
B
Urea degradation associated genes (Meta1_4096 and Meta1_4097)
Figure S5: Related to Figure 4. Fitness of adaptive mutations in E1 and E2 in the WT background. Competitive fitness of WT, a mutant strain of WT containing the nmgRW173* allele from E1, and a mutant strain of WT containing the kefBQ446* allele from E2 relative on 20 mM methylamine hydrochloride. The error bars indicate the 95% CI of the mean fitness value of three replicate competition assays.
0
0.2
0.4
0.6
0.8
1
1.2
WT WT nmgRevo WT kefBevo
Fitn
ess
WTnmgRW173*
WTkefBQ446*
Table S1: Related to Figure 1. Mutations in A1, E1, and E2. A list of mutations in A1 relative to the M. extroquens AM1 reference genome and in E1 and E2 relative to their respective ancestors. Each of these mutations were verified by Sanger sequencing.
A1 E1 E2
G420551C (R73P)Meta_0397
G1617391A (W173*)Meta1_1544 or nmgR
G2837255A(Q446*)kefB
2308994 (+T)Meta1_2237
ISmex4 insertion in ykkC-yxkD
Deletion (28679 bp-30272 bp)Meta1_2751-Meta1_2783
(Δmau gene cluster)
G3724230A(intergenic)Meta1_3582/Meta1_3583
New JunctionPosition 162893, Meta1_1554
or nmgR, and position 1616982, Meta1_1544 or
nmgR
A4125088G (intergenic)Meta1_4038/rffH
A11165G(R31R)Meta2_1054
T144186G(Q24H)Meta2_0154
C5903G (intergenic)p2meta_006/p2meta_007
Deletion (28679bp -30281 bp)Meta1_2751 -Meta1_2783
(Δmau gene cluster)
Deletion (61658bp -61678bp)Meta1_3749-Meta1_tRNA33
Deletion (~0.6Mb)Meta2_0933 – Meta1_0277
ISmex4 insertionMeta1_2101/Meta1_2102
ISmex4 insertionglnE
ISmex4 insertionureA/icuA
Table S2: Related to Figure 5. Growth on ammonium versus methylamine as nitrogen source. Growth rate (h-1) of WT, E1, and E2 in media with 3.5 mM disodium succinate and either 7.66 mM ammonia as the sole nitrogen source (blue column) or 7.66 mM methylamine as the sole nitrogen source (green column). The values indicate the mean of three replicate growth measurements and the 95% CI.
Strain k(succinate, ammonia)
k(succinate, methylamine)
Ratio k(succinate,
methylamine)k(succinate, ammonia)
WT 0.207±0.001 0.220±0.001 1.059±0.013
E1 0.271±0.001 0.245±0.001 0.906±0.011
E2 0.251±0.004 0.222±0.001 0.884±0.021
Table S3. M.extorquens strains and plasmids used in this study
Strains/Plasmids Description Reference
Strains
CM501 M. extorquens AM1 S1
CM1054 A1 S2
CM2351 Δmau in M. extorquens AM1 This study
CM2720 Δcel in M. extorquens AM1 S3
CM2770 Δfae in CM2720 (A2) This study
CM2986 E2 This study
CM3014 E1 This study
CM3488 CM2986 + pAYC139 This study
CM3807 ΔmptG in CM2720 This study
CM3811 ΔmptG in CM2986 This study
CM3813 ΔmptG in CM3014 This study
CM4135 kefBQ446* in CM2770 This study
CM4140 ΔkefB in CM2986 This study
CM4142 kefBWT in CM2986 This study
CM4149 ΔmgdABCD in CM2986 This study
CM4152 ΔmgdABCD in CM3014 This study
CM4153 nmgRWT in CM3014 This study
CM4168 nmgRW173* in CM1054 This study
CM4169 kefBQ446* in CM2720 This study
CM4187 ΔmgdABCD in CM4149 This study
CM4189 ΔmgdABCD in CM2720 This study
CM4194 nmgRW173* in CM2720
This study
CM4311 Δfae in CM2351 This study
Plasmids
pCM433 Allelic exchange vector (TetR, SucroseS) S1
pDN50 pCM433 with Δ fae upstream and downstream flanks S4
pDN69 pCM433 with ΔmptG upstream and downstream flanks This study
pDN112 pCM433 with ΔmgdABCD upstream and downstream flanks This study
pDN136 pCM433 with kefB allele from E2 This study
pDN137 pCM433 with kefB allele from WT This study
pDN142 pCM433 with ΔkefB upstream and downstream flanks This study
pDN150 pCM433 with nmgR allele from E1 This study
pDN151 pCM433 with nmgR allele from WT This study
pAYC139 Plasmid with mau gene cluster from M. extorquens AM1 S5
pRK2073 Conjugative helper plasmid (IncP, tra, StrR) S6
Table S4 List of primers used for q-RT PCR in this study
Primer Description Sequence
rpsB_qPCR_f (internal standard) TGACCAACTGGAAGACCATCTCC
rpsB_qPCR_r (internal standard) TTGGTGTCGATCACGAACAGCAG
qRT-DNA_contamination_check_f TCTGAGGTTCCAGGCTCGCTC
qRT-DNA_contamination_check_r TGCGACACTACGCCTTGGCAC
Meta1_4101_f GTGATCGAGTTCGTGCGCTA
Meta1_4101_r GAAGAAGGTGCCGATGACGA
Meta1_4103_f AGGGCTGGGAGACGAAGTAT
Meta1_4103_r GTCTTCTTGAGCTTGCGCTG
Supplemental Experimental Procedures
Experimental Evolution
Experimental Evolution I: Eight replicate populations of Methylobacterium extorquens AM1 (CM501 and CM502) were evolved in Hypho medium with 1.75 mM disodium succinate and 7.5 mM methanol for 1500 generations in an orbital, shaking incubator maintained at a constant speed (225 rpm) and temperature (30 ºC) [S7]. An evolved isolate (CM1054 or A1) from population C1 that could not grow on methylamine [S7] was subsequently evolved in 10 mL of modified Hypho minimal medium with 20 mM methylamine hydrochloride in an orbital, shaking incubator maintained at a constant speed (225 rpm) and temperature (30 ºC). The culture was allowed to grow for ~14 days till the media turned cloudy and was then plated on Hypho, 20 mM methylamine, agar (2% w/v) plates to obtain single colonies. Strain CM3014 (E1) was isolated from this plate and was used for further studies.
Experimental Evolution II: A single population of a ∆fae ∆cel strain of Methylobacterium extorquens AM1 (CM2770 or A2) was experimentally evolved in one well of a 48-well microtiter plates (CoStar-3548) containing 640 μl of modified Hypho minimal medium containing 20 mM methylamine hydrochloride. Plates were maintained in a shaking incubator at 650 rpm (Liconic USA LTX44 with custom fabricated cassettes) in a room that was constantly maintained at 30 ºC and 80% humidity and the culture was allowed to grow for ~7 days till the media turned cloudy. A 1/32 dilution was serially passaged every 3.5 days for 150 generations. Strain CM2986 (i.e. E2) was isolated as a single colony when the evolved population was plated on Hypho, 20 mM methylamine, agar (2% w/v).
Competitive fitness assays
Growth of the test strain and M. extorquens AM1 ∆cel-∆katA::Ptac-mCherry (CM3120) was initiated by inoculating 10 µL of the freezer stock of each strain into 10 mL of Hypho medium with 3.5 mM disodium succinate. Cultures were incubated in an orbital, shaking incubator maintained at a constant speed (225 rpm) and temperature (30 ºC). Upon reaching stationary phase, cultures were transferred 1:64 in 48-well microtiter plates (CoStar-3548) containing 640 μl of the modified Hypho medium with 20 mM methylamine hydrochloride. Plates were maintained in a shaking incubator at 650 rpm (Liconic USA LTX44 with custom fabricated cassettes) in a room that was constantly maintained at 30 ºC and 80% humidity and allowed to reach saturation in this ‘acclimation’ phase. The acclimation phase for strains that did not (or could barely) grow on methylamine consisted of growth in modified Hypho medium with 3.5 mM disodium succinate and 20 mM methylamine hydrochloride. At the end of the acclimation phase, CM3120 and the test strain were mixed in equal proportions by volume and a 64-fold dilution of this initial mix (T0) was transferred into fresh 48-well microtiter plates (CoStar-3548) containing 640 μl of the modified Hypho medium with 20 mM methylamine hydrochloride. The remaining 450 µL of the T0 sample was mixed with 10% DMSO and frozen at -80 ºC. Plates containing the T0 mix were incubated under the same conditions as the ‘acclimation’ phase. At the onset of saturation phase, a 500 µL sample of the culture (T1) was collected. The ratio of CM3120 and the test strain before and after growth was ascertained using flow cytometry. Cells were diluted appropriately such that at a flow rate of 0.5 μl/sec on the LSR Fortesssa (BD, Franklin Lakes, NJ) ~1000 events/second would be recorded. Fluorescent mCherry was excited at 561
nm and measured at 620/10 nm. The competitive fitness was calculated as 𝑊 =!"# (!!∗!!! )
!"# ( !!!! ∗!!!!! )
where R1 and
R0 represent the population fraction of the test strain before and after mixed growth, and N represents the fold increase in the population density.
Supplemental References
S1. Marx CJ. Development of a broad-host-range sacB-based vector for unmarked allelic exchange. BMC
Res Notes 2008;1:1. doi:10.1186/1756-0500-1-1.
S2. Lee MC, Marx CJ. Repeated, selection-driven genome reduction of accessory genes in experimental populations. PLoS Genet 2012;8:2–9. doi:10.1371/journal.pgen.1002651.
S3. Delaney NF, Kaczmarek ME, Ward LM, Swanson PK, Lee MC, Marx CJ. Development of an
optimized medium, strain and high-throughput culturing methods for Methylobacterium extorquens. PLoS One 2013;8. doi:10.1371/journal.pone.0062957.
S4. Nayak DD, Marx CJ. Genetic and phenotypic comparison of facultative methylotrophy between
Methylobacterium extorquens strains PA1 and AM1. PLoS One 2014;9:e107887. doi:10.1371/journal.pone.0107887.
S5. Chistoserdov AY, Chistoserdova L V., McIntire WS, Lidstrom ME. Genetic organization of the mau
gene cluster in Methylobacterium extorquens AM1: Complete nucleotide sequence and generation and characteristics of mau mutants. J Bacteriol 1994;176:4052–65.
S6. Figurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid RK2 dependent
on a plasmid function provided in trans. Proc Natl Acad Sci U S A 1979;76:1648–52. S7. Lee MC, Chou HH, Marx CJ. Asymmetric, bimodal trade-offs during adaptation of Methylobacterium
to distinct growth substrates. Evolution 2009;63:2816–30. doi:10.1111/j.1558-5646.2009.00757.x.