Discovery, taxonomic distribution, and phenotypic
characterization of a gene required for 3-methylhopanoid
production
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Citation
Welander, P. V., and R. E. Summons. “Discovery, Taxonomic
Distribution, and Phenotypic Characterization of a Gene
Required for 3-methylhopanoid Production.” Proceedings of the
National Academy of Sciences 109.32 (2012): 12905–12910.
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http://dx.doi.org/10.1073/pnas.1208255109
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Detailed Terms
Discovery, taxonomic distribution, and phenotypic
characterization of a gene required for
3-methylhopanoid production
Paula V. Welander1 and Roger E. Summons
Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue, E25-629, Cambridge,
MA 02139
radical SAM ∣ bacteriohopanepolyols ∣ molecular markers
H
opanoids are pentacyclic triterpenoid lipids produced by a
variety of bacteria that are often utilized as geological
proxies or biomarkers for certain bacterial species and their
metabolisms. Among the various hopanoid structures produced
by bacteria (1), those methylated at the C-3 position and those
with a penta- and hexafunctionalized amino polar side group
are thought to be primarily produced by Type I and Type X
methanotrophs (Fig. 1A) (2). As such, the occurrence of these
hopanoids in conjunction with their significant 13 C-depletion in
modern ecosystems are often utilized as an indicator of methanotrophic communities (3). In particular, environmental lipid
analyses have uncovered the existence of aerobic methanotrophy
in a variety of environments including, for example, the surface
sediments of an active marine mud volcano in the Barents Sea
and in the oxic-anoxic transition zone of the Black Sea water
column (4, 5). Furthermore, the recalcitrant nature of hopanoid
hydrocarbons allows for their preservation in ancient sediments,
which may provide evidence for aerobic metabolisms deep in
Earth’s history. Although the functionalized amino side group
is lost over time, methylation of the A-ring is retained (6). Thus,
the presence of C-3 methylated hopanes in sediments 2.5–2.7 billion years old has been used as one of several lines of molecular
and isotopic evidence for Neoarchean aerobiosis (7–11).
The effectiveness of these specific hopanoids as indicators
for aerobic methanotrophy rests partly on the premise that the
www.pnas.org/cgi/doi/10.1073/pnas.1208255109
majority of C-3 methylated hopanoid producers are aerobic
methanotrophs. However, the production of 3-methylhopanoids
has also been demonstrated in the acetic acid bacteria (12) indicating that the taxonomic distribution of 3-methylhopanoids is
not restricted to methanotrophs. Furthermore, recent studies
utilizing molecular approaches to identify hopanoid biosynthesis
genes in sequenced genomes have highlighted that the diversity of
bacteria capable of producing a specific hopanoid structure could
be underestimated (13–15). These studies have also shown that a
more precise interpretation of hopane hydrocarbon signatures in
both ancient and modern ecosystems requires not only a grasp
of the taxonomic distribution of methylhopanoid producers but
also a deeper understanding of their physiological function in
extant bacteria (16, 17).
To this end, we employed a combination of microbial genetics,
microbial physiology, and bioinformatics analysis to begin to
understand the biosynthesis and function of C-3 methylated
hopanoids in Methylococcus capsulatus. A genetic system for
constructing unmarked in-frame deletion mutants was utilized to
identify a methylase required for the production of 3-methylhopanoids. Bioinformatics analysis of this methylase revealed a
diverse taxonomic distribution beyond the methanotrophic and
acetic acid bacteria. Furthermore, phenotypic analysis of the
C-3 methylase mutant uncovered a potential role for 3-methylhopanoids in late stationary phase survival. These studies highlight
the power of combining gene discovery with bioinformatics and
physiological analyses to potentially enhance our understanding
of biomarker signatures in the rock record.
Results and Discussion
Identification of a C-3 Methylase in the M. capsulatus Genome. To
identify a protein required for the methylation of hopanoids
at the C-3 position, the genome of M. capsulatus was examined
for possible C-3 methylase candidates utilizing search criteria
based on two previous findings. First, bacterial feeding studies
done with labeled methionine have posited that S-adenosylmethionine (AdoMet) is a potential methyl donor in the
biosynthesis of both 2-methyl and 3-methylhopanoids (12). Second, it was recently discovered that a B-12 binding radical
AdoMet protein, HpnP, is required for the production of 2methylhopanoids in the α-Proteobacterium Rhodopseudomonas
palustris (14). Accordingly, we hypothesized that the methylase
responsible for 3-methylhopanoid production was also a radical
AdoMet protein possibly containing a B-12 binding domain.
Author contributions: P.V.W. designed research; P.V.W. performed research; R.E.S.
contributed new reagents/analytic tools; P.V.W. and R.E.S. analyzed data; and P.V.W.
and R.E.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: welander@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1208255109/-/DCSupplemental.
PNAS ∣
August 7, 2012 ∣
vol. 109 ∣
no. 32 ∣
12905–12910
MICROBIOLOGY
Hopanoids methylated at the C-3 position are a subset of bacterial
triterpenoids that are readily preserved in modern and ancient sediments and in petroleum. The production of 3-methylhopanoids
by extant aerobic methanotrophs and their common occurrence
in modern and fossil methane seep communities, in conjunction
with carbon isotope analysis, has led to their use as biomarker
proxies for aerobic methanotrophy. In addition, these lipids are
also produced by aerobic acetic acid bacteria and, lacking carbon
isotope analysis, are more generally used as indicators for aerobiosis in ancient ecosystems. However, recent genetic studies have
brought into question our current understanding of the taxonomic
diversity of methylhopanoid-producing bacteria and have highlighted that a proper interpretation of methylhopanes in the rock
record requires a deeper understanding of their cellular function. In
this study, we identified and deleted a gene, hpnR, required for
methylation of hopanoids at the C-3 position in the obligate
methanotroph Methylococcus capsulatus strain Bath. Bioinformatics analysis revealed that the taxonomic distribution of HpnR
extends beyond methanotrophic and acetic acid bacteria. Phenotypic analysis of the M. capsulatus hpnR deletion mutant demonstrated a potential physiological role for 3-methylhopanoids; they
appear to be required for the maintenance of intracytoplasmic
membranes and cell survival in late stationary phase. Therefore,
3-methylhopanoids may prove more useful as proxies for specific
environmental conditions encountered during stationary phase
rather than a particular bacterial group.
EARTH, ATMOSPHERIC,
AND PLANETARY SCIENCES
Edited by John M. Hayes, Woods Hole Oceanographic Institution, Berkeley, CA, and approved July 2, 2012 (received for review May 15, 2012)
OH
A
OH
OH
OH
OH
A
NH2
I
3
R = H aminobacteriohopanepentol (I)
R = CH 3 3-methylaminobacteriohopanepentol (III)
OH
OH
OH
OH
NH2
3
R
II
Relative Intensity
R
III IV
R = H aminobacteriohopanetetrol (II)
R = CH3 3-methylaminobacteriohopanetetrol (IV)
B
rfA
O
rfB
18
35
07
07
36
O
nR
hp
38
07
yf
iA
hy
p
40
39
07
07
07
41
rp
oN
ISMca3
20
22
24
26
28
30
32
24
26
28
30
32
30
32
B
1 kb
Using InterPro (http://www.ebi.ac.uk/interpro/), an integrated
database of predictive protein signatures, 21 proteins with a
radical AdoMet motif were identified in the M. capsulatus genome. These 21 proteins were queried against the Acetobacter pasterurianus genome, an acetic acid bacterium known to produce
3-methylhopanoids (12). Of these 21 proteins, MCA0738 was
the only protein annotated as a B-12 binding radical AdoMet that
also had a homologue in A. pasterurianus (e-value of 0). A Basic
Local Alignment Search Tool (BLAST) search of MCA0738
revealed a homologue in other acetic acid bacterial genomes.
Although the MCA0738 gene was not surrounded by other
hopanoid biosynthesis genes on the chromosome (Fig. 1B), the
occurrence of this particular gene in both M. capsulatus and
all sequenced acetic acid bacterial genomes made it an attractive
candidate for encoding the C-3 methylase.
To determine if MCA0738 did encode for a C-3 methylase, an
unmarked in-frame deletion of MCA0738 was attempted by
adapting a counter selection protocol that has been used in a
variety of bacterial species (18, 19). This allelic exchange method
involves integrating a suicide plasmid at the locus of interest
by homologous recombination and subsequently excising the
plasmid from the chromosome, which can result in the deletion
of the gene of interest (Fig. S1). To delete MCA0738, a deletion
plasmid containing a replacement allele missing the MCA0738
gene was transferred into M. capsulatus via conjugation and integrated onto the chromosome by homologous recombination.
The plasmid was forced to excise from the chromosome through
nonselective growth and several potential deletion colonies were
screened by PCR for deletion of MCA0738. One strain was found
to be devoid of this gene and was picked for further characterization (Fig. S1).
To verify that MCA0738 was required for C-3 methylation,
a total lipid extract (TLE) was isolated from the MCA0738 deletion mutant and analyzed for its complement of bacteriohopanepolyols. As shown in Fig. 2, the MCA0738 deletion mutant is
able to produce both the desmethyl aminobacteriohopanepentol
and aminobacteriohopanetetrol but not their C-3 methylated
counterparts as confirmed by detailed mass spectral analysis
(Fig. S2). Furthermore, introduction of a copy of the MCA0738
gene on a self-replicating plasmid into the deletion strain restores
production of the methylated hopanoids (Fig. 2). These data in12906 ∣
www.pnas.org/cgi/doi/10.1073/pnas.1208255109
II
Relative Intensity
I
18
20
22
C
I
Relative Intensity
Fig. 1. Identification of a putative 3-methylhopanoid methylase in M. capsulatus. (A) The 3-methyl and desmethyl aminobacteriohopanepolyols produced by M. capsulatus. The roman numerals in parenthesis correspond to
the structures identified in Fig. 2. (B) Genomic context of the C-3 methylase
gene hpnR (MCA0738). Upstream of the gene there is a hypothetical protein
(hyp), a putative Sigma-54 modulation protein (yfiA), and the RNA polymerase factor Sigma-54 (rpoN). Downstream are two genes that are annotated as
ISMca3 transposase genes (OrfA and OrfB).
II
III
IV
18
20
22
24
26
28
Time (min)
Fig. 2. Deletion of hpnR results in loss of 3-methylhopanoid production.
LC-MS extracted ion chromatograms of acetylated total lipid extracts from
(A) wild type M. capsulatus, (B) ΔhpnR, and (C) ΔhpnR complemented with
a copy of hpnR on a self-replicating plasmid (pPVW100). The chromatograms
are a combination of ions m/z 830 (I, aminobacteriohopanepentol), 772 (II,
aminobacteriohopanetetrol), 844 (III, 3-methylaminobacteriohopanepentol),
and 786 (IV, 3-methylaminobacteriohopanetetrol). Hopanoids were identified based on their mass spectra shown in Fig. S2.
dicate that MCA0738 is the only gene required for C-3 methylation of hopanoids in M. capsulatus and we propose to rename this
locus hpnR based on a previously established nomenclature in
Zymomonas mobilis (20).
Identification of Putative HpnR Homologues. The radical AdoMet
protein family encompasses a diverse set of proteins that catalyze
a variety of biochemical reactions. The proteins in this family are
primarily identified by the short amino acid sequence motif
CxxxCxxC. As a result, BLASTanalyses of HpnR return a variety
of radical AdoMet proteins that may or may not be involved in
3-methylhopanoid biosynthesis. To determine which of these
Welander and Summons
1.00
0.1
Fig. 3. Maximum likelihood phylogenetic tree of putative HpnR sequences.
A total of 192 radical AdoMet proteins were used to generate the tree: Fiftytwo HpnR sequences plus 140 radical AdoMets with an e-value less than e −17
when queried against the M. capsulatus HpnR sequence. The tree was rooted
by using the 140 sequences as an out group to the 52 HpnR sequences shown.
The full unrooted tree is shown in Fig. S3.
Welander and Summons
3-Methylhopanoids Play a Role in Late Stationary Phase Survival. The
diverse and sporadic distribution of HpnR introduces further
ambiguity in our ability to correlate specific bacterial taxa to
3-methylhopanes in the environment. As a result, a proper analysis of the presence of 3-methylhopanes in the rock record needs
to move beyond simply understanding which organisms produce
these molecules. A more nuanced interpretation may be achieved
if we better understand the physiological function of 3-methylhopanoids in bacteria as well as the environmental factors that induce their production in the cell. To this end, we have begun
physiological characterization of the M. capsulatus hpnR mutant
to identify any potential phenotype(s) associated with the loss of
3-methylhopanoid production.
PNAS ∣ August 7, 2012 ∣
vol. 109 ∣
no. 32 ∣
12907
EARTH, ATMOSPHERIC,
AND PLANETARY SCIENCES
Candidatus Koribacter
Candidatus Methylomirabilis oxyfera
Nitrococcus mobilis
1.00
Frankia sp. EAN1
Frankia sp. CcI3
0.50 0.99
Streptomyces sp. e14
Streptomyces chartreusis
1.00
1.00
Streptomyces griseoflavus
Streptomyces ghanaensis
0.98
Streptomyces xinghaiensis
0.72
Streptomyces fradiae
1.00
Streptomyces
pristinaespiralis
0.45
0.81 1.00 Streptomyces cattleya DSM 46488
1.00
Streptomyces cattleya NRRL 8057
Nitrosococcus halophilus
1.00
Nitrosococcus watsoni
1.00 Nitrosococcus oceani AFC27
0.95
Nitrosococcus oceani ATCC 19707
Methylococcus capsulatus
0.99
1.00
Methylomicrobium alcaliphilum
Methylomicrobium album
Soil Metagenome AAFX01006490
0.89
0.95
Leptospirillum ferrooxidans
Mine Drainage Metagenome ACXJ01008692.1
0.99
1.00 Leptospirillum sp. Group II
0.60
Leptospirillum rubarum
Mine Drainage Metagenome ACXJ01008849.1
0.81
Burkholderia sp. H160
1.00
Burkholderia xenovorans
0.90
Burkholderia phymatum
0.96
Methylobacterium nodulans
Gluconacetobacter
diazotrophicus
0.55
Gluconacetobacter hansenii
Gluconacetobacter sp. SXCC1
0.89
1.00
Gluconacetobacter xylinus
Gluconacetobacter oboediens
Gluconacetobacter europaeus LMG 18494
1.00
Gluconacetobacter europaeus LMG 18890
Gluconacetobacter europaeus 5P3
Acetobacter aceti
0.15
Gluconobacter oxydans
Gluconobacter morbifer
Acetobacter tropicalis
0.97
Acetobacter pomorum
0.98
Acetobacter pasteurianus IFO 3283-01
1.00 Acetobacter pasteurianus IFO 3283-01-42C
Acetobacter pasteurianus IFO 3283-03
Acetobacter pasteurianus IFO 3283-07
Acetobacter pasteurianus IFO 3283-12
Acetobacter pasteurianus IFO 3283-22
Acetobacter pasteurianus IFO 3283-26
Acetobacter pasteurianus IFO 3283-32
0.89
copy of this protein suggesting that only a subset of methanotrophs may be capable of producing 3-methylhopanoids. The inconsistent distribution of HpnR among methanotrophs is also
observed in the genomes of other hopanoid-producing bacterial
genera. For example, 38 Burkholderia, 43 Streptomyces, and 8
Methylobacterium genomes have been sequenced to date. All of
these genomes, except for Burkholderia pseudomallei MSHR346,
contain a copy of the squalene hopene cyclase gene and at least
one species from each of these groups has been shown to produce
hopanoids. Yet, only three Burkholderia, eight Streptomyces, and
one Methylobacterium contain the HpnR methylase suggesting
that only a subset of species from certain genera may be able
to produce 3-methylhopanoids. This observation is particularly
critical when we consider that our current understanding of which
bacteria produce certain hopanoid molecules is often based on
lipid analysis of a few culturable species of a certain genus.
The sporadic phylogenetic distribution of HpnR also suggests
a potentially complex evolutionary history of this methylase. Two
evolutionary scenarios seem plausible: either HpnR was present
in the ancestor of all HpnR-containing bacteria and was repeatedly lost or it could have been acquired through horizontal gene
transfer (HGT). For most of these taxa, the HpnR phylogeny is
congruent with that of the species phylogeny based on 16S rRNA
sequence (22). This consistency between the HpnR phylogeny
and species phylogeny is evident for the acetic acid bacterial clade
suggesting a vertical descent within this group. However, the
Methylobacteium nodulans and Nitrococcus mobilis HpnR sequences tend to cluster outside their expected species phylogeny
clade suggesting acquisition through HGT in these organisms.
Thus, the evolutionary history of this protein remains unclear
and more robust analyses are needed to better resolve it.
Given the taxonomic diversity of potential 3-methylhopanoid
producers uncovered by our analysis, the detection of 3-methylhopanoids in both modern and ancient sediments cannot be attributed specifically to aerobic methanotrophic bacteria without
other lines of evidence (e.g., carbon isotope data). However, all of
the bacterial species that contain an HpnR homologue in their
genomes utilize a form of aerobic metabolism. Thus, it seems
reasonable to continue to employ 3-methylhopanes in the rock
record as indicators for the existence of aerobic metabolisms deep
in time. The one organism that might be considered an exception
to this rule is Candidatus Methylomirabilis oxyfera which was isolated from anoxic sediments and grows anaerobically by coupling
nitrite reduction to methane oxidation (23). Although M. oxyfera
is an anaerobe, it is also an oxygenic organism as it produces its
own supply of oxygen through the dismutation of nitrite. It subsequently uses this oxygen for the oxidation of methane through
the same methanotrophic pathway utilized by other aerobic
methanotrophs (23). Whereas M. oxyfera is encountered in anaerobic environments, it still requires oxygen for its metabolism.
Therefore, the distribution of the C-3 methylase in oxygen-demanding bacteria seems robust for now and can be seen as further
evidence for the use of 3-methylhopanes as proxies for the occurrence of aerobic metabolisms in ancient environments.
MICROBIOLOGY
radical AdoMet proteins are genuine C-3 methylases, we constructed an unrooted maximum likelihood tree of 192 radical
AdoMet proteins retrieved through a protein BLAST search of
the M. capsulatus HpnR sequence against the Kyoto Encyclopedia of Genes and Genomes (KEGG) and National Center for
Biotechnology Information (NCBI) databases (e-value cut-off <
e −17 ). This analysis shows that those radical AdoMet proteins
with an e-value lower than e −100 cluster together (Fig. S3). Within
this clade, we find HpnR from M. capsulatus and homologues
from the acetic acid bacteria, the only currently known producers
of 3-methylhopanoids (Fig. 3). Further, all of the strains in this
clade also contain a copy of the squalene hopene cyclase gene,
which is required for hopanoid biosynthesis, as well as several
other hopanoid biosynthesis genes in their genomes (17, 21).
Therefore, it seems reasonable to propose that the cutoff for a
bona fide C-3 methylase is a value lower than e −100 .
Using this criteria, there are 52 putative homologues of HpnR
in the genomic and metagenomic databases (Fig. 3). The species
that contain these HpnR homologues are from a diverse set of
bacterial phyla: 33 strains of Proteobacteria (22 α-, 3 β-, and 8
γ-Proteobacteria), 11 Actinobacteria, 3 Nitrospirae, 1 Acidobacterium, 1 candidate NC10 phylum organism, and 3 metagenomic
sequences. In agreement with our current understanding of 3methylhopanoid distribution in bacteria, all 21 of the partially
or completed acetic acid bacterial genomes have an HpnR homologue. However, only three of the nine methanotrophic genomes
sequenced to date (two complete and seven incomplete) have a
A previous study in M. capsulatus demonstrated that 3-methylhopanoids accumulate preferentially in stationary phase cells
(24). To test whether 3-methylhopanoids play a role in stationary
phase physiology, we grew both the wild type and ΔhpnR strains
in batch culture for fourteen days. Cultures were supplemented
with methane only at the point of inoculation (day 0) to ensure
that they would become nutrient-limited and enter stationary
phase. Cell growth was monitored by following the OD at 600 nm.
Under these growth conditions, it was determined that the cells
were entering stationary phase and ceased oxidizing methane on
day 2 of growth (Fig. S4). Given that the methane in the headspace was not depleted (Fig. S4), we presumed that the cessation
of growth on day 2 resulted from the depletion of oxygen. These
experiments also demonstrated that ΔhpnR cells exhibited similar
growth characteristics to the wild type strain during exponential
growth. However, upon entering stationary phase, the methylase
mutant seemed to experience a larger drop in OD than the wild
type indicating a potential loss in viability under these conditions.
To better assess cell viability in stationary phase, the number of
cfu on day 2, 7, and 14 of growth were determined. As shown in
Fig. 4, the wild type strain maintains the same number of cfu
throughout stationary phase. The ΔhpnR strain sustains a similar
number of viable cells on day 2 and 7, even though the observed
drop in OD occurs immediately upon entering stationary phase at
day 2. However, the number of cfu drops approximately six orders
of magnitude on day 14 suggesting a role for 3-methylhopanoids
in late stationary phase (Fig. 4). To more directly show that the
lack of 3-methylhopanoids was responsible for this reduction in
viability, we also determined the cfu values for the ΔhpnR strain
complemented with a copy of the methylase gene (Fig. 4;
ΔhpnR þ pPVW100). The overall cfu values for the complemented strain were approximately two orders of magnitude lower
than the wild type on each day tested. This reduction in cfu
was most likely a result of the sluggish growth observed in the
presence of the kanamycin antibiotic necessary to maintain the
complementing plasmid. Nevertheless, reinstating 3-methylhopanoid production in the ΔhpnR strain did result in sustained cell
viability through day 14.
3-Methylhopanoids and Intracytoplasmic Membrane Formation in
Late Stationary Phase. The decreased viability of the methylase
mutant suggests that it may be deficient in mechanisms necessary
to cope with the stresses encountered in stationary phase. M. capsulatus is one of several methanotrophs that are capable of forming Azotobacter-like cyst structures. Cyst formation has been
shown to be important in enhancing the survival of a bacterium
under adverse environmental conditions such as those experienced in stationary phase (25). Accordingly, we hypothesized that
Colony Forming Units (cfu)
1.0E+09
1.0E+08
1.0E+07
1.0E+06
1.0E+05
1.0E+04
1.0E+03
1.0E+02
1.0E+01
1.0E+00
Day 2
Day 7
Day 14
wild type
Fig. 4. Deletion of hpnR results in decreased survival in late stationary
phase. The cfu for M. capsulatus strains were determined on day 2, 7, and
14 of growth by spot plating serial dilutions on NMS agar plates. Each bar
represents the average cfu of three separate experiments and the error bars
are standard deviations.
12908 ∣
www.pnas.org/cgi/doi/10.1073/pnas.1208255109
the lack of 3-methylhopanoids in the methylase mutant may result in inadequate cyst formation which, in turn, compromises viability during prolonged stationary phase incubation.
To test this hypothesis, transmission electron microscopy
(TEM) of both wild type and mutant cells on day 2 and day 14
of growth were analyzed for the formation of cyst-like structures.
As shown in Fig. 5, no cyst-like cells were found to be produced
either by the wild type or the methylase mutant throughout stationary phase suggesting that 3-methylhopanoids play a role in
stationary phase survival independent of cyst formation. On
the other hand, the images revealed formation of extensive intracytoplasmic membranes (ICM) in late stationary phase. In particular, both the wild type and ΔhpnR were capable of forming
these membranes in early stationary phase (day 2). But by day
14, the wild type had significantly more ICM present than on day
2 whereas the 3-methylhopanoid deletion mutant was no longer
producing these membranes. The ability of the methylase mutant
to produce lamellar membranes upon entering stationary phase
but not deep into stationary phase suggests that 3-methylhopanoids play a role in maintaining these membranes rather than
a role in forming them. Complementation of the deletion strain
with the hpnR gene partially restored the production of ICM on
day 14 further indicating that 3-methylhopanoids may be important in maintaining ICM formation during stationary phase.
The decreased viability of the methylase mutant along with its
inability to maintain ICM formation leads us to speculate that ICM
maintenance is necessary for survival in late stationary phase.
Based on our physiological data, we presume that the cells in our
cultures are limited for oxygen rather than methane (Fig. S4). Previous studies have shown that ICM formation and methane oxidation rates in methanotrophs increase under similar high methane/
low oxygen growth conditions (26). Because the methane monooxygenase is a membrane-bound enzyme localized to the ICM it
is thought that the increase in ICM formation allows for increased
methane oxidation at low oxygen levels. This strategy may then aid
survival in low oxygen environments encountered in nature.
Interestingly, these high methane/low oxygen growth conditions are reminiscent of the modern and ancient seafloor
methane seeps in which 3-methylhopanes and 3-methylhopanoidproducing methanotrophs have been detected (4, 27). The physical conditions at these seafloor methane seeps are quite transitory, particularly in terms of the availability of methane and
oxygen (28). The methanotrophs in these communities must be
adapted to survive persistent low oxygen levels as well as to respond rapidly to methane pulses (28). Thus, our late stationary
phase studies could be demonstrating a role for ICMs and 3methylhopanoids in the persistence of certain methanotrophic
communities in their natural environments. This hypothesis is
particularly appealing when we consider a recent study on the fate
of spilled methane from the 2010 Deepwater Horizon oil spill
(29). In this study, the idea was put forward that aerobic methanotrophic communities may act as dynamic biofilters of largescale methane inputs into the ocean (29). These methanotrophic
communities persist for the most part in nutrient-limited environments yet seem poised to respond to sudden influxes of
methane. Some of the methanotrophs identified from this potential methanotrophic bloom after the Deepwater Horizon disaster
were γ-Proteobacteria of the Methylococcaceae family that is
known to contain 3-methylhopanoid-producing methanotrophic
species such as M. capsulatus. Therefore, it is possible that the
ability to survive in these transient methanotrophic environments
may be linked to 3-methylhopanoid production.
The observations presented here point to a potential role
of 3-methylhopanoids (and ICM formation) in cell viability
under nutrient-limited conditions. These findings are pertinent
given that low nutrients and harsh conditions are known to be
ubiquitous in natural environments and as a result, microbes
in nature are thought to persist in a type of stationary phase
Welander and Summons
Day 2
wild type
∆hpnR
∆hpnR +
pPVW100
∆hpnR
∆hpnR +
pPVW100
Day 14
wild type
Fig. 5. Deletion of 3-methylhopanoid production results in reduced intracytoplasmic membranes during stationary phase. TEM images show ICM formation
(black arrows) by all cells on day 2 of growth. On day 14 of growth the ΔhpnR mutant has significantly lower ICM formation than the wild type and
complemented strains (ΔhpnR þ pPVW100). (Black scale, 0.5 μ.)
Bacterial Strains, Media, and Growth Conditions. Bacterial strains used in this
study are listed in Table S1. Escherichia coli strains were grown in lysogeny
broth (LB) and M. capsulatus strains were grown in nitrate minimal salts
(NMS) medium supplemented with 10 μM CuSO4 (31) at 37 °C while shaking
at 250 rpm. M. capsulatus batch cultures were sealed in serum vials without
removing the ambient air and given methane: carbon dioxide mix (95∶5) at
60 kPa over ambient pressure. For growth on solid medium, LB or NMS was
solidified with 1.5% agar and supplemented, if necessary, with 15 μg∕mL
gentamicin (Gm), 50 μg∕mL kanamycin (Km), 600 μM diaminopimelic acid
(DAP), or 5% sucrose. M. capsulatus plates were incubated in Vacu-Quik Jars
(Almore International, Inc.) and supplied with methane: carbon dioxide mix
at 20 kPa over ambient pressure. Additional details are described in SI
Materials and Methods.
Bioinformatics Analysis. InterPro (http://www.ebi.ac.uk/interpro) was used to
identify putative radical AdoMet proteins in the M. capsulatus genome. HpnR
homologues were identified in the KEGG and the NCBI databases by a translated Basic Local Assignment Search Tool (TBLASTN) (33) and were aligned
using the Multiple Sequence Comparison by Log-Expectation (MUSCLE) program (34). Maximum likelihood trees were constructed by phylogenetic estimation using maximum likelihood (PhyML) (35) using the LG þ gamma model,
four gamma rate categories, ten random starting trees, Nearest Neighbor Interchange (NNI) branch swapping, and substitution parameters estimated
from the data. The HpnR tree was generated and edited by importing the resulting PhyML tree into the Interactive Tree of Life tool (iTOL) (http://
itol.embl.de/) (36).
DNA Methods, Transformation, and Mutant Construction. All plasmid constructs
and the sequences of oligonucleotide primers used in this study are described
in Table S1. Construction of the hpnR deletion mutant and complemented
strain is described in SI Materials and Methods. DNA sequences of all cloning
intermediates were confirmed by sequencing at the GENEWIZ Boston
Laboratory. E. coli strains were transformed by electroporation. Plasmids
were mobilized from E. coli BW20767 into M. capsulatus by conjugation
as described in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Dr. Florence Schubotz for technical advice
and assistance, Prof. Tanja Bosak and Prof. Shuhei Ono for providing lab
space and equipment, and Prof. Martin Klotz for his generous gift of Methylococcus capsulatus strain Bath. This work was supported by the National
Science Foundation (NSF) Program on Emerging Trends in Biogeochemical
Cycles (OCE-0849940) supported, in turn, by NSF programs in Chemical
Oceanography and Geobiology-Low Temperature Geochemistry (R.E.S.),
and a National Aeronautics and Space Administration Postdoctoral Program
Fellowship (P.V.W.).
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EARTH, ATMOSPHERIC,
AND PLANETARY SCIENCES
Materials and Methods
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MICROBIOLOGY
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