Discovery, taxonomic distribution, and phenotypiccharacterization of a gene required for3-methylhopanoid productionPaula 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

Edited by John M. Hayes, Woods Hole Oceanographic Institution, Berkeley, CA, and approved July 2, 2012 (received for review May 15, 2012)

Hopanoids methylated at the C-3 position are a subset of bacterialtriterpenoids that are readily preserved in modern and ancient se-diments and in petroleum. The production of 3-methylhopanoidsby extant aerobic methanotrophs and their common occurrencein modern and fossil methane seep communities, in conjunctionwith carbon isotope analysis, has led to their use as biomarkerproxies for aerobic methanotrophy. In addition, these lipids arealso produced by aerobic acetic acid bacteria and, lacking carbonisotope analysis, are more generally used as indicators for aerobio-sis in ancient ecosystems. However, recent genetic studies havebrought into question our current understanding of the taxonomicdiversity of methylhopanoid-producing bacteria and have high-lighted that a proper interpretation of methylhopanes in the rockrecord requires a deeper understanding of their cellular function. Inthis study, we identified and deleted a gene, hpnR, required formethylation of hopanoids at the C-3 position in the obligatemethanotroph Methylococcus capsulatus strain Bath. Bioinfor-matics analysis revealed that the taxonomic distribution of HpnRextends beyond methanotrophic and acetic acid bacteria. Phenoty-pic analysis of the M. capsulatus hpnR deletion mutant demon-strated a potential physiological role for 3-methylhopanoids; theyappear to be required for the maintenance of intracytoplasmicmembranes and cell survival in late stationary phase. Therefore,3-methylhopanoids may prove more useful as proxies for specificenvironmental conditions encountered during stationary phaserather than a particular bacterial group.

radical SAM ∣ bacteriohopanepolyols ∣ molecular markers

Hopanoids are pentacyclic triterpenoid lipids produced by avariety of bacteria that are often utilized as geologicalproxies or biomarkers for certain bacterial species and theirmetabolisms. Among the various hopanoid structures producedby bacteria (1), those methylated at the C-3 position and thosewith a penta- and hexafunctionalized amino polar side groupare thought to be primarily produced by Type I and Type Xmethanotrophs (Fig. 1A) (2). As such, the occurrence of thesehopanoids in conjunction with their significant 13C-depletion inmodern ecosystems are often utilized as an indicator of metha-notrophic communities (3). In particular, environmental lipidanalyses have uncovered the existence of aerobic methanotrophyin a variety of environments including, for example, the surfacesediments of an active marine mud volcano in the Barents Seaand in the oxic-anoxic transition zone of the Black Sea watercolumn (4, 5). Furthermore, the recalcitrant nature of hopanoidhydrocarbons allows for their preservation in ancient sediments,which may provide evidence for aerobic metabolisms deep inEarth’s history. Although the functionalized amino side groupis 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 bil-lion years old has been used as one of several lines of molecularand isotopic evidence for Neoarchean aerobiosis (7–11).

The effectiveness of these specific hopanoids as indicatorsfor aerobic methanotrophy rests partly on the premise that the

majority of C-3 methylated hopanoid producers are aerobicmethanotrophs. However, the production of 3-methylhopanoidshas also been demonstrated in the acetic acid bacteria (12) indi-cating that the taxonomic distribution of 3-methylhopanoids isnot restricted to methanotrophs. Furthermore, recent studiesutilizing molecular approaches to identify hopanoid biosynthesisgenes in sequenced genomes have highlighted that the diversity ofbacteria capable of producing a specific hopanoid structure couldbe underestimated (13–15). These studies have also shown that amore precise interpretation of hopane hydrocarbon signatures inboth ancient and modern ecosystems requires not only a graspof the taxonomic distribution of methylhopanoid producers butalso a deeper understanding of their physiological function inextant bacteria (16, 17).

To this end, we employed a combination of microbial genetics,microbial physiology, and bioinformatics analysis to begin tounderstand the biosynthesis and function of C-3 methylatedhopanoids in Methylococcus capsulatus. A genetic system forconstructing unmarked in-frame deletion mutants was utilized toidentify a methylase required for the production of 3-methylho-panoids. Bioinformatics analysis of this methylase revealed adiverse taxonomic distribution beyond the methanotrophic andacetic acid bacteria. Furthermore, phenotypic analysis of theC-3 methylase mutant uncovered a potential role for 3-methylho-panoids in late stationary phase survival. These studies highlightthe power of combining gene discovery with bioinformatics andphysiological analyses to potentially enhance our understandingof biomarker signatures in the rock record.

Results and DiscussionIdentification of a C-3 Methylase in the M. capsulatus Genome. Toidentify a protein required for the methylation of hopanoidsat the C-3 position, the genome of M. capsulatus was examinedfor possible C-3 methylase candidates utilizing search criteriabased on two previous findings. First, bacterial feeding studiesdone with labeled methionine have posited that S-adenosyl-methionine (AdoMet) is a potential methyl donor in thebiosynthesis of both 2-methyl and 3-methylhopanoids (12). Sec-ond, it was recently discovered that a B-12 binding radicalAdoMet protein, HpnP, is required for the production of 2-methylhopanoids in the α-Proteobacterium Rhodopseudomonaspalustris (14). Accordingly, we hypothesized that the methylaseresponsible for 3-methylhopanoid production was also a radicalAdoMet 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.1To 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.

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Using InterPro (http://www.ebi.ac.uk/interpro/), an integrateddatabase of predictive protein signatures, 21 proteins with aradical AdoMet motif were identified in the M. capsulatus gen-ome. These 21 proteins were queried against the Acetobacter pas-terurianus genome, an acetic acid bacterium known to produce3-methylhopanoids (12). Of these 21 proteins, MCA0738 wasthe only protein annotated as a B-12 binding radical AdoMet thatalso had a homologue in A. pasterurianus (e-value of 0). A BasicLocal Alignment Search Tool (BLAST) search of MCA0738revealed a homologue in other acetic acid bacterial genomes.Although the MCA0738 gene was not surrounded by otherhopanoid biosynthesis genes on the chromosome (Fig. 1B), theoccurrence of this particular gene in both M. capsulatus andall sequenced acetic acid bacterial genomes made it an attractivecandidate for encoding the C-3 methylase.

To determine if MCA0738 did encode for a C-3 methylase, anunmarked in-frame deletion of MCA0738 was attempted byadapting a counter selection protocol that has been used in avariety of bacterial species (18, 19). This allelic exchange methodinvolves integrating a suicide plasmid at the locus of interestby homologous recombination and subsequently excising theplasmid from the chromosome, which can result in the deletionof the gene of interest (Fig. S1). To delete MCA0738, a deletionplasmid containing a replacement allele missing the MCA0738gene was transferred into M. capsulatus via conjugation and in-tegrated onto the chromosome by homologous recombination.The plasmid was forced to excise from the chromosome throughnonselective growth and several potential deletion colonies werescreened by PCR for deletion of MCA0738. One strain was foundto be devoid of this gene and was picked for further character-ization (Fig. S1).

To verify that MCA0738 was required for C-3 methylation,a total lipid extract (TLE) was isolated from the MCA0738 dele-tion mutant and analyzed for its complement of bacteriohopane-polyols. As shown in Fig. 2, the MCA0738 deletion mutant isable to produce both the desmethyl aminobacteriohopanepentoland aminobacteriohopanetetrol but not their C-3 methylatedcounterparts as confirmed by detailed mass spectral analysis(Fig. S2). Furthermore, introduction of a copy of the MCA0738gene on a self-replicating plasmid into the deletion strain restoresproduction of the methylated hopanoids (Fig. 2). These data in-

dicate that MCA0738 is the only gene required for C-3 methyla-tion of hopanoids inM. capsulatus and we propose to rename thislocus hpnR based on a previously established nomenclature inZymomonas mobilis (20).

Identification of Putative HpnR Homologues. The radical AdoMetprotein family encompasses a diverse set of proteins that catalyzea variety of biochemical reactions. The proteins in this family areprimarily identified by the short amino acid sequence motifCxxxCxxC. As a result, BLASTanalyses of HpnR return a varietyof radical AdoMet proteins that may or may not be involved in3-methylhopanoid biosynthesis. To determine which of these

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Fig. 1. Identification of a putative 3-methylhopanoid methylase in M. cap-sulatus. (A) The 3-methyl and desmethyl aminobacteriohopanepolyols pro-duced by M. capsulatus. The roman numerals in parenthesis correspond tothe structures identified in Fig. 2. (B) Genomic context of the C-3 methylasegene hpnR (MCA0738). Upstream of the gene there is a hypothetical protein(hyp), a putative Sigma-54 modulation protein (yfiA), and the RNA polymer-ase factor Sigma-54 (rpoN). Downstream are two genes that are annotated asISMca3 transposase genes (OrfA and OrfB).

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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 witha copy of hpnR on a self-replicating plasmid (pPVW100). The chromatogramsare a combination of ions m/z 830 (I, aminobacteriohopanepentol), 772 (II,aminobacteriohopanetetrol), 844 (III, 3-methylaminobacteriohopanepentol),and 786 (IV, 3-methylaminobacteriohopanetetrol). Hopanoids were identi-fied based on their mass spectra shown in Fig. S2.

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radical AdoMet proteins are genuine C-3 methylases, we con-structed an unrooted maximum likelihood tree of 192 radicalAdoMet proteins retrieved through a protein BLAST search ofthe M. capsulatus HpnR sequence against the Kyoto Encyclope-dia of Genes and Genomes (KEGG) and National Center forBiotechnology Information (NCBI) databases (e-value cut-off <e−17). This analysis shows that those radical AdoMet proteinswith an e-value lower than e−100 cluster together (Fig. S3). Withinthis clade, we find HpnR from M. capsulatus and homologuesfrom the acetic acid bacteria, the only currently known producersof 3-methylhopanoids (Fig. 3). Further, all of the strains in thisclade also contain a copy of the squalene hopene cyclase gene,which is required for hopanoid biosynthesis, as well as severalother hopanoid biosynthesis genes in their genomes (17, 21).Therefore, it seems reasonable to propose that the cutoff for abona fide C-3 methylase is a value lower than e−100.

Using this criteria, there are 52 putative homologues of HpnRin the genomic and metagenomic databases (Fig. 3). The speciesthat contain these HpnR homologues are from a diverse set ofbacterial phyla: 33 strains of Proteobacteria (22 α-, 3 β-, and 8γ-Proteobacteria), 11 Actinobacteria, 3 Nitrospirae, 1 Acidobac-terium, 1 candidate NC10 phylum organism, and 3 metagenomicsequences. In agreement with our current understanding of 3-methylhopanoid distribution in bacteria, all 21 of the partiallyor completed acetic acid bacterial genomes have an HpnR homo-logue. However, only three of the nine methanotrophic genomessequenced to date (two complete and seven incomplete) have a

copy of this protein suggesting that only a subset of methano-trophs may be capable of producing 3-methylhopanoids. The in-consistent distribution of HpnR among methanotrophs is alsoobserved in the genomes of other hopanoid-producing bacterialgenera. For example, 38 Burkholderia, 43 Streptomyces, and 8Methylobacterium genomes have been sequenced to date. All ofthese genomes, except for Burkholderia pseudomallei MSHR346,contain a copy of the squalene hopene cyclase gene and at leastone species from each of these groups has been shown to producehopanoids. Yet, only three Burkholderia, eight Streptomyces, andone Methylobacterium contain the HpnR methylase suggestingthat only a subset of species from certain genera may be ableto produce 3-methylhopanoids. This observation is particularlycritical when we consider that our current understanding of whichbacteria produce certain hopanoid molecules is often based onlipid analysis of a few culturable species of a certain genus.

The sporadic phylogenetic distribution of HpnR also suggestsa potentially complex evolutionary history of this methylase. Twoevolutionary scenarios seem plausible: either HpnR was presentin the ancestor of all HpnR-containing bacteria and was repeat-edly lost or it could have been acquired through horizontal genetransfer (HGT). For most of these taxa, the HpnR phylogeny iscongruent with that of the species phylogeny based on 16S rRNAsequence (22). This consistency between the HpnR phylogenyand species phylogeny is evident for the acetic acid bacterial cladesuggesting a vertical descent within this group. However, theMethylobacteium nodulans and Nitrococcus mobilis HpnR se-quences tend to cluster outside their expected species phylogenyclade suggesting acquisition through HGT in these organisms.Thus, the evolutionary history of this protein remains unclearand more robust analyses are needed to better resolve it.

Given the taxonomic diversity of potential 3-methylhopanoidproducers uncovered by our analysis, the detection of 3-methyl-hopanoids in both modern and ancient sediments cannot be at-tributed specifically to aerobic methanotrophic bacteria withoutother lines of evidence (e.g., carbon isotope data). However, all ofthe bacterial species that contain an HpnR homologue in theirgenomes utilize a form of aerobic metabolism. Thus, it seemsreasonable to continue to employ 3-methylhopanes in the rockrecord as indicators for the existence of aerobic metabolisms deepin time. The one organism that might be considered an exceptionto this rule is Candidatus Methylomirabilis oxyfera which was iso-lated from anoxic sediments and grows anaerobically by couplingnitrite reduction to methane oxidation (23). Although M. oxyferais an anaerobe, it is also an oxygenic organism as it produces itsown supply of oxygen through the dismutation of nitrite. It sub-sequently uses this oxygen for the oxidation of methane throughthe same methanotrophic pathway utilized by other aerobicmethanotrophs (23). Whereas M. oxyfera is encountered in anae-robic environments, it still requires oxygen for its metabolism.Therefore, the distribution of the C-3 methylase in oxygen-de-manding bacteria seems robust for now and can be seen as furtherevidence for the use of 3-methylhopanes as proxies for the occur-rence of aerobic metabolisms in ancient environments.

3-Methylhopanoids Play a Role in Late Stationary Phase Survival. Thediverse and sporadic distribution of HpnR introduces furtherambiguity in our ability to correlate specific bacterial taxa to3-methylhopanes in the environment. As a result, a proper ana-lysis of the presence of 3-methylhopanes in the rock record needsto move beyond simply understanding which organisms producethese molecules. A more nuanced interpretation may be achievedif we better understand the physiological function of 3-methylho-panoids in bacteria as well as the environmental factors that in-duce their production in the cell. To this end, we have begunphysiological characterization of the M. capsulatus hpnR mutantto identify any potential phenotype(s) associated with the loss of3-methylhopanoid production.

Acetobacter pasteurianus IFO 3283-03Acetobacter pasteurianus IFO 3283-07Acetobacter pasteurianus IFO 3283-12Acetobacter pasteurianus IFO 3283-22Acetobacter pasteurianus IFO 3283-26Acetobacter pasteurianus IFO 3283-32

Acetobacter pasteurianus IFO 3283-01-42CAcetobacter pasteurianus IFO 3283-01Acetobacter pomorum

Acetobacter tropicalis

Gluconobacter oxydansGluconobacter morbifer

Acetobacter aceti

Gluconacetobacter europaeus LMG 18494Gluconacetobacter europaeus LMG 18890Gluconacetobacter europaeus 5P3

Gluconacetobacter oboediens

Gluconacetobacter sp. SXCC1Gluconacetobacter xylinus

Gluconacetobacter hanseniiGluconacetobacter diazotrophicus

Burkholderia xenovoransBurkholderia phymatum

Burkholderia sp. H160

Methylobacterium nodulans

Leptospirillum ferrooxidansMine Drainage Metagenome ACXJ01008692.1

Leptospirillum rubarumMine Drainage Metagenome ACXJ01008849.1

Leptospirillum sp. Group II

Soil Metagenome AAFX01006490

Methylomicrobium alcaliphilumMethylomicrobium album

Methylococcus capsulatus

Nitrosococcus watsoniNitrosococcus oceani AFC27Nitrosococcus oceani ATCC 19707

Nitrosococcus halophilus

Candidatus Koribacter

Nitrococcus mobilisFrankia sp. EAN1

Frankia sp. CcI3Streptomyces sp. e14

Streptomyces pristinaespiralisStreptomyces cattleya DSM 46488Streptomyces cattleya NRRL 8057

Streptomyces xinghaiensisStreptomyces fradiae

Streptomyces griseoflavusStreptomyces ghanaensis

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Candidatus Methylomirabilis oxyfera

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when queried against theM. capsulatus HpnR sequence. The tree was rootedby using the 140 sequences as an out group to the 52 HpnR sequences shown.The full unrooted tree is shown in Fig. S3.

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A previous study inM. capsulatus demonstrated that 3-methyl-hopanoids accumulate preferentially in stationary phase cells(24). To test whether 3-methylhopanoids play a role in stationaryphase physiology, we grew both the wild type and ΔhpnR strainsin batch culture for fourteen days. Cultures were supplementedwith methane only at the point of inoculation (day 0) to ensurethat they would become nutrient-limited and enter stationaryphase. Cell growth was monitored by following the OD at 600 nm.Under these growth conditions, it was determined that the cellswere entering stationary phase and ceased oxidizing methane onday 2 of growth (Fig. S4). Given that the methane in the head-space was not depleted (Fig. S4), we presumed that the cessationof growth on day 2 resulted from the depletion of oxygen. Theseexperiments also demonstrated thatΔhpnR cells exhibited similargrowth characteristics to the wild type strain during exponentialgrowth. However, upon entering stationary phase, the methylasemutant seemed to experience a larger drop in OD than the wildtype indicating a potential loss in viability under these conditions.

To better assess cell viability in stationary phase, the number ofcfu on day 2, 7, and 14 of growth were determined. As shown inFig. 4, the wild type strain maintains the same number of cfuthroughout stationary phase. The ΔhpnR strain sustains a similarnumber of viable cells on day 2 and 7, even though the observeddrop in OD occurs immediately upon entering stationary phase atday 2. However, the number of cfu drops approximately six ordersof magnitude on day 14 suggesting a role for 3-methylhopanoidsin late stationary phase (Fig. 4). To more directly show that thelack of 3-methylhopanoids was responsible for this reduction inviability, we also determined the cfu values for the ΔhpnR straincomplemented with a copy of the methylase gene (Fig. 4;ΔhpnRþ pPVW100). The overall cfu values for the complemen-ted strain were approximately two orders of magnitude lowerthan the wild type on each day tested. This reduction in cfuwas most likely a result of the sluggish growth observed in thepresence of the kanamycin antibiotic necessary to maintain thecomplementing plasmid. Nevertheless, reinstating 3-methylhopa-noid production in the ΔhpnR strain did result in sustained cellviability through day 14.

3-Methylhopanoids and Intracytoplasmic Membrane Formation inLate Stationary Phase. The decreased viability of the methylasemutant suggests that it may be deficient in mechanisms necessaryto cope with the stresses encountered in stationary phase.M. cap-sulatus is one of several methanotrophs that are capable of form-ing Azotobacter-like cyst structures. Cyst formation has beenshown to be important in enhancing the survival of a bacteriumunder adverse environmental conditions such as those experi-enced in stationary phase (25). Accordingly, we hypothesized that

the lack of 3-methylhopanoids in the methylase mutant may re-sult in inadequate cyst formation which, in turn, compromises via-bility 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 14of growth were analyzed for the formation of cyst-like structures.As shown in Fig. 5, no cyst-like cells were found to be producedeither by the wild type or the methylase mutant throughout sta-tionary phase suggesting that 3-methylhopanoids play a role instationary phase survival independent of cyst formation. Onthe other hand, the images revealed formation of extensive intra-cytoplasmic membranes (ICM) in late stationary phase. In parti-cular, both the wild type and ΔhpnR were capable of formingthese membranes in early stationary phase (day 2). But by day14, the wild type had significantly more ICM present than on day2 whereas the 3-methylhopanoid deletion mutant was no longerproducing these membranes. The ability of the methylase mutantto produce lamellar membranes upon entering stationary phasebut not deep into stationary phase suggests that 3-methylhopa-noids play a role in maintaining these membranes rather thana role in forming them. Complementation of the deletion strainwith the hpnR gene partially restored the production of ICM onday 14 further indicating that 3-methylhopanoids may be impor-tant in maintaining ICM formation during stationary phase.

The decreased viability of the methylase mutant along with itsinability to maintain ICM formation leads us to speculate that ICMmaintenance is necessary for survival in late stationary phase.Based on our physiological data, we presume that the cells in ourcultures are limited for oxygen rather than methane (Fig. S4). Pre-vious studies have shown that ICM formation and methane oxida-tion rates in methanotrophs increase under similar high methane/low oxygen growth conditions (26). Because the methane mono-oxygenase is a membrane-bound enzyme localized to the ICM itis thought that the increase in ICM formation allows for increasedmethane oxidation at low oxygen levels. This strategy may then aidsurvival in low oxygen environments encountered in nature.

Interestingly, these high methane/low oxygen growth condi-tions are reminiscent of the modern and ancient seafloormethane seeps in which 3-methylhopanes and 3-methylhopanoid-producing methanotrophs have been detected (4, 27). The phy-sical conditions at these seafloor methane seeps are quite tran-sitory, particularly in terms of the availability of methane andoxygen (28). The methanotrophs in these communities must beadapted to survive persistent low oxygen levels as well as to re-spond rapidly to methane pulses (28). Thus, our late stationaryphase studies could be demonstrating a role for ICMs and 3-methylhopanoids in the persistence of certain methanotrophiccommunities in their natural environments. This hypothesis isparticularly appealing when we consider a recent study on the fateof spilled methane from the 2010 Deepwater Horizon oil spill(29). In this study, the idea was put forward that aerobic metha-notrophic communities may act as dynamic biofilters of large-scale methane inputs into the ocean (29). These methanotrophiccommunities persist for the most part in nutrient-limited envir-onments yet seem poised to respond to sudden influxes ofmethane. Some of the methanotrophs identified from this poten-tial methanotrophic bloom after the Deepwater Horizon disasterwere γ-Proteobacteria of the Methylococcaceae family that isknown to contain 3-methylhopanoid-producing methanotrophicspecies such as M. capsulatus. Therefore, it is possible that theability to survive in these transient methanotrophic environmentsmay be linked to 3-methylhopanoid production.

The observations presented here point to a potential roleof 3-methylhopanoids (and ICM formation) in cell viabilityunder nutrient-limited conditions. These findings are pertinentgiven that low nutrients and harsh conditions are known to beubiquitous in natural environments and as a result, microbesin nature are thought to persist in a type of stationary phase

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(30). We are currently pursuing molecular and physiological stu-dies to better pinpoint the factors that induce ICM formation instationary phase, the potential role of 3-methylhopanoids in ICMmaintenance, and how 3-methylhopanoids this might aid cell via-bility. If a relationship between 3-methylhopanoids and cell via-bility during stationary phase can be further established, then 3-methylhopanoids have the potential to be proxies for the parti-cular environmental stressors (e.g., low oxygen) encountered dur-ing stationary phase.

Materials and MethodsBacterial Strains, Media, and Growth Conditions. Bacterial strains used in thisstudy are listed in Table S1. Escherichia coli strains were grown in lysogenybroth (LB) and M. capsulatus strains were grown in nitrate minimal salts(NMS) medium supplemented with 10 μM CuSO4 (31) at 37 °C while shakingat 250 rpm. M. capsulatus batch cultures were sealed in serum vials withoutremoving the ambient air and given methane: carbon dioxide mix (95∶5) at60 kPa over ambient pressure. For growth on solid medium, LB or NMS wassolidified with 1.5% agar and supplemented, if necessary, with 15 μg∕mLgentamicin (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 mixat 20 kPa over ambient pressure. Additional details are described in SIMaterials and Methods.

DNAMethods, Transformation, and Mutant Construction. All plasmid constructsand the sequences of oligonucleotide primers used in this study are describedin Table S1. Construction of the hpnR deletion mutant and complementedstrain is described in SI Materials and Methods. DNA sequences of all cloningintermediates were confirmed by sequencing at the GENEWIZ BostonLaboratory. E. coli strains were transformed by electroporation. Plasmidswere mobilized from E. coli BW20767 into M. capsulatus by conjugationas described in SI Materials and Methods.

Analysis of Hopanoid Production. M. capsulatus strains were grown in 50 mLNMS at 37 °C for 3 d. Lipids were extracted and analyzed by liquid chroma-tography-mass spectrometry (LC-MS) as previously described (17, 21). The LC-MS system comprises a 1200 Series HPLC (Agilent Technologies) equippedwith an autosampler and a binary pump linked to a Q-TOF 6520mass spectro-meter (Agilent Technologies) via an atmospheric pressure chemical ionizationinterface (Agilent Technologies). Hopanoids were identified on the basis ofaccurate mass measurements of their protonated molecular ions, fragmenta-tion patterns in MS-MS mode, and by comparison of relative retention timeand the mass spectra with published data (32).

Bioinformatics Analysis. InterPro (http://www.ebi.ac.uk/interpro) was used toidentify putative radical AdoMet proteins in the M. capsulatus genome. HpnRhomologues were identified in the KEGG and the NCBI databases by a trans-lated Basic Local Assignment Search Tool (TBLASTN) (33) and were alignedusing the Multiple Sequence Comparison by Log-Expectation (MUSCLE) pro-gram (34). Maximum likelihood trees were constructed by phylogenetic esti-mation usingmaximum likelihood (PhyML) (35) using the LGþ gammamodel,four gamma rate categories, ten random starting trees, Nearest Neighbor In-terchange (NNI) branch swapping, and substitution parameters estimatedfrom the data. The HpnR tree was generated and edited by importing the re-sulting PhyML tree into the Interactive Tree of Life tool (iTOL) (http://itol.embl.de/) (36).

ACKNOWLEDGMENTS. We thank Dr. Florence Schubotz for technical adviceand assistance, Prof. Tanja Bosak and Prof. Shuhei Ono for providing labspace and equipment, and Prof. Martin Klotz for his generous gift of Methy-lococcus capsulatus strain Bath. This work was supported by the NationalScience Foundation (NSF) Program on Emerging Trends in BiogeochemicalCycles (OCE-0849940) supported, in turn, by NSF programs in ChemicalOceanography and Geobiology-Low Temperature Geochemistry (R.E.S.),and a National Aeronautics and Space Administration Postdoctoral ProgramFellowship (P.V.W.).

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Day 2

wild type ∆∆hpnR + pPVW100

∆hpnR

Day 14∆hpnR + pPVW100

wild type ∆hpnR

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 andcomplemented strains (ΔhpnRþ pPVW100). (Black scale, 0.5 μ.)

Welander and Summons PNAS ∣ August 7, 2012 ∣ vol. 109 ∣ no. 32 ∣ 12909

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