Discovery, Taxonomic Distribution, and Phenotypic Characterization of a Gene Required for 3-Methylhopanoid Production

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. CrossRef. Web.

As Published http://dx.doi.org/10.1073/pnas.1208255109

Publisher National Academy of Sciences (U.S.)

Version Final published version

Citable link http://hdl.handle.net/1721.1/77589

Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. 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

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 bacterial majority of C-3 methylated hopanoid producers are aerobic triterpenoids that are readily preserved in modern and ancient se- methanotrophs. However, the production of 3-methylhopanoids diments and in petroleum. The production of 3-methylhopanoids has also been demonstrated in the acetic acid bacteria (12) indi- by extant aerobic methanotrophs and their common occurrence cating that the taxonomic distribution of 3-methylhopanoids is in modern and fossil methane seep communities, in conjunction not restricted to methanotrophs. Furthermore, recent studies with carbon isotope analysis, has led to their use as biomarker utilizing molecular approaches to identify hopanoid biosynthesis proxies for aerobic methanotrophy. In addition, these lipids are genes in sequenced genomes have highlighted that the diversity of also produced by aerobic acetic acid bacteria and, lacking carbon bacteria capable of producing a specific hopanoid structure could isotope analysis, are more generally used as indicators for aerobio- be underestimated (13–15). These studies have also shown that a sis in ancient ecosystems. However, recent genetic studies have more precise interpretation of hopane hydrocarbon signatures in brought into question our current understanding of the taxonomic both ancient and modern ecosystems requires not only a grasp diversity of methylhopanoid-producing bacteria and have high- of the taxonomic distribution of methylhopanoid producers but lighted that a proper interpretation of methylhopanes in the rock also a deeper understanding of their physiological function in record requires a deeper understanding of their cellular function. In extant bacteria (16, 17). hpnR this study, we identified and deleted a gene, , required for To this end, we employed a combination of microbial genetics, methylation of hopanoids at the C-3 position in the obligate microbial physiology, and bioinformatics analysis to begin to Methylococcus capsulatus methanotroph strain Bath. Bioinfor- understand the biosynthesis and function of C-3 methylated matics analysis revealed that the taxonomic distribution of HpnR hopanoids in Methylococcus capsulatus. A genetic system for extends beyond methanotrophic and acetic acid bacteria. Phenoty- constructing unmarked in-frame deletion mutants was utilized to M. capsulatus hpnR pic analysis of the deletion mutant demon- identify a methylase required for the production of 3-methylho- strated a potential physiological role for 3-methylhopanoids; they panoids. Bioinformatics analysis of this methylase revealed a appear to be required for the maintenance of intracytoplasmic diverse taxonomic distribution beyond the methanotrophic and membranes and cell survival in late stationary phase. Therefore, acetic acid bacteria. Furthermore, phenotypic analysis of the 3-methylhopanoids may prove more useful as proxies for specific C-3 methylase mutant uncovered a potential role for 3-methylho- environmental conditions encountered during stationary phase panoids in late stationary phase survival. These studies highlight rather than a particular bacterial group. the power of combining gene discovery with bioinformatics and physiological analyses to potentially enhance our understanding radical SAM ∣ bacteriohopanepolyols ∣ molecular markers of biomarker signatures in the rock record.

opanoids are pentacyclic triterpenoid lipids produced by a Results and Discussion EARTH, ATMOSPHERIC, AND PLANETARY SCIENCES Hvariety of bacteria that are often utilized as geological Identification of a C-3 Methylase in the M. capsulatus Genome. To proxies or biomarkers for certain bacterial species and their identify a protein required for the methylation of hopanoids metabolisms. Among the various hopanoid structures produced at the C-3 position, the genome of M. capsulatus was examined by bacteria (1), those methylated at the C-3 position and those for possible C-3 methylase candidates utilizing search criteria with a penta- and hexafunctionalized amino polar side group based on two previous findings. First, bacterial feeding studies are thought to be primarily produced by Type I and Type X done with labeled methionine have posited that S-adenosyl- methanotrophs (Fig. 1A) (2). As such, the occurrence of these 13 methionine (AdoMet) is a potential methyl donor in the MICROBIOLOGY hopanoids in conjunction with their significant C-depletion in biosynthesis of both 2-methyl and 3-methylhopanoids (12). Sec- modern ecosystems are often utilized as an indicator of methanotrophic communities (3). In particular, environmental lipid ond, it was recently discovered that a B-12 binding radical analyses have uncovered the existence of aerobic methanotrophy AdoMet protein, HpnP, is required for the production of 2- methylhopanoids in the α-Proteobacterium Rhodopseudomonas in a variety of environments including, for example, the surface palustris sediments of an active marine mud volcano in the Barents Sea (14). Accordingly, we hypothesized that the methylase and in the oxic-anoxic transition zone of the Black Sea water responsible for 3-methylhopanoid production was also a radical column (4, 5). Furthermore, the recalcitrant nature of hopanoid AdoMet protein possibly containing a B-12 binding domain. hydrocarbons allows for their preservation in ancient sediments, which may provide evidence for aerobic metabolisms deep in Author contributions: P.V.W. designed research; P.V.W. performed research; R.E.S. Earth’s history. Although the functionalized amino side group contributed new reagents/analytic tools; P.V.W. and R.E.S. analyzed data; and P.V.W. is lost over time, methylation of the A-ring is retained (6). Thus, and R.E.S. wrote the paper. the presence of C-3 methylated hopanes in sediments 2.5–2.7 bil- The authors declare no conflict of interest. lion years old has been used as one of several lines of molecular This article is a PNAS Direct Submission. and isotopic evidence for Neoarchean aerobiosis (7–11). 1To whom correspondence should be addressed. E-mail: [email protected]. The effectiveness of these specific hopanoids as indicators This article contains supporting information online at www.pnas.org/lookup/suppl/ for aerobic methanotrophy rests partly on the premise that the doi:10.1073/pnas.1208255109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1208255109 PNAS ∣ August 7, 2012 ∣ vol. 109 ∣ no. 32 ∣ 12905–12910 A OH OH OH A

OHOH NH2 3 I R R = H aminobacteriohopanepentol (I) II R = CH 3 3-methylaminobacteriohopanepentol (III) OH OH

OHOH NH2 3 III IV

R Relative Intensity R = H aminobacteriohopanetetrol (II)

R = CH 3 3-methylaminobacteriohopanetetrol (IV)

B ISMca3

yfiA hyp OrfB 18 20 22 24 26 28 30 32 rpoN hpnR OrfA B 0741 0740 0739 0738 0736 0735 1 kb

Fig. 1. Identification of a putative 3-methylhopanoid methylase in M. cap- II I sulatus.(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 polymer- ase factor Sigma-54 (rpoN). Downstream are two genes that are annotated as ISMca3 transposase genes (OrfA and OrfB). Relative Intensity

Using InterPro (http://www.ebi.ac.uk/interpro/), an integrated database of predictive protein signatures, 21 proteins with a M. capsulatus radical AdoMet motif were identified in the gen- 18 20 22 24 26 28 30 32 ome. 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 C the only protein annotated as a B-12 binding radical AdoMet that A. pasterurianus also had a homologue in (e-value of 0). A Basic I 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 II B hopanoid biosynthesis genes on the chromosome (Fig. 1 ), the III 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. IV Relative Intensity 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 18 20 22 24 26 28 30 32 involves integrating a suicide plasmid at the locus of interest Time (min) by homologous recombination and subsequently excising the plasmid from the chromosome, which can result in the deletion Fig. 2. Deletion of hpnR results in loss of 3-methylhopanoid production. of the gene of interest (Fig. S1). To delete MCA0738, a deletion LC-MS extracted ion chromatograms of acetylated total lipid extracts from (A) wild type M. capsulatus,(B) ΔhpnR, and (C) ΔhpnR complemented with plasmid containing a replacement allele missing the MCA0738 M. capsulatus a copy of hpnR on a self-replicating plasmid (pPVW100). The chromatograms gene was transferred into via conjugation and in- are a combination of ions m/z 830 (I, aminobacteriohopanepentol), 772 (II, tegrated onto the chromosome by homologous recombination. aminobacteriohopanetetrol), 844 (III, 3-methylaminobacteriohopanepentol), The plasmid was forced to excise from the chromosome through and 786 (IV, 3-methylaminobacteriohopanetetrol). Hopanoids were identi- nonselective growth and several potential deletion colonies were fied based on their mass spectra shown in Fig. S2. screened by PCR for deletion of MCA0738. One strain was found to be devoid of this gene and was picked for further character- dicate that MCA0738 is the only gene required for C-3 methyla- M. capsulatus ization (Fig. S1). tion of hopanoids in and we propose to rename this To verify that MCA0738 was required for C-3 methylation, locus hpnR based on a previously established nomenclature in a total lipid extract (TLE) was isolated from the MCA0738 dele- Zymomonas mobilis (20). tion mutant and analyzed for its complement of bacteriohopanepolyols. As shown in Fig. 2, the MCA0738 deletion mutant is Identification of Putative HpnR Homologues. The radical AdoMet able to produce both the desmethyl aminobacteriohopanepentol protein family encompasses a diverse set of proteins that catalyze and aminobacteriohopanetetrol but not their C-3 methylated a variety of biochemical reactions. The proteins in this family are counterparts as confirmed by detailed mass spectral analysis primarily identified by the short amino acid sequence motif (Fig. S2). Furthermore, introduction of a copy of the MCA0738 CxxxCxxC. As a result, BLASTanalyses of HpnR return a variety gene on a self-replicating plasmid into the deletion strain restores of radical AdoMet proteins that may or may not be involved in production of the methylated hopanoids (Fig. 2). These data in- 3-methylhopanoid biosynthesis. To determine which of these

12906 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1208255109 Welander and Summons radical AdoMet proteins are genuine C-3 methylases, we con- copy of this protein suggesting that only a subset of methano- structed an unrooted maximum likelihood tree of 192 radical trophs may be capable of producing 3-methylhopanoids. The in- AdoMet proteins retrieved through a protein BLAST search of consistent distribution of HpnR among methanotrophs is also the M. capsulatus HpnR sequence against the Kyoto Encyclope- observed in the genomes of other hopanoid-producing bacterial dia of Genes and Genomes (KEGG) and National Center for genera. For example, 38 Burkholderia,43Streptomyces, and 8 Biotechnology Information (NCBI) databases (e-value cut-off < Methylobacterium genomes have been sequenced to date. All of e−17). This analysis shows that those radical AdoMet proteins these genomes, except for Burkholderia pseudomallei MSHR346, with an e-value lower than e−100 cluster together (Fig. S3). Within contain a copy of the squalene hopene cyclase gene and at least this clade, we find HpnR from M. capsulatus and homologues one species from each of these groups has been shown to produce from the acetic acid bacteria, the only currently known producers hopanoids. Yet, only three Burkholderia, eight Streptomyces, and of 3-methylhopanoids (Fig. 3). Further, all of the strains in this one Methylobacterium contain the HpnR methylase suggesting clade also contain a copy of the squalene hopene cyclase gene, that only a subset of species from certain genera may be able which is required for hopanoid biosynthesis, as well as several to produce 3-methylhopanoids. This observation is particularly other hopanoid biosynthesis genes in their genomes (17, 21). critical when we consider that our current understanding of which Therefore, it seems reasonable to propose that the cutoff for a bacteria produce certain hopanoid molecules is often based on −100 bona fide C-3 methylase is a value lower than e . lipid analysis of a few culturable species of a certain genus. Using this criteria, there are 52 putative homologues of HpnR The sporadic phylogenetic distribution of HpnR also suggests in the genomic and metagenomic databases (Fig. 3). The species a potentially complex evolutionary history of this methylase. Two that contain these HpnR homologues are from a diverse set of evolutionary scenarios seem plausible: either HpnR was present bacterial phyla: 33 strains of Proteobacteria (22 α-, 3 β-, and 8 in the ancestor of all HpnR-containing bacteria and was repeat- γ-Proteobacteria), 11 Actinobacteria, 3 Nitrospirae, 1 Acidobac- edly lost or it could have been acquired through horizontal gene terium, 1 candidate NC10 phylum organism, and 3 metagenomic transfer (HGT). For most of these taxa, the HpnR phylogeny is sequences. In agreement with our current understanding of 3- congruent with that of the species phylogeny based on 16S rRNA methylhopanoid distribution in bacteria, all 21 of the partially sequence (22). This consistency between the HpnR phylogeny or completed acetic acid bacterial genomes have an HpnR homo- and species phylogeny is evident for the acetic acid bacterial clade logue. However, only three of the nine methanotrophic genomes suggesting a vertical descent within this group. However, the sequenced to date (two complete and seven incomplete) have a Methylobacteium nodulans and Nitrococcus mobilis HpnR sequences tend to cluster outside their expected species phylogeny Candidatus Koribacter 0.89 Candidatus Methylomirabilis oxyfera clade suggesting acquisition through HGT in these organisms. 1.00 Nitrococcus mobilis Thus, the evolutionary history of this protein remains unclear 1.00 Frankia sp. EAN1 and more robust analyses are needed to better resolve it. 0.50 Frankia sp. CcI3 0.99 Streptomyces sp. e14 Given the taxonomic diversity of potential 3-methylhopanoid 1.00 1.00 Streptomyces chartreusis Streptomyces griseoflavus producers uncovered by our analysis, the detection of 3-methyl- 0.98 Streptomyces ghanaensis hopanoids in both modern and ancient sediments cannot be at- Streptomyces xinghaiensis 0.72 1.00 Streptomyces fradiae tributed specifically to aerobic methanotrophic bacteria without 0.45 Streptomyces pristinaespiralis 1.00 Streptomyces cattleya DSM 46488 other lines of evidence (e.g., carbon isotope data). However, all of 1.00 0.81 Streptomyces cattleya NRRL 8057 the bacterial species that contain an HpnR homologue in their 1.00 Nitrosococcus halophilus Nitrosococcus watsoni genomes utilize a form of aerobic metabolism. Thus, it seems Nitrosococcus oceani AFC27 reasonable to continue to employ 3-methylhopanes in the rock 0.95 1.00 Nitrosococcus oceani ATCC 19707 0.99 Methylococcus capsulatus record as indicators for the existence of aerobic metabolisms deep 1.00 Methylomicrobium alcaliphilum Methylomicrobium album in time. The one organism that might be considered an exception 0.89 Soil Metagenome AAFX01006490 to this rule is Candidatus Methylomirabilis oxyfera which was iso- 0.95 Leptospirillum ferrooxidans 0.99 Mine Drainage Metagenome ACXJ01008692.1 lated from anoxic sediments and grows anaerobically by coupling Leptospirillum sp. Group II M. oxyfera 0.60 1.00 nitrite reduction to methane oxidation (23). Although EARTH, ATMOSPHERIC,

Leptospirillum rubarum AND PLANETARY SCIENCES 0.81 Mine Drainage Metagenome ACXJ01008849.1 is an anaerobe, it is also an oxygenic organism as it produces its 1.00 Burkholderia sp. H160 own supply of oxygen through the dismutation of nitrite. It sub- 0.90 Burkholderia xenovorans 0.96 Burkholderia phymatum sequently uses this oxygen for the oxidation of methane through Methylobacterium nodulans Gluconacetobacter diazotrophicus the same methanotrophic pathway utilized by other aerobic 0.55 Gluconacetobacter hansenii methanotrophs (23). Whereas M. oxyfera is encountered in anae- Gluconacetobacter sp. SXCC1 0.89 1.00 Gluconacetobacter xylinus robic environments, it still requires oxygen for its metabolism. Gluconacetobacter oboediens Therefore, the distribution of the C-3 methylase in oxygen-de- 1.00 Gluconacetobacter europaeus LMG 18494 MICROBIOLOGY Gluconacetobacter europaeus LMG 18890 manding bacteria seems robust for now and can be seen as further Gluconacetobacter europaeus 5P3 evidence for the use of 3-methylhopanes as proxies for the occur- 0.15 Acetobacter aceti Gluconobacter oxydans rence of aerobic metabolisms in ancient environments. Gluconobacter morbifer 0.97 Acetobacter tropicalis Acetobacter pomorum 3-Methylhopanoids Play a Role in Late Stationary Phase Survival. The 0.98 Acetobacter pasteurianus IFO 3283-01 1.00 Acetobacter pasteurianus IFO 3283-01-42C diverse and sporadic distribution of HpnR introduces further Acetobacter pasteurianus IFO 3283-03 ambiguity in our ability to correlate specific bacterial taxa to Acetobacter pasteurianus IFO 3283-07 Acetobacter pasteurianus IFO 3283-12 3-methylhopanes in the environment. As a result, a proper ana- Acetobacter pasteurianus IFO 3283-22 lysis of the presence of 3-methylhopanes in the rock record needs Acetobacter pasteurianus IFO 3283-26 Acetobacter pasteurianus IFO 3283-32 to move beyond simply understanding which organisms produce 0.1 these molecules. A more nuanced interpretation may be achieved if we better understand the physiological function of 3-methylho- Fig. 3. Maximum likelihood phylogenetic tree of putative HpnR sequences. panoids in bacteria as well as the environmental factors that in- A total of 192 radical AdoMet proteins were used to generate the tree: Fifty- −17 duce their production in the cell. To this end, we have begun two HpnR sequences plus 140 radical AdoMets with an e-value less than e M. capsulatus hpnR when queried against the M. capsulatus HpnR sequence. The tree was rooted physiological characterization of the mutant by using the 140 sequences as an out group to the 52 HpnR sequences shown. to identify any potential phenotype(s) associated with the loss of The full unrooted tree is shown in Fig. S3. 3-methylhopanoid production.

Welander and Summons PNAS ∣ August 7, 2012 ∣ vol. 109 ∣ no. 32 ∣ 12907 A previous study in M. capsulatus demonstrated that 3-methyl- the lack of 3-methylhopanoids in the methylase mutant may re- hopanoids accumulate preferentially in stationary phase cells sult in inadequate cyst formation which, in turn, compromises via- (24). To test whether 3-methylhopanoids play a role in stationary bility during prolonged stationary phase incubation. phase physiology, we grew both the wild type and ΔhpnR strains To test this hypothesis, transmission electron microscopy in batch culture for fourteen days. Cultures were supplemented (TEM) of both wild type and mutant cells on day 2 and day 14 with methane only at the point of inoculation (day 0) to ensure of growth were analyzed for the formation of cyst-like structures. that they would become nutrient-limited and enter stationary As shown in Fig. 5, no cyst-like cells were found to be produced phase. Cell growth was monitored by following the OD at 600 nm. either by the wild type or the methylase mutant throughout sta- Under these growth conditions, it was determined that the cells tionary phase suggesting that 3-methylhopanoids play a role in were entering stationary phase and ceased oxidizing methane on stationary phase survival independent of cyst formation. On day 2 of growth (Fig. S4). Given that the methane in the head- the other hand, the images revealed formation of extensive intra- space was not depleted (Fig. S4), we presumed that the cessation cytoplasmic membranes (ICM) in late stationary phase. In parti- of growth on day 2 resulted from the depletion of oxygen. These cular, both the wild type and ΔhpnR were capable of forming experiments also demonstrated that ΔhpnR cells exhibited similar these membranes in early stationary phase (day 2). But by day growth characteristics to the wild type strain during exponential 14, the wild type had significantly more ICM present than on day growth. However, upon entering stationary phase, the methylase 2 whereas the 3-methylhopanoid deletion mutant was no longer mutant seemed to experience a larger drop in OD than the wild producing these membranes. The ability of the methylase mutant type indicating a potential loss in viability under these conditions. to produce lamellar membranes upon entering stationary phase To better assess cell viability in stationary phase, the number of but not deep into stationary phase suggests that 3-methylhopa- cfu on day 2, 7, and 14 of growth were determined. As shown in noids play a role in maintaining these membranes rather than Fig. 4, the wild type strain maintains the same number of cfu a role in forming them. Complementation of the deletion strain throughout stationary phase. The ΔhpnR strain sustains a similar with the hpnR gene partially restored the production of ICM on number of viable cells on day 2 and 7, even though the observed day 14 further indicating that 3-methylhopanoids may be impor- drop in OD occurs immediately upon entering stationary phase at tant in maintaining ICM formation during stationary phase. day 2. However, the number of cfu drops approximately six orders The decreased viability of the methylase mutant along with its of magnitude on day 14 suggesting a role for 3-methylhopanoids inability to maintain ICM formation leads us to speculate that ICM in late stationary phase (Fig. 4). To more directly show that the maintenance is necessary for survival in late stationary phase. lack of 3-methylhopanoids was responsible for this reduction in Based on our physiological data, we presume that the cells in our viability, we also determined the cfu values for the ΔhpnR strain cultures are limited for oxygen rather than methane (Fig. S4). Pre- complemented with a copy of the methylase gene (Fig. 4; vious studies have shown that ICM formation and methane oxida- ΔhpnR þ pPVW100). The overall cfu values for the complemen- tion rates in methanotrophs increase under similar high methane/ ted strain were approximately two orders of magnitude lower low oxygen growth conditions (26). Because the methane mono- than the wild type on each day tested. This reduction in cfu oxygenase is a membrane-bound enzyme localized to the ICM it was most likely a result of the sluggish growth observed in the is thought that the increase in ICM formation allows for increased presence of the kanamycin antibiotic necessary to maintain the methane oxidation at low oxygen levels. This strategy may then aid complementing plasmid. Nevertheless, reinstating 3-methylhopa- survival in low oxygen environments encountered in nature. noid production in the ΔhpnR strain did result in sustained cell Interestingly, these high methane/low oxygen growth condi- viability through day 14. tions are reminiscent of the modern and ancient seafloor methane seeps in which 3-methylhopanes and 3-methylhopanoid- 3-Methylhopanoids and Intracytoplasmic Membrane Formation in producing methanotrophs have been detected (4, 27). The phy- Late Stationary Phase. The decreased viability of the methylase sical conditions at these seafloor methane seeps are quite tran- mutant suggests that it may be deficient in mechanisms necessary sitory, particularly in terms of the availability of methane and to cope with the stresses encountered in stationary phase. M. cap- oxygen (28). The methanotrophs in these communities must be sulatus is one of several methanotrophs that are capable of form- adapted to survive persistent low oxygen levels as well as to re- ing Azotobacter-like cyst structures. Cyst formation has been spond rapidly to methane pulses (28). Thus, our late stationary shown to be important in enhancing the survival of a bacterium phase studies could be demonstrating a role for ICMs and 3- under adverse environmental conditions such as those experi- methylhopanoids in the persistence of certain methanotrophic enced in stationary phase (25). Accordingly, we hypothesized that communities in their natural environments. This hypothesis is particularly appealing when we consider a recent study on the fate 1.0E+09 of spilled methane from the 2010 Deepwater Horizon oil spill 1.0E+08 (29). In this study, the idea was put forward that aerobic metha- 1.0E+07 notrophic communities may act as dynamic biofilters of large- scale methane inputs into the ocean (29). These methanotrophic 1.0E+06 communities persist for the most part in nutrient-limited envir- 1.0E+05 onments yet seem poised to respond to sudden influxes of 1.0E+04 methane. Some of the methanotrophs identified from this poten- 1.0E+03 tial methanotrophic bloom after the Deepwater Horizon disaster were γ-Proteobacteria of the Methylococcaceae family that is 1.0E+02 known to contain 3-methylhopanoid-producing methanotrophic

Colony Forming Units (cfu) 1.0E+01 species such as M. capsulatus. Therefore, it is possible that the 1.0E+00 ability to survive in these transient methanotrophic environments Day 2 Day 7 Day 14 may be linked to 3-methylhopanoid production. wild type The observations presented here point to a potential role of 3-methylhopanoids (and ICM formation) in cell viability 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 under nutrient-limited conditions. These findings are pertinent 14 of growth by spot plating serial dilutions on NMS agar plates. Each bar given that low nutrients and harsh conditions are known to be represents the average cfu of three separate experiments and the error bars ubiquitous in natural environments and as a result, microbes are standard deviations. in nature are thought to persist in a type of stationary phase

12908 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1208255109 Welander and Summons Day 2 wild type ∆hpnR ∆hpnR + pPVW100

Day 14 wild type ∆hpnR ∆hpnR + pPVW100

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.)

(30). We are currently pursuing molecular and physiological stu- Analysis of Hopanoid Production. M. capsulatus strains were grown in 50 mL dies to better pinpoint the factors that induce ICM formation in NMS at 37 °C for 3 d. Lipids were extracted and analyzed by liquid chroma- stationary phase, the potential role of 3-methylhopanoids in ICM tography-mass spectrometry (LC-MS) as previously described (17, 21). The LC- maintenance, and how 3-methylhopanoids this might aid cell via- MS system comprises a 1200 Series HPLC (Agilent Technologies) equipped bility. If a relationship between 3-methylhopanoids and cell via- with an autosampler and a binary pump linked to a Q-TOF 6520 mass spectro- bility during stationary phase can be further established, then 3- meter (Agilent Technologies) via an atmospheric pressure chemical ionization methylhopanoids have the potential to be proxies for the parti- interface (Agilent Technologies). Hopanoids were identified on the basis of cular environmental stressors (e.g., low oxygen) encountered dur- accurate mass measurements of their protonated molecular ions, fragmenta- ing stationary phase. tion patterns in MS-MS mode, and by comparison of relative retention time and the mass spectra with published data (32). Materials and Methods Bioinformatics Analysis. InterPro (http://www.ebi.ac.uk/interpro)wasusedto Bacterial Strains, Media, and Growth Conditions. Bacterial strains used in this identify putative radical AdoMet proteins in the M. capsulatus genome. HpnR study are listed in Table S1. Escherichia coli strains were grown in lysogeny homologues were identified in the KEGG and the NCBI databases by a trans- broth (LB) and M. capsulatus strains were grown in nitrate minimal salts lated Basic Local Assignment Search Tool (TBLASTN) (33) and were aligned (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 using the Multiple Sequence Comparison by Log-Expectation (MUSCLE) pro- removing the ambient air and given methane: carbon dioxide mix (95∶5)at gram (34). Maximum likelihood trees were constructed by phylogenetic esti- 60 kPa over ambient pressure. For growth on solid medium, LB or NMS was mation using maximum likelihood (PhyML) (35) using the LG þ gamma model, 15 μ ∕ EARTH, ATMOSPHERIC, solidified with 1.5% agar and supplemented, if necessary, with g mL four gamma rate categories, ten random starting trees, Nearest Neighbor In- AND PLANETARY SCIENCES gentamicin (Gm), 50 μg∕mL kanamycin (Km), 600 μM diaminopimelic acid terchange (NNI) branch swapping, and substitution parameters estimated (DAP), or 5% sucrose. M. capsulatus plates were incubated in Vacu-Quik Jars from the data. The HpnR tree was generated and edited by importing the re- (Almore International, Inc.) and supplied with methane: carbon dioxide mix sulting PhyML tree into the Interactive Tree of Life tool (iTOL) (http:// at 20 kPa over ambient pressure. Additional details are described in SI itol.embl.de/)(36). Materials and Methods. ACKNOWLEDGMENTS. We thank Dr. Florence Schubotz for technical advice DNA Methods, Transformation, and Mutant Construction. All plasmid constructs and assistance, Prof. Tanja Bosak and Prof. Shuhei Ono for providing lab MICROBIOLOGY and the sequences of oligonucleotide primers used in this study are described space and equipment, and Prof. Martin Klotz for his generous gift of Methy- in Table S1. Construction of the hpnR deletion mutant and complemented lococcus capsulatus strain Bath. This work was supported by the National strain is described in SI Materials and Methods. DNA sequences of all cloning Science Foundation (NSF) Program on Emerging Trends in Biogeochemical intermediates were confirmed by sequencing at the GENEWIZ Boston Cycles (OCE-0849940) supported, in turn, by NSF programs in Chemical Laboratory. E. coli strains were transformed by electroporation. Plasmids Oceanography and Geobiology-Low Temperature Geochemistry (R.E.S.), were mobilized from E. coli BW20767 into M. capsulatus by conjugation and a National Aeronautics and Space Administration Postdoctoral Program as described in SI Materials and Methods. Fellowship (P.V.W.).

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12910 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1208255109 Welander and Summons