Discovery, taxonomic distribution, and phenotypic characterization of a gene required for 3-methylhopanoid production

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 2-methylhopanoids 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. 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 indicate 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, BLAST analyses 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 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.

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Fig. 3.

Maximum likelihood phylogenetic tree of putative HpnR sequences. A total of 192 radical AdoMet proteins were used to generate the tree: Fifty-two 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.

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 3-methylhopanoid 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 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.

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.

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 + 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.

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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.

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 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.

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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 + pPVW100). (Black scale, 0.5 μ.)

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-methylhopanoid-producing 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 3-methylhopanoids 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 large-scale 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 (30). We are currently pursuing molecular and physiological studies to better pinpoint the factors that induce ICM formation in stationary phase, the potential role of 3-methylhopanoids in ICM maintenance, and how 3-methylhopanoids this might aid cell viability. If a relationship between 3-methylhopanoids and cell viability during stationary phase can be further established, then 3-methylhopanoids have the potential to be proxies for the particular environmental stressors (e.g., low oxygen) encountered during stationary phase.