Take a Deep Breath of... Methane

by Christoph
 

If you were faced with the task of preparing a lecture series on "general bacterial physiology", you could easily choose Pseudomonas aeruginosa  as your companion, your model organism, to cover (almost) every topic. Yet, when it comes to metabolic versatility, you could also think of Rhodo­pseu­domonas palustris  as an alternative. This Aphaproteobacterium of the Order Rhizobiales – despite its similar name it is only distantly related to P. aeruginosa (Gammaproteobacteria) – can flexibly switch among any of four metabolic 'life styles': photoautotrophic, photoheterotrophic, chemo­au­to­tro­ph­ic, and chemoheterotrophic. Plus, it can fix atmospheric nitrogen. That's hard to swallow...
 

A whiff of physiology

Figure 1. Rhodopseudomonas palustris streak­ed on a plate that was incubated anaerobically. Source. Front­page: EM of a bunch of R. palustris cells. Note that the grayscale of the original elec­­tron micrograph was con­vert­ed to shades of red (no scale given) Source

Let me dilute these highly-concentrated pills into more drinkable sips by citing what Carrie Harwood's lab at the University of Washington, Seattle, WA, tells about this bacterium on their webpage:

Rhodopseudomonas palustris  is a purple facul­ta­ti­ve­ly photosynthetic bacterium that lives in environ­ments like the surface layers of water logged soils that tend to straddle oxic to anoxic transition zones. When R. palustris  is exposed to atmospheric levels of oxygen, it oxidizes carbon compounds such as acetate for carbon and energy by aerobic respi­ra­tion. When it is exposed to oxygen depleted (2 – 6% O₂) or anaerobic conditions, R. palustris turns a cha­racteristic deep purple color (Figure 1) as it synthesi­zes the light absorbing pigments (bacteriochlorophyll) needed to carry out photosynthesis. It generates ATP anaerobically and under low oxygen tensions by anoxygenic photosynthesis, which does not generate O₂ and is not obligatorily linked to CO₂ fixation. R. palustris prefers to use organic compounds for cell carbon when it is generating ATP by anoxygenic pho­to­syn­the­sis. This growth mode is referred to as photoheterotrophic. Under microaerobic or anae­ro­bic growth conditions R. palustris can use nitrogen gas (N₂) from the atmosphere as a sole nitrogen source for growth by the process of nitrogen fixation, generating hydrogen (H₂) along with ammonium (NH₃/NH₄⁺) as products.
 

From Nitrogen fixation to Methane production

Nitrogen fixation is performed in Archaea and Bacteria by three homologous, multi-subunit enzy­me complexes, whose structures and biochemistry are well understood, and which differ in their requirements for two metal cofactors each: molybdenum (Mo) and iron (Fe) ions for 'Mo nitro­ge­na­­ses', vanadium (V) and iron ions for 'V nitrogenases', and iron ions for 'Fe-only nitrogenases'. Genes for all three nitrogenases are encoded in the R. palustris  genome (major components: NifD, VnfD, and AnfD, respectively). As enzymes, nitrogenases are notoriously "promiscuous" in the re­duction reactions that they catalyse. Mo and V nitrogenases, for example, reduce N₂ to NH₃/NH₄⁺ but can, as by-products, reduce carbon monoxide (CO), and "concatenate" methylene inter­me­di­at­es (–CH2–) to various hydrocarbon products in vitro. In a very recent study from the Harwood lab, Zheng et al. demonstrated that purified R. palustris  Fe-only nitrogenase AnfDGKH (precisely: re­constituted with the AnfH subunit of A. vinelandii, which is highly ­homologous to the cognate AnfH) produced small but detectable amounts of methane (CH₄) in addition to NH₃/NH₄⁺ and H₂ (see here). The production of H₂ by nitrogenases in this reaction has long been a riddle for biochemists yet it was partially solved recently (and as it is not connected to CH₄ production I won't go into the details; if you're interested you can learn more here).
 

(Click to enlarge )
Figure 2. Metabolic route of CH₄ production by R. pa­lus­tris  expressing wild-type Fe-only nitro­ge­nase. ATP is produced by cyclic photophos­pho­­rylation, in which elec­­trons energized by light are cycled through a pro­ton-pumping elec­tron transport chain. Electrons derived from oxi­­dation of the inorganic compound thiosulfate (A) or the organic compound acetate (B) are used in bio­synthesis and in CO₂ fixation and are also di­verted to nitrogenase. CO₂ generated from bi­car­­bonate or from the oxidation of acetate and N₂ from the atmosphere are converted by Fe-only nitrogenase to CH₄ and NH₃, respectively. Not shown here is H₂, which is also a product of nitrogenase. CBB cycle, Calvin-Benson-Bass­ham cycle; hv, light photons. Source

To verify that the CH₄ produced by the Fe-only nitro­ge­na­se of R. palustris  came from CO₂ reduction, they turned to labeling substrates for in vivo  experiments with ¹³C, a stable, non-radioactive carbon isotope present in 'nor­mal' carbon compounds at 1.1%, that is, at a low per­cent­age, and which would allow them to easily detect the source of carbon atoms in the products. They incubated cell suspensions of R. palustris CGA7553 (ΔnifHDK Δvnf HDGK) anaerobically, under light and N₂ as sole nitrogen source, with thiosulfate (S₂O₃²⁻) as a source of electrons, and ¹³C-labeled sodium bicarbonate (NaH¹³CO₃) as a source of ¹³CO₂ (R. palustris has a carbonic anhydrase that converts HCO₃^– to CO₂). Gas chromatography of headspace gases from the cultures revealed a compound with an m/z (mass/charge) ratio of 17 and a retention time (t_r) corresponding to ¹³CH₄. The "lighter" NaH¹²CO₃ as control gave a product with the same t_r  but an m/z value of 16, proving that they had correctly identified CH₄. In their reconstruction of the most likely pathway(s), Zheng et al.  propose that ATP is produced by cyclic pho­tophosphorylation, in which electrons energized by light are cycled through a proton-pumping electron transport chain. Electrons derived from oxidation of the inorganic compound thiosulfate are used for CO₂ fixation and are also diverted to nitroge­nase. CO₂ generated from bicarbonate and N₂ from the atmosphere are converted by Fe-only ni­trogenase to CH₄ and NH₃/NH₄⁺, respectively (Figure 2A). In another experimental setup, they found that Fe-only nitrogenase-expressing R. palustris  (ΔnifHDK ΔvnfHDGK) cells grown with ace­tate (C₂H₃O₂⁻) as a carbon and electron source also produced CH₄. Most likely the CH₄ was de­ri­ved in this case from CO₂ that is released when acetate is metabolized by the cells (Figure 2B).
 

Zheng et­ al.  were curious to learn whether the small amounts of methane obtained as by-product of nitrogen fixation by the Fe-only nitrogenase of the wild-type strain R. palustris CGA010 were suf­ficient to support growth of an aerobic methanotrophic (= obligate CH₄-utilizing) bacterium. They set up a co-culture of R. palustris  with Methylomonas sp. LW13, an aerobe Gamma­pro­teo­bac­te­ri­um isolated from a freshwater lake. The anaerobe co-culture was incubated in Mo‑depleted me­dium (–Mo) where Mo nitrogenase is repressed (wild-type V nitrogenase does not produce CH₄). To enable Methylomonas cells to convert CH₄ to methanol (CH₃OH) the co-culture was spiked with fresh air (O₂) in time intervals. Methylomonas cell numbers increased more than three-fold in 15 days, suggesting that the cells were growing on CH₄ produced by R. palustris. In control experi­ments, Methylomonas did not increase in cell number when incubated with R. palustris in medium supplemented with Mo (+Mo), where the Fe-only nitrogenase is repressed, but grew under this condition (+Mo) when CH₄ was added (Figure 3).
 

Figure 1. Co-culture of Methylomonas sp. LW13 and R. palustris. Methylomonas increased in cell numbers when the co-culture was incuba­ted in Mo-depleted me­dium, a condition that derepresses expression of the R. palustris Fe-only nitrogenase. The initial inoculation ratio of R. palustris to Methylomonas was ~50:1. O₂ was provided for the growth of Methylomonas by ad­ding 10% filtered air every 48 hours into a 250 ml serum bottle containing 50 ml co-cultu­re. The relative cell numbers were determined by quantitative PCR. These data are the average of two independent experiments, and the error bars represent the range of values. Source

To really "nail down" the 'microbial community interac­tion' found by the co-culture experiment, Zheng et al.  grew Methylomonas sp. LW13 in nitrate (NO₃⁻) mineral salts medium in air-tight culture vials in which part of the air (O₂) in the headspace gases was repeatedly replaced by gases collected from cultures of wild-type strain R. pa­lustris CGA010 that had been grown phototrophically in Mo-free minimal medium supplemented with acetate (C₂H₃O₂⁻), thiosulfate (S₂O₃²⁻), and ¹³C-labeled bi­car­bo­na­te (H¹³CO₃⁻). After 5 days with daily exchange of the headspace gases, they found by mass spectrometry ¹³C labelled glucose-6-phosphate and ¹³C fructose-6-phos­phate in Methylomonas cell lysates. This supported their conclusion that ¹³CH₄ produced by the Fe-only nitroge­na­se of R. palustris  was incorporated into Methylomonas cellular components (clearly not a case of "spooky in­ter­action at a distance" here!). The authors deem it likely that microbial interactions like these occur in nature be­cause CH₄ is produced concomitantly with NH₃/NH₄⁺ by Fe-only nitrogenases, which are known to be active in na­tural habitats of bacteria like R. palustris, and available to support growth of methanotrophic (CH₄-oxidizing) bacteria in the anoxic zones and, by diffusion, in the oxic zones.
 

Not a detour: The global carbon cycle

It may appear as a stretch to jump from methane-producing R. palustris and their local commu­ni­ty interactions to the 'global carbon cycle'. But actually it's not. Climate scientists consider metha­ne as one of the primary greenhouse gases, and nailing anthropogenic and biogeochemical sour­ces of at­mo­sphe­ric CH₄ together with determining their proportional quantitative  contributions provide cru­cial parame­ters for increasingly reliable climate models. In a not-quite-recent yet still valid review (open ac­cess PDF), James Ferry from Penn State University, Pennsylvania, PA, summa­ri­zed the pre­sent knowl­­edge about the position of methane in the bio­lo­gi­cal carbon cycle as follows:

Earth's biosphere contains diverse oxygen-free (anaerobic) environments where complex or­ganic matter is decomposed to CH₄ in a process called biomethanation that is an essential link in the global carbon cycle (Figure 4). In the cycle, atmospheric CO₂ is fixed into [cya­no­bac­te­ri­al and] plant bio­mass primarily via oxygenic photosynthesis (step 1). Microbes in aerobic environments digest the biomass consuming O₂ and returning CO₂ to the atmosphere (step 2). A portion of the biomass also enters diverse O₂-free en­vi­ron­ments (step 3) such as the lower intestinal tract of humans, wetlands, rice paddy soils, and the rumen of livestock where anaerobic microbes digest the organic matter producing CO₂ and CH₄ (steps 4 – 6) in a ratio of approximately 1:1 (biogas). (Addition in square brackets by the author.)

Figure 4. The global carbon cycle. See text for the nine numbered steps. Source

In freshwater environments, biomethanation in­vol­ves a minimum of three metabolic groups from the domains Bacteria and Archaea. The fermentative group decomposes biomass primarily to butyrate, propionate, acetate, formate, and H₂ plus CO₂ (step 4). The obligate proton-reducing acetogenic group (acetogens) converts the butyrate and propionate to acetate, CO₂, and H₂ or HCO₂⁻ (formate) (step 5). The thermodynamics of these conversions are un­favorable under standard conditions of equimolar reactants and products, requiring a symbiosis with the methane-producing group (methanogens) that metabolizes the products to thermodynamically fa­vorable levels (step 6). The methanogens produce CH₄ by two major pathways. In the CO₂-reduction pathway, formate or H₂ is oxidized and CO₂ is reduced to CH₄. In the aceticlastic pathway, C₂H₃O₂⁻ (acetate) is cleaved with the carbonyl group oxidized to CO₂ and the methyl group reduced to CH₄. In most freshwater environ­ments, the aceticlastic group is responsible for approximately two-thirds with most of the re­main­ing one-third produced by CO₂-reducing methanogens. Smaller, yet significant amounts of CH₄, are produced from the methyl groups of methanol, methylamines, and dimethyl­sul­fide. Some of the CH₄ is oxidized to CO₂ (step 7) by a consortia of anaerobes that reduce ei­ther sulfate, nitrate, manganese, or iron. The CH₄ also diffuses into aerobic environments (step 8) where O₂-requiring methanotrophic microbes oxidize it to CO₂ (step 9).
 

Figure 5. Average CH₄ production rates (μmol· g_DW^–1·h^–1) obtained from multiple long-term measure­ments (2 – 5 days) with a membrane inlet mass spec­trometer. The rates are shown by colour according to the environment from which the Cyanobacteria were originally iso­la­ted; dark blue, light blue, and green for marine, freshwater, and soil environments, respecti­ve­ly. The rates are presented in comparison to three known methanogens. Rates for the methano­gens were obtained from references given in the publication. Modified from Source

Within this grand scheme, R. palustris would have a role in "step 6" when it finds itself in a low-O₂ or anoxic en­vi­ronment (they have flagella and are thus motile). But this Alphaproteobacterium is probably not an eccentric intru­der into the realm of the methanogenic Archaea. Zheng et al. found by data mining anfD gene homologs for Fe-only ni­trogenases in ~9% of the sequenced genomes of all bac­terial phyla, notably among Proteobacteria and Firmi­cu­tes (genomes from these both groups are over­represented in the genome collections though). Homo­logs of anfD were not (yet) detected in their genomes but the ubiquitous Cyanobacteria are apparently never­the­less "on board" when it comes to bacterial methane pro­duc­tion. Mina Bižić-Ionescu and her colleagues from the IGB Leibnitz Institute, Neuglobsow, Germany, could show in a recent study (pre-publication in bioRχiv) that a num­ber of them produce CH₄ directly – that is, not by their known capacity to metabolize methylphosphonates un­der phos­phate-limiting conditions – under oxic growth con­di­tions with or without light (Figure 5). Although the Cyanos cannot compete with the Archaea for efficiency in methane production, their contribution to the entire "bio­­genic methane budget" is certainly not negligible as they thrive in much higher numbers than the latter. The authors estimate (conservatively) a mean contribution of 9.5 Tg·y^–1 (2.2 – 16.8 Tg·y^–1) of the cyanos to the global 190 ± 25 Tg·y^–1 biogenic methane in 2006 (1 Tg = 10¹² gram = 1 million tons). As far as I'm aware, nobody has yet calculated how much of these 190 Tg·y^–1 is attributable to R. palustris  and related purple non-sulfur bacteria (Rhodospirillaceae).