Evolving understanding of rumen methanogen ecophysiology.

Production of methane by methanogenic archaea, or methanogens, in the rumen of ruminants is a thermodynamic necessity for microbial conversion of feed to volatile fatty acids, which are essential nutrients for the animals. On the other hand, methane is a greenhouse gas and its production causes energy loss for the animal. Accordingly, there are ongoing efforts toward developing effective strategies for mitigating methane emissions from ruminant livestock that require a detailed understanding of the diversity and ecophysiology of rumen methanogens. Rumen methanogens evolved from free-living autotrophic ancestors through genome streamlining involving gene loss and acquisition. The process yielded an oligotrophic lifestyle, and metabolically efficient and ecologically adapted descendants. This specialization poses serious challenges to the efforts of obtaining axenic cultures of rumen methanogens, and consequently, the information on their physiological properties remains in most part inferred from those of their non-rumen representatives. This review presents the current knowledge of rumen methanogens and their metabolic contributions to enteric methane production. It also identifies the respective critical gaps that need to be filled for aiding the efforts to mitigate methane emission from livestock operations and at the same time increasing the productivity in this critical agriculture sector.

Nitrite reductase activity in F420-dependent sulphite reductase (Fsr) from Methanocaldococcus jannaschii.

Methanocaldococcus jannaschii (Mj), a hyperthermophilic and evolutionarily deeply rooted methanogenic archaeon from a deep-sea hydrothermal vent, produces F420-dependent sulphite reductase (Fsr) in response to exposure to sulphite. This enzyme allows Mj to detoxify sulphite, a potent inhibitor of methyl coenzyme-M reductase (Mcr), by reducing it to sulphide with reduced coenzyme F420 (F420H2) as an electron donor; Mcr is essential for energy production for a methanogen. Fsr allows Mj to utilize sulphite as a sulphur source. Nitrite is another potent inhibitor of Mcr and is toxic to methanogens. It is reduced by most sulphite reductases. In this study, we report that MjFsr reduced nitrite to ammonia with F420H2 with physiologically relevant K m values (nitrite, 8.9 µM; F420H2, 9.7 µM). The enzyme also reduced hydroxylamine with a K m value of 112.4 µM, indicating that it was an intermediate in the reduction of nitrite to ammonia. These results open the possibility that Mj could use nitrite as a nitrogen source if it is provided at a low concentration of the type that occurs in its habitat.

A Reduced F420-Dependent Nitrite Reductase in an Anaerobic Methanotrophic Archaeon.

Anaerobic methanotrophic archaea (ANME), which oxidize methane in marine sediments through syntrophic associations with sulfate-reducing bacteria, carry homologs of coenzyme F420-dependent sulfite reductase (Fsr) of Methanocaldococcus jannaschii, a hyperthermophilic methanogen from deep-sea hydrothermal vents. M. jannaschii Fsr (MjFsr) and ANME-Fsr belong to two phylogenetically distinct groups, FsrI and FsrII, respectively. MjFsrI reduces sulfite to sulfide with reduced F420 (F420H2), protecting methyl coenzyme M reductase (Mcr), an essential enzyme for methanogens, from sulfite inhibition. However, the function of FsrIIs in ANME, which also rely on Mcr and live in sulfidic environments, is unknown. We have determined the catalytic properties of FsrII from a member of ANME-2c. Since ANME remain to be isolated, we expressed ANME2c-FsrII in a closely related methanogen, Methanosarcina acetivorans. Purified recombinant FsrII contained siroheme, indicating that the methanogen, which lacks a native sulfite reductase, produced this coenzyme. Unexpectedly, FsrII could not reduce sulfite or thiosulfate with F420H2. Instead, it acted as an F420H2-dependent nitrite reductase (FNiR) with physiologically relevant Km values (nitrite, 5 µM; F420H2, 14 µM). From kinetic, thermodynamic, and structural analyses, we hypothesize that in FNiR, F420H2-derived electrons are delivered at the oxyanion reduction site at a redox potential that is suitable for reducing nitrite (E0' [standard potential], +440 mV) but not sulfite (E0', -116 mV). These findings and the known nitrite sensitivity of Mcr suggest that FNiR may protect nondenitrifying ANME from nitrite toxicity. Remarkably, by reorganizing the reductant processing system, Fsr transforms two analogous oxyanions in two distinct archaeal lineages with different physiologies and ecologies. IMPORTANCE Coenzyme F420-dependent sulfite reductase (Fsr) protects methanogenic archaea inhabiting deep-sea hydrothermal vents from the inactivation of methyl coenzyme M reductase (Mcr), one of their essential energy production enzymes. Anaerobic methanotrophic archaea (ANME) that oxidize methane and rely on Mcr, carry Fsr homologs that form a distinct clade. We show that a member of this clade from ANME-2c functions as F420-dependent nitrite reductase (FNiR) and lacks Fsr activity. This specialization arose from a distinct feature of the reductant processing system and not the substrate recognition element. We hypothesize FNiR may protect ANME Mcr from inactivation by nitrite. This is an example of functional specialization within a protein family that is induced by changes in electron transfer modules to fit an ecological need.

Reduced protein sequence patterns in identifying key structural elements of dissimilatory sulfite reductase homologs.

Methanogenic archaea carry homologs of dissimilatory sulfite reductase (Dsr), called Dsr Like proteins (DsrLP). Dsr reduces sulfite to sulfide, a key step in an Earth's ancient metabolic process called dissimilatory sulfate reduction. The DsrLPs do not function as Dsr, and a computational approach is needed to develop hypotheses for guiding wet bench investigations on DsrLP's function. To make the computational analysis process efficient, the DsrLP amino acid sequences were transformed using only eight alphabets functionally representing twenty amino acids. The resultant reduced amino acid sequences were analyzed to identify conserved signature patterns in DsrLPs. Many of these patterns mapped on critical structural elements of Dsr and some were associated tightly with particular DsrLP groups. A search into the UniProtKB database identified several proteins carrying DsrLP's signature patterns; cysteine desulfurase, nucleosidase, and uroporphyrinogen III methylase were such matches. These outcomes provided clues to the functions of DsrLPs and highlighted the utility of the computational approach used.