User:Eesakguee/Methanococcus maripaludis

Methanococcus maripaludis is a species of methanogenic archaea found in marine environments, predominantly salt marshes. M. maripaludis is a non-pathogenic, gram-negative, weakly motile, non-spore-forming, and strictly anaerobic mesophile with a pleomorphic coccoid-rod shape of 1.2 by 1.6 μm, in average size.   The genome of M. maripaludis consists of 1,661,137 bp and has been sequenced, leading to the identification of over 1,700 protein-coding genes. In ideal conditions, M. maripaludis grows quickly and can double every two hours.

Metabolism[edit]
The metabolic landscape of M. maripaludis, a hydrogenotrophic methanogen, consists of eight major subsystems, providing pathways for energy generation and cell growth. These subsystems include amino acid metabolism, glycolysis/glycogen metabolism, methanogenesis, nitrogen metabolism, non-oxidative pentose phosphate pathway (NOPPP), nucleotide metabolism and reductive citric acid (RTCA) cycle.

Methanogenesis, the process of reducing carbon dioxide to methane, serves as the primary pathway for energy generation using unique numbers of coenzymes and membrane-bound enzyme complexes. The methanogenesis pathway utilizes the same carbon source as the remaining seven subsystems for cell growth. Additionally, the subsystems use two essential intermediates, acetyl CoA and pyruvate, to produce precursors critical for cell growth.

Amino Acid Metabolism
M. maripaludis uses carbon dioxide and acetate as substrates for amino acid biosynthesis. Each of these substrates can produce Acetyl-CoA through various mechanisms. Using carbon dioxide, M. maripaludis can generate Acetyl-CoA from methyl-THMPT, an intermediate of methanogenesis, and carbon monoxide, produced from the reduction of carbon dioxide. Using acetate, Acetyl-CoA is synthesized from the AMP-forming acetate CoA ligase. Acetyl-CoA then acts as a precursor to pyruvate, which promotes methanogenesis and alanine biosynthesis. Pyruvate can be converted to L-alanine via alanine dehydrogenase, which is a reversible reaction. Once alanine is synthesized, it can be transported into the microbe via alanine permease.

Glucose/Glycogen Metabolism
M. maripaludis has a modified Embden Meyerhof-Parnas (EMP) pathway, also known as glycolysis, responsible for the catabolic breakdown of glucose into pyruvate. Dissimilarly to other organisms that reduce NAD to NADH in the EMP Pathway, M. maripaludis reduces ferrodoxins. Additionally, the protein kinases, responsible for transferring phosphate groups between compounds, uniquely rely on ADP rather than ATP. M. maripaludis is also capable of synthesizing glycogen. Due to experimentally observed activities of enzymes involved in both the catabolic and anabolic directions of the EMP Pathway, the latter is utilized to a higher extent, resulting in the formation of glycogen stores.

Methanogenesis
In M. maripaludis, the primary carbon source for methanogenesis is carbon dioxide, although alternatives such as formate are also used. Though all methanogens utilize certain key coenzymes, cofactors, and intermediates to produce methane, M. maripaludis undergoes the Wolfe cycle, which converts CO2 and hydrogen gas into methane and H2O. 7 different hydrogenases are present in M. maripaludis that allow for the usage of H2 as an electron donor to reduce CO2. Some strains and mutants of M. maripaludis have been shown to be capable of methanogenesis in the absence of hydrogen gas, though this is uncommon.

Methanogenesis in M. maripaludis occurs in the following steps:


 * 1) Reduction of CO2 via methanofuran and reduced ferredoxins
 * 2) Oxidation and subsequent reduction of the coenzyme F420 in the presence of H2
 * 3) Transfer of a methyl group from methyl-THMPT to coenzyme M (HS-CoM), driving translocation of 2Na+ across membrane to strengthen the proton gradient
 * 4) Demethylation of methyl-S-CoM to form methane and generate additional energy via subsequent reduction of byproducts with H2

Nitrogen Metabolism
M. maripaludis utilizes three sources of nitrogen: ammonia, alanine, and dinitrogen with ammonia. Nitrogen assimilation occurs in the bacteria through ammonia. Nitrogen assimilation is when an inorganic nitrogen compound is converted to an organic nitrogen compound. In M. maripaludis, glutamine synthetase is used to make glutamine from glutamate and ammonia. The glutamine created then is sent to continue through protein synthesis.

M. maripaludis utilizes alanine racemase and alanine permease for alanine uptake. A racemase enzyme is used to convert the inversion of stereochemistry within the molecule while a permease is a protein that catalyzes the transport of a molecule across the membrane.

Free N2 fixation is well established in ''M. maripaludis. M. maripaludis'' contains a multiprotein nitrogen complex containing a Fe protein and a MoFe. The ferredoxin is reduced and reduces the oxidized Fe, stripping the Fe of its electrons in the presence of N2. The now reduced Fe protein is oxidized by ATP, reducing the MoFe protein. The MoFe protein then reduces N2 to ammonia. This reductive step uses the electrons from the reduced ferredoxin which requires high amounts of energy. N2 fixation is unfavorable in M. maripaludis because of the high energy demand, so it is common for a cell to not activate this fixation when ammonia and alanine are available.

Non-Oxidative Pentose Phosphate Pathway (NOPPP)
The pentose phosphate pathway is essential for M. maripaludis to make nucleotides and nucleic acids. The non-oxidative pentose phosphate pathway (NOPPP) is regulated and used through substrate availability. In M. maripaludis, ribulose-5-phosphate is converted to erythrose-4-phosphate and fructose-6-phosphate. Four enzymes are used in this conversion: transketoloase, ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and translaldolase.

Nucleotide Metabolism
Nucleotide metabolism for M. maripaludis is well understood. In order to accomplish nucleic acid biosynthesis, the methanogen must produce pyrimidines, such as uridine triphosphate (UTP) and cytidine triphosphate (CTP), as well as purines such as guanine triphosphate (GTP) and adenosine triphosphate (ATP). In order to synthesize the pyrimidines, phosphoribosyl pyrophosphate (PRPP) combines with bicarbonate, L-glutamine, or orotate. This combination synthesizes uridine monophosphate, which can then be converted into uridine triphosphate (UTP). UMP also functions as a precursor to CTP.

In order to synthesize the purines, inosinic acid (IMP) is first made via a series of reactions, which include PRPP combining with glutamine to form 5-phosphoribosylamine. This reaction is catalyzed by PRPP synthetase. Once IMP is synthesized, it can be further converted into adenosine monophosphate (AMP) and guanine monophosphate (GMP). To synthesize AMP, IMP combines with adenylosuccinate. To synthesize GMP, IMP is converted into xanthine monophosphate (XMP) which can then be converted into GMP.

Reductive Citric Acid (RTCA) Cycle
The tricarboxylic acid cycle serves as a central metabolic pathway in aerobic organisms, playing an essential role in energy production and biosynthesis by generating electron carriers such as NADH and FAD. This is performed by oxidizing acetyl-CoA, derived from various nutrients and complex carbon molecules, to CO2 and H2O.

M. maripaludis, a strictly anerobic mesophile, undergoes an incomplete Reductive Citric Acid (RTA) Cycle to reduce CO2 and H2O and synthesize complex carbon molecules. Lacking several steps and essential enzymes, including phosphoenolpyruvate carboxykinase, citrate synthase, aconitate, and isocitrate dehydrogenase, hinders the completion of this cycle. Pyruvate, produced from glycolysis/gluconeogenesis, is an initial metabolite in M. maripaludis for the Tricarboxylic Acid Cycle.

Cell Structure [edit]
The irregular-shaped weakly-motile coccus, Methanococcus maripaludis, has a diameter of 0.9-1.3 µm with a single, electron-dense S-layer lacking peptidoglycan. Commonly found in methanogens, their cell walls lack murein and ether-linked membrane lipids, among other biochemical properties. The S-layer is composed of glycoproteins that enclose the entire cell, and it allows for protection from direct interactions with the environment. More specifically, it provides archaeal cells a stabilization barrier that is resistant to environmental changes. With these properties, M. maripaludis grows best in conditions between 20°C and 45°C with a pH of 6.5 to 8.0. Additionally, M. maripaludis consists of two surface appendages assisting in motility: flagella and pili.

Flagella and Pili
Archaeal flagella contain distinctive prokaryotic motility structures that are similar to bacterial type IV pili (T4P). They are constructed from proteins bearing class III signal peptides that are cleaved by specific signal peptidases. They also possess homologous genes, which encode an ATPase, and conserved membrane proteins for appendage assembly. The flagellum of M. maripaludis is composed of three flagellin glycoproteins, which are all modified with an N-linked tetrasaccharide. This is critical for continued attachment to surfaces, cell-to-cell contact, and locomotion. Both flagella and pili structures are used to attach to surfaces, allowing them the ability to remain in desirable environments.

The archaeal flagellum has a unique motility mechanism carried out through flagella-associated genes. Specifically, M. maripaludis encompasses a complete set of fla genes with three distinct flagellin genes, flaB1, flaB2 and flaB3, and a remaining eight genes, flaC-J. From the flagella locus, there are two major flagellin proteins required for flagella filaments, flaB1 and flaB2. Flagellin export also requires two specific proteins including flaH and flaI. The hook-like protein in M. maripaludis is strongly indicated from the minor flagellin protein, flaB3. The flagellins in numerous archaea undergo post-translational modifications, including glycosylation. Consequently, these flagella exhibit larger proteins than their expected gene sequence.

Similar to the flagella, the proteins involved in the pilus assembly of M. maripaludis exhibit resemblance to bacterial Type IV pili due to the presence of an N-terminal signal peptide and an anticipated N-terminal hydrophobic α-helix. The two pilin-like genes, MMP0236 (epdB) and MMP0237 (epdC), possess a short, atypical signal peptide ending in a conserved glycine. This is then succeeded by a hydrophobic segment, resulting to a distinct quaternary structure and pilus formation.

Genetic characteristics[edit]
Methanococcus maripaludis is one of four hydrogenotrophic methanogens, along with Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri, to have its genome sequenced. Of these three, Methanocaldococcus jannaschii is the closest living, known relative of M. maripaludis. M. maripaludis, like many other archaea, has one single, circular chromosome. According to the number of BlastP hits, or similar protein sequences identified by the Basic Local Alignment Search Tool (BLAST), in the genome sequence, M. maripaludis is similar to most other methanogens. However, M. maripaludis is missing common features, such as the ribulose bisphosphate carboxylase enzyme.

To date, 21 different strains of M. maripaludis have had their genomes sequenced, and each genome includes many copies of the chromosome in the singular cell, ranging from 5 to 55. Of its 1,722 protein coding genes, 835 ORFs, or open reading frames, have unknown functions and 129 ORFs are unique to M. maripaludis. Certain genes have been identified using in vivo transposon mutagenesis that have a chance of being essential for growth, making up approximately 30% of the genome. The sequenced genome also revealed about 48 protein transporter systems, largely dominated by ABC transporters followed by iron transporters.

As a subject of metabolic engineering, M. maripaludis has been genetically altered to produce non-native, desired products, such as geraniol and polyhydroxybutyrate. Recent research has also shown that M. maripaludis can be used to sequence a variety of promoters and ribosome-binding sites using CRISPR/Cas9 technology. Large deletions in the DNA can also be facilitated by a CRISPR/Cas9 system specifically designed for a strain named S0001. This technology can further be used to improve recombinant protein and gene expression. In addition, gene tagging and multiple nucleotide mutagenesis have been used for genetic manipulation in M. maripaludis.

Environmental roles[edit]
Methanogens play important roles in waste water treatment, carbon conversion, hydrogen production, and many other environmental processes. In terms of waste water treatment, methanogens have been used to anaerobically degrade waste into methane utilizing a symbiotic relationship with syntrophic bacteria. M. maripaludis, in addition to other methanogens, has the potential for generating fuels, such as methane and methanol, from CO2 emissions due to native CO2 uptake. CO2 emissions are currently one of the leading sources of global warming. The ability of M. maripaludis to uptake CO2 from the environment in the presence of N2 allows for a potential route for conversion of CO2 emissions to a useful fuel like methane. It is able to capture and convert CO2 from power and chemical plant emissions as well. Despite the many potential applications, the need for large amounts of hydrogen is an issue with using any methanogen for biomethane production.

Methanogens also play a role in biotechnology production. As previously discussed, they have been metabolically engineered to produce the bioplastic polymer polyhydroxybutyrate (PHB). This conversion reduces methane emissions and provides a biodegradable polymer substitute for plastic production. It is estimated that over 311 million tons of plastics are produced annually, and non-degradable, primary plastics are contributing 18% of the total atmospheric radiation. Methanotrophs are able to accumulate PHB when their nutrient supply is imbalanced, yielding 67% PHB. PHB materials are currently in demand for usage in packing supplies and materials to eliminate harmful waste. This discovery of using methanogens to produce polyhydroxybutyrate can help diminish green house gas pollution in the atmosphere and reduce the cost of PHB production.