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 complex. The methanogenesis pathway utilizes the same carbon source as the remaining seven subsystems for cell growth. The even 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 acetate, Acetyl-CoA is synthesized from the AMP-forming acetate CoA ligase. Using carbon dioxide, M. maripaludis can generate Acetyl-CoA from carbon monoxide, produced from the reduction of carbon dioxide, and methyl-THMPT, produced as an intermediate of methanogenesis during biosynthesis. 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 CO_2. 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 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 ${\ce {N2}}$ 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 ${\ce {N2}}$. The now reduced Fe protein is then oxidized by ATP and reducing the MoFe protein. The MoFe protein then reduces ${\ce {N2}}$ to ammonia. This reductive step requires high energy to be carried out as it uses the electrons from the reduced ferredoxinn. ${\ce {N2}}$ 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 in 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 will be converted to Erythrose-4-phosphate and Fructose-6-phosphate. Four enzymes are used in this conversion: Transketoloase, ribulose-phosphate 3-epimerase, ribose-r-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

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, 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 to synthesize complex carbon molecules. Several steps and lacking 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 Notes:

Methanococcus maripaludis is a weakly-motile coccus specie of methanogenic archaea of 0.9–1.3 µm diameters. It grows best in conditions of 20°C and 45°C with a pH of 6.5 to 8.0.

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 in methanogens, their cell walls lack murein and ether-linked membrane lipids and other biochemical properties. The S-layer is composed of glycoproteins that encloses the entire cell associated with protection from direct interactions with the environment. Additionally, it provides archaea cells a stabilization barrier that is resistant to environmental changes.

Flagella and Pili

Methanococcus maripaludis consists of two surface appendages, flagella and pili. Archaeal flagella contains distinctive prokaryotic motility structure that is similar to bacterial type IV pili (T4P) such as constructed from proteins bearing class III signal peptides that are cleaved by specific signal peptidase and possess homologous genes encoding an ATPase and conserved membrane protein for appendage assembly. The flagella of M. maripaludis is composed of three flagellin glycoproteins modified with a tetrasaccharide N-linked to multiple positions and responsible for locomotion, critical for continued attachment to surfaces, and cell-to-cell contacts. 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, and flagellin export, 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, commonly glycosylation. Consequently, these proteins exhibit larger proteins than their expected gene sequence.

M. maripaludis contains flagella for motility and pili. The pili's function for this microbe is not fully known but could include cellular attachment, motility, and biofilm 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 four, 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 certain features present in most methanogens, 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]

${\ce {CO2}}$ emissions are a growing problem in the world today being the leading source of global warming. M. maripaludis' ability to uptake ${\ce {CO2}}$ from the environment in the presence of ${\ce {N2}}$ allows for a potential route for conversion of ${\ce {CO2}}$ emissions to a useful fuel like methane. It is able to capture and convert ${\ce {CO2}}$ from power and chemical plant emissions as well.