Ralstonia eutropha
Encyclopedia
Ralstonia eutropha is a gram-negative
soil
bacterium of the betaproteobacteria
class.
organisms were clustered into the group Hydrogenomonas. R. eutropha was originally named Hydrogenomonas eutrophus because it fell under the Hydrogenomonas classification and was “well nourished and robust”. Some of the original H. eutrophus cultures isolated were by Bovell and Wilde. After characterizing cell morphology
, metabolism
and GC content
, the Hydrogenomonas nomenclature was disbanded because it comprised many species of microorganisms. R. eutropha at this time was renamed Alcaligenes eutropha because it was a microorganism with degenerate peritrichous flagellation
. Investigating pheynotype
, lipid
composition, fatty acid
composition and 16S rRNA
analysis, A. eutropha was found to belong to the genus Ralstonia and named Ralstonia eutropha. Upon further study of the Ralstonia genus, Ralstonia was found to comprise two phenotypically distinct clusters. The new genus Wautersia was created from one of these clusters which included R. eutropha. In turn R. eutropha was renamed Wautersia eutropha. Looking at DNA-DNA hybridization and phyenotype comparision with Cupridavidus necator, W. eutropha was found to be the same species as previously described C. necator. Because C. necator was named in 1987 far before the name change to R. eutropha and W. eutropha, the name C. necator was assigned to R. eutropha according to Rule 23a of the International Code of Nomenclature of Bacteria
.
ic lifestyle and an autotroph
ic lifestyle. Both organic compounds and hydrogen can be used as a source of energy R. eutropha can perform aerobic respiration
or anaerobic respiration
by denitrification
of nitrate and/or nitrite to nitrogen gas. When growing under autotrophic conditions R. eutropha fixes carbon through the pentose phosphate pathway
. R. eutropha is known to produce and sequester polyhydroxyalkanoate
(PHA) plastics when exposed to excess amounts of sugar substrate. PHA can accumulate to levels of approximately 90% of the cell's dry weight. In order to better characterize the lifestyle of R. eutropha, the genome
s of two strains have been sequenced
.
s that have [Ni-Fe] active sites
and all perform the following reaction:
The hydrogenases of R. eutropha are like other typical [Ni-Fe] hydrogenases because they are made up of a large and small subunit. The large subunit is where the [Ni-Fe] active site resides and the small subunit is composed of [Fe-S] clusters
. However, the hydrogenases of R. eutropha are different from typical [Ni-Fe] hydrogenases because they are tolerant to oxygen and are not inhibited by CO
. While the three hydrogenases perform the same reaction in the cell, each hydrogenase is linked to a different cellular process. The differences between the regulatory hydrogenase, membrane bound hydrogenase and soluble hydrogeanse in R. eutropha are described below.
subunit. The hydrogen gas is oxidized at the [Ni-Fe] center in the large subunit and in turn reduces the [Fe-S] clusters in the small subunit. It is unknown whether the electrons are transferred from the [Fe-S] clusters to the protein kinase domain. The histidine protein kinase activates a response regulator. The response regulator is active in the dephosphorylated form. The dephosphorylated response regulator promotes the transcription of the membrane bound hydrogenase and soluble hydrogenase.
through a specific cytochrome b
related protein in R. eutropha. Hydrogen gas is oxidized at the [Ni-Fe] active site in the large subunit and the electrons are shuttled through the [Fe-S] clusters in the small subunit to the cytochrome b-like protein. The MBH is located on the outer cytoplasmic membrane
. It recovers energy for the cell by funneling electrons into the respiratory chain and by increasing the proton gradient
. The MBH in R. eutropha is not inhibited by CO and is tolerant to oxygen.
reducing equivalence by oxidizing hydrogen gas. The SH is a heterodimer protein
with two subunits making up the large and small subunits of the [Ni-Fe] hydrogenase and the other two subunits comprising a protein similar to Complex I
(as shown in the accompanying figure). The [Ni-Fe] active site oxidized hydrogen gas which transfers electrons to a FMNa
cofactor, then to [Fe-S] clusters of the small hydrogenase subunit, then to another FMNb cofactor and finally to NAD+. The reducing equivalence provide a means for fixing carbon dioxide when R. eutropha is growing autotrophically.
The [Ni-Fe] hydrogenase from Desulfovibrio vulgaris
and Desulfovibrio gigas have similar protein structure to each other and represent typical [Ni-Fe] hydrogenases. The large subunit contains the [Ni-Fe] active site buried deep in the core of the protein and the small subunit contains [Fe-S] clusters. The Ni atom is coordinated to the Desulfovibrio hydrogenase by 4 cysteine
ligand
s. Two of these same cysteine ligands also bridge the Fe of the [Ni-Fe] active site. The Fe atom also contains three ligands, one CO and two CN
that complete the active site. It is predicted these additional ligands might contribute to the reactivity or help stabilize the Fe atom in the +2 oxidation state. Typical [NiFe] hydrogenases like those of D. vulgaris and D. gigas are poisioned by oxygen because an oxygen atom binds strongly to the NiFe active site.
The soluble [Ni-Fe] hydrogenases in R. eutropha are unique for other organisms because it is oxygen tolerant. The active site of the SH has been studied to learn why this protein is tolerant to oxygen. A major difference of the [Ni-Fe] hydrogenases of R. eutropha is it has more coordinating ligands then in typical [Ni-Fe] hydrogenases. Two cystine ligands bridge the Ni atom and Fe atom of the active site in R. eutropha. Two modified cysteine sulfenate ligands also are predicted to coordinate the Ni atom. An additional CN ligand is added to the Ni atom in R. eutropha and removing this CN ligand makes the enzyme suseptable to oxygen. Also, the Fe has an additional CN ligand bound in addition to the 2 CN ligands and 1 CO ligand present in typical NiFe hydrogenases (see the above figure for a schematic of the active site). These additional ligands are predicted to help the protein be more oxygen tolerant because it stabilizes the Ni atom in the active, +2 oxidation state.
in urine as a nitrogen source and use hydrogen as an energy source in order to create dense cultures that could be used as a source of protein. Also, the electrolysis of water
was one way of creating oxygenic atmosphere in space and R. eutropha was investigated to recycle the hydrogen produced during this process. Today the oxygen tolerant hydrogenases are being used to investigate biofuels. Hydrogenases from R. eutropha have been used to coat electrode surfaces to create hydrogen fuel cell
s tolerant to oxygen and carbon monoxide and to design hydrogen producing light complexes
. In addition, the hydrogenases from R. eutropha have been used to create hydrogen sensors.
Gram-negative
Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counterstain is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color...
soil
Soil
Soil is a natural body consisting of layers of mineral constituents of variable thicknesses, which differ from the parent materials in their morphological, physical, chemical, and mineralogical characteristics...
bacterium of the betaproteobacteria
Betaproteobacteria
Betaproteobacteria is a class of Proteobacteria. Betaproteobacteria are, like all Proteobacteria, gram-negative.The Betaproteobacteria consist of several groups of aerobic or facultative bacteria which are often highly versatile in their degradation capacities, but also contain chemolithotrophic...
class.
Taxonomy
R. eutropha has gone through a series of name changes. In the first half of the 20th century many microorganisms were isolated for their ability to utilize hydrogen. Hydrogen metabolizing chemolithotrophicLithotroph
A lithotroph is an organism that uses an inorganic substrate to obtain reducing equivalents for use in biosynthesis or energy conservation via aerobic or anaerobic respiration. Known chemolithotrophs are exclusively microbes; No known macrofauna possesses the ability to utilize inorganic...
organisms were clustered into the group Hydrogenomonas. R. eutropha was originally named Hydrogenomonas eutrophus because it fell under the Hydrogenomonas classification and was “well nourished and robust”. Some of the original H. eutrophus cultures isolated were by Bovell and Wilde. After characterizing cell morphology
Morphology (biology)
In biology, morphology is a branch of bioscience dealing with the study of the form and structure of organisms and their specific structural features....
, metabolism
Metabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
and GC content
GC-content
In molecular biology and genetics, GC-content is the percentage of nitrogenous bases on a DNA molecule that are either guanine or cytosine . This may refer to a specific fragment of DNA or RNA, or that of the whole genome...
, the Hydrogenomonas nomenclature was disbanded because it comprised many species of microorganisms. R. eutropha at this time was renamed Alcaligenes eutropha because it was a microorganism with degenerate peritrichous flagellation
Flagellum
A flagellum is a tail-like projection that protrudes from the cell body of certain prokaryotic and eukaryotic cells, and plays the dual role of locomotion and sense organ, being sensitive to chemicals and temperatures outside the cell. There are some notable differences between prokaryotic and...
. Investigating pheynotype
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
, lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...
composition, fatty acid
Fatty acid
In chemistry, especially biochemistry, a fatty acid is a carboxylic acid with a long unbranched aliphatic tail , which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from...
composition and 16S rRNA
16S ribosomal RNA
16S ribosomal RNA is a component of the 30S subunit of prokaryotic ribosomes. It is approximately 1.5kb in length...
analysis, A. eutropha was found to belong to the genus Ralstonia and named Ralstonia eutropha. Upon further study of the Ralstonia genus, Ralstonia was found to comprise two phenotypically distinct clusters. The new genus Wautersia was created from one of these clusters which included R. eutropha. In turn R. eutropha was renamed Wautersia eutropha. Looking at DNA-DNA hybridization and phyenotype comparision with Cupridavidus necator, W. eutropha was found to be the same species as previously described C. necator. Because C. necator was named in 1987 far before the name change to R. eutropha and W. eutropha, the name C. necator was assigned to R. eutropha according to Rule 23a of the International Code of Nomenclature of Bacteria
International Code of Nomenclature of Bacteria
The International Code of Nomenclature of Bacteria or Bacteriological Code governs the scientific names for bacteria, including Archaea. It denotes the rules for naming taxa of bacteria, according to their relative rank...
.
Metabolism
R. eutropha is a hydrogen-oxidizing bacterium (“knallgas” bacterium) capable of growing at the interface of anaerobic and aerobic environments. It can easily adapt between a heterotrophHeterotroph
A heterotroph is an organism that cannot fix carbon and uses organic carbon for growth. This contrasts with autotrophs, such as plants and algae, which can use energy from sunlight or inorganic compounds to produce organic compounds such as carbohydrates, fats, and proteins from inorganic carbon...
ic lifestyle and an autotroph
Autotroph
An autotroph, or producer, is an organism that produces complex organic compounds from simple inorganic molecules using energy from light or inorganic chemical reactions . They are the producers in a food chain, such as plants on land or algae in water...
ic lifestyle. Both organic compounds and hydrogen can be used as a source of energy R. eutropha can perform aerobic respiration
Cellular respiration
Cellular respiration is the set of the metabolic reactions and processes that take place in the cells of organisms to convert biochemical energy from nutrients into adenosine triphosphate , and then release waste products. The reactions involved in respiration are catabolic reactions that involve...
or anaerobic respiration
Cellular respiration
Cellular respiration is the set of the metabolic reactions and processes that take place in the cells of organisms to convert biochemical energy from nutrients into adenosine triphosphate , and then release waste products. The reactions involved in respiration are catabolic reactions that involve...
by denitrification
Denitrification
Denitrification is a microbially facilitated process of nitrate reduction that may ultimately produce molecular nitrogen through a series of intermediate gaseous nitrogen oxide products....
of nitrate and/or nitrite to nitrogen gas. When growing under autotrophic conditions R. eutropha fixes carbon through the pentose phosphate pathway
Pentose phosphate pathway
The pentose phosphate pathway is a process that generates NADPH and pentoses . There are two distinct phases in the pathway. The first is the oxidative phase, in which NADPH is generated, and the second is the non-oxidative synthesis of 5-carbon sugars...
. R. eutropha is known to produce and sequester polyhydroxyalkanoate
Polyhydroxyalkanoates
Polyhydroxyalkanoates or PHAs are linear polyesters produced in nature by bacterial fermentation of sugar or lipids. They are produced by the bacteria to store carbon and energy. More than 150 different monomers can be combined within this family to give materials with extremely different properties...
(PHA) plastics when exposed to excess amounts of sugar substrate. PHA can accumulate to levels of approximately 90% of the cell's dry weight. In order to better characterize the lifestyle of R. eutropha, the genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
s of two strains have been sequenced
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
.
Hydrogenases
R. eutropha can use hydrogen gas as a source of energy when growing under autotrophic conditions. It contains three different hydrogenaseHydrogenase
A hydrogenase is an enzyme that catalyses the reversible oxidation of molecular hydrogen . Hydrogenases play a vital role in anaerobic metabolism....
s that have [Ni-Fe] active sites
Nickel-dependent hydrogenase
Hydrogenases are enzymes that catalyze the reversible activation of hydrogen and which occur widely in prokaryotes as well as in some eukaryotes. There are various types of hydrogenases, but all of them seem to contain at least one iron-sulphur cluster...
and all perform the following reaction:
- H2
2H+ + 2e-
The hydrogenases of R. eutropha are like other typical [Ni-Fe] hydrogenases because they are made up of a large and small subunit. The large subunit is where the [Ni-Fe] active site resides and the small subunit is composed of [Fe-S] clusters
Iron-sulfur protein
Iron-sulfur proteins are proteins characterized by the presence of iron-sulfur clusters containing sulfide-linked di-, tri-, and tetrairon centers in variable oxidation states...
. However, the hydrogenases of R. eutropha are different from typical [Ni-Fe] hydrogenases because they are tolerant to oxygen and are not inhibited by CO
Carbon monoxide
Carbon monoxide , also called carbonous oxide, is a colorless, odorless, and tasteless gas that is slightly lighter than air. It is highly toxic to humans and animals in higher quantities, although it is also produced in normal animal metabolism in low quantities, and is thought to have some normal...
. While the three hydrogenases perform the same reaction in the cell, each hydrogenase is linked to a different cellular process. The differences between the regulatory hydrogenase, membrane bound hydrogenase and soluble hydrogeanse in R. eutropha are described below.
Regulatory Hydrogenase (RH)
The first hydrogenase is a regulatory hydrogenase (RH) that signals to the cell hydrogen is present. The RH is a protein containing large and small [Ni-Fe] hydrogenase subunits attached to a histidine protein kinaseProtein kinase
A protein kinase is a kinase enzyme that modifies other proteins by chemically adding phosphate groups to them . Phosphorylation usually results in a functional change of the target protein by changing enzyme activity, cellular location, or association with other proteins...
subunit. The hydrogen gas is oxidized at the [Ni-Fe] center in the large subunit and in turn reduces the [Fe-S] clusters in the small subunit. It is unknown whether the electrons are transferred from the [Fe-S] clusters to the protein kinase domain. The histidine protein kinase activates a response regulator. The response regulator is active in the dephosphorylated form. The dephosphorylated response regulator promotes the transcription of the membrane bound hydrogenase and soluble hydrogenase.
Membrane Bound Hydrogenase (MBH)
The membrane bound hydrogenase (MBH) is linked to the respiratory chainElectron transport chain
An electron transport chain couples electron transfer between an electron donor and an electron acceptor with the transfer of H+ ions across a membrane. The resulting electrochemical proton gradient is used to generate chemical energy in the form of adenosine triphosphate...
through a specific cytochrome b
Cytochrome b
Cytochrome b/b6 is the main subunit of transmembrane cytochrome bc1 and b6f complexes. In addition, it commonly refers to a region of mtDNA used for population genetics and phylogenetics.- Function :...
related protein in R. eutropha. Hydrogen gas is oxidized at the [Ni-Fe] active site in the large subunit and the electrons are shuttled through the [Fe-S] clusters in the small subunit to the cytochrome b-like protein. The MBH is located on the outer cytoplasmic membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
. It recovers energy for the cell by funneling electrons into the respiratory chain and by increasing the proton gradient
Electrochemical gradient
An electrochemical gradient is a spatial variation of both electrical potential and chemical concentration across a membrane; that is, a combination of the membrane potential and the pH gradient...
. The MBH in R. eutropha is not inhibited by CO and is tolerant to oxygen.
Soluble Hydrogenase (SH)
The soluble hydrogenase (SH) creates a NADHNicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide, abbreviated NAD, is a coenzyme found in all living cells. The compound is a dinucleotide, since it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine base and the other nicotinamide.In metabolism, NAD is involved...
reducing equivalence by oxidizing hydrogen gas. The SH is a heterodimer protein
Protein dimer
In biochemistry, a dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules like proteins or nucleic acids...
with two subunits making up the large and small subunits of the [Ni-Fe] hydrogenase and the other two subunits comprising a protein similar to Complex I
NADH dehydrogenase
NADH dehydrogenase is an enzyme located in the inner mitochondrial membrane that catalyzes the transfer of electrons from NADH to coenzyme Q...
(as shown in the accompanying figure). The [Ni-Fe] active site oxidized hydrogen gas which transfers electrons to a FMNa
Flavin mononucleotide
Flavin mononucleotide , or riboflavin-5′-phosphate, is a biomolecule produced from riboflavin by the enzyme riboflavin kinase and functions as prosthetic group of various oxidoreductases including NADH dehydrogenase as well as cofactor in biological blue-light photo receptors...
cofactor, then to [Fe-S] clusters of the small hydrogenase subunit, then to another FMNb cofactor and finally to NAD+. The reducing equivalence provide a means for fixing carbon dioxide when R. eutropha is growing autotrophically.
Soluble Hydrogenase Active Site
This section highlights the differences between R. eutropha SH with other anaerobic [Ni-Fe] hydrogenases that are poisoned by oxygen. The active site of the SH of R. eutropha H16 has been extensively studied because R. eutropha H16 can be produced in large amounts, can be genetically manipulated and can be analyzed with spectrographic techniques. However, no crystal structure is currently available for the R. eutropha H16 soluble hydrogenase in the presence of oxygen to determine the interactions of the active site with the rest of the protein.Typical Anaerobic [Ni-Fe] Hydrogenases
The [Ni-Fe] hydrogenase from Desulfovibrio vulgaris
Desulfovibrio vulgaris
Desulfovibrio vulgaris is a species of Gram-negative sulfate-reducing bacteria in the Desulfovibrionaceae family....
and Desulfovibrio gigas have similar protein structure to each other and represent typical [Ni-Fe] hydrogenases. The large subunit contains the [Ni-Fe] active site buried deep in the core of the protein and the small subunit contains [Fe-S] clusters. The Ni atom is coordinated to the Desulfovibrio hydrogenase by 4 cysteine
Cystine
Cystine is a dimeric amino acid formed by the oxidation of two cysteine residues that covalently link to make a disulfide bond. This organosulfur compound has the formula 2. It is a white solid, and melts at 247-249 °C...
ligand
Ligand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...
s. Two of these same cysteine ligands also bridge the Fe of the [Ni-Fe] active site. The Fe atom also contains three ligands, one CO and two CN
Cyanide
A cyanide is a chemical compound that contains the cyano group, -C≡N, which consists of a carbon atom triple-bonded to a nitrogen atom. Cyanides most commonly refer to salts of the anion CN−. Most cyanides are highly toxic....
that complete the active site. It is predicted these additional ligands might contribute to the reactivity or help stabilize the Fe atom in the +2 oxidation state. Typical [NiFe] hydrogenases like those of D. vulgaris and D. gigas are poisioned by oxygen because an oxygen atom binds strongly to the NiFe active site.
R. eutropha Oxygen Tolerant Soluble Hydrogenase
The soluble [Ni-Fe] hydrogenases in R. eutropha are unique for other organisms because it is oxygen tolerant. The active site of the SH has been studied to learn why this protein is tolerant to oxygen. A major difference of the [Ni-Fe] hydrogenases of R. eutropha is it has more coordinating ligands then in typical [Ni-Fe] hydrogenases. Two cystine ligands bridge the Ni atom and Fe atom of the active site in R. eutropha. Two modified cysteine sulfenate ligands also are predicted to coordinate the Ni atom. An additional CN ligand is added to the Ni atom in R. eutropha and removing this CN ligand makes the enzyme suseptable to oxygen. Also, the Fe has an additional CN ligand bound in addition to the 2 CN ligands and 1 CO ligand present in typical NiFe hydrogenases (see the above figure for a schematic of the active site). These additional ligands are predicted to help the protein be more oxygen tolerant because it stabilizes the Ni atom in the active, +2 oxidation state.
Applications
The oxygen tolerant hydrogenases of R. eutropha have been studied for many diverse purposes. R. eutropha was studied as an attractive organism to help support life in space. It could fix carbon dioxide as a carbon source, use the ureaUrea
Urea or carbamide is an organic compound with the chemical formula CO2. The molecule has two —NH2 groups joined by a carbonyl functional group....
in urine as a nitrogen source and use hydrogen as an energy source in order to create dense cultures that could be used as a source of protein. Also, the electrolysis of water
Electrolysis of water
Electrolysis of water is the decomposition of water into oxygen and hydrogen gas due to an electric current being passed through the water.-Principle:...
was one way of creating oxygenic atmosphere in space and R. eutropha was investigated to recycle the hydrogen produced during this process. Today the oxygen tolerant hydrogenases are being used to investigate biofuels. Hydrogenases from R. eutropha have been used to coat electrode surfaces to create hydrogen fuel cell
Fuel cell
A fuel cell is a device that converts the chemical energy from a fuel into electricity through a chemical reaction with oxygen or another oxidizing agent. Hydrogen is the most common fuel, but hydrocarbons such as natural gas and alcohols like methanol are sometimes used...
s tolerant to oxygen and carbon monoxide and to design hydrogen producing light complexes
Photosystem I
Photosystem I is the second photosystem in the photosynthetic light reactions of algae, plants, and some bacteria. Photosystem I is so named because it was discovered before photosystem II. Aspects of PS I were discovered in the 1950s, but the significances of these discoveries was not yet known...
. In addition, the hydrogenases from R. eutropha have been used to create hydrogen sensors.