Do bacteria exist which live exclusively on non-organic matter?

Do bacteria exist which live exclusively on non-organic matter?

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Are there bacteria which consume exclusively non-organic matter? Meaning that they do not require organic matter from other organisms to stay alive and reproduce? A completely pacifistic bacterium.

Or phrased differently, are there bacteria which can stay alive and reproduce indefinitely with only non-organic matter present (plus sunlight or thermal vents etc.)?

I'm asking specifically, to name one or more bacteria species which can do this.

Sulfate reducing bacteria get their energy converting sulfate to hydrogen sulfide. They are relatively common and may have been the source to turn some oil/gas deposits "sour". Specifics in Wikipedia.

Organic matter

Organic matter, organic material, or natural organic matter refers to the large source of carbon-based compounds found within natural and engineered, terrestrial, and aquatic environments. It is matter composed of organic compounds that have come from the remains of organisms such as plants and animals and their waste products in the environment. [1] Organic molecules can also be made by chemical reactions that don't involve life. [2] Basic structures are created from cellulose, tannin, cutin, and lignin, along with other various proteins, lipids, and carbohydrates. Organic matter is very important in the movement of nutrients in the environment and plays a role in water retention on the surface of the planet. [3]


R. pachyptila was discovered in 1977 on an expedition of the American bathyscaphe DSV Alvin to the Galápagos Rift led by geologist Jack Corliss. [4] The discovery was unexpected, as the team was studying hydrothermal vents and no biologists were included in the expedition. Many of the species found living near hydrothermal vents during this expedition had never been seen before.

At the time, the presence of thermal springs near the midoceanic ridges was known. Further research uncovered aquatic life in the area, despite the high temperature (around 350 – 380 °C). [5] [6]

Many samples were collected, for example, bivalves, polychaetes, large crabs, and R. pachyptila. [7] [8] It was the first time that species was observed.

R. pachyptila develops from a free-swimming, pelagic, nonsymbiotic trochophore larva, which enters juvenile (metatrochophore) development, becoming sessile, and subsequently acquiring symbiotic bacteria. [9] [10] The symbiotic bacteria, on which adult worms depend for sustenance, are not present in the gametes, but are acquired from the environment through the skin in a process akin to an infection. The digestive tract transiently connects from a mouth at the tip of the ventral medial process to a foregut, midgut, hindgut, and anus and was previously thought to have been the method by which the bacteria are introduced into adults. After symbionts are established in the midgut, they undergo substantial remodelling and enlargement to become the trophosome, while the remainder of the digestive tract has not been detected in adult specimens. [11]

Isolating the vermiform body from white chitonous tube, a small difference exists from the classic three subdivisions typical of phylum Pogonophora: [12] the prosoma, the mesosoma, and the metasoma.

The first body region is the vascularized branchial plume, which is bright red due to the presence of hemoglobin that contain up to 144 globin chains (each presumably including associated heme structures). These tube worm hemoglobins are remarkable for carrying oxygen in the presence of sulfide, without being inhibited by this molecule, as hemoglobins in most other species are. [13] [14] The plume provides essential nutrients to bacteria living inside the trophosome. If the animal perceives a threat or is touched, it retracts the plume and the tube is closed due to the obturaculum, a particular operculum that protects and isolates the animal from the external environment. [15]

The second body region is the vestimentum, formed by muscle bands, having a winged shape, and it presents the two genital openings at the end. [16] [17] The heart, extended portion of dorsal vessel, enclose the vestimentum. [18]

In the middle part, the trunk or third body region, is full of vascularized solid tissue, and includes body wall, gonads, and the coelomic cavity. Here is located also the trophosome, spongy tissue where a billion symbiotic, thioautotrophic bacteria and sulfur granules are found. [19] [20] Since the mouth, digestive system, and anus are missing, the survival of R. pachyptila is dependent on this mutualistic symbiosis. [21] This process, known as chemosynthesis, was recognized within the trophosome by Colleen Cavanaugh. [21]

The soluble hemoglobins, present in the tentacles, are able to bind O2 and H2S, which are necessary for chemosynthetic bacteria. Due to the capillaries, these compounds are absorbed by bacteria. [22] During the chemosynthesis, the mitochondrial enzyme rhodanase catalyzes the disproportionation reaction of the thiosulfate anion S2O3 2- to sulfur S and sulfite SO3 2- . [23] [24] The R. pachyptila’s bloodstream is responsible for absorption of the O2 and nutrients such as carbohydrates.

Nitrate and nitrite are toxic, but are required for biosynthetic processes. The chemosynthetic bacteria within the trophosome convert nitrate to ammonium ions, which then are available for production of amino acids in the bacteria, which are in turn released to the tube worm. To transport nitrate to the bacteria, R. pachyptila concentrates nitrate in its blood, to a concentration 100 times more concentrated than the surrounding water. The exact mechanism of R. pachyptila’s ability to withstand and concentrate nitrate is still unknown. [14]

In the posterior part, the fourth body region, is the opistosome, which anchors the animal to the tube and is used for the storage of waste from bacterial reactions. [25]

The discovery of bacterial invertebrate chemoautotrophic symbiosis, particularly in vestimentiferan tubeworms R. pachyptila [21] and then in vesicomyid clams and mytilid mussels revealed the chemoautotrophic potential of the hydrothermal vent tube worm. [26] Scientists discovered a remarkable source of nutrition that helps to sustain the conspicuous biomass of invertebrates at vents. [26] Many studies focusing on this type of symbiosis revealed the presence of chemoautotrophic, endosymbiotic, sulfur-oxidizing bacteria mainly in R. pachyptila, [27] which inhabits extreme environments and is adapted to the particular composition of the mixed volcanic and sea waters. [28] This special environment is filled with inorganic metabolites, essentially carbon, nitrogen, oxygen, and sulfur. In its adult phase, 'R. pachyptila lacks a digestive system. To provide its energetic needs, it retains those dissolved inorganic nutrients (sulfide, carbon dioxide, oxygen, nitrogen) into its plume and transports them through a vascular system to the trophosome, which is suspended in paired coelomic cavities and is where the intracellular symbiotic bacteria are found. [20] [29] [30] The trophosome [31] is a soft tissue that runs through almost the whole length of the tube's coelom. It retains a large number of bacteria on the order of 10 9 bacteria per gram of fresh weight. [32] Bacteria in the trophosome are retained inside bacteriocytes, thereby having no contact with the external environment. Thus, they rely on R. pachyptila for the assimilation of nutrients needed for the array of metabolic reactions they employ and for the excretion of waste products of carbon fixation pathways. At the same time, the vestimentiferan depends completely on the microorganisms for the byproducts of their carbon fixation cycles that are needed for its growth.

Initial evidence for a chemoautotrophic symbiosis in R. pachyptila came from microscopic and biochemical analyses showing Gram-negative bacteria packed within a highly vascularized organ in the tubeworm trunk called the trophosome. [21] Additional analyses involving stable isotope, [33] enzymatic, [34] [26] and physiological [35] characterizations confirmed that the end symbionts of R. pachyptila oxidize reduced-sulfur compounds to synthesize ATP for use in autotrophic carbon fixation through the Calvin cycle. The host tubeworm enables the uptake and transport of the substrates required for thioautotrophy, which are HS − , O2, and CO2, receiving back a portion of the organic matter synthesized by the symbiont population. The adult tubeworm, given its inability to feed on particulate matter and its entire dependency on its symbionts for nutrition, the bacterial population is then the primary source of carbon acquisition for the symbiosis. Discovery of bacterial–invertebrate chemoautotrophic symbioses, initially in vestimentiferan tubeworms [21] [26] and then in vesicomyid clams and mytilid mussels, [26] pointed to an even more remarkable source of nutrition sustaining the invertebrates at vents.

A wide range of bacterial diversity is associated with symbiotic relationships with R. pachyptila. Many bacteria belong to the class Epsilonproteobacteria [36] as supported by the recent discovery in 2016 of the new species Sulfurovum riftiae belonging to the class Epsilonproteobacteria, family Helicobacteraceae isolated from R. pachyptila collected from the East Pacific Rise. [37] Other symbionts belong to the class Delta-, Alpha- and Gamma- proteobacteria. [36] The Candidatus Endoriftia persephone is a facultative R. pachyptila symbiont and has been shown to be a mixotroph, thereby exploiting both Calvin Benson cycle and reverse TCA cycle (with an unusual ATP citrate lyase) according to availability of carbon resources and whether it is free living in the environment or inside a eukaryotic host. The bacteria apparently prefer a heterotrophic lifestyle when carbon sources are available. [31]

Evidence based on 16S rRNA analysis affirms that R. pachyptilachemoautotrophic bacteria belong to two different phyla of Proteobacteria superphylum: Gammaproteobacteria phylum [38] [20] and Epsilonproteobacteria phylum (e.g. Sulfurovum riftiae) [37] that get energy from the oxidation of inorganic sulfur compounds such as hydrogen sulfide (H2S, HS − , S 2- ) to synthetize ATP for carbon fixation via the Calvin cycle. [20] Unfortunately, most of these bacteria are still uncultivable. Symbiosis works so that R. pachyptila provides nutrients such as HS − , O2, CO2 to bacteria, and in turn it receives organic matter from them. Thus, because of lack of a digestive system, R. pachyptila depends entirely on its bacterial symbiont to survive. [39] [40]

In the first step of sulfide-oxidation, reduced sulfur (HS − ) passes from the external environment into R. pachyptila blood, where, together with O2, it is bound by hemoglobin, forming the complex Hb-O2-HS − and then it is transported to the trophosome, where bacterial symbionts reside. Here, HS − is oxidized to elemental sulfur (S 0 ) or to sulfite (SO3 2- ). [20]

In the second step, the symbionts make sulfite-oxidation by the "APS pathway", to get ATP. In this biochemical pathway, AMP reacts with sulfite in the presence of the enzyme APS reductase, giving APS (adenosine 5'-phosphosulfate). Then, APS reacts with the enzyme ATP sulfurylase in presence of pyrophosphate (PPi) giving ATP (substrate-level phosphorylation) and sulfate (SO4 2- ) as end products. [20] In formulas:

The electrons released during the entire sulfide-oxidation process enter in an electron transport chain, yielding a proton gradient that produces ATP (oxydative phosphorylation). Thus, ATP generated from oxidative phosphorylation and ATP produced by substrate-level phosphorylation become available for CO2 fixation in Calvin cycle, whose presence has been demonstrated by the presence of two key enzymes of this pathway: phosphoribulokinase and RubisCO. [26] [41]

To support this unusual metabolism, R. pachyptila has to absorb all the substances necessary for both sulfide-oxidation and carbon fixation, that is: HS − , O2 and CO2 and other fundamental bacterial nutrients such as N and P. This means that the tubeworm must be able to access both oxic and anoxic areas.

Oxidation of reduced sulfur compounds requires the presence of oxidized reagents such as oxygen and nitrate. Hydrothermal vents are characterized by conditions of high hypoxia. In hypoxic conditions, sulfur-storing organisms start producing hydrogen sulfide. Therefore, the production of in H2S in anaerobic conditions is common among thiotrophic symbiosis. H2S can be damaging for some physiological processes as it inhibits the activity of cytochrome c oxidase, consequentially impairing oxidative phosphorilation. In R. pachyptila the production of hydrogen sulfide starts after 24h of hypoxia. In order to avoid physiological damage some animals, including Riftia pachyptila are able to bind H2S to haemoglobin in the blood to eventually expel it in the surrounding environment.

Unlike metazoans, which respire carbon dioxide as a waste product, R. pachyptila-symbiont association has a demand for a net uptake of CO2 instead, as a cnidarian-symbiont associations. [42] Ambient deep-sea water contains an abundant amount of inorganic carbon in the form of bicarbonate HCO3 − , but it is actually the chargeless form of inorganic carbon, CO2, that is easily diffusible across membranes. The low partial pressures of CO2 in the deep-sea environment is due to the seawater alkaline pH and the high solubility of CO2, yet the pCO2 of the blood of R. pachyptila may be as much as two orders of magnitude greater than the pCO2 of deep-sea water. [42]

CO2 partial pressures are transferred to the vicinity of vent fluids due to the enriched inorganic carbon content of vent fluids and their lower pH. [20] CO2 uptake in the worm is enhanced by the higher pH of its blood (7.3 – 7.4), which favors the bicarbonate ion and thus promotes a steep gradient across which CO2 diffuses into the vascular blood of the plume. [43] [20] The facilitation of CO2 uptake by high environmental pCO2 was first inferred based on measures of elevated blood and coelomic fluid pCO2 in tubeworms, and was subsequently demonstrated through incubations of intact animals under various pCO2 conditions. [30]

Once CO2 is fixed by the symbionts, it must be assimilated by the host tissues. The supply of fixed carbon to the host is transported via organic molecules from the trophosome in the hemolymph, but the relative importance of translocation and symbiont digestion is not yet known. [30] [44] Studies proved that within 15 min, the label first appears in symbiont-free host tissues, and that indicates a significant amount of release of organic carbon immediately after fixation. After 24 h, labeled carbon is clearly evident in the epidermal tissues of the body wall. Results of the pulse-chase autoradiographic experiments were also evident with ultrastructural evidence for digestion of symbionts in the peripheral regions of the trophosome lobules. [44] [45]

In deep-sea hydrothermal vents, sulfide and oxygen are present in different areas. Indeed, the reducing fluid of hydrothermal vents is rich in sulfide, but poor in oxygen, whereas sea water is richer in dissolved oxygen. Moreover, sulfide is immediately oxidized by dissolved oxygen to form partly, or totally, oxidized sulfur compounds like thiosulfate (S2O3 2- ) and ultimately sulfate (SO4 2- ), respectively less, or no longer, usable for microbial oxidation metabolism. [46] This causes the substrates to be less available for microbial activity, thus bacteria are constricted to compete with oxygen to get their nutrients. In order to avoid this issue, several microbes have evolved to make symbiosis with eukaryotic hosts. [47] [20] In fact, R. pachyptila is able to cover the oxic and anoxic areas to get both sulfide and oxygen. [48] [49] [50] Thanks to its hemoglobin that can bind sulfide reversibly and apart from oxygen by means of two cysteine residues, [51] [52] [53] and then transport it to the trophosome, where bacterial metabolism can occur.

The acquisition of a symbiont by a host can occur in these ways:

  • Environmental transfer (symbiont acquired from a free-living population in the environment)
  • Vertical transfer (parents transfer symbiont to offspring via eggs)
  • Horizontal transfer (hosts that share the same environment)

Evidence suggests that R. pachyptila acquires its symbionts through its environment. In fact, 16S rRNA gene analysis showed that vestimentiferan tubeworms belonging to three different genera: Riftia, Oasisia, and Tevnia, share the same bacterial symbiont phylotype. [54] [55] [56] [57] [58]

This proves that R. pachyptila takes its symbionts from a free-living bacterial population in the environment. Other studies also support this thesis, because analyzing R. pachyptila eggs, 16S rRNA belonging to the symbiont was not found, showing that the bacterial symbiont is not transmitted by vertical transfer. [59]

Another proof to support the environmental transfer comes from several studies conducted in the late 1990s. [60] PCR was used to detect and identify a R. pachyptila symbiont gene whose sequence was very similar to the fliC gene that encodes some primary protein subunits (flagellin) required for flagellum synthesis. Analysis showed that R. pachyptila symbiont has at least one gene needed for flagellum synthesis. Hence, the question arose as to the purpose of the flagellum. Flagellar motility would be useless for a bacterial symbiont transmitted vertically, but if the symbiont came from the external environment, then a flagellum would be essential to reach the host organism and to colonize it. Indeed, several symbionts use this method to colonize eukaryotic hosts. [61] [62] [63] [64]

Thus, these results confirm the environmental transfer of R. pachyptila symbiont.

R. pachyptila [65] is a dioecious vestimentiferan. [66] Individuals of this species are sessile and are found clustered together around deep-sea hydrothermal vents of the East Pacific Rise and the Galapagos Rift. [67] The size of a patch of individuals surrounding a vent is within the scale of tens of metres. [68]

The male's spermatozoa are thread-shaped and are composed of three distinct regions: the acrosome (6 μm), the nucleus (26 μm) and the tail (98 μm). Thus, the single spermatozoa is about 130 μm long overall, with a diameter of 0.7 μm, which becomes narrower near the tail area, reaching 0.2 μm. The sperm is arranged into an agglomeration of around 340-350 individual spermatozoa that create a torch-like shape. The cup part is made up of acrosomes and nucleus, while the handle is made up by the tails. The spermatozoa in the package are held together by fibrils. Fibrils also coat the package itself to ensure cohesion. [ citation needed ]

The large ovaries of females run within the gonocoel along the entire length of the trunk and are ventral to the trophosome. Eggs at different maturation stages can be found in the middle area of the ovaries, and depending on their developmental stage, are referred to as: oogonia, oocytes, and follicular cells. When the oocytes mature, they acquire protein and lipid yolk granules. [ citation needed ]

Males release their sperm into sea water. While the released agglomerations of spermatozoa, referred to as spermatozeugmata, do not remain intact for more than 30 seconds in laboratory conditions, they may maintain integrity for longer periods of time in specific hydrothermal vent conditions. Usually, the spermatozeugmata swim into the female's tube. Movement of the cluster is conferred by the collective action of each spermatozoon moving independently. Reproduction has also been observed involving only a single spermatozoon reaching the female's tube. Generally, fertilization in R. pachyptila is considered internal. However, some argue that, as the sperm is released into sea water and only afterwards reaches the eggs in the oviducts, it should be defined as internal-external. [ citation needed ]

R. pachyptila is completely dependent on the production of vulcanic gases and the presence of sulfide-oxidizing bacteria. Therefore, its metapopulation distribution is profoundly linked to vulcanic and tectonic activity that create active hydrothermal vent sites with a patchy and ephemeral distribution. The distance between active sites along a rift or adjacent segments can be very high, reaching hundreds of km. [67] This raises the question regarding larval dispersal. R. pachytpila is capable of larval dispersal across distances of 100 to 200 km [67] and cultured larvae show to be viable for 38 days. [69] Though dispersal is considered to be effective, the genetic variability observed in R. pachyptila metapopulation is low compared to other vent species. This may be due to high extinction events and colonization events, as R. pachyptila is one of the first species to colonize a new active site. [67]

The endosymbionts of R. pachyptila are not passed to the fertilized eggs during spawning, but are acquired later during the larval stage of the vestimentiferan worm. R. pachyptila planktonic larvae that are transported through sea-bottom currents until they reach active hydrothermal vents sites, are referred to as trophocores. The trophocore stage lacks endosymbionts, which are acquired once larvae settle in a suitable environment and substrate. Free-living bacteria found in the water column are ingested randomly and enter the worm through a ciliated opening of the branchial plume. This opening is connected to the trophosome through a duct that passes through the brain. Once the bacteria are in the gut, the ones that are beneficial to the individual, namely sulfide- oxidizing strains are paghocytized by epithelial cells found in the midgut are then retained. Bacteria that do not represent possible endosymbionts are digested. This raises questions as to how R. pachyptila manages to discern between essential and nonessential bacterial strains. The worm's ability to recognise a beneficial strain, as well as preferential host-specific infection by bacteria have been both suggested as being the drivers of this phenomenon. [70]

R. pachyptila has the fastest growth rate of any known marine invertebrate. These organisms have been known to colonize a new site, grow to sexual maturity, and increase in length to 4.9 feet (1.5 m) in less than two years. [71]

Some Benefits of Saprophytic Bacteria

As a primary organism needed for decomposition, saprophytic bacteria have the obvious benefit of returning nutrients like iron, phosphorus and calcium to the Earth, where plants absorb them to produce life-sustaining hydrogen, nitrogen, carbon and other vitamins and minerals. Many beneficial types of bacteria live inside the human body to help keep humans healthy. Hundreds of organisms work to break down the nonliving organisms humans eat. Without them, food would never digest properly, and harmful bacteria would quickly multiply.

P lants contain cellulose, a component that humans can’t actually digest. The bacteria Spirochaeta cytophaga uses absorptive nutrition to break down the cellulose, unlocking the power of plant nutrition for humans. Another example, Lactobacillus, breaks down milk to make yogurt, while other types of bacteria break it down to make all the different types of cheeses.

B oth aerobic bacteria and anaerobic bacteria are critical components in breaking down toxins in sewage, which is critical for the ongoing safety of our environment. Additionally, the methane gas produced during the process is sometimes used as a cheap biogas in place of fuel. The biofuel ethanol also comes from a process developed by biotechnology experts that uses anaerobic bacteria to break down cane sugar and corn.

How bacteria break down human food

Last weeks post on the changing composition of bacteria in the vagina generated a lot of interest, and as there's been quite a of talk about the human microbiome (all the bacteria that live on the human body) at the moment I thought I'd stick with the theme. This weeks post is about how bacteria break down the nutrients that humans eat and use them to create their own food.

The paper (reference 1 below) from PLoS One focuses on carbohydrates. Starting with some biochemistry background: carbohydrates are molecules made exclusively from carbon, hydrogen and oxygen (hence the name). These three molecules are arranged into a ring structure for the simple carbohydrates such as glucose, and those rings are put together into long complex branching chains for the complex carbohydrates such as starch and cellulose.

Simple carbohydrates, like the glucose shown in the picture above, are fairly easy to metabolise and can be used to power-up ATP (the molecules that the cell uses for energy) or in the synthesis of proteins. More complex carbohydrates like starch or cellulose (shown below) take a bit more effort, as they need to be broken down into their component simple sugars before they can be processed. To break them down bacteria use a specific group of enzymes called CAZymes which stands for “Carbohydrate-active enzymes". As enzymes are very specialised in the molecules that they break down, different CAZymes exist for different complex carbohydrates.

Different bacteria will have different CAZymes, but an intriguing question the PLoS paper set out to answer is how the pattern of CAZymes changes throughout the body. There isn't just one bacterial species inside you, but many, each species differently related to the ones surrounding it. It's less a community of bacteria inside you and more like a badly organised safari park, with different species all milling around in close proximity to each other, relying on the resources available in whichever part of the body they happen to live in.

The researchers compared the carbohydrate digesting abilities of 493 bacterial genomes, associated with five different sites on the exterior and interior of the human body. When they tried to work out the number and distribution of CAZymes by species they very quickly ran into difficulties. Some bacterial families, such as Bacillaceae, had an average of number of 25 sugar-cleaving enzymes, with a respectable standard deviation of 3.3 (for the uninitiated, the standard deviation measures how likely each individual is to be close to the average). The bacterial family Clostridiaceae on the other hand had an average of 56 sugar-cleaving enzymes but with a standard deviation of 79! As well as showing the large inter-species variation, this also makes it difficult to predict relatedness between bacteria based on their carbohydrate-digesting abilities.

As comparing species didn't seem to be yielding particularly concise results, the researchers then moved onto comparing CAZymes by bacterial habitat. Unlike humans, and indeed pretty much all eukaryotes, bacteria don't just pass genes down to their offspring, they can also pass genes across to a nearby friend. Unsurprisingly, bacteria living in the same place on the body tended to have more similar carbohydrate-digesting enzymes than bacteria that were more related by species. Overall the researchers found four major patterns of carbohydrate-use:

1) Bacteria in the nose and nasal cavities - unsurprisingly these bacteria tended to have very little carbohydrate metabolising ability (very few people inhale starch)

2) Bacteria in the vagina - These bacteria tended to be breaking down more simple sugars, and also had carbohydrate forming enzymes in order to build up biofilms

3) Bacteria in the mouth - these bacteria have a wide range of carbohydrate digesting enzymes in order to break down the bits of food which get trapped in your teeth. The researchers also identified three enzymes used for metabolising dextran, which may be unique for mouth-bacteria and seemed to be a marker for plaque formation.

4) Bacteria in the gut - this is where the big carbohydrate-digesting muscle lies! Not only do gut bacteria have plenty of CAZymes for human carbohydrates, they also have range that deal with plant carbohydrates. Many of these bacteria have the ability to form a cellulosome - a large complex of cellulose digesting enzymes all held together by scaffold proteins.

It may be slightly weird to think of bacteria living in so many parts of your body - colonising your spaces and eating your food - but really, it would have been much more of a surprise to find they weren't. Pretty much every surface on earth has bacteria living on it, and humans are such a warm, moist, nutrient-rich surface that they provide a great living environment for a huge number of bacterial species.

Cantarel BL, Lombard V, & Henrissat B (2012). Complex carbohydrate utilization by the healthy human microbiome. PloS one, 7 (6) PMID: 22719820

The views expressed are those of the author(s) and are not necessarily those of Scientific American.


A biochemist with a love of microbiology, the Lab Rat enjoys exploring, reading about and writing about bacteria. Having finally managed to tear herself away from university, she now works for a small company in Cambridge where she turns data into manageable words and awesome graphs.

Microbial processes

Anaerobic Respiration

Due to lack of oxygen the deep subsurface, the only way for life to carry on is to engage in anaerobic respiration. Anaerobic respiration is characterized by the use of alternative compounds as terminal electron acceptors,


Microbes use reduced inorganic substances, such as iron, sulfur, or magnesium from which to derive the chemical energy necessary to conduct biosynthesis. H2 gas is the primary electron donor, though other compounds such as SO3 2− S4O62 − , S 0 , Fe 2+ , and Mn(II) are utilized as well [2].


Methanogenesis is the production of methane by microbes via anaerobic respiration. Methanogens as they are effectively named, have only been identified within the domain Archaea. Deep subsurface archaea are known to metabolize available organic carbon sources and are responsible for the production of large pockets of methane trapped within the earths crust.

Hydrocarbon Degredation

Metabolic activity in areas rich in hydrocarbon substances support large numbers of anaerobic heterotrophic microorganisms. These microbes metabolize the hydrocarbons as both an energy source as well as for a carbon source. These organisms have special relevance in today's day and age, as petroleum products become increasingly more and more prevalent. These organisms facilitate the breakdown of these substances, and have been used to clean up oil spills and to help degrade other petroleum distillates [4].

Figure 4: This light micrograph shows a 100× magnification of red blood cells infected with P. falciparum (seen as purple). (credit: modification of work by Michael Zahniser scale-bar data from Matt Russell)

Members of the genus Plasmodium must infect a mosquito and a vertebrate to complete their life cycle. In vertebrates, the parasite develops in liver cells and goes on to infect red blood cells, bursting from and destroying the blood cells with each asexual replication cycle ([Figure 4]). Of the four Plasmodium species known to infect humans, P. falciparum accounts for 50 percent of all malaria cases and is the primary cause of disease-related fatalities in tropical regions of the world. In 2010, it was estimated that malaria caused between 0.5 and 1 million deaths, mostly in African children. During the course of malaria, P. falciparum can infect and destroy more than one-half of a human’s circulating blood cells, leading to severe anemia. In response to waste products released as the parasites burst from infected blood cells, the host immune system mounts a massive inflammatory response with delirium-inducing fever episodes, as parasites destroy red blood cells, spilling parasite waste into the blood stream. P. falciparum is transmitted to humans by the African malaria mosquito, Anopheles gambiae. Techniques to kill, sterilize, or avoid exposure to this highly aggressive mosquito species are crucial to malaria control.

This movie depicts the pathogenesis of Plasmodium falciparum, the causative agent of malaria.

Looking for LUCA, the last universal common ancestor

A hydrothermal vent in the north-east Pacific Ocean, similar to the kind of environment in which LUCA seems to have lived. Credit: NOAA

Around 4 billion years ago there lived a microbe called LUCA: the Last Universal Common Ancestor. There is evidence that it could have lived a somewhat 'alien' lifestyle, hidden away deep underground in iron-sulfur rich hydrothermal vents. Anaerobic and autotrophic, it didn't breathe air and made its own food from the dark, metal-rich environment around it. Its metabolism depended upon hydrogen, carbon dioxide and nitrogen, turning them into organic compounds such as ammonia. Most remarkable of all, this little microbe was the beginning of a long lineage that encapsulates all life on Earth.

If we trace the tree of life far enough back in time, we come to find that we're all related to LUCA. If the war cry for our exploration of Mars is 'follow the water', then in the search for LUCA it's 'follow the genes'. The study of the genetic tree of life, which reveals the genetic relationships and evolutionary history of organisms, is called phylogenetics. Over the last 20 years our technological ability to fully sequence genomes and build up vast genetic libraries has enabled phylogenetics to truly come of age and has taught us some profound lessons about life's early history.

For a long time it was thought that the tree of life formed three main branches, or domains, with LUCA at the base – eukarya, bacteria and archaea. The latter two – the prokaryotes – share similarities in being unicellular and lack a nucleus, and are differentiated from one another by subtle chemical and metabolic differences. Eukarya, on the other hand, are the complex, multicellular life forms comprised of membrane-encased cells, each incorporating a nucleus containing the genetic code as well as the mitochondria 'organelles' powering the cell's metabolism. The eukarya are considered so radically different from the other two branches as to necessarily occupy its own domain.

However, a new picture has emerged that places eukarya as an offshoot of bacteria and archaea. This "two-domain tree" was first hypothesized by evolutionary biologist Jim Lake at UCLA in 1984, but only got a foothold in the last decade, in particular due to the work of evolutionary molecular biologist Martin Embley and his lab at the University of Newcastle, UK, as well as evolutionary biologist William Martin at the Heinrich Heine University in Düsseldorf, Germany.

Bill Martin and six of his Düsseldorf colleagues (Madeline Weiss, Filipa Sousa, Natalia Mrnjavac, Sinje Neukirchen, Mayo Roettger and Shijulal Nelson-Sathi) published a 2016 paper in the journal Nature Microbiology describing this new perspective on LUCA and the two-domain tree with phylogenetics.

Previous studies of LUCA looked for common, universal genes that are found in all genomes, based on the assumption that if all life has these genes, then these genes must have come from LUCA. This approach has identified about 30 genes that belonged to LUCA, but they're not enough to tell us how or where it lived. Another tactic involves searching for genes that are present in at least one member of each of the two prokaryote domains, archaea and bacteria. This method has identified 11,000 common genes that could potentially have belonged to LUCA, but it seems far-fetched that they all did: with so many genes LUCA would have been able to do more than any modern cell can.

Bill Martin and his team realized that a phenomenon known as lateral gene transfer (LGT) was muddying the waters by being responsible for the presence of most of these 11,000 genes. LGT involves the transfer of genes between species and even across domains via a variety of processes such as the spreading of viruses or homologous recombination that can take place when a cell is placed under some kind of stress.

A growing bacteria or archaea can take in genes from the environment around them by 'recombining' new genes into their DNA strand. Often this newly-adopted DNA is closely related to the DNA already there, but sometimes the new DNA can originate from a more distant relation. Over the course of 4 billion years, genes can move around quite a bit, overwriting much of LUCA's original genetic signal. Genes found in both archaea and bacteria could have been shared through LGT and hence would not necessarily have originated in LUCA.

The field of hydrothermal vents known as Loki’s Castle, in the North Atlantic Ocean, where scientists found archaea believed to be related to the archaea that created eukaryotes through endosymbiosis with bacteria. Credit: R B Pedersen/Centre for Geobiology

Knowing this, Martin's team searched for 'ancient' genes that have exceptionally long lineages but do not seem to have been shared around by LGT, on the assumption that these ancient genes should therefore come from LUCA. They laid out conditions for a gene to be considered as originating in LUCA. To make the cut, the ancient gene could not have been moved around by LGT and it had to be present in at least two groups of archaea and two groups of bacteria.

"While we were going through the data, we had goosebumps because it was all pointing in one very specific direction," says Martin.

Once they had finished their analysis, Bill Martin's team was left with just 355 genes from the original 11,000, and they argue that these 355 definitely belonged to LUCA and can tell us something about how LUCA lived.

Such a small number of genes, of course, would not support life as we know it, and critics immediately latched onto this apparent gene shortage, pointing out that essential components capable of nucleotide and amino acid biosynthesis, for example, were missing. "We didn't even have a complete ribosome," admits Martin.

However, their methodology required that they omit all genes that have undergone LGT, so had a ribosomal protein undergone LGT, it wouldn't be included in the list of LUCA's genes. They also speculated that LUCA could have gotten by using molecules in the environment to fill the functions of lacking genes, for example molecules that can synthesize amino acids. After all, says Martin, biochemistry at this early stage in life's evolution was still primitive and all the theories about the origin of life and the first cells incorporate chemical synthesis from their environment.

What those 355 genes do tell us is that LUCA lived in hydrothermal vents. The Düsseldorf team's analysis indicates that LUCA used molecular hydrogen as an energy source. Serpentinization within hydrothermal vents can produce copious amounts of molecular hydrogen. Plus, LUCA contained a gene for making an enzyme called 'reverse gyrase', which is found today in extremophiles existing in high-temperature environments including hydrothermal vents.

Martin Embley, who specializes in the study eukaryotic evolution, says the realization of the two-domain tree over the past decade, including William Martin's work to advance the theory, has been a "breakthrough" and has far-reaching implications on how we view the evolution of early life. "The two-domain tree of life, where the basal split is between the archaea and the bacteria, is now the best supported hypothesis," he says.

It is widely accepted that the first archaea and bacteria were likely clostridia (anaerobes intolerant of oxygen) and methanogens, because today's modern versions share many of the same properties as LUCA. These properties include a similar core physiology and a dependence on hydrogen, carbon dioxide, nitrogen and transition metals (the metals provide catalysis by hybridizing their unfilled electron shells with carbon and nitrogen). Yet, a major question remains: What were the first eukaryotes like and where do they fit into the tree of life?

A schematic of the two-domain tree, with eukaryotes evolving from endosymbiosis between members of the two original trunks of the tree, archaea and bacteria. Credit: Weiss et al/Nature Microbiology

Phylogenetics suggests that eukaryotes evolved through the process of endosymbiosis, wherein an archaeal host merged with a symbiont, in this case a bacteria belonging to the alphaproteobacteria group. In the particular symbiosis that spawned the development of eukarya, the bacteria somehow came to thrive within their archaeal host rather than be destroyed. Hence, bacteria came to not only exist within archaea but empowered their hosts to grow bigger and contain increasingly large amounts of DNA. After aeons of evolution, the symbiont bacteria evolved into what we know today as mitochondria, which are little battery-like organelles that provide energy for the vastly more complex eukaryotic cells. Consequently, eukaryotes are not one of the main branches of the tree-of-life, but merely a large offshoot.

A paper that appeared recently in Nature, written by a team led by Thijs Ettema at Uppsala University in Sweden, has shed more light on the evolution of eukaryotes. In hydrothermal vents located in the North Atlantic Ocean – centered between Greenland, Iceland and Norway, known collectively as Loki's Castle– they found a new phylum of archaea that they fittingly named the 'Asgard' super-phylum after the realm of the Norse gods. The individual microbial species within the super-phylum were then named after Norse gods: Lokiarchaeota, Thorarchaeota, Odinarchaeota and Heimdallarchaeota. This super-phylum represents the closest living relatives to eukaryotes, and Ettema's hypothesis is that eukaryotes evolved from one of these archaea, or a currently undiscovered sibling to them, around 2 billion years ago.

If it's possible to date the advent of eukaryotes, and even pinpoint the species of archaea and bacteria they evolved from, can phylogenetics also date LUCA's beginning and its split into the two domains?

It must be noted that LUCA is not the origin of life. The earliest evidence of life dates to 3.7 billion years ago in the form of stromatolites, which are layers of sediment laid down by microbes. Presumably, life may have existed even before that. Yet, LUCA's arrival and its evolution into archaea and bacteria could have occurred at any point between 2 to 4 billion years ago.

Phylogenetics help narrow this down, but Martin Embley isn't sure our analytical tools are yet capable of such a feat. "The problem with phylogenetics is that the tools commonly used to do phylogenetic analysis are not really sophisticated enough to deal with the complexities of molecular evolution over such vast spans of evolutionary time," he says.

Embley believes this is why the three-domain tree hypothesis lasted so long – we just didn't have the tools required to disprove it. However, the realization of the two-domain tree suggests that better techniques are now being developed to handle these challenges.

These techniques include examining the ways biochemistry, as performed in origin-of-life experiments in the lab, can coincide with the realities of what actually happens in biology.

This is a concern for Nick Lane, an evolutionary biochemist at University College of London, UK. "What I think has been missing from the equation is a biological point of view," he says. "It seems trivially easy to make organic [compounds] but much more difficult to get them to spontaneously self-organize, so there are questions of structure that have largely been missing from the chemist's perspective."

Jupiter’s moon Europa has a subterranean ocean, a rocky seabed, and geothermal heat produced by Jupiter’s gravitational tides. Water, rock and heat were all that were required by LUCA, so could similar life also exist on Europa? Credit: NASA/JPL–Caltech/SETI Institute

For example, Lane highlights how lab experiments routinely construct the building blocks of life from chemicals like cyanide, or how ultraviolet light is utilized as an ad hoc energy source, yet no known life uses these things. Although Lane sees this as a disconnect between lab biochemistry and the realities of biology, he points out that William (Bill) Martin's work is helping to fill the void by corresponding to real-world biology and conditions found in real-life hydrothermal vents. "That's why Bill's reconstruction of LUCA is so exciting, because it produces this beautiful, independent link-up with real world biology," Lane says.

The biochemistry results in part from the geology and the materials that are available within it to build life, says Martin Embley. He sees phylogenetics as the correct tool to find the answer, citing the Wood–Ljungdahl carbon-fixing pathway as evidence for this.

Carbon-fixing involves taking non-organic carbon and turning it into organic carbon compounds that can be used by life. There are six known carbon-fixing pathways and work conducted over many decades by microbiologist Georg Fuchs at the University of Freiburg has shown that the Wood–Ljungdahl pathway is the most ancient of all the pathways and, therefore, the one most likely to have been used by LUCA. Indeed, this is corroborated by the findings of Bill Martin's team.

In simple terms the Wood–Ljundahl pathway, which is adopted by bacteria and archaea, starts with hydrogen and carbon dioxide and sees the latter reduced to carbon monoxide and formic acid that can be used by life. "The Wood–Ljungdahl pathway points to an alkaline hydrothermal environment, which provides all the things necessary for it – structure, natural proton gradients, hydrogen and carbon dioxide," says Martin. "It's marrying up a geological context with a biological scenario, and it has only been recently that phylogenetics has been able to support this."

Astrobiological implications

Understanding the origin of life and the identity of LUCA is vital not only to explaining the presence of life on Earth, but possibly that on other worlds, too. Hydrothermal vents that were home to LUCA turn out to be remarkably common within our solar system. All that's needed is rock, water and geochemical heat. "I think that if we find life elsewhere it's going to look, at least chemically, very much like modern life," says Martin.

Moons with cores of rock surrounded by vast global oceans of water, topped by a thick crust of water-ice, populate the Outer Solar System. Jupiter's moon Europa and Saturn's moon Enceladus are perhaps the most famous, but there is evidence that hints at subterranean oceans on Saturn's moons Titan and Rhea, as well as the dwarf planet Pluto and many other Solar System bodies. It's not difficult to imagine hydrothermal vents on the floors of some of these underground seas, with energy coming from gravitational tidal interactions with their parent planets. The fact that the Sun does not penetrate through the ice ceiling does not matter – the kind of LUCA that Martin describes had no need for sunlight either.

"Among the astrobiological implications of our LUCA paper is the fact that you do not need light," says Martin. "It's chemical energy that ran the origin of life, chemical energy that ran the first cells and chemical energy that is present today on bodies like Enceladus."

As such, the discoveries that are developing our picture of the origin of life and the existence of LUCA raise hopes that life could just as easily exist in a virtually identical environment on a distant locale such as Europa or Enceladus. Now that we know how LUCA lived, we know the signs of life to look out for during future missions to these icy moons.

Laura Eme et al. Archaea and the origin of eukaryotes, Nature Reviews Microbiology (2017). DOI: 10.1038/nrmicro.2017.133

Defining Life

What is life, exactly? This is a question that keeps biologists up at night. The science of biology is the study of life, yet scientists can’t agree on an absolute definition. Are the individual cells of your body, with all their complex machinery, “alive?” What about a computer program that learns and evolves? Can a wild fire – which feeds, grows, and reproduces – be considered a living entity?

Trying to define life is not just a philosophical exercise. We need to understand what separates living creatures from non-living matter before we can claim to find life elsewhere in the Universe.

In 1944, the physicist Erwin Shrodinger defined living matter as that which “avoids the decay into equilibrium.” This definition refers to the Second Law of Thermodynamics, which says that entropy always increases. Entropy is often referred to as chaos or disorder, but really it is the spreading out of energy towards a state of uniformity. This law can be seen in a cold glass of water that slowly grows warmer until it is the same temperature as the surrounding air. Because of this trend toward equilibrium, the Universe eventually will have a complete lack of structure, consisting of evenly spread atoms of equal warmth.

But living things, said Shrodinger, are able to postpone this trend. Consider: while you are alive your body maintains its structure, but once you die your body begins to break down through bacterial action and chemical processes. Eventually the atoms of your body are evenly spread out, recycled by the Earth. To die is to submit your body to the entropy of the Universe.

Living things resist entropy by taking in nutrients. This biochemical process of taking in energy for activities and expelling waste byproducts is known as a “metabolism.” If metabolism is a sign of life, scientists can look for the waste byproducts of a metabolism when searching for life on other worlds.

Image of the Viking Lander.
Credit: NSSDC Photo Gallery

At least, that was the idea behind the Viking Lander‘s Labeled Release Experiment, conducted on Mars in 1976. This experiment tested for metabolic clues to life by adding radioactively labeled liquid nutrients to a sample of Martian soil. If these nutrients were consumed by life forms, any gases released as waste byproducts would also be radioactively labeled.

After the nutrient was injected, there was a rapid increase in carbon dioxide (CO2) gas. Because this gas had the radioactive label, scientists at first concluded that organisms in the Martian soil were eating the nutrient and releasing the CO2 as a waste byproduct. However, the Martian soil turned out to have a unique soil chemistry that could produce a metabolic-like reaction. Although the test remains inconclusive, most scientists believe that non-living, chemical processes in the Martian soil caused the “metabolic” reaction. The Viking experiments showed that while metabolism may be a quality of life, it is not a narrow enough guideline to search for life elsewhere.

Another quality of all life on Earth is a dependence on water. Since water plays such a crucial role in all known life forms, many scientists believe that water-use will be a quality universal to all life. But Benton Clark, an astrobiologist with the University of Colorado and Lockheed Martin, says that water is really a side issue.

“Water doesn’t define life, it is just an aspect of our environment,” says Clark.

Life on Earth evolved with water, and so today life on Earth is dependent on that resource. But we cannot say that without water, life is impossible. On Earth, life has been able to adapt to the harshest environments, so it is possible that life may have found a way to survive on worlds that have no liquid water.

Steven Benner, an astrobiologist with the University of Florida, agrees that water is not necessarily a universal quality of life.

“We can conceive of chemistries that might occur in sulfuric acid as a solvent – as on Venus – or in methane-ammonia mixtures – as on Jupiter,” says Benner. “Discovering these would have a profound impact on our view of life, however, as well as the way that NASA looks for it.”

A recent definition of life (often created to Gerald Joyce of the Scripps Research Institute) doesn’t mention either metabolism or water. This definition says that life is “a self-sustaining system capable of Darwinian evolution.”

But Clark says most life forms technically are not self-sustaining. Animals feed on plants or other animals, plants need microorganisms at their roots to take up nutrients, and bacteria often live inside other organisms, relying on the internal environment of their host. He says the only truly self-sustaining organisms are chemolithotrophs and photolithotrophs, and they are relatively rare.

Clark says that Darwinian evolution is another problematic criteria. How could you tell if something has undergone Darwinian evolution? The time scales involved are enormous – scientists would need a complete understanding of an organism’s fossil history before being able to declare that the object is, indeed, alive.

According to Clark, living organisms exhibit at least 102 observable qualities. Adding all these qualities together into a single – if exceedingly long – definition still does not capture the essence of life. But Clark has picked out three qualities from this list that he considers universal, creating a new definition of life. This definition says that “life reproduces, and life uses energy. These functions follow a set of instructions embedded within the organism.”

The instructions are the DNA and RNA “letters” that make up the genetic code in all organisms on Earth. A wild fire, one might say, reproduces and uses energy. So do crystals and various chemical reactions. In fact, Benner says that, “every spontaneous chemical process must expend free energy, living or not.”

“Every spontaneous chemical process must expend free energy, living or not,” Benner says. The formation of these crystals is an example.
Credit: National Ignition Facility Programs

But Clark says none of these phenomena are “alive” because none of them have the embedded instructions of a genetic code. We know there are no instructions, because there has not been any mutation over the years. They follow the rules of physics rather than embedded instructions, and so they behave the same every time. Mutation, says Clark, is the key to understanding whether or not something has embedded instructions.

Not all living things are capable of reproduction, however. Mules are born sterile. Most honeybees do not reproduce: only the Queen bee has that honor. Many human beings live their entire lives without producing offspring, and no one would argue that such people were not therefore alive.

But Clark says that reproduction and energy-use need not both occur for life to exist. He divides life into two categories: “organisms” and “Lifeforms.” Organisms channel energy according to embedded instructions, and this energy allows the organism to perform certain activities. A Lifeform, says Clark, is a broader category that encompasses organisms and makes reproduction possible.

“What I am proposing is that the individual physical entities should be called ‘organisms,’ but it sometimes takes a collection of organisms, the ‘Lifeform,’ to achieve reproduction,” says Clark.

There have been many definitions of life created over the years, but there has yet to be a single definition accepted by all. Every definition has had to face down challenges to its validity. According to Carol Cleland of the University of Colorado, this is because definitions are concerned only with language and concepts they can’t expand our understanding of the world. We can only define things we already understand.

Cleland says that scientists in the seventeenth century had the same problem trying to define water. There are many descriptions of water – it’s wet, thirst-quenching, it freezes and turns into vapor – but other substances also have these qualities. Once scientists discovered molecular chemistry, they could define water to everyone’s satisfaction as one oxygen atom coupled with two hydrogen atoms (H2O). Perhaps we need a similar revolution in scientific thought in order to define life.

Image of a water molecule, 2 hydrogen atoms and 1 oxygen atom.
Credit: FTC

“Current attempts to answer the question, ‘What is life?’ by defining life in terms of features like metabolism or reproduction – features that we ordinarily use to recognize samples of terrestrial life – are unlikely to succeed,” says Cleland. “What we need to answer the question, ‘What is life?’ is a general theory of living systems.”

Could we use Clark’s definition to find life on other worlds? The Viking Lander already looked for energy-use in the form of a metabolism, and the results were inconclusive. To search for this criterion as a means for finding life, we would need to consider other ways life could use energy.

The problem with searching for life forms with embedded instructions, says Clark, is that the criteria may be too specific. The only instructions we know of are DNA and RNA – there may be other genetic systems possible in the Universe that do not resemble the system found here on Earth.

Lotus japonicus: A Model Plant for the Legume Family☆

The Model Plant Features

Many cultivated legumes such as pea and soybean have complex genomes or are, for other reasons, not amenable to modern molecular genetic methods. Its favorable biological properties made L. japonicus the model plant of choice for classical and molecular genetic analysis of legumes. The qualities of L. japonicus include a small genome size of approximately 450 Mb, diploid genetics, six chromosome pairs, genomic colinearity to different legume crops, self-fertile flowers, a short seed-to-seed generation time, ample seed production, small seeds, simple straight (nonspiral) seed pod like soybean, large flowers enabling manual crossing, described transformation procedures using Agrobacterium tumefaciens or Agrobacterium rhizogenes, described in vitro tissue culture and regeneration procedures, effective nodulation and mycorrhization, perennial life cycle, regrowth from stem base, propagation from nodal cuttings, and production of cyanogenic glucosides, isoflavonoids, tannin, and other secondary metabolites ( Fig. 1 ).

Fig. 1 . Lotus japonicus plants grown for seed production in the greenhouse.


Most legumes develop root nodules in symbiosis with nitrogen-fixing soil bacteria collectively called rhizobia, and nodulated legume plants can use atmospheric dinitrogen as their sole nitrogen source. The interaction between the bacterial microsymbionts and legumes is selective. Individual species of rhizobia have a characteristic host range allowing nodulation of a particular set of legume plants. Mesorhizobium loti and the broad host range Rhizobium sp. NGR234 induce root nodules on L. japonicus. Roots of L. japonicus are also effectively colonized by symbiotic arbuscular mycorrhizal fungi, for example, Glomus intraradices and Gigaspora margarita. These fungi invade the root tissue by intercellular and intracellular hyphal growth and form arbuscules in cortical cells where metabolic interchanges take place. Mycorrhizal hypha increase the root surface and improves phosphor uptake. Identification of single gene plant mutations impairing both colonization by mycorrhiza and rhizobial invasion demonstrates that the two interactions share common steps during the early infection processes. Extending this observation may open a broader approach to the understanding of plant–microbe interactions, where symbiotic studies contribute not only to realization of the potential of symbiosis, but also to our understanding of, for example, plant–pathogen interactions. One of the interests of the plant science community is to use L. japonicus in the molecular genetic analysis of symbiosis. For this purpose, tools and resources for molecular analysis have been established. Insertion mutagenesis is possible with T-DNA or the maize transposon Ac. Ethyl methanesulfonate (EMS) is effective for chemical mutagenesis and a TILLING population for reverse genetics has been established as a service for the legume community and the plant community at large (TILLING, targeting-induced local lesions in genomes). More recently, an endogenous retransposon, LORE1, was shown to generate independent new insertions in the pollen germ line of regenerated plants and a large LORE1 insertion population has been established. This population is a new resource for forward genetics screening for interesting phenotypes and reverse genetics to identify insertion mutants in genes of interest.

Equally important, it can be used for identifying insertions in genes of interest using reverse genetics based on large-scale sequencing of insertion sites in a structured population connecting insertion sites in genes with plants and seeds carrying the individual insertions.

Genetics of Symbiotic Pathways

Like soybean, L. japonicus develops the determinate type of nodules. In contrast to, for example, pea nodules with a persistent meristem, the meristematic activity ceases early in determinate nodules developing on L. japonicus . After the initial phase with meristematic cell proliferation, determinate nodules grow by expansion giving a typical spherical shape. All developmental stages, from root hair curling to nodule senescence, are consequently phased in time. Root nodule development is a rare example of induced and dispensable organ formation in plants. Nodulation mutants can be rescued on nitrogen-containing nutrient solution, and developmental control genes that would compromise plant development and completion of the life cycle in other organogenic processes could thus be identified from nodulation mutants. Screening of the EMS, T-DNA, LORE1 and Ac populations of L. japonicus has so far identified more than 40 symbiotic loci. The phenotypes of these developmental plant mutants divide them roughly into three classes: non-nodulating mutants arrested in bacterial recognition or nodule initiation nodule development mutants arrested at consecutive stages of the organogenic process and autoregulatory mutants where the plant control of root nodule numbers is nonfunctional and spontaneous nodulating mutant plant developing root nodules in the absence of the rhizobial microsymbiont. Development of root nodules can thus be divided into a series of genetically separable steps. Taking advantage of the genetic and genomic resources available, genes involved in these different steps have been cloned and characterized. A novel class of LysM receptor kinases perceiving the rhizobial Nod-factor signals triggering the development of root nodules has been identified and shown to be involved in deciphering the structure of the Nod-factor signal molecule, thus contributing to the determination of the plant–bacterial host specificity, which in older literature is described as cross-inoculation groups. Another LysM receptor kinase was recently shown to distinguish between compatible and incompatible bacterial exopolysaccharides thus regulating rhizobial infection.

Likewise, a signal transduction pathway shared with mycorrhizal infection frequently called the common pathway has been defined. This knowledge inspired the cloning of the chitin receptor from the Arabidopsis model plant and the role of this receptor in plant–pathogen interaction is now studied intensively. In a similar development, the information from identification and functional analysis of plant genes required for Nod-factor perception, downstream signaling, or functional symbiosis has been used to identify the corresponding genes in cultivated legumes such as pea, bean, and soybean. More recently, the availability of both loss-of-function and gain-of-function mutants made it possible to investigate the role of individual genes in the highly synchronized infection and organogenic pathways leading to the development of functional nitrogen fixing root nodules. Combining loss-of-function and gain-of-function mutations in synthetic mutants showed that L. japonicus possess three different infection routes reflecting the different bacterial entry pathways in the legume family. The genetic requirement for single-cell peg entry, crack entry, and infection thread infection was defined by this analysis, which provides support for the origin of rhizobial infection through direct intercellular epidermal invasion and subsequent evolution of crack entry and root hair invasions observed in most extant legumes.

Translational Genetics and Genomics

Several resources for genetic and genomic research in L. japonicus have been developed, and an integrated genetic map of Lotus has been assembled. The Gifu and Miyakojima ecotypes of L. japonicus were used to develop the genetic linkage map together with the closely related cross-fertile Lotus burtii and Lotus filicaulis. In addition, recombinant inbreed lines have been developed from a Gifu×Miyakojima cross, from a L. japonicus Gifu×Lotus burtii cross and from an L. japonicus Gifu×Lotus filicaulis cross. In order to transfer this genetic information and gene discoveries to crops unfit for genetic analysis, a bioinformatics-based legume anchor marker design was developed and applied to bean (Phaseolus vulgaris) and groundnut (Arachis). This has revealed a high degree of colinearity, and a substantial synteny exists toward the genomes of other legumes such as pea, common bean, and peanut. Another observation from these studies suggests that the gene space in Papilionoid legumes may be divided into two broadly defined components: more conserved regions, which tend to have low retrotransposon densities and are relatively stable during evolution and, variable regions, which tend to have high retrotransposon densities and whose frequent rearrangement may contribute toward evolution of some gene families.

Genome and Proteome Resources

For further studies, the following genome resources have been developed: a general genetic map and bacterial artificial chromosome (BAC) libraries for positional cloning of untagged mutants recombinant inbred lines and inventories of expressed sequence tags (ESTs) sampling the gene expression profiles from several tissues and growth conditions. Deep sequencing is now contributing to defining transcribed regions, intron–exon structures, alternative splicing, and microRNAs (miRNAs), thus annotating the genome. Sequencing of the gene space of the L. japonicus genome is almost complete and the latest release of genome sequence provides 98% coverage with updates still to come. The full genome sequence of the bacterial M. loti MAFF 303099 strain and the symbiosis island of the M. loti R7A strain, including bacterial genes required for nodulation and nitrogen fixation, are available, together with a wide selection of rhizobial mutants. Lotus has over the last decade contributed to the discovery of novel genes involved in diverse biological processes. High-throughput transcriptome, proteome, and metabolome studies have been performed and the information made available in public databases. As an example, a large-scale proteomics analysis of seeds and seed pods through the different developmental stages into maturation of the seeds and senescence of the seed pod has been performed in order to improve our understanding of the developmental and metabolic processes leading to the production of the protein- and oil-rich legume seeds that is a major source of food and feed.