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Introduction
Rhizobia are estimated to fix over half of all biologically reduced N2 in the ecosystem, yet many theories predict all mutualisms should be rare due to factors such as cheating. Considering these Rhizobia are extremely abundant it is important to understand the costs/benefits of these symbiotic interactions in order to better describe their ecological importance. Industrial agriculture heavily relies upon artificially fixed nitrogen via the Haber-Bosch process which is very inefficient since 30-50% of the fertilizer will not be absorbed by plants. This excess will eventually be transported by water and can cause massive environmental damage such as toxic algal blooms. Though the majority of food comes from non-legumes, understanding nitrogen fixation in a legume model may eventually allow us to modify the bacteria for use in non-legumes ameliorating the waste and damage produced by current fertilization methods. These interactions are not as simple as just pure nutrient exchange and understanding these mechanisms could prove to be greatly beneficial in application to non-legume food crops. Most individual rhizobia cells will not infect a host, thus the limiting factor in rhizobia reproduction is the amount of receptive root hairs capable of nodulation. When nitrogen fixing bacteria engage in symbiosis they are essentially “the lucky few that win the lottery” when they infect plant host. In order to “win the lottery” rhizobia have evolved mechanisms that increase their chances of being able to form nodules within legume roots and wait for the opportune time to participate in the symbiosis.

Benefits to Rhizobium
Amino acids and carbon sources are not the only benefits provided to rhizobia in legume symbiosis. The type of molecules exchanged between the rhizobia and host plant depend on the species involved, but in general host plants exchange energetic molecules such as sugars or citric acid cycle (TCA) metabolites for nitrogen. Depending on the symbiosis, essential nutrients like amino acids, vitamins and metals can also be provided to the bacteria. The bacteria also benefit from protection against predation and greatly increase their ability to reproduce during the symbiosis.

Nutrients
When legumes engage in symbiosis, key metabolic pathways are upregulated to compensate for the increased energy demand from the bacteria. Hexoses, primarily sucrose, which is made in the leaves of plants is transported to root cells where the plant will perform glycolysis and TCA, ultimately turning the sugars into malate. The majority of carbon sources provided to rhizobia bacteroids are dicarboxylic acids from the TCA such as malate. These energy sources bind to the transporter, DctA, which will bring the malate into the bacterial cell, where it is then shuttled to the TCA or undergoes gluconeogenesis providing energy for all cellular processes, including N-fixation. Endosymbionts such as rhizobia typically rely on their host for all essential nutrients; iron, sulfur, vanadium and molybdenum are all crucial elements for nitrogengases are provided to the bacteria by its host. Iron can be transported to the bacteria via divalent metal ion transporters and Sulfur can be shuttled via other special transporter proteins. Cobalt is also required by rhizobia to synthesize vitamin B12 and many cobalt transporters have been discovered.

Protection
Protozoans that engulf rhizobia are found in nearly all soils. Engaging in symbiosis effectively isolates the rhizobia into nodules protecting them from possible protozoan predation. This also provides an anaerobic environment for the bacteria since oxygen is toxic to nitrogenases. Furthermore, some legumes will secrete phytochemicals that inhibit microbial growth, yet many rhizobia can endure these chemicals. For example, Mesorhizobium tianshanense, a rhizobium associated with licorice plants can withstand and even catabolize toxic amino acids derivatives, mimosine and canavanine, which would inhibit growth of most root microbes.

Reproduction
When rhizobia engage in symbiosis they can greatly increase their numbers compared to living freely in soil. One or a few founding rhizobium cell(s) can reproduce into 108 to 109 cells within a nodule providing great incentive to form a symbiosis. This is a change of over one million-fold which likely causes strong selection for Rhizobia to engage in symbiosis. Some rhizobia cannot reproduce after turning into bacteroids within their host, I.e. S. meliloti, yet in one study a nodule can still have up to 106 cells that have not differentiated into bacteroids and are capable of reproduction. If it is approximated 10% of the reproductively viable cells will migrate back into soil, the study concluded reproduction from symbiosis is still increased by 105, compared to living freely in soil.

Costs to Rhizobia: Predators/Competition
Common predators/competitors such as protozoa, bacteriophages, and bacteriocin producing rhizobia are likely to be more abundant in areas where rhizobia will try to infect hosts. It was found bacteriophage released by Rhizobium trifolii decreased the population of a strain vulnerable to the phage by 98%. Also, a rhizobia strain susceptible to bacteriocins was reduced by 99% in the presence of a R. trifolii bacteriocin producer. Since it is not a guarantee rhizobia will be able to infect a host, the cost of predation must be considered. There will be less cost of predation if the bacteria are in close proximity to a receptive root hair.

Chemical Sensing
Considering a major cost to rhizobia when engaging in symbiosis is death from predators such as protozoa or bacteriocin producing bacteria that will have high densities around receptive nodules, it would make sense that the rhizobia have mechanisms to lessen this cost. Rhizobia use QS and other chemical sensing mechanisms to determine distance from receptive nodulation area, density of predators and density of competitors. This also increases the chances of rhizobia being able to successfully infect the proper host plant and allows them to invest at the right time.

a. Quorum sensing
Research into QS involvement in the rhizobia-legume interactions is still in its infancy. Yet, it has been found that specific ligands, such as N-acylated-L-homoserine’s (AHL’s) that are common regulators of QS in many gram-negative bacteria can help influence rate of nodulation, number of nodules, and frequency of nodulation. Many of QS systems found in gram negative bacteria rely on AHL synthase (LuxI proteins) and AHL receptors (LuxR proteins). When AHL binds its LuxR receptor it can then bind to specific promoters to increase transcription of certain proteins. For example, biofilm formation of the infection thread is crucial for bacteria to successfully enter the plant epidermis and is regulated by QS. Specifically, the infection thread in the rhizobia species Sinorhizobium meliloti is mediated by the LuxI/LuxR pair SinI/SinR. Furthermore, naturally occurring AHL’s with longer acyl chains (>12 carbons), which are common to the rhizobia, can be detected by a host plant and ultimately increases the total number of nodules permitted within the roots. In terms of sensing density of competitors, it has been found that nodulation decreases with high rhizobia populations and mutants with hindered QS abilities will show more nodulation.

b. Nod-D pathway

The major signaling pathway between plant hosts and rhizobia is the Nod-D pathway. Plants will secrete flavonoids and isoflavonoids that the rhizobia sense and then begin expressing Nod factor genes. The Nod factors cause the root hairs to curl and envelop the bacteria where the rhizobia will eventually turn into nitrogen-fixing bacteroids. These factors are lipo-chito-oligosaccharides and are species specific which can help the bacteria to lessen the costs of predation. With specific flavonoid signals the bacteria will be enticed to engage in nodulation with the plant host. Rather than some random event, a specific signaling cascade occurs so bacteria know it is the correct species to infect, the right time to induce nodulation and not waste energy or risk predation at an inopportune time. This helps to maximize the chances of rhizobia infecting a host, considering legume-rhizobia symbiosis is analogous to “winning the lottery” for the bacteria.

c. Other signals
Sidephores are used by microorganisms to obtain iron from the environment. Rhizobia can sense competitors release of the siderophore, bradyoxetin, to determine competitor density. In vitro and in planta experiments of Bradyrhizobium show that at high concentrations of bradyoxetin, transcription of nodulation genes is inhibited. Again, similar to QS and specific Nod factors, these signals help bacteria to determine the best time to infect a root.

Save for a rainy day
Many rhizobia can live for over a year without external nutrients or symbiosis with a legume. This is important for the bacteria allowing them to live freely until they receive the chemical ques that conditions for nodulation are optimal. They do this by saving enough lipids in the form of PHB (poly-3-hydroxybutyrate) and phosphate for multiple generations to live off the excess resources. Up to 50% dry weight of a rhizobium can be in PHB. The symbiont of alfalfa plants, Sinorhizobium meliloti, can triple its population using stores of PHB as its sole carbon source. S. meliloti is considered a “persister” since it can live up to 528 days on its own energy reserves compromised of mostly PHB. In a phosphate free culture, B. japonicum, a soybean symbiont, can sustain around 5 generations with its phosphate reserves. Clearly, this has fitness impacts beyond the immediate cell lines helping to support future generations engagement in symbiosis, increasing the chances that the rhizobia species will eventually infect a host.

Biofilm formation
Not only are biofilms a crucial component for rhizobia to enter the plant epidermis, multi-species soil surface biofilms can also help rhizobia survive without a host until conditions are optimal to engage in symbiosis with a plant. Though it is unknown how often these biofilms on soil surfaces occur and clusters of a few rhizobium cells may be more common; biofilm formation is still a mechanism rhizobium can engage in to lessen costs. These biofilms can help prevent desiccation, help the bacteria live in non-ideal pH’s, and can also gain protection from certain species in the biofilm secreting antibiotics.

Amino Acid shuttling prevents plant dominance
Within the legume-rhizobia interaction, the bacteria become dependent on the plant for all amino acids and cease their own amino acid biosynthesis. In order for the bacteria to get the amino acids they will have to provide the plant with reduced nitrogen so the plant can synthesize the amino acid's. Yet, pea bacteroids can turn dicarboxylic acids such as malate into aspartate after also getting glutamine from the plant. The host plant can become dependent on this source of aspartate which is necessary for asparagine synthesis within the plant. This makes proper protein synthesis in the host plant dependent on the rhizobia for the two amino acids, aspartate and arginine. Thus, amino-acid cycling prevents plant or bacterial dominance in the symbiosis.

Alteration of the Rhizosphere
A well studied model shows certain strains of agrobacterium can alter the rhizosphere in favor of future generations. These bacteria contain plasmids that provide opine synthesis genes to the host plant. The plant will begin producing opines once infected and release the molecules into the rhizosphere. Since the majority of microorganisms cannot catabolize opines, it provides selective advantage for the agrobacterium that use the opines as a source of carbon and nitrogen. Host plants producing opines have higher densities of agrobacterium compared to wild-type host plants that have not been transfected by the plasmid containing genes for opine synthesis. Agrobacterium species cannot fix nitrogen, but many are closely related to rhizobia and both are in the family Rhizobiaceae . In two species of rhizobia, R. meliloti and R. leguminosarum, some bacteroids can produce rhizopines that the same free-living rhizobia species can then catabolize for energy. In terms of competition, when a rhizopine producing strain and non-rhizopine producing strains of rhizobium are present, the strain that can catabolize the rhizopines will be more abundant within nodules. This evidence suggests mutualistic rhizobia have the ability to provide selective advantage to their free-living relatives.

Summary/Ideal Conditions for Rhizobia Nodule Formation
This is very context dependent, but in general the risk of predation is higher near receptive root hairs. In order to lessen these costs rhizobia can sense distance from hair, competitor density and predator density. With the ability to store nutrients, bacteria can survive in the soil without a host and wait until chemical ques signal the conditions are optimal for nodulation. Though it is costly for the bacteria to produce excess nitrogen, rhizobia can benefit greatly from the interaction. The bacteria not only obtain energetic molecules, but other essential nutrients as well. Within a nodule a single bacterium can reproduce into millions of cells and be protected from pathogens providing great pressure for symbiosis. Amino acid shuttling can prevent plant dominance since the plant will be reliant on the bacteria for aspartate. By expanding our knowledge of legume-rhizobia symbiosis we may eventually use these bacteria for agriculture rather than the current methods of nitrogen fertilization which are inefficient and have negative consequences for the environment.