Azorhizobium caulinodans ORS571 (hereafter designated A. caulinodans) are Gram-negative, motile (flagellated), 0.5-0.6 um by 1.5-2.5 um in size, rod-shaped, obligate aerobes (8). A. caulinodans has the ability to fix atmospheric dinitrogen N2 in free living states as well as in mutualistic symbiotic association with its host plant Sesbania rostrata (2). This association is a rather complex, step-wise process that includes chemotactic migration of A. caulinodans towards its host plant, colonization, infection, and the initiation of nodule formation (2). The motile alpha-proteobacterium A. caulinodans can form nodules not only on roots but also on the stem of S. rostrata by crack-entry invasion at the site of adventitious roots (3). Sesbanians form a unique group of legumes that inhabit tropical and subtropical regions. Their habitats differ in terms of soil type, nutrient content, as well as the various biotic and abiotic factors (O2., temperature, pH) (1). To help rice grow, farmers plant Sesbanians, but it's not really the Sesbanians (Riverhemp is one common name) that help the rice, it's the microbes that fix the nitrogen for the river hemp that consequently help the rice. The most notable metabolic activity of A. caulinodans is nitrogen-fixation, which also highlights its ecological significance. Plants are only able to use nitrogen in its reduced forms, such as ammonia, NH3 (6). In host-rhizobial mutualism, the plant (S. rostrata) provides sugars derived from photosynthesis to its mutualist microsymbionts (A. caulinodans), which then use them for energy and fix nitrogen. So far, we have heavily relied on the use of conventional fertilizers to grow majority of our crops which have negative impacts on the global climate due to increased CO2 emission and aquatic “dead-zones” formation (6). Most farmers in Africa are unable to buy fertilizers and can instead benefit from the use of these stem-nodulated legumes (7). These nitrogen-fixing microbes, A. caulinodans, can help farmers reduce their dependence on chemical fertilizers. Due to their primitive features and easy visualization, stem-nodulating bacteria such as our A. caulinodans can be used as effective model organisms (7) for discovering more details involved in nodulation of different host plants. With short life-spans, higher mutation rates, and horizontal gene transfer to rapidly adapt to the environmental stresses, microbes truly are one of the most amazing, adaptive, and successful organisms that inhabit the Earth.
Electron micrograph of negatively stained Azorhizobium caulinodans ORS 571T grown in liquid medium. From a research article by Dreyfus et al. (8), published in INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Jan. 1988, p. 89-98. Source for image: Dreyfus B, Garcia J L, Gillis M, et al. Characterization of Azorhizobium caulinodans gen. nov. sp. nov., a Stem-Nodulating Nitrogen-Fixing Bacterium Isolated from Sesbania rostrata [Internet]. Microbiologyresearch.org. [cited 2020 Nov 6]. Available from: https://www.microbiologyresearch.org/docserver/fulltext/ijsem/38/1/ijs-38-1-89.pdf?expires=1604723413&id=id&accname=guest&checksum=785FFD853C6D77DC55462FCD89B3509B
Higher order taxa Superkingdom: Bacteria Phylum: Proteobacteria Class: Alphaproteobacteria Order: Rhizobiales Family: Xanthobacteraceae Genus: Azorhizobium Species: Azorhizobium caulinodans Taxonomy of Azorhizobium caulinodans ORS 571 (10) (NCBI) Azorhizobium caulinodans ORS 571 GOLD organism information (11) (JGI)
A. caulinodans was originally included in Rhizobium sp. but then, inspired by its stem-nodulation (cauli-) and diazotrophic, free-living state, strain ORS571 was renamed Azorhizobium caulinodans. The authors used 108 core proteins of 45 alpha-proteobacterial strains to construct a maximum likelihood phylogenetic tree. NCBI Microbial Genome Resource database was used for the sequence data of these bacterial strains. Core proteins in each strain were only present as single copies. A total of 32, 327 amino acids were included in the alignment and A. caulinodans 5.37 Mb genome was used as the reference (4). Xanthobacter autotrophicus was determined to be the closest relative, for A. caulinodans strain via protein expression pattern, DNA-DNA, and DNA-rRNA hybridizations. Xanthobacter sp. occupy diverse soil habitats, fix atmospheric nitrogen to ammonia, and associate with rice roots – of Oryza sativa. Upon 16S rRNA sequences comparison, X. flavus was found to be strongly related to A. caulinodans. The synteny was reported to be poor between the genomes of X. autotrophicus and A. caulinodans (4); synteny provides a method to determine conserved homologous genes between genomes of two different species being compared (9).
Maximum likelihood tree (4) of 45 alpha-proteobacterial genome sequences, reference genome was A. caulinodans (highlighted). Image from Lee et al. (4) Source for image: Lee K-B, De Backer P, Aono T, Liu C-T, Suzuki S, Suzuki T, et al. The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571. BMC Genomics. 2008;9:271. Available from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2443382/
A. caulinodans can be found in a wide array of habitats where it forms mutual symbiotic interactions, via nodulation, with its host plant Sesbania rostrata – a submergence-tolerant tropical legume with relatively fast growth rate (4). Naturally, ecological habitat of S. rostrata includes marshy areas, floodplains, muddy banks of various rivers and even the edges of the pools. They have been observed to grow in savannas as well (13) - transitional biomes intermediate between forest and desert, characterized by patches of grass and low-growing shrubs (14). An important biophysical limit to S. rostrata would be that it cannot grow beyond the altitude of 1600m (13). Though this plant species is native to the Sahel region of Africa (15), it has been introduced to several regions of Asia as well such as India, Bangladesh, Philippines, and Sri Lanka (13) S. rostrata has a fast growth rate; it can reach a height of 2 meter within 2 months. Where this legume has not been grown before, one needs to apply only a solution of its rhizobial mutual-symbiont; A. caulinodans for example. This increases the nitrogen fixation rate and allows for better survival under both flooded and dry conditions (13). This can be viewed as a contribution to the environment as now you are using mutualist biological microsymbionts, instead of fertilizers, to allow for the growth of certain plants. We get enhanced nitrogen fixation coupled with reduced pollution.
This is a waterlogged habitat, where Sesbania rostrata can thrive well. Waterlogged soil can be easily found across the globe; especially in Africa and Asia (India, Sri Lanka). S. rostrata have also been seen growing in swamps and savannas (13) (Not shown) Note: this does not show Sesbania rostrata, but Sesbania cannabina (another species of the same genus). Image from: Mondal et al. (16)
Azorhizobium genus has consisted primarily of two species that have been linked to the Sesbania host plant genus; Azorhizobium caulinodans that can associate with Sesbania rostrata and Azorhizobium doebereinerae that forms root nodules with Sesbania virgata (17). As mentioned earlier, S. rostrata can survive in a variety of habitats including savannas and swampy, marshy areas (13). S. virgata is a woody shrub with fast growth rate, reaching up to 4 meters in height and is highly adaptive in flooded environments. It can be found in various parts of Brazil, Argentina, and Uruguay (18). Currently, researchers have added Azorhizobium oxalatiphilum to the genus as well, and that strain was isolated from petioles of Rumex sp. (17). Rumex sp. are widely distributed in North America (19). In summary, the information about the possible habitats and associative partners for Azorhizobium is incomplete. So far, we have seen diverse habitat occupation, association with two distinct host plant species that can be found throughout North and South America (18, 19), Africa and in South Asia (13, 15). Sesbania legumes growing in marshy, swampy areas in Africa or Asia could indicate the presence of A. caulinodans (13), whereas Sesbania legumes in flooded areas of Brazil would show association with A. doebereinerae (18). The recently discovered A. oxalatiphilum can be found associated with Rumex sp. in beaches, saline marshes, and streambanks across North America (19). These could be the macroscopic markers one can look for to seek habitats of Azorhizobium sp. The level of nitrogen fixation depends on several factors, such as soil temperature, oxygen level in the rhizosphere environment, and how effectively nitrogenase enzyme can catalyze nitrogen fixation (6). The environmental physical and chemical abiotic factors necessary for optimal growth of host plants, will ensure stable association with the microsymbionts (mutualist) and continued exchange of photosynthates for fixed nitrogen (6). For A. caulinodans, its host plant S. rostrata grows best at a temperature above 25 deg. C, in swampy places, and in pH range from 5.5 to slightly basic. They are naturally found in waterlogged, clayey soils and can be tolerant of low to moderate salinity. Natural moisture requirements lie between 600-1000 mm rainfall (20). S. rostrata can grow up to an altitude of 1600 m from sea-level (13). These plants can grow in water, at 30 cm depth or more, and be cultivated in moist soil to be used as green manure for rice crops (20). Moist soils tend to have relatively less oxygen content in them as the soil pores get saturated with water (21). This aligns with the fact that nitrogenase activity of mutualist microsymbionts is inhibited by oxygen (6), so it is ideal for these plants to live in moist conditions. The microbes that associate with them no doubt, are quite capable of living in very diverse and dynamic environments. Relatively moist soil and alkaline content also characterize the physical abiotic factors of several plants of the Rumex sp. (19); the host for newly discovered Azorhizobium oxalatiphilum (17).
Significance to the Environment
Biological nitrogen fixation has a pivotal role in the cycling of nitrogen globally (4). Biological nitrogen fixation relies on nitrogenase; an enzyme complex that reduces molecular dinitrogen into ammonia (4). Free-living nitrogen fixers (diazotrophs) use the fixed nitrogen directly for growth while symbionts (mutualistic) provide that ammonia to a host plant. In return, they get sugars for growth from the plant and this can support the population better. The mutualistic interaction that occurs between leguminous crops and their mutualist microsymbionts, has an integral role in agriculture (4) and ecological success of the leguminous species (1). Plants require nitrogen for their growth and various metabolic processes. It is an integral component of amino acids, which are the subunits required for protein synthesis, as well as chlorophyll – crucial pigment involved in photosynthesis. ATP and nucleic acids also contain nitrogen, which are the energy currency and genetic information carriers of the cell, respectively. The most abundant element in Earth’s atmosphere is nitrogen gas but plants are not capable to use it; they require its reduced form, ammonia. This explains the importance of biological nitrogen fixation. Other sources of ammonia include decaying organic matter, lightning, and fertilizers (harmful!) (6). Important Biological Nitrogen Fixation organisms include cyanobacteria, Rhizobium, and Bradyrhizobium. Nitrogenase enzyme breaks high energy covalent bonds between dinitrogen gas and adds three hydrogen atoms to each nitrogen atom to form ammonia (NH3). Nitrogen fixing microbes require 16 moles of adenosine triphosphate (ATP) for reducing one mole of nitrogen – that is a lot of energy. For nitrogenase energy requirements, organic molecules are oxidized that the mutualist microsymbionts such as A. caulinodans, receive from their host plant. Mutualiostic bacteria that reside in plant nodules undergo significant morphological changes that make them entirely dependent on their host plant for energy, benefitting their hosts in return by fixing nitrogen (6). All prokaryotes that can fix nitrogen contain the enzyme, nitrogenase. The genes for this enzyme complex seemed to have been shared among many microbial populations by horizontal gene transfer, using plasmids (transformation), or natural evolutionary events (22). Balanced chemical equation is given as: N2 + 8H+ + 8e- + 16 ATP 2NH3 + H2 + 16ADP + 16 Pi (MicrobeWiki) Each nitrogenase complex catalyzes the formation of 2 ammonia per reaction, which get converted to ammonium (NH¬4+) by addition of a proton (22).
Image of nitrogen cycle. From MicrobeWiki (22)
It is worth mentioning that the balance in nitrogen content is essential for both, plant growth and climate health. Lack of nitrogen will result in plants characterized by yellowish color and smaller fruits. They show stunted growth. Adding too much nitrogen for plants will turn out to be toxic to them. If we use fertilizers to aid in plant growth, we risk global climate change as additional nitrogen can contaminate underground water sources (leaching), or it can run off and pollute the aquatic systems. This leads to a process called “eutrophication” which means that additional N2 in water will promote growth of plants and algae, phytoplankton. This is what makes the water bodies appear green. When the algae die and get decomposed, O2 levels in aquatic systems get depleted. As a result, we get ”dead zones” which cannot support life; consequently, aquatic organisms suffocate to death (23). For more information and easy graphical representations, visit Aczel et al (23) Environmental changes mediated by fossil fuel use, greenhouse gases emission, eutrophication have a negative impact overall on agricultural production. Not much research has been done to observe the impact of human activities on (non-staple) vegetables and legumes. One study however, reported in 2018 as to how the predicted changes in the environment would lead to reduced yields for non-staple vegetables and legumes. The limited adaptation of these crops in context of consistent global warming will likely result in their altered availability; they will be hard to afford and thus consumed less often. This will have negative health consequences. It is therefore important to emphasize on agricultural development sooner rather than later (24) and prevent food shortage in a world already facing hunger or severe malnutrition in various parts of the world. Studies have not shown yet a direct impact of human activity on A. caulinodans. No evolutionary events or horizontal gene transfer have been observed thus far in response to global climate change that would increase their adaptability. It is not clear how increasing CO2 levels will affect Azorhizobium microbes that thrive otherwise, well in O2 depleted microenvironment of host-nodules. One study has shown that the elevated levels of CO2 do have an impact on both abundance and diversity in nitrogen cycling functional genes, in certain soil microbes. This has resulted in low nitrogen content in soil (25).
Ecological Lifestyle and Interactions
In terms of ecological interactions, A. caulinodans show specificity and do not associate with a wide variety of plant hosts. They mainly form mutualistic interactions with plants of the Sesbania species, specifically in the roots or stem of S. rostrata and S. punctata. Stem-nodulating bacteria are more susceptible to varying environmental conditions than root-associated ones, where they must be able to survive and proliferate. High O2 levels, lack of combined nitrogen, and direct light exposure present unique challenges at the stem-level. This can explain how A. caulinodans has free-living nitrogen fixing ability unique to other symbiotic rhizobia, as the environmental conditions for stem-nodulation are not much different. Researchers sometimes consider them as aerial or epiphytic bacteria and not traditional soil bacteria (7). There are some similarities that exist in root and stem nodulation; both of these processes have been studied in extensive detail in S. rostrata. Nodulation sites are fixed and bound by the cavities that form in stem-located primordia or below the lateral roots. The Azorhizobia penetrate by direct intercellular infection of the plant basal cells, also called the crack-entry method. Upon entry, they multiply effectively, and fill up the intercellular basal cell spaces. The intercellular infection threads gradually protrude toward the meristematic cells of the cortex. Azorhizobia move via the infection threads into the cell cytoplasm, where a peribacteroid membrane forms around them. This marks the early stage of nodule development which happens 5 days after infection. At the center of the infected zone, a red color starts to develop which is characteristic of leghemoglobin. Now, the Azorhizobia can start their mutualistic N2-fixation for their host plant. The nodules of S. rostrata are round-shaped which is observed for the legumes typically found in the tropics. Sesbania stem nodules show a green cortex due to the presence of chloroplasts in the vicinity of Azorhizobia, in contrast to their dark-developed root nodules. Higher concentration of intercellular glycoprotein in the cortex of stem nodules have been hypothesized to protect the Azorhizobia against the major problem of high oxygen levels (7). Plants express certain genes during symbiosis called nodulin genes. The S. rostrata Enod2 gene showed varying expression patterns in root vs. stem nodules. There was transient expression in root nodules while stem nodules showed stable Enod2 expression. This could be due to a continuous need for protecting the microsymbionts against high-O2 tension (7). The nod genes encode proteins that allow for the developmental changes leading to the formation of the nodules (26). In low nitrogen conditions, legumes secrete specific flavonoids to attract host-specific rhizobia. This enhances nodulation and supports plant growth (43). Flavonoids are important determinants for A. caulinodans mutualistic interactions with its host plants. Free-living A. caulinodans could be induced to express nodulation genes upon flavonoid naringenin exposure (44). In fact, upon naringenin flavonoid introduction, the tropical legume-fixers A. caulinodans were able to colonize wheat roots (45) and increased colonization for Brassica napus was also observed (46). B. napus or canola is an important plant for oil and livestock (47). Moreover, they can form epiphytic associations with rice, forming nodules without the need of any prior enzyme or hormone treatment (7). Some nitrogen fixing genes have been found in the rhizosphere of sugar-cane that showed ≥90% sequence similarity to A. caulinodans (48). A. caulinodans also exhibits microbial interactions within its host plant. Naturally, it was determined that S. rostrata had 50 to 60% of the root nodules formed by Sinorhizobium strains. Interestingly, Sinorhizobium were only present in 10% of the stem nodules; the rest 90% stem-nodules were occupied by Azorhizobium. Sinorhizobium is also an important group of microbial nitrogen fixers that exhibit a narrow host range for Sesbania sp. (7). This highlights A. caulinodans to be mainly stem-nodulating and Sinorhizobium to be a more typical soil bacteria (7), and also illustrates how microbial biological interactions result in differential niche exploitation. Multiple niches allow more resources to be used and offer different microbes to live in harmony without having to compete. For S. rostrata, this provides more nitrogen fixation as both mutualist microsymbionts can “share” the host plant at the root vs. stem level. Unlike most Sinorhizobia, it has been found that Azorhizobia can nodulate stem even if the roots were previously nodulated for S. rostrata (7). It must have been an adaptive strategy evolved by the Azorhizobia strains. Many microbes live in biofilm communities; in case of A. caulinodans, it has certain genes and signaling pathways that allow it to transition from a motile form to establishing a sessile biofilm (4). This can in turn help with increased stability and better colonization of its host plants, benefiting their mutualistic relation and enhancing microbe-mediated nitrogen-cycling in the environment (49).
Significance to Humans
(a) S. rostrata + A. caulinodans as Green Manure Lowland rice yield in rainy areas can be profoundly increased, thanks to the high nitrogen fixing ability of S. rostrata in conjunction with its mutualistic microbial partner – A. caulinodans. Enhanced soil fertility, resistance to soil-flooding, and faster growth rates make these legumes valuable to humans native to flooded habitats (7). The basic principle behind green manuring is to enhance soil productivity by adding organic matter and nitrogen content. The practice of green manuring is ancient and dates back to the use of broad beans for this purpose by the Greeks, 300 B. C. (50). Up to 200 kg of Nitrogen content/hectare can be added to the soil in just 6-8 weeks, with S. rostrata serving as green-manure (7). Majority of this nitrogen content comes from the mutualistic Biological Nitrogen Fixer, A. caulinodans. This is equivalent to the use of 80-100 kg/hectare worth of mineral fertilizer. The average kg of rice grain yield, for every kg of N added in soil with current input levels, has been found to be higher with S. rostrata when compared to mineral fertilizer (7). In summary, reports from both Asia and Africa suggest that such green manuring can more than double the current rice yield. Most of such green-manuring has been restricted to research purposes only and it remains unclear whether stem-nodulating legumes should be adapted for this purpose in context of large-scale, rice-farming systems. In Asia, majority of small farmers can buy chemical fertilizers for their rice, limiting the use and application of S. rostrata green-manuring (7). Potential reductions in fertilizer subsidies and increased awareness about green manure benefits by the governments of major rice-producing countries can help pave the path for reintroducing this traditional method of increased soil productivity. This will undoubtedly improve agriculture and economy for developing nations. (b) Take A. caulinodans, directly nodulate the rice-plant? Host plants get directly benefited from their nitrogen fixers that reside in their nodules. Free-living nitrogen fixers cannot benefit the rice crop efficiently; there is colonizing competition for the rhizosphere environment and denitrification-mediated loss of nitrogen content. Understanding the molecular basis of nodulation has sparked interest in direct mutualistic interactions of nitrogen fixers with food crops. Larger populations of A. caulinodans were observed in rice rhizosphere following green-manuring with S. rostrata. High nitrogenase activity was detected for A. caulinodans in the rice-rhizosphere. A. caulinodans have been observed to create nodular structures in rice-roots without any hormone or enzyme treatment, using the same “crack-entry” method as seen for its host plant S. rostrata. However, this has been attributed to the first step in infection, not to a mutualistic symbiosis interaction (7). Possible experimentations to genetically mutate either the rice crop or A. caulinodans might help establish mutualism between the two. This might aid in enhanced manure-independent nitrogen fixation and tackle the issue of increased food-demand, in conjunction with less fertilizer use. (c) Wheat and canola interactions Researchers have observed wheat plant root colonization by A. caulinodans. Here, A. caulinodans resided in the intercellular spaces among the cortical root cells. Wheat colonization was significantly enhanced by a specific flavonoid “naringenin” – highest effectiveness was seen for 10mmol/m3 dosage. This helps us better understand nitrogen fixing microbial interactions with non-legumes (45). The lateral roots of canola (Brassica napus sp.) plant are also colonized by A. caulinodans. For canola plant (third most broadly cultivated crop next to wheat and barley in UK), the percentage of colonization was dependent on glucosinolate content in seeds; high- and low-glucosinolate-seed plants had low and higher colonization by A. caulinodans, respectively (an inverse correlation). Glucosinolates are potentially harmful chemicals for human and animal consumption and canola varieties low in glucosinolate have been grown. A. caulinodans colonization further increased with naringenin flavonoid treatment, without any change in glucosinolate content. This gives us two important chemical signals that independently alter A. caulinodans association with non-legumes. This also raises the question regarding how naringenin can increase the colonization without changing glucosinolate content, maybe by making A. caulinodans more resistant to glucosinolates (46)? (d) Less pollution, more “agriculture” Let us discuss a common problem; fertilizer use leads to pollution. Plants need reduced nitrogenous compounds to grow and industries use Haber-Bosch process to reduce nitrogen and manufacture commercial fertilizers (6). Haber-Bosch process involves conversion of nitrogen and hydrogen in ammonia, leading to rapid access of ammonia fertilizers. These fertilizers increase crop yield, but Haber-Bosch contributes to more CO2 emission than any other industrial chemical reaction (51). Increased fertilizer use disrupts the natural nitrogen cycle, run-off of these fertilizers to aquatic ecosystems leads to eutrophication that expand algal populations. These algae upon dying, increase microbial respiration which reduces the level of oxygen dissolved in water. With reduced oxygen, aquatic organisms die creating “dead-zones” (6). A possible solution to this problem: Biological Nitrogen Fixation. There are many microbes that fix significant levels of nitrogen in soil, either free-living or by mutualistic host plant interactions. Drastic effects and poor growth occur if legumes for example, are not nodulated with their nitrogen fixing partners (6). With such natural nitrogen fixers, we can easily cut short fertilizer-mediated marine ecosystem destruction and greenhouse gas emission. The future of agriculture might heavily depend on genetically engineered nitrogen fixers, each associating with a major crop and helping them grow; all with the added benefit of reduced pollution! Indeed, this would be the correct “utilization” of some of the most amazing microbes, A. caulinodans.
A. caulinodans are Gram-negative nitrogen-fixing bacteria. Their cells are rod shaped with measurements of 0.5 by 1.5-2.5 um for their dimensions. Interestingly, they had differences in the type of flagella according to the growth media. Peritrichous flagella was seen on solid medium and only a single lateral flagellum was observed in liquid medium (8). It is due to their flagella that they can move towards their host plant following chemotactic gradient. This allows for more competitive colonization, nodule formation, and biofilm synthesis on their host plant (42). The transition from a motile to static biofilm condition is advantageous if the organism is looking for more stable mutual interactions with S. rostrata. This is accomplished by A. caulinodans genes that encode for GGDEF/EAL domain containing proteins (4). Colonies were grown on agar plate and the characteristics of their morphology were circular with a creamy color. They were 1-2 mm in diameter and lacked the formation of slime. A. caulinodans strains were grown on both yeast-extract mannitol medium and lactate medium. Growth can occur for them at a wide temperature range of 12 – 43 deg. C. and a pH range of 5.5 – 7.8 (8). This range can account for their success with S. rostrata which itself inhabits a wide range of habitats including marshy areas, floodplains, muddy banks of various rivers, and savannas (13). They are obligate aerobes which rely on the availability of O2 for their metabolic activities and cellular respiration.
A. caulinodans has oxidase and catalase enzymes (8). Oxidases are the enzymes that simply transfer electrons from a donor to oxygen yielding hydrogen peroxide (H2O2) in the process (36). Both aerobes and anaerobes require oxidases in cellular respiration; they may just use different types of them (37). Hydrogen peroxide is a reactive oxygen species. In order to neutralize it, catalase decomposes H2O2 and keeps just about enough levels of it to establish homeostasis and maintain cell-signaling (38). Urease was not present for Azorhizobium (8) which converts urea to ammonia and carbamate. Carbamate then further spontaneously decomposes into ammonia and bicarbonate. A. caulinodans cannot catalyze this reaction, which is otherwise an important step of global nitrogen cycle (39). There were 4 azide-resistant mutant A. caulinodans strains reported in a research that showed significantly enhanced nitrogen fixation and induction of nodulation on host plants. When compared to parent strains, they either had the same or relatively higher symbiotic interactions (32). A potential target for antibiotic-treatment of these otherwise beneficial microbes could be their LPS molecules, which are normally found in Gram-negative bacteria. Below is a table of substrates to show what substrates A. caulinodans can metabolize: Growth on (A. Caulinodans) +/- D-Ribose, D-xylose, D-mannose, D-galactose, D-arabinose, L-arabinose - D-Maltose, D-cellobiose, lactose, inositol, raffinose - L-Xylose - D-Fructose, sucrose, trehalose - Acidification on glucose - β-Alanine, ethanolamine, L-arginine - Azelate, maleate, adipate, pimelate, suberate + Alkalization on glucose + L-alpha- Alanine + Citrate + Pyruvate + DL-malate + Succinate, 2-ketoglutarate +
Note: This table is from Dreyfus et al. (8). The complete table can also be accessed here: Dreyfus et al. (8). They proposed the new genus Azorhizobium and the first species identified was A. caulinodans. (type strain ORS 571) (8). We now know there are more - Azorhizobium oxalatiphilum associated with Rumex sp.(17) and Azorhizobium doebereinerae associated with Sesbania virgata (18). There is conflicting information regarding glucose metabolism for A. caulinodans. It is mentioned in Dreyfus et al. (8) that glucose is oxidized whereas a more recent study by Lee et al. (4) suggested that glucose cannot be metabolized. Both studies do agree on no metabolism for fructose, sucrose, xylose, ribose, and lactose (4, 8). Organic acids (succinate e. g.) are their primary carbon-source (8) which is verified by the occurrence of numerous C4-dicarboxylic acid transport systems (4). Interestingly, Dreyfus et al. (8) has shown them not capable of growing on ethanol (8) whereas Lee et al. (4) proposed that ethanol could be a primary carbon-source in case of flood as they house 16 putative alcohol dehydrogenase genes (4). A. caulinodans fix atmospheric nitrogen using nitrogenase enzyme (4). Nitrogenase enzyme functioning can be prevented by O2 (6) so the nitrogen fixation process is carried out in low O2 conditions. Successful symbiotic/mutualistic interaction also requires that the host species provides a rhizosphere to its nitrogen-fixing partner that is low in O2 pressure (6). This sort of presents a dilemma in a sense that a very efficient terminal electron acceptor is rendering the process of nitrogen fixation (40). In an attempt to answer that, researchers have looked at the nitrogenase complex and its encoding genes (41), and also hypothesized a few phenomena, such as respiratory protection and auto-protection (40). In respiratory protection, the bacteria simply increase their respiration rate when O2 concentration is high to use up more O2. This brings the O2 content low enough for the nitrogenase to carry out its function. Study showed that this concept does not sufficiently address the impact increasing O2 concentration has on A. caulinodans growth (40). Auto-protection better explains the relationship between A. caulinodans nitrogenase activity and altering O2¬ concentration. In this process, nitrogenase actually reduces (adds H+) O2 instead of dinitrogen (N2). This results in a significant loss of free energy, resulting in reduced growth of A. caulinodans and more demand for ATP. ATP-shortage induces more ATP-production, and this cleverly protects the nitrogenase by consuming more O2. (40). Researchers have tried to establish better understanding between O2 content and nitrogenase functioning, by looking at nitrogen-fixation gene deletions. Nitrogenase complex is composed of dinitrogenase and dinitrogenase reductase. Dinitrogenase is a heterodimer that contains gene products of nifD and nifK genes. Dinitrogenase reductase contains the nifH gene product only. The evolutionary history of nitrogenase complex hints at the occurrence of gene duplication. This allowed for them to adapt to the oxygenic environment – nitrogen fixation evolved prior to oxygenic photosynthesis. nifH1 deletion affected nitrogen fixation in semi-aerobic conditions and nifH2 deletion mostly influenced the same fixation process in microaerobic conditions. This implied a beneficial impact that nifH gene duplication had for A. caulinodans; the organism can now carry out its functions at a wider range of O2 requirements (41). Gene duplication is a process of evolutionary essence as it can add more genetic material and thus, by increasing the genetic toolbox for organisms, can allow for their increased adaptability.
Genome Structure, Content, and/or Gene Expression
Plants are unable to use atmospheric free nitrogen and need a way to get it in its reduced form. A. caulinodans can fix nitrogen for the host S. rostrata and this important function possibly led to the sequencing of A. caulinodans (35). Researchers investigated the genes for establishing the interactions with the host plant (26). The genome (34) and symbiosis island sizes are the smallest for A. caulinodans among the rhizobia sequenced thus far (26). Yet, they contain genes necessary for many vital functions and a high coding density (4). The genomic information has provided insights about its diverse metabolic capabilities, host interactions, habitat exploitation, as well as the need for further investigation to determine the function of hypothetical genes (4). Horizontal Gene Transfer has conferred the ability for this microbe to form mutualistic-symbiotic interactions with host plant S. rostrata (4). Together, the duo can provide potential environmental and agricultural benefits, such as Biological Nitrogen Fixation for decreased fertilizer needs (6) and legume manuring for higher crop yield (7). It has also been observed that S. rostrata plant flavonoids can signal its A. caulinodans to conjugatively transfer its symbiosis island to surrounding rhizobia in the rhizosphere (31). This results in higher frequency of HGT, symbiosis island transfer, and more nitrogen fixing bacteria to nodulate S. rostrata. This shows how the eukaryotic host affects bacterial horizontal gene transfer to enhance its own fixed nitrogen supply (31). Furthermore, scientists have discovered azide-resistant A. caulinodans mutants, as compared to their wild-type parents (32). Azide inhibits protein export while azide-resistant mutants demonstrate higher resistance to such inhibition (33). abeS, a gene that encodes for quaternary ammonium compound-resistance protein, has been found in A. caulinodans (53). A. caulinodans has a single circular chromosome, its genome size is 5.37 Mb, and is characterized by an average of 67% GC content. Various genomic islands have also been observed (4), which are genetic regions that appear to have been the products of horizontal gene transfer (27). Research found 4717 genes that encoded a protein product and a relatively high coding density of 89% which meant that there was one gene in every 1123 base pairs. There were many genes found that had a role in amino acid synthesis (2.8%), fatty acid metabolism (2.9%), transcription (1%), translation (4.8%), DNA replication and recombination (1.7%), and other regulatory functions (8.1%). About 175 genes (3.7%) however, showed no significant similarity with any of the registered genes. Least number of genes were involved for signal transduction (0.8%) and the highest number of genes belonged to the hypothetical group (20.2%) (4). A hypothetical gene does contain a start-stop codon and an ORF, but its product of expression has not yet been identified. Genes were also found, scattered throughout the genome, that encoded 44 tRNA species for all of the 20 amino acids (4). About half of the ribosomal protein genes (30 out of 57) occurred in a cluster, the others were dispersed throughout the genome (4). A. caulinodans follow chemoattractant signals to migrate to their host plant. There were a significant number of genes found for A. caulinodans that had a role in chemotaxis and showed its capability to respond to different types of molecules. Different mechanisms and proteins were also discovered that would provide protection to A. caulinodans against toxic environmental factors. These included antibiotic modifying enzymes, efflux pumps, and pathways for breaking down harmful plant toxins. In order to communicate with the host plant, hormones were found along with several exopolysaccharides and lipopolysaccharides on the cell-surface for nodulation, recognition, and attachment. Proteins with certain domains (GGDEF/EAL) were found that allowed the A. caulinodans to transition from a motile form to sessile biofilm, aiding in its attachment to the host (4). Using genome analyses, we can get an understanding of how microbes have evolved, the niches they occupy, and how they communicate with their hosts and other microbes. In order to survive and grow, A. caulinodans rely on transporter system for the uptake of amino acids, sugars, and other substrates, similar to those of other soil bacteria. Several sugars such as lactose, sucrose, glucose, were unable to be metabolized, whereas multiple C4-dicarboxylic acid transport systems indicated that A. caulinodans relied on dicarboxylic acids for their primary carbon source. Under flooded conditions, a switch to using ethanol for carbon was suggested evidenced by the occurrence of alcohol dehydrogenase genes. Glycolysis, Citric Acid Cycle, and the Entner-Doudoroff (5) pathways were encoded by the genome to derive energy for cell metabolism (4). Not surprisingly, due to the dual nature of A. caulinodans that makes it such an effective and versatile nitrogen-fixing microbe, genes were found for nitrogen fixation in both free-living and host-associated state. Their functions included formation of the nitrogenase complex, its stabilization, electron transport, and host symbiosis (4). nif genes occurred in multiple copies, spread throughout the genome, along with the several types of fix genes (4). nif genes, found in both free-living and symbiotic/mutual nitrogen fixing bacteria and cyanobacteria, encode for nitrogenase enzymes and various regulatory proteins that allow for nitrogen fixation. The expression of nitrogen fixing complex is also regulated by the levels of O2 and reduced nitrogen (28), reflecting how external factors influence what gene products are to be expressed. fix genes are important for nitrogen fixation in symbiotic/mutualistic interactions with the host plant, occurring normally in rhizobial species (29). Horizontal Gene Transfer resulted in the acquisition of an 87.6 kb long symbiosis region by A. caulinodans characterized by a relatively lower GC content and integrase sequences (4). Genes for the synthesis of Nod factors and chemotaxis are also found in this symbiosis island (4). Nod factors or nodulation factors are signaling molecules that rhizobia secrete in response to the flavonoids from the host plant, due to depleted soil nitrogen levels. The Nod factors will then cause the induction of nodule formation (30), which allows for the internalization of rhizobia, providing them an isolated microenvironment to differentiate into specialized nitrogen-fixing bacteroid cells (4). HGT is an excellent example as to how functioning can be altered by changes in the genome sequences, like the ability to form mutualistic interactions in an otherwise free-living microbe. For transfer of genetic information to fellow microbes, genes were also discovered for plasmid stability and transfer (via conjugation), in the symbiosis island. Some were homologous to the genes that allowed for the expression of a type-IV secretion system. It is used for transferring a tumor-inducing plasmid to the bacterial species of Agrobacterium tumefaciens (4).
The most interesting feature of A. caulinodans is its dual-nature for nitrogen fixation; it can do that on its own (free-living) or in conjunction with its host plant (mutualistic symbiosis). This is not observed for most other nitrogen fixing rhizobia. (4, 41, 52). Moreover, we have seen A. caulinodans nodulating both stem and roots. Stem-nodulation provides them a competitive advantage over other rhizobial bacteria (7). There is evidence for horizontal gene transfer of their symbiosis island (4) possibly due to plant-bacteria coevolution (7). S. rostrata mutualism with A. caulinodans, mostly on stem, and Sinorhizobia on roots (7) illustrates this concept of differential niche advantage. This not only ensures a higher survival potential of A. caulinodans via protective S. rostrata stem nodule microenvironment, but when looked at a broader context, leads to a better growth overall. When used as manure, environment can benefit from a higher soil nitrogen content, thanks to the amazing duo (7). This interesting feature of stem-nodulation can be helpful for humans, specifically for research purposes, using A. caulinodans as an effective model organism. Because of their exceptional characteristics (free-living, mutualistic N-fixation, root as well as stem nodulation, survival in flooded regions) (7), A. caulinodans can be used to discover more about “nodulation” in general, and “stem-nodulation” specifically. A. caulinodans can be used to observe colonization of non-legume cereal crops (wheat, rice, canola plants), unique flavonoids and other signals secreted can be discovered, analyzed, and exploited for higher yields (46). Lastly, we can use mutant A. caulinodans strains (genetically recombined, mutated, HGT etc.) to see whether we can establish direct nitrogen-fixing mutualisms with food crops such as rice, wheat, canola (7, 45, 46) and investigate the effects of anti-biotics, pesticides, and various enzymes in lab settings for predicting how human interactions might affect them. We need to determine how plants allow or inhibit certain microbial interactions, how flavonoid signals work both independently and in an “interdependent manner” and broaden the study for an extensive comparative analysis of legume and non-legume “symbiosis”. A. caulinodans can surely revolutionize our current agronomical plant-microbial knowledge and applications.
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