Mesorhizobium loti

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A Microbial Biorealm page on the genus Mesorhizobium loti

Classification

Higher order taxa

(Domain)Bacteria; (Phylum) Proteobacteria; (Class) Alphaproteobacteria; (Order) Rhizobiales; (family) Phyllobacteriaceae; (genus) Mesorhizobium; (species) loti) [Link for M. loti - use [1] NCBI link to find]

Species

Mesorhizobium loti, formally known as Rhizobium loti

Description and significance

Though this bacteria was vaguely studied starting in 1982 by B.D. Jarvis, it was finally genomically sequenced by the Kazusa DNA Research institute in 2000. The purpose of studying the Mesorhizobium species was to further understand the process of symbiotic nitrogen fixation as well as horizontal gene transfer among natural microsymbionts. M. loti was isolated from the rhizosphere of a legume called Lotus japonicus in the grassland areas of New Zealand and parts of China. This bacteria can also be found on several other Lotus species. Here the growth of the plants was studied and compared to other plants in the surrounding fields. Several plants were obtained and the bacteria found was knocked out through genome sequencing and analysis and was then compared to a control plant to see the function and role the bacteria played. From this, strain MAFF303099, Mesorhizobium loti, was discovered. Mesorhizobium loti is a member of Rhizobia. Rhizobia is a collective name for the genera of Rhizobium, Sinorhizobium, Mesorhizobium, and Bradyrhizobium; rhiza, Greek for the word root, and bios, Greek for the word life. M. loti are soil and rhizosphere (zone that surrounds the roots of plants) bacteria that undergo nitrogen-fixing symbiosis with leguminous plants: such as clover, beans, and soy). It is a motile, gram-negative bacteria, with non-sporulating rods and cannot perform nitrogen fixation without a plant host. Nitrogen fixation begins at the root hair and increases in abundance when the air is deficient in plant-usable nitrogen. To enter into the plant, M. loti travels down through an anoxic center of the root hair cell, at this point it proliferates and forms a nodule. Here, the bacteria differentiates into a bacteroid (in definition: a rod-shaped or branched bacteria in the root nodules of nitrogen-fixing plants). M. loti fixes the nitrogen from the atmosphere into ammonium (NH4+) with the enzyme nitrogenase. This is described as a symbiotic relationship because this nitrogen is fixed to a plant-usable form; in return, the plant supplies M.loti with carbohydrates, proteins, and oxygen. Mesorhizobium loti is also very useful for research anaylsis of other related species as well as those whose pathologies are related. Also, the quest for better understanding horizontal gene transfer and the evolutionary changes that occur over the years is still being examined. For example, comparative sequence analysis of the genomic similarities between Mesorhizobium loti and Escherichia coli was done to better understand the effects of different bacteria on certain plants. Since Mesorhizobium loti survives in a beneficial symbiotic relationship with other plants, bacteria which have a detrimental pathology to plants can be studied and compared to non-harmful bacteria in order to further understand its effects. Functional assignment of the different genes was performed based on the similarity of genes of known function. For genes that encoded 100 amino acids residues or more, a BLAST score was taken and an e-20 value was taken. Those with a high e-20 value were considered to encode smaller proteins.

Genome structure

The complete nucleotide sequence of the genome of a symbiotic bacterium Mesorhizobium loti strain MAFF303099 was determined in 2000. Much of the sequencing was found through whole-genome shotgun strategy combined with “birdging shotgun” methods. Four shotgun libraries with three types of cloning vectors were obtained through this linear bacterial strain. Further analysis found that the genome of M. loti consisted of a single chromosome (7,036,071 bp) and two circular plasmids, designated as pMLa (351,911 bp) and pMLb (208, 315 bp). The chromosome was made up of 6752 potential protein-coding genes, two sets of rRNA genes and 50 tRNA genes representing 47 tRNA species. 3675 (54%) of the protein genes showed sequence similarity to genes of known function, 1423 (21%) to hypothetical genes, and the remaining 1654 (25%) had no apparent similarity to reported genes. 63.7% of the genomic sequence was found to consist of GC content. Also, a 611 kb DNA segment, highly probable that it is a candidate of a symbiotic island, was identified as well as 30 genes for nitrogen fixation and 24 genes for nodulation were assigned in this region. Codon usage analysis suggested that the symbiotic island as well as the plasmids originated and were transmitted from other genetic systems through horizontal transfer, though this is still being investigated. The genomes of two plasmids, pMLa and pMLb, contained 320 and 209 potential protein-coding genes, respectively, for a variety of biological functions. These include genes for the ABC-transporter system, phosphate assimilation, two-component system, DNA replication and conjugation, but only one gene for nodulation was identified. Some genes have already been identified to code for those of amino acid biosynthesis, cell envelope, energy metabolism, transcription, translation, replication, biochemical synthesis, and many others that are important for bacterial survival.

Cell structure and metabolism

Because M. loti undergoes nitrogen fixation, many useful compounds and enzymes arise. The process occurs first from the nitrogen limiting conditions of plants from the pea family Fabaceae in which it forms a symbiotic relationship with Mesorhizobium loti. Within these legumes the nitrogen from the atmosphere turns into ammonia (NH4+) and then taken in by the plant in order to form amino acids, nucleotides, and things such as vitamins, flavones, and hormones. The amino acids are used to create specific proteins and the nucleotides to build DNA, RNA, and ATP. In order to fix nitrogen for the necessary enzymes, certain structures and steps need to come about. First, the nitrogen gas in the nodule comes in and it needs to be split. To split the gas, energy created by photosynthesis is taken from the leaf. Also, sucrose is broken down into malate and provides a carbon source for the bacteroid. Not only do the nodules have the nitrogen, they also store the iron containing protein hemoglobin which aids in the nitrogen fixation process. As nitrogen enters into the roots the legumes also release compounds called flavnoids. This stimulates a cascade in which M. loti produces nod factors which trigger other biochemical and morphological changes to happen. For example, cell division is triggered in the root to create the nodule, and the root hair redirected to grow around the bacteria until it fully encapsulates it. To further this cascade, the bacterium, once encapsulated, divides multiple times, forming a microcolony that enters into the nodule through a structure called an infection thread. This infection thread grows through the root hair into the basal part of the epidermal cell, then going on towards the root cortex. Finally, the bacterium is surrounded by the legumes membrane and is differentiated into bacteroids that then fix nitrogen. Interestingly, this nodulation process is controlled by a variety of other things that occur both externally (heat, acidic soils, drought, nitrate) and internally (autoregulation of nodulation, ethylene). The internal autoregulation of nodulation controls nodule numbers per plant through a systemic process involving the leaf. Again, this autoregulation causes a cascade of events to occur. First the leaf tissue senses the early nodulation events in the root through an unknown chemical signal. This then restricts further nodule development in newly developing root tissue. The Leucine rich repeat (LRR) receptor kinases, called NARK, in soybean and HAR1 in Lotus japonicus, are essential for autoregulation of nodulation. Without autoregulation of the nodulation, nodulation would overproliferate.

Ecology

The ecology of Mesorhizobium loti is still under development. Its specific evolutionary history as well as specific locations where they are found are still uncertain. It is known that Mesorhizobium loti comes from Rhizobia but how or when it evolved is still undetermined. M. loti is known to contribute to nitrogen usage in plants by transforming gaseous nitrogen into usable mineral nitrogen, ammonia, thus allowing plants to undergo necessary processes, using and recycling many elements in the environment. Most of the plants Mesorhizobium are found in are those of the Lotus species. The symbiotic nitrogen fixation which M. loti undergoes is important because it benefits the environment by avoiding the use of once necessary agricultural inputs and ensures flexibility of sources and needs of the plants and thus is a major contribution in soil fertility. Using nitrogen fertilizers consumes fossil energy since two tons of fuel are needed to spread each ton of the fertilizer. Using nitrogen-fixing plants such a legumes to produce plant proteins results in a decrease of the consumption of fossil energy, therefore decreasing the effect agriculture has on global warming. There are no known interactions with humans or animals.

Pathology

Mesorhizobium loti is not known to cause diseases in humans, animals, or plants. In fact, the novelty of aiding in nitrogen use in plants is widely praised by scientists and environmentalists for its positive symbiotic lifestyle. Its lack of pathogenicity is also appreciated by researchers because of its ability to be used as a comparison to other pathogenic bacteria discovered.

Application to Biotechnology

Mesorhizobium loti has many applications to biotechnology today. As described earlier, this bacteria undergoes an important process of nitrogen fixation which makes legumes an ideal agricultural organism for people around the world. Through this process, many useful compounds and enzymes are created, most importantly the byproduct of ammonia. Since Mesorhizobium loti helps bring in plant-usable nitrogen, plants are able to survive in low nitrogen areas, and also reduce the need for nitrogen fertilizer, a costly expense for farmers. It was also found that Mesorhizobium loti encodes haloalkane dehalogenases, a key enzyme in degrading halogenated aliphatic pollutants. The open reading frame mlr5323 found in this bacteria is also found in crude extracts of the organism Escherichia coli, both of which are known to dehalogenate 18 halogenated compounds. With this in mind, researchers are able to use M. loti to degrade pollutants in the air and soil through plant usage. This process is still in its initial steps.

Current Research

Currently there are studies in bacterial genome research in order to learn more about bacteria–plant interactions. At the Protection and Food Research Centre in London there are undergoing studies about the invasion of Lotus japonicus root hairless 1 by M. loti. In many legumes, including Lotus japonicus and Medicago truncatula, susceptible root hairs are the primary sites for the initial signal perception and physical contact between the host plant and the compatible nitrogen-fixing bacteria that leads to the initiation of root invasion and nodule growth. However, there are many mechanisms of nodulation have been described several legume species that do not rely on root hairs. To clarify the significance of root hairs during the L. japonicus-Mesorhizobium loti symbiosis, the labs have isolated and performed an analysis of four independent L. japonicus root hair developmental mutants. Through that, it is speculated that although it is important for the efficient colonization of roots, the presence of wild-type root hairs is not required for the initiation of nodule growth and the colonization of the nodule structures. At the FraLin Biotechnology Center located at Virginia Tech, Mesorhizobium loti is being used to find out more about the formation of essential nitrogen containing compounds. The Dean lab uses molecular biology approaches such as site directed mutagenesis to determine where and how N2 becomes bound to an iron cofactor to be prepared for reduction to ammonia. So far the lab has discovered that iron and sulfur are processed by specific proteins NifU and NifS. Using genetic defects that affect normal activities may allow specific functions to be studied in hopes of being able to replicate the function of nitrogen fixation for future uses in plants and crops that do not contain these nitrogen fixating bacteria. There is also current research in which Mesorhizobium loti increases root-specific expression of a calcium-binding protein homologue. This research is being done through a promoter tagging program in Lotus japonicus in order to identify plant genes which are involved. Scientists are inoculating M. loti in the roots of mutant promoterless reporter genes uidA in several legumes. When sequencing the genome of these legumes, specific calcium-binding proteins named LjCbp1 (calcium-binding protein) are shown to have formed motifs. Through Northern analysis between different legumes it was found that this protein arose specifically in the roots of the Lotus japonicus and no others. Further comparison and analysis of these results are currently in process.

References

1. Webb, Scot, Nicolson, Judith, Leif, Margarette. "Mesorhizobium loti Increases Root-Specific Calcium Binding Proteins." Institute of Grassland and Environmental Research, February 24 2000 1-11. <http://apsjournals.apsnet.org/doi/pdfplus/10.1094/MPMI.2000.13.6.606?cookieSet=1>. 2. Toshiki, Takuji, Itakura, Nukui, Uchiumi, Ohwada, Hisayuki , Noriyuki . "Expression Islands Clustered on the Symbiosis Island of the Mesorhizobium loti Genome." Journal of Bacteriology April 2004 1-3. <http://jb.asm.org/cgi/content/abstract/186/8/2439?etoc>. 3. Jarvis, BDW. "Mesorhizobium loti." NCBI. 1997. 26 Aug 2007 <http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=381&lvl=3&p=mapview&p=has_linkout&p=blast_url&p=genome_blast&lin=f&keep=1&srchmode=1&unlock>. 4. “Mesorhizobium loti." GENEQIZ. June 2001. 25 Aug 2007 <http://jura.ebi.ac.uk:8765/ext-genequiz//genomes/mel0103/index.html>. 5. Kaneko T, Nakamura Y, Sato S, Asamizu E, Kato T, Sasamoto S, Watanabe A, Idesawa K, Ishikawa A, Kawashima K, Kimura T, Kishida Y, Kiyokawa C, Kohara M, Matsumoto M, Matsuno A, Mochizuki Y, Nakayama S, Nakazaki N, Shimpo S, Sugimoto M, Takeuchi C, Yamada M, Tabata S., "Kazusa DNA Research Institute, Kisarazu, Chiba , Japan.." omplete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. NCBIDec 31 2000 3-6. <http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=11214968&dopt=AbstractPlus>. 6. "RhizoBase." The Genome Database for Rhizobia. Aug 1, 2007. 26 Aug 2007 <http://bacteria.kazusa.or.jp/rhizobase/>. 7. Szczyglowski K, Shaw RS, Wopereis J, Copeland S, Hamburger D, Kasiborski B, Dazzo FB, de Bruijn FJ. 1998. Nodule organogenesis and symbiotic mutants of the model legume Lotus japonicus. Molecular Plant–Microbe Interactions 11, 684–697. 8. Wopereis J, Pajuelo E, Dazzo FB, Jiang Q, Gresshoff PM, de Bruijn FJ, Stougaard J, Szczyglowski K. 2000. Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype. The Plant Journal 23, 97–114. 9. Dasharath P. Lohar and Kathryn A. VandenBosch, "Grafting between model legumes." Journal of Experimental Botany . 9 March 2005. Department of Plant Biology, University of Minnesota. 26 Aug 2007 <http://jxb.oxfordjournals.org/cgi/content/full/56/416/1643#SEC4>. 10. Gene Lay, 2000. www.biolegend.com , BioLegend Inc.

Edited by student of Rachel Larsen

Edited by KLB