Rhizobium etli

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A Microbial Biorealm page on the genus Rhizobium etli

Classification

Higher order taxa

Bacteria (Domain); Proteobacteria (Phylum); Alphaproteobacteria (Class); Rhizobiales (Order); Rhizobiaceae (family)(1)

Species

Rhizobium etli

Description and significance

Rhizobium Etli is one of the many soil-living bacteria able to live in conditions of nitrogen limitation due to its distinctive ability to settle onto root nodules of legumes. Like other rhizobia, it is characterized as aerobic, gram-negative, and able to form symbiotic relationship with legumes. (3, 4) In specific, Rhizobium etli is the predominant bacteria found in legumes such as the common bean, P. Vulgaris.(5) Some of the different strains include Rhizobium etli bv. mimosae, Rhizobium etli bv. phaseoli, and Rhizobium etli CFN 42(2)

Rhizobium Etli is found world wide and discovered as early as 16th century. Due to its early existence, attempts to identify origin of the species were performed by identifying its molecular marker. This was conducted by searching a diversity of microbes within different Rhizobium etli species from P. Vulgaris. Isolation of the rhizobia strain from the nodule of the root of the plant was removed, sterilized with ethanol and hydrogen peroxides, and grown on YEM-Congo red agar medium. Isolation and identification was done by 16S rRNA-encoding DNA-RFLP which demonstrated most to be from species Rhizobium etli. The nodC gene was identified and isolated to be used as a molecular marker. Results from the experiment showed Rhizobium etli is not only found in the America’s but also identified in parts of Africa, Asia, and Europe. (5)

Rhizobium Etli is important enough to have its genome sequence because of its unique ability to form symbiotic relationship with legumes. The detail in which it performs this are in the following two sections (genome structure, cell structure and metabolism). To give a general idea of its importance, the host benefits by being provided nitrogen in the form of ammonia from the bacteria, while the bacteria is provided carbon and nutrients from the host. (4)

Genome structure

Rhizobium Etli has a complete genome sequence of 6,530,228 base pairs. It contains 4,381,608 circular chromosomes averaging 61.27% GC content. (7) Six plasmids contain the complete metabolic pathways. Two of the plasmids, p42a and p42d, are abnormal as it contains a lower GC value of 58% as compared to the other four plasmids at 61.5%. Also, the complete genome sequence reveals identical of more then 100 nucleotide repeats which are located in plasmid p42a and p42d. The plasmids appear to have been acquired at some point of divergence which is unknown. (7) The plasmids are also characterized by having a Rep ABC replicator which allows stability with distinct initiators and origins of replication. An advantage of the replicator is faster duplication to replicate its genome which is beneficial to its survival. (7)

As for its transcriptional regulation, 536 transcriptional factors are found, and 331 which are one-component regulators. The majority (65%) of the one component regulators are near ABC transporters or permease genes which may activate in response to the environmental stress in the soil.(7) Furthermore, 23 sigma factors are found. Though most of the roles are unknown, they are thought to required for gene expression under the different environmental conditions faced by the microbe. (7) While Rhizobium Etli has many genome structures and regulation for surviving in stressful environments, it is also characterized by having an unusually high number of specific genes and replicons.

Rhizobium Etli contains the most abundant number of replicons compared to other known nitrogen-fixing bacteria and its protein-coding genes, classified as COGs, are indicated to be overrepresented. Carbohydrate transport and metabolism, amino acid metabolism and transcription are vital to its niche of obtaining products from its plant host, which may be the reason these COGs are overrepresented. (7)

Cell structure and metabolism

An interesting feature of rhizobium etli is its ability to swarm as a mean of motility and colonization onto plant roots. The swarming is produced by flagella movement located in its extracellular slime layer. The organism contains a quorum sensing genes which binds a protein called N-acylhomoserine lactones. Its function is responsible for swarming, promoting surface colonization, and the ability to sense areas of low oxygen. Since the microbe is aerobic, sensing areas of low oxygen is an important component to its survival. (8) Another important aspect of the microbe is its metabolic pathways.

Though the exact number of pathways is not known, 263 metabolic pathways composing of 1,340 enzymatic reactions are thought to exist. A unique ability of rhizobia is its capability of switching from an ammonium assimilation metabolism to nitrogen fixation when it undergoes symbiosomes. The end product from its metabolism is used as a precursor by the plant. In exchange, the microbe receives nutrients and energy from the plant. (9) Pathways such as glycerol metabolism, thiamine biosynthesis, cobalamine biosynthesis, and the incomplete denitrification pathway are all located in the plasmids. Also, it contains high number of fermentation pathways, catabolism and anabolism pathways of amino acids, and polysaccharides. These pathway are important for the metabolism of the products it receives from the host plant. (8)

Ecology

Rhizobium Etli is a soil bacteria which interacts with the root of legumes. The microbe forms a specialized structure called a nodule in the plant's root, and differentiates into a bacteroid. The plant's cell membrane surrounds the bacteroids in the nodules, to form a peribacteroidal membrane. (9) At this point, the free living microbe has moved from the soil to a low-pH environment which contains certain carbon and nitrogenous compounds, as well as oxidative stress. (4) In the plants cells, it adapts by losing its ability to undergo cell division, and also by switching its metabolism focused on nitrogen fixation. (9) Adaptation is a fast process, which is critical to allowing the organism to colonize on the plant of roots. (4) The interaction between the two organisms to form the bacteroid, peribacteroidal membrane, and peribacterial space is a process called symbiosomes. (9)

Pathology

Rhizobium etli is not known to be a pathogen to humans. Instead, it forms a symbiotic relationship by binding the root of legumous plants. In the plants cells, it forms specialized structures called nodules in the plants root and differentiate into bacteroids which fix nitrogen. The plant is not harmed, but benefits from the presence of the microbe. (9)

Application to Biotechnology

In agriculture, crop rotation and soil fumigation is performed each year to prevent fungal and bacterial diseases. (6) Crop rotation is a tool in which plant species are not allowed to be at the same location year after year. Rotating the crops allows management of soil pathogens which prevents many plants to be infected. (10) While natural selection has allowed many crops to have genetic resistance for pathogens above the ground, there is few genetic resistance for soil pathogens. Knowledge of Rhizobium etli has allowed research to utilize its specialty of binding onto to roots to provide plants with antibiotics. Bioengineering has been successful in producing phenazine-producing bacteria which may one day be used to fight off soil pathogens.(11)

Current Research

Phenazine producing bacteria

In agriculture, crop rotation is performed each year to prevent fungal and bacterial diseases. (6) While natural selection has allowed many crops to have genetic resistance for pathogens above ground, there is few genetic resistance for soil pathogens. Since "Rhizobium etli", fungal and bacterial diseases all bind the same region on the root, the presence of an antibiotic would prevent unwanted pathogens from colonizing. (11) As reported earlier in the biotechnology section, successful gene expression of phenazine has been produced on rhizobium etli. Phenazine is a compound found with some Pseudomonas species and has been shown to work as an antibiotic against fungal activity. Drawbacks exist though, as the antibiotic-producing bacteria cannot fix nitrogen nor inhibit its bacterial growth. These drawbacks cause the wildtype to outcompete the bioengineered bacteria, and researchers are suggesting follow up work to finguring out if phenazine toxicity is the cause of the membrane damage.(11)


Sigma 70 factor

A study has been done to determine the sigma 70 factor of Rhizobium etli. The sigma 70 factor is the main part which controls genes involved with RNA polymerase and has been determined to be promiscuous as it was able to recognize promoters from other species such as E. Coli. The promiscuous nature of the sigma 70 factor allows it to recognize more variable structures. In specific, there is high variability in the G+C regions located in 10 boxes. The flexibility in recognizing promotors are thought to result from the biological diversity Rhizobium etli faces. Further research is needed to confirm if a more promiscuous factor makes it easier for adapting to different environmental conditions. In specific, researching the effects Sigma 70 has on lateral gene transfer needs to be investigated. (12)


Outer membrane of Rhizobium Etli

Extracelullar polysaccharides, capsular polysaccharides, and lipopolysaccharides make up the outer surface of Rhizobium Etli. The lipopolysaccharide (LPS) is composed of three regions: lipid A, core, and O-antigen polysaccharide. The article proves that the presence and number of O-antigen in the LPS is necessary for invasion and formation of root nodules on plants such as P. Vulgaris. Since Rhizobium Etli interacts with the cell surface of roots, it is exposed to cationic peptides. The plasma membrane is found to provide protection to the bacteroid during such interaction, allowing survival through structural changes. Follow up research is to be performed to determine the role the plasma membranes plays for survival of the bacteroids. The interaction between the LPS, and bacteroid may give increased knowledge of the molecular basis and increased sensitivity to cationic peptides. (13)

References

1. NCBI: Rhizobium etli, Accessed August 24, 2007.

2. NCBI: Rhizobium etli, Accessed August 24, 2007.

3. Encarnacion, S., Dunn, M., Willms, K., Mora, J. “Fermentative and Aerobic Metabolism in ‘’Rhizobium etli’’. Journal of Bacteriology. June 1995, p. 3058-3066.

4. Moris, M., Braeken, K., Schoeters, E., Verreth, C., Beullens, S., Vanderleyden, J., Michiels, J., “Effective Symbiosis between ‘’Rhizobium etli’’ and ‘’Phaseolus vulgaris’’ Requires Alarmone ppGpp.” Journal of Bacteriology. August 2005, p. 5460-5469

5.Aguilar, M., Riva O., Peltzer, E. “Analysis of ‘’Rhizobium etli’’ and of its symbiosis with wild ‘’Phaseolus vulgaris’’ supports coevolution in centers of host diversification.” Proceedings of the National Academy of Sciences of the United states of America. 2004 p.13548-13553

6. Cook, R., Thomashow, LS., Weller, DM., Fujimoto, D., Mazzola, M., Bangera, G., Kim, D. “Molecular Mechanisms of Defense by Rhizobacteria Against Root Disease” National Academy of Sciences. May 1995. Vol. 92. pp. 4197-4201.

7. González, V., Santamaría, R., Bustos, P., Hernández-González, I., Medrano-Soto, A., Moreno-Hagelsieb, G., Chandra Janga, S., Ramírez, M., Jiménez-Jacinto, V., Collado-Vides, J., Dávila, G. “The partitioned ‘’Rhizobium etli’’ genome: genetic and metabolic redundancy in seven interacting replicons” Proceedings of the National Academy of Sciences of the United states of America. March 2006. p. 3834-3839

8. Daniels, R., Reynaert, S., Hoekstra, H., Verreth, C., Janssens, J., Braeken, K., Fauvart, M., Beullens, S., Heusdens, C., Lambrichts, I., De Vos, D., Vanderleyden, J., Vermant, J., Michiels, J. “Quorum signal molecules as biosurfactants affecting swarming in Rhizobium etli” ” Proceedings of the National Academy of Sciences of the United states of America. September 2006. p. 14965-14970

9. Calderón-Flores, A., Du Pont, G., Huerta-Saquero, A., Merchant-Larios, H., Servín-González, L., Durán, S. “ The stringent response is required for amino acid and nitrate utilization, Nod Factor regulation, Nodulation, and nitrogen fixation in Rhizobium etli” Journal of Bacteriology. August 2005. p. 5075-5083.

10. Cook, J., Thomashow, L., Weller, D., Fujimoto, D., Mazzola, M., Bangera, G., Kim, D. “Molecular mechanisms of defense of rhizobacteria against root disease” Proceedings of the National Academy of Sciences of the United states of America. May 1995. p. 4197-4201

11.Krishnan, H., Kang, B., Krishnan, A., Kim, K., Kim, Y., “Rhizobium etli USDA 9032 Engineered to Produce a phenazine antibiotic inhibits growth of fungal pathogens but is impaired in symbiotic performance.” Applied and Environmental Microbiology. January 2007. p. 327-330

12. Ramierz, R., Masulis, I., Cevallos, M., Gonzalez, V., Davila, G. “ The Rhizobium etli sigma 70 (Sig A) factor recognizes a lax consensus promoter” Nucleic Acids Research. March 2006. Volume 34 p. 1470-1480

13. [ http://www.jbc.org/cgi/reprint/M611669200v1 D’Haeze, W., Leoff, C., Freshour, G., Noel, D., Carlson, R. “ Rhizobium etli CE3 bacteroid lipopolysaccharides are structurally similar but not identical to those produced by cultured CE3 bacteria” JBC Journals. April 2007. Manuscript M61166900]

Edited by Stephane Ly, student of Rachel Larsen