Memoria para optar al Título Profesional de Ingeniero Agrónomo Mención: Fitotecnia

The Rhizobia are a group of free-living soil bacteria known for their ability to symbiotically infect the roots of specific host plants as well as to produce siderophores in order to compete with other microorganisms for the limited availability of iron in the rhizosphere. In this study, Rhizobium leguminosarum ATCC 14479, which preferentially infects the red clover Trifolium pratense, was found to produce the trihydroxamate siderophore vicibactin (C 33H55N6O15) under iron restricted conditions. In addition, two other iron-binding, siderophore-like compounds: C20H36N4O10, C31H 55N6O15, were isolated and purified from the culture media. Due to the structural similarity of the latter compounds to vicibactin based on electrospray-mass spectrometry and nuclear magnetic resonance data, these heretofore unreported molecules are thought to be either modified or degraded products of vicibactin. Although vicibactin has previously been found to be commonly produced by other rhizobial strains, this is the first time it has been chemically characterized from a clover infecting strain of R. leguminosarum.

The Rhizobia are a group of free-living soil bacteria known for their ability to symbiotically infect the roots of specific host plants as well as to produce siderophores in order to compete with other microorganisms for the limited availability of iron in the rhizosphere. In this study, Rhizobium leguminosarum ATCC 14479, which preferentially infects the red clover Trifolium pratense, was found to produce the trihydroxamate siderophore vicibactin (C 33H55N6O15) under iron restricted conditions. In addition, two other iron-binding, siderophore-like compounds: C20H36N4O10, C31H 55N6O15, were isolated and purified from the culture media. Due to the structural similarity of the latter compounds to vicibactin based on electrospray-mass spectrometry and nuclear magnetic resonance data, these heretofore unreported molecules are thought to be either modified or degraded products of vicibactin. Although vicibactin has previously been found to be commonly produced by other rhizobial strains, this is the first time it has been chemically characterized from a clover infecting strain of R. leguminosarum.

Many gram-negative bacteria produce and secrete siderophores under iron-deficient conditions. Siderophores are low molecular weight compounds (600-1500 Daltons), which chelate ferric iron with an extremely high affinity, and the complex is actively transported across the outer and inner membranes of gram-negative bacteria. There are two main classes of siderophores: catechol and hydroxamate. Catechol-type siderophores chelate ferric iron via hydroxyl groups, and hydroxamate-type siderophores chelate ferric iron via a carbonyl group with an adjacent nitrogen. Rhizobia fix atmospheric nitrogen symbiotically in leguminous plants using the iron-containing enzyme nitrogenase. To satisfy their iron requirements, many rhizobia are known to produce siderophores. Rhizobium leguminosarum Strain IARI 312 is known to infect pigeon pea plants. R. leguminosarum Strain IARI 312 produces both a catechol-type and a hydroxamate-type siderophore when grown under iron deficient conditions. The catechol-type siderophore has been purified and chemically characterized, and is consistent with that of enterobactin.

Siderophores are small, iron chelating molecules produced by many bacteria to help meet the iron requirements of the cell. Multiple metabolic functions require iron as it serves as a cofactor in many enzymes and cellular processes. However, in the presence of oxygen and at physiologic pH, iron forms insoluble ferric complexes that cause the nutrient to be unavailable to bacterial cells. Siderophores alleviate this limitation by chelating the ferric iron, rendering it soluble and available for uptake. One group of microorganisms known for their ability to produce siderophores is the rhizobia. These bacteria are characterized both by their formation of symbiotic relationships with leguminous plants and their ability to fix atmospheric nitrogen. Rhizobium leguminosarum ATCC 14479, which infects the red clover Trifolium pratense, was found to produce a trihydroxamate siderophore. Purification and chemical characterization identified this siderophore as Vicibactin that has been found to be produced by other rhizobial strains.

Rhizobium leguminosarum is a Gram negative nitrogen-fixing soil bacterium. Due to the limited bioavailability of iron, bacteria utilize siderophores that scavenge and bind available iron. The transport of iron-siderophore complexes is achieved by the TonB-ExbB-ExbD complex. We have previously shown that a functional TonB protein is necessary for iron transport by creating ΔtonB mutants and assessing their growth and 55Fe-siderophore transport ability. We attempted to identify and characterize the roles of putative exbB and exbD genes using a similar approach. Growth curves and sequence analyses suggest putative exbB and exbD may be the tolpal-associated genes tolQ and tolR. Phenotypic and sensitivity assays showed mutants do not exhibit the characteristic tol phenotype and are not sensitive to detergents or changes in ionic strength of the growth medium. We also expressed and purified the 120 amino acid fragment of the TonB C-terminus for further physical and chemical characterization.

Siderophores are low molecular weight, iron chelating compounds produced by many bacteria for uptake of iron in case of iron scarcity. Vicibactin is a trihydroxamate type siderophore produced by Rhizobium leguminosarum bv. trifolii ATCC 14479. This work focuses on identifying an outer membrane receptor involved in the transport of vicibactin. We have confirmed the presence of the putative fhuA gene in R. leguminosarum bv. trifolii ATCC 14479. This bacteria shows mutualistic symbiosis with the red clover plant Trifoliium prantense. Leghemoglobin, with its cofactor heme is present in the plant root nodules that surrounds the infecting organism present in the nodules. This work attempts to elucidate the ability of Rhizobium leguminosarum bv. trifolii ATCC 14479 to utilize heme-bound iron and genes involved in the transport. We have also elucidated the role of energy transducing proteins TonB- ExbB-ExbD on the heme-bound iron uptake system.

No description available.

Apios priceana is a native endangered species plant found in the Southeast United States. It is characterized as a leguminous species that bears wisteria like clusters with pea like flowers, a large tuberous root and four to six inch long seed pods. It is believed the Native Americans and early European settlers relied on this species as a source of protein and utilized the seeds for cultivation of the tuberous “potato” which formed. Apios priceana contains an average of 13% fiber, 6.9% protein, 71% carbohydrate and 9 of the 11 essential amino acids needed in human diets (Walter et al.,1986). In addition, A. priceanatuberous roots contain anti-carcinogenic properties known to be used to treat prostate and breast cancer as well as lowering blood pressure and cholesterol with an added use for diabetes. If removed from the endangered species list it could prove to be a valuable agronomic crop. Its use spans human and animal consumption, bio fuel, medicinal and horticultural purposes. This research was initiated to investigate a biological symbiosis between A. priceana and known beneficial soil bacteria which may indicate growth potential of known colonies. Experimental treatments were 1) no inoculation 2) Azospirillum brasilense inoculate 3) Bradyrhizobium japonicum inoculate and 4) Rhizobium leguminosarum biovar viceae inoculate. Specimens were evaluated at 30, 60 and 90 day’s growth from emergence for taproot length, number of lateral roots and taproot girth. Due to non-germination of seeds, data presented is for treatments 2 and 3. The correlation coefficient for average taproot length, number of lateral roots developed and taproot girth per treatment was as follows: taproot length to number of lateral roots, positive correlation coefficient 0.996; taproot length to taproot girth, positive correlation coefficient 0.999; and number of lateral roots to taproot girth, positive correlation coefficient 0.991. All correlation coefficients are significant at the 0.01 level.

Reproduction de : Thèse de doctorat : Biosciences végétales : Toulouse 3 : 2008. / Titre provenant de l'é215-232.