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No description available.

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.

The genome of 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 contains two genes for malic enzymes. One uses NAD⁺ as a cofactor (𝘥𝘮𝘦) and one utilizes NADP⁺ (𝘵𝘮𝘦). The two enzymes have been purified and the genes cloned and sequenced. Loss of TME enzyme function gives no detectable phenotype in either 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 grown in culture or in bacteroids. Loss of DME function gives no detectable phenotype in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 grown in culture but does result in bacteroids that are unable to fix nitrogen (Fix⁻). Expression of 𝘵𝘮𝘦 is reduced in bacteroids whereas 𝘥𝘮𝘦 expression remains unchanged. In order to overexpress 𝘵𝘮𝘦 in bacteroids a fusion gene was constructed with the 𝘥𝘮𝘦 promoter driving expression of the 𝘵𝘮𝘦 structural gene (𝘥𝘵𝘮𝘦). The 𝘥𝘵𝘮𝘦 gene was expressed and functional in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 cells grown in culture, but alfalfa plants inoculated with strains expressing only the 𝘥𝘵𝘮𝘦 gene were Fix⁻. In addition the NAD⁺-dependent malic enzyme gene from 𝘚𝘵𝘳𝘦𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘣𝘰𝘷𝘪𝘴 (𝘮𝘢𝘦𝘌) was similarly cloned downstream of the 𝘥𝘮𝘦 promoter. The fusion gene 𝘥𝘮𝘢𝘦𝘌 was expressed in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 cells grown in culture, surprisingly plants inoculated with strains expressing only the 𝘥𝘮𝘢𝘦𝘌 gene showed a Fix⁻ phenotype. A truncated 𝘥𝘮𝘦 gene was constructed which contained only the N-terminal, malic enzyme domain of the protein (𝘥𝘮𝘦Δ𝘗𝘴𝘵). The truncated enzyme was expressed and active in 𝘙. 𝘮𝘦𝘭𝘪𝘭𝘰𝘵𝘪 cells grown in culture and gave a Fix⁺ phenotype when inoculated onto alfalfa plants. / Thesis / Master of Science (MS)

R. meliloti carrying defined deletions that remove fix114 form Fix- nodules which are devoid of intracellular bacteria. Occasionally strains which carry these deletions form pink nodules which appear effective in contrast to the normal white ineffective nodules formed by strains carrying fix114 mutations. Bacteria isolated from these pink nodules retain the original deletion and form effective pink nodules when reinoculated onto alfalfa. It is hypothesized that these isolates carry second site mutations which enable the bacteria to overcome the symbiotic block associated with the fix114 mutation. In this work, five independent isolates were examined and were shown to carry second site mutations that suppress the symbiotic ineffectiveness completely on alfalfa and incompletely on sweet clover. The five independent second site revertants can be divided into two classes based on genetic data and on their sensitivity to detergents and both classes were localized to the chromosome of the wild type Rm1021. One such second site revertant allele, sfx-1, was cloned and localized to a large 18 kb BamHI fragment. / Thesis / Master of Science (MS)

Grapevine crown gall causes significant economic damage in vineyards and nurseries worldwide. Infected vines are not curable due to irreversible genomic transformation by the tumorigenic bacterium Rhizobium vitis. Crown gall results in a gradual decline in yield and vine vigor, then the complete collapse of the vine. R. vitis ARK-1, an antagonistic and non-tumorigenic strain, has been shown to inhibit gall formation when equal cell concentrations of antagonist and tumorigenic strains are co-inoculated in planta. In this study, we tested the efficacy of ARK-1 against higher cell numbers of tumorigenic isolates and evaluated timing and methods of ARK-1 application in in-planta assays in grapevine (Vitis vinifera 'Chardonnay', 'Cabernet Sauvignon', 'Merlot') and tomato (Solanum lycopersicum 'Beefsteak'). ARK-1 significantly (P ≤ 0.01) reduced gall incidence and gall diameter against four times higher tumorigenic bacterial cell numbers. Inoculation of ARK-1 up to 48 hours before or within 6 hours after inoculation with tumorigenic isolates significantly (P ≤ 0.01) reduced gall incidence and gall diameter. Root dipping of grapevine for 24 hours and tomato for 1 hour in the formulated ARK-1 suspension (Kumiai Chemical Industry Co. Ltd., Japan) prior to transplanting in tumorigenic bacteria-infested soil completely inhibited gall formation in the roots. These results are promising and support the development of ARK-1 as a biological control agent to manage grapevine crown gall. / Master of Science in Life Sciences / Grapevine crown gall is an economically significant disease in vineyards and nurseries worldwide. The pathogen of grapevine crown gall alters plant genome and causes tumor-like gall formation. Infected vines lose yield and vigor and eventually get killed. In this study, we tested the efficacy of a biological control agent called ARK-1. We challenged ARK-1 against higher cell numbers of pathogenic bacteria, evaluated timings, and practical methods of ARK-1 application. We conducted our greenhouse studies in grapevine (Vitis vinifera) cultivars 'Chardonnay', 'Cabernet Sauvignon', 'Merlot' and tomato (Solanum lycopersicum) cultivar 'Beefsteak'. In efficacy test, ARK-1 was effective and reduced 68% gall incidence against a four times higher pathogenic bacteria mixture. In timing assay, the application of ARK-1 up to 48 hours before inoculation of pathogenic bacteria reduced gall formation by more than 95%. Also, ARK-1 was effective when it was applied within 6 hours after inoculation of pathogenic bacteria. Root soaking of grapevine and tomato in the formulated ARK-1 suspension (KUF-1511, Kumiai Chemical Industry Co. Ltd., Japan), prior to transplanting in pathogenic bacteria-infested soil, completely inhibited gall formation in the roots. The promising result of this study is a step forward towards use of ARK-1 as a biological control agent to manage grapevine crown gall disease.

Crown gall of grapevine (Vitis spp.) is a serious and economically important disease caused by the bacterial pathogen Rhizobium vitis, which transforms healthy plant cell genomes leading to hypertrophic and hyperplastic growth of affected plant cells. Recent studies have documented a strong inhibitory effect against Japanese tumorigenic R. vitis isolates by a newly identified non-tumorigenic strain of R. vitis, ARK-1. We conducted co-inoculation assays in tomato (Solanum lycopersicum) and wine grape cultivars (Vitis vinifera) with four tumorigenic isolates of R. vitis from Virginia. These tumorigenic isolates were co-inoculated with ARK-1 in various ratios and resulting gall incidence and gall size were measured. Analysis was conducted with the generalized linear mixed model (GLIMMIX) in SAS (ver. 9.4). ARK-1 significantly reduced both the mean probability of gall formation and the mean gall size (P < 0.05). ARK-1 efficacy against combinations of two or four tumorigenic isolates and up to twice as many cells of tumorigenic isolates was also significant. However, there was an indication of a loss of efficacy when ARK-1 was challenged with four isolates at four times the cell number of ARK-1. Also, the efficacy of ARK-1 was influenced by both the specific isolate and host plant used in the study. Our results suggest that ARK-1 has promising potential as an effective biological control agent for grapevine crown gall in the United States. / MSLFS / Crown gall of grapevine (Vitis species) is a serious and economically important disease to the grape production industry caused by the bacterium Rhizobium vitis. This pathogen alters the genetic material of plant cells leading to cancer-like growth of affected plant cells (tumors); which may lead to the death of a grapevine. Recently, a non-tumor inducing strain of R. vitis (ARK-1) was documented to inhibit gall induction by tumor-inducing R. vitis strains in Japan. To see if ARK-1 would be a good candidate for biological control of the tumor-causing strains of R. vitis in the US, we tested it against four tumor-inducing strains in both tomato (Solanum lycopersicum) and grapevine (Vitis vinifera). The tumor-inducing strains were mixed with ARK-1, injected into plant stems in various ratios, and resulting gall incidence and gall size were measured 42 to 90 days later. ARK-1 significantly reduced both the probability of gall formation and the gall size in both tomatoes and grapevines. ARK-1 was an effective agent against mixtures of one, two, or four tumor-inducing strains and provided control even when outnumbered two to one by cells of tumor-inducing strains. The efficacy of ARK-1 was influenced by the specific strain, relative cell number to tumor-inducing strains, and host plant used in the study. Our results suggest that ARK-1 has promising potential as an effective biological control agent for grapevine crown gall in the United States.

Anaerobic chemolithotrophic growth of Rhizobium japonicum occurred in the presence of carbon monoxide with nitrate serving as the electron acceptor. Under conditions where the atmospheric concentrations of CO were varied; the cells grew in up to 50% (v/v) CO. Optimum growth was observed in the presence of 20-30% CO. Under these conditions absorbance (660 nm) reached a maximum of 0.33 after 15 days of growth, after which no further increase was noted. Colony forming units increased from 1 X 10⁶ cells per ml to a maximum of 2.0 X 10⁸ cells per ml of culture medium. Growth in the absence of CO was substantially less indicating that CO was required for growth. The rate of disappearance of NO₃⁻-nitrogen from the culture medium during growth was correlated with the growth rate. Hydrogen uptake was measured amperometrically with cells grown in the presence of CO with nitrate, nitrite, nitrous oxide or oxygen serving as the electron acceptor. Addition of acetylene, in the presence of N₂O resulted in a 92.5% inhibition of N₂O-dependent H₂ uptake. Demonstration of H₂ uptake activity with NO₃⁻, NO₂⁻ or N₂O as the only electron acceptor substantiated the presence of dissimilatory nitrate, nitrite, and nitrous oxide reductase(s). Hydrogenase activity with O₂ as the electron acceptor in CO grown cultures showed the presence of cytochrome components necessary for transferring electrons from H₂ to O₂. Increasing the H₂ concentration above 0.5% (v/v) resulted in repression of growth in CO grown cultures. CO:oxidoreductase, the enzyme responsible for oxidation of CO was demonstrated in anaerobic, CO grown cultures. / M.S.

Rhizobium meliloti mutants defective in C₄-dicarboxylic acid transport (Dct⁻) were previously isolated by Tn5 mutagenesis, and divided into two groups based on complementation of Dct- with cosmid clones. In this work further characterization was carried out on the two loci. Group I mutants were found to be defective in dicarboxylate transport (Dct⁻), nitrate utilization, and symbiotic nitrogen fixation. Subcloning and complementation work confined the Group I mutations to a 3.5 kbp BamHI-EcoRI fragment containing the ntrA gene. Group V mutants were defective in dicarboxylate transport and demonstrated varying levels of nitrogen fixation. Complementation and site-directed Tn5 mutagenesis revealed three transcriptional units, corresponding to dctA, dctB, and dctD, localized within a 6 kbp HindIII fragment. The use of dctA::TnphoA fusions determined the expression of dctA to be ntrA, dctB, and dctD dependent. Dct+ revertants of dctB and dctD mutants were selected which carried second-site mutations responsible for restoring the Dct⁺ phenotype. / Thesis / Master of Science (MS)