No description available.

No description available.

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)

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)

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)

No description available.

Many bacteria accumulate carbon stores as poly-3-hydroxybutyrate (PHB) when growth is limited but carbon availability is not. This stored carbon can then be utilized during conditions of limited carbon availability. The net PHB accumulation in the cell is dependent on the balance between PHB synthesis and degradation. Sinorhizobium meliloti accumulates PHB in the free-living stage but not in the symbiotic stage. The physiological role of the PHB cycle in S. meliloti is unknown. As a first step to understand the genetics of PHB degradation, transposon-generated mutants that were not able to use PHB degradation intermediates, such as 3-hydroxybutyrate and acetoacetate, as a sole carbon source, were isolated. Genetic mapping revealed that there were at least three chromosomal loci involved in acetoacetate metabolism. Identification of these three loci determined that in S. meliloti: (1) acetoacetyl-CoA synthetase (AcsA), encoded by acsA2 gene, rather than the enzyme acetoacetate:succinyl-CoA transferase, is the enzyme that catalyzes activation of acetoacetate to acetoacetyl-CoA; (2) PHB synthase, encoded by phbC, is required for acetoacetate utilization; (3) a putative transporter protein encoding gene, aau-3, may also be involved in acetoacetate metabolism. acsA2 and aau-3 were 78% linked in co-transduction, while phbC was mapped to somewhere else on the chromosome. Biochemical analysis revealed that acsA2::Tn5 mutants lacked AcsA activity but not acetoacetate:succinyl-CoA transferase activity, while phbC::Tn5 maintained similar level of AcsA activity as wild type in vitro. PHB was absent in the phbC mutant. / One transposon-generated mutant, age-1, showed enhanced growth rate on acetoacetate medium. Genetic mapping and transductional analysis indicated that the location of the mutation in age-1 is tightly linked to acsA2. Fine mapping with PCR and DNA sequence techniques showed that Tn5 in age-1 was located at 132 by upstream of the putative translation start site of acsA2. Gene expression analysis indicated that age-1 insertion results in elevated transcription of acsA2. Thus enhanced growth rate on acetoacetate was due to the increased gene expression. acsA2 transcription was induced by acetoacetate and 3-hydroxybutyrate, and repressed by glucose and acetate. / All mutants formed root nodules that fixed nitrogen with varying decrease of impairment. Acetoacetate metabolism and the PHB degradation are not essential for symbiosis.

Alfalfa, Medicago sativa L., cultivars, breeding lines and germplasm releases (populations) and Rhizobium meliloti L. strains that exhibit nitrogen fixation efficiency and tolerance to salinity and drought stresses should enhance seedling establishment, increase yields, reduce nitrogen dependency on petroleum-based nitrogen fertilizers, and allow wider use of irrigated lands in semiarid and a rid regions. In vitro experiments were conducted to determine relative salt (sodium chloride -- NaCl) tolerance: l) during germination and early seedling growth of 229 alfalfa populations from North Africa, the Middle East and the United States, 2) survival and growth of 92 rhizobial strains obtained from different laboratories in the United States or isolated from host plants growing in saline and arid regions of the Intermountain west, 3) compatibility, nodulation and nitrogen fixation efficiency of the most salt-tolerant alfalfa populations and rhizobial strains, and 4) survival, nodulation and nitrogen fixation efficiency of the most salt-tolerant alfalfa populations and rhizobial strain tested under drought conditions. Results from screening of alfalfa at salinity levels of 0 to 3.2 S m-1 (0.1 S m-1 = 640 mg L-1 = 10 mM NaCl at 25° C) indicated 29 populations exhibited salt tolerance at 2.8 S m-1. At salinity levels of 0 to 7.2 S m-1, 14 rhizobial strains exhibited salt tolerance at 6.4 s m-1. Compatibility, nodulation and nitrogen fixation efficiency in the 29 alfalfa populations and the 14 rhizobial strains under controlled conditions showed that four of the rhizobial strains were highly compatible with all 29 populations. These 29 populations and four strains were then exposed to 0 to 1.6 S m-1 NaCl, with the result that nitrogen was fixed with highly compatible symbionts at 1.6 S m-1 NaCl. From these results, the six most highly compatible alfalfa populations and the best overall rhizobial strain were combined and tested under simulated drought stress. These populations were able to fix more nitrogen under drought stress (-0.76 MPa) (0.088 nmol seedling-1 s-1) than they did under salt stress (1.6 S m-1) (0.066 nmol seedling-1 s-1). The in vitro screening technique for acetylene reduction appears to be a useful tool for detecting physiological changes due to salinity and water stresses and for measuring seedling nitrogen fixation efficiency. Breeding for drought and salinity tolerance in conjunction with high nitrogen fix ing potential may be more realistic than breeding strictly for nitrogen fixation without regard for environmental adaptation.

Universiẗat, Diss., 2006--Frankfurt (Main).