Rozprawy doktorskie na temat „Rhizobium leguminosarum Molecular aspects”

Corrigendum attached to back cover. Includes bibliographical references (leaves 160-190). Investigates the role of rhizopines in rhizobial competition for nodulation, and to isolate the rhizopine synthesis genes in Rhizobium leguminosarum bv. viciae.

This study describes the use of monoclonal antibodies to investigate molecular components of the Rhizobium cell surface that might be important for symbiotic interactions with the host legume. Components that have been identified include lipopolysaccharide and both membrane-associated and secreted proteins. Differences were observed in the structure and antigenicity of lipopolysaccharide (LPS) from free-living rhizobia compared with that of endosymbiotic bacteroids. Culture pH, oxygen concentration and carbon source were all found to be important factors that could affect the expression of LPS antigens within the nodule. Mutants with altered LPS demonstrated that complete LPS structures are necessary for effective symbiosis. Monoclonal antibody nM 25 identified a protease-sensitive epitope that appeared to be attached to a particular species of LPS macromolecule (identified by MAC 114 antibody). Immunocytochemical localization studies of pea nodule sections revealed that JIM 25 antigen, present on the cell surface, was expressed in infection threads but its expression was low in the symbiotic zone containing mature bacteroids. A 38kDa secreted protein was identified by JIM 24 antibody. Fractionation of nodule extracts by differential centrifugation suggested that the protein was present in the peribacteroid space. A 55kDa membrane protein recognized by MAC 115 antibody proved to be a species-specific marker for R. leguminosarum. The structural gene(s) for this protein are encoded on a 2.8kb EcoRI fragment. Localised mutagenesis of this DNA region with the transposon TnPhoA (which carries a promoter-less gene for alkaline phosphatase) provided evidence that the 55kDa protein was' membraneassociated. However, attempts to "marker-exchange" the transposon-induced mutations from cosmid DNA into the Rhizobium genome were unsuccessful, suggesting that the 55kDa protein is essential for growth of free-living rhizobia. In a DNA region adjoining this 2.8kb EcoRI fragment that encodes the 55kDa protein, a new gene has been described, termed muc. When present on a cosmid the muc gene from R. leguminosarum conferred non-mucoid colony morphology on R. meliloti strain B287. "Marker-exchange" of muc::Tn5 mutations from the plasmid to R. ieguminosarum 8002 (bv. phaseo/i) resulted in derivatives that had lost the ability to nodulate Phaseolus beans. However, marker-exchange into R. leguminosarum B556 (bv. viciae) resulted in mutants that showed no abnormal symbiotic phenotypes on peas. The cosmid carrying the muc and 55kDa protein determinants (pIJ1639) was subjected to saturation transposon mutagenesis with TnPhoA This study revealed several new genes that probably encode membrane-associated or secreted proteins. In some cases gene transcription was dependent on the presence of hesperitin [which is known to be an activator of Rhizobium nodulation (nod) genes]. A 4.6kb EcoRI fragment adjacent to the 2.8kb fragment described above was also found to encode essential functions that prevented the construction of chromosomal mutants by marker-exchange.

Vicibactin is a small, high-affinity iron chelator produced by Rhizobium leguminosarum ATCC 14479. Previous work has shown that vicibactin is produced and secreted from the cell to sequester ferric iron from the environment during iron-deplete conditions. This ferric iron is then transported into the cell to be converted into ferrous iron. This study uses UV-Vis spectroscopy as well as ion trap-time of flight mass spectroscopy to determine that vicibactin does form a complex with copper(II) ions, however, at a much lower affinity than for iron(III). Stability tests have shown that the copper(II)-vicibactin complex is stable over time. The results of this study show that vicibactin could be used in order to remove copper(II) ions from the soil or other media if they are present in toxic amounts. It also suggests that vicibactin’s purpose for the rhizobia could be expanded to include both copper sequestering and to reduce extracellular copper concentrations to prevent toxicity.

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.

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.

In this study, lipo-chitooligosaccharide (NodBj-V (C 18:1, MeFuc); LCO) 10-7M, extracted from Bradyrhizobium japonicum, was sprayed on the leaves of soybean cv. OAC Bayfield soybean and Evans x L66-2470 (carrying the rj1 mutation, and unable to nodulate). Leaf SA level and activities of the PR proteins chitinase, beta-1,3-glucanase and guaiacol peroxidase (GPOX) were quantified. Phenylalanine ammonia-lyase 1 (PAL1) and isoflavone synthase 2 (IFS2) relative gene expression levels in the sprayed leaves were quantified using quantitative real-time PCR. Messenger RNA abundance was quantified using microarrays. The treatment caused a transient increase in local salicylate levels 24 h after exposure, and a systemic increase in GPOX activity 48 h after exposure, in both soybean types. Of the selected 38 genes affected by the LCO treatment, 25 were stress-related. There were no significant differences in (A) chitinase and beta-1,3-glucanase activity, or (B) in PAL1 and IFS2 gene expression.

Corrigendum attached to back cover.
Includes bibliographical references (leaves 160-190).
x, 190, [20] leaves : ill. ; 30 cm.
Investigates the role of rhizopines in rhizobial competition for nodulation, and to isolate the rhizopine synthesis genes in Rhizobium leguminosarum bv. viciae.
Thesis (Ph.D.) -- University of Adelaide, Dept. of Crop Protection, 1999

Bibliography: leaves 101-121.
vii, 122, [77] leaves, [21] leaves of plates : ill. ; 30 cm.
Reports the characterisation of mos genes and preliminary studies on the mos genes in R. leguminosarum br. viciae strain 1a.
Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1998?

Recently, it was found that strains of the soil bacterium Rhizobium leguminosarum biovar trifolii, which normally infect and nodulate clovers, can also associate and colonise different rice culutivars. When rice seedlings were inoculated with these rhizobia, there was a strain-specific response in the growth of the seedlings. This thesis examines the interaction of Rhizobium strains with rice. The following approaches were investigated to increase our understanding of the effects of Rhizobium on the early growth and development of this rice-Rhizobium association by: (a) using of a reporter GFP construct in colonisation studies and following the progress of colonisation of GFP-labelled rhizobia; (b) locating the rhizobia genes involved in the interaction by deleting and curing the plasmids and comparing these changes with the genomic sequence of Sinorhizobium meliloti Sm1021. These studies were further refined by; (c)the use of R-primes, cosmid libraries and Tn5-Mob constructs; (d) the application of exogenous phytohormones and compounds regulating the auxin phytohormone pathway; (e) GC/MS quantification of IAA produced by different bacterial strains; and (f) the use of a plant bioassay to mimic rice growth under paddy field conditions. It was found that legume-associated Rhizobium strains can intimately associate with and enter rice roots within 48 h. However, the rice-Rhizobium association is a complex interaction between the medium, the non-legume host and rhizobia. Under certain conditions, rhizobia could either promote (eg., strain R4), inhibit (eg., strains ANU843 and E4) or have no effect on rice growth. By using a series of plasmid-cured strains of rhizobia, it was found that genes associated with plasmids of Rhizobium strains were involved in the inhibition or the stimulation of rice seedling growth and at least four replicons were needed to cause the inhibition of rice growth. High concentrations of auxin, cytokinin and nitrate mimicked the effect of inhibitory Rhizobium strains on rice root growth and development by forming short lateral roots. Similar observations were made with rice seedlings treated with the sequenced wildtype strain S. meliloti Sm1021 and its closely related strain Rm2011 when they were inoculated onto rice cv. Pelde and cv. Calrose. All pSymA deleted and cured derivatives of strain Rm2011 had no effect on rice growth while pSymB deleted derivatives partially inhibited rice seedling growth. To examine the effect of genes associated with the megaplasmids pSymA and pSymB a series of transconjugant experiments were done. By mobilising the megaplasmids pSymA and pSymB into the non-inhibitory Rhizobium strains of rice growth, it was found that the inhibition of the rice seedlings was associated with the pSymA. Complementation studies with RP4 plasmids containing regions of the pSymA plasmid suggested that genes involved in rice growth inhibition were located in the 450Kb deleted region of pSymA that is deleted from strain SmA146. This region has genes involved in nitrate reduction and IAA biosynthesis. However, Tn5-mutagenesis of the nitrate transport and nitrate reduction genes in megaplasmid pSymA of Rm2011 did not abolish rice growth inhibition. Use of GC/MS demonstrated that IAA was synthesised by all S. meliloti strains, Sm1021 (Rm2011) and pSymA and pSymB deleted and cured derivatives of Rm2011, and rice-associated strains R4 and E4. In addition, it was shown that the addition of agar, the form of nitrogen in the plant medium, and the light could affect the association of Rhizobium with rice seedlings cv. Pelde. This thesis will present evidence from these studies suggesting that IAA and nitrate are not the cause for rice growth inhibition in cv. Pelde. It is proposed that (a) the plasmid-associated inhibition phenomenon in Rhizobium-rice interaction is linked to the pattern of root growth and development; (b) a high level of nitrite in the medium may be the cause of inhibition due to the formation of NO in the plant medium and rice root tissues, and (c) IAA may indirectly be involved in rice-Rhizobium inhibition phenomenon. Therefore, the association of Rhizobium with rice seedlings i.e., the colonization of rice roots by rhizobia and the strain-specific response in the growth of the seedlings are controlled by plasmid-associated genes. Such plasmid-associated genes, under laboratory conditions affected the growth of the rice seedlings and under field conditions may be an important factor in stimulating or inhibiting rice growth and subsequently rice yields.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1991.
Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 113-126).