Characterization of a major cluster of genes involved in nitrogen fixation and another required for indole-3-acetic acid biosynthesis in the sugarcane endophyte, Acetobacter diazotrophicus
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PublisherThe University of Arizona.
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AbstractAcetobacter diazotrophicus is a true endophyte of sugarcane and is often found in plants grown in agricultural areas of low nitrogen fertilizer input. Results from our laboratory, using mutant strains of A. diazotrophicus unable to fix nitrogen, have shown that there are two beneficial effects of A. diazotrophicus on sugarcane: one dependent on nitrogen fixation, and the other independent of nitrogen fixation. A plant growth promoting substance like indole-3-acetic acid (IAA) may represent the latter effect that accounts for improved plant growth. My first project was to characterize the genes responsible for nitrogen fixation, and determine their regulation. In summary, I have isolated, sequenced, and analyzed the major 31.5 kb nif gene cluster, including both nif and associated genes of A. diazotrophicus. This cluster of 33 genes represents the largest and most complete assembly of contiguous nif/fix and associated genes characterized in any diazotrophic bacterial species. My second project has been to determine whether nitrogen fixation and/or IAA production are important for the ability of A. diazotrophicus to stimulate plant growth. In order to determine the role of IAA directly, mutants of A. diazotrophicus producing reduced amounts of IAA were generated by Tn5 mutagenesis. Among IAA - candidates, one excreting less than 6% of IAA compared to the parent strain was further characterized. The mutation was mapped to genes involved in cytochrome c biogenesis (ccm genes-c&barbelow;ytochrome c&barbelow; m&barbelow;aturation genes). A Nif -/Iaa- double mutant and Nif- mutant were constructed by inserting a chloramphenicol cassette into nifD region. Plant inoculation experiments using mutant strains also demonstrated that A. diazotrophicus could stimulate plant growth regardless of N availability, as evidenced by the significant growth difference between plants inoculated with wild type and uninoculated plants. Under N-limiting conditions plants inoculated with wild type had greater height and biomass than plants inoculated with Nif- or Nif -/Iaa- mutants, suggesting nitrogen fixation by A. diazotrophicus stimulates sugarcane growth. Plants inoculated with Iaa- mutants were always comparable to uninoculated plants regardless of N availability, indicating that IAA biosynthesis is a major bacterial factor influencing sugarcane growth, particularly under N-sufficient conditions.
Degree ProgramGraduate College