THE MOLECULAR GENETIC ANALYSIS OF SYMBIOTIC NITROGEN FIXATION (NIF) GENES FROM RHIZOBIUM MELILOTI

The subject of this thesis is the molecular genetic analysis of the symbiotic nitrogen fixation (nif) genes of Rhizobium meliloti, the symbiont of alfalfa (Medicago sativa L.).; Using cloned K. pneumoniae nif genes as a hybridization probe in Southern blot analysis I showed that there is DNA homology to these genes in 19 of 19 evolutionarily divergent nitrogen fixing bacterial strains including r. meliloti but no DNA homology in 10 non-nitrogen fixing species tested. The nitrogenase genes from R. meliloti were cloned by screening a R. meliloti clone bank for homology to cloned K. pneumoniae nif genes.; Transposon Tn5 insertions were introduced at random in the cloned R. meliloti restriction fragments, then the wild type genomic parental R. meliloti nif region was replaced by the homologous cloned DNA fragment containing a Tn5 insertion. The resulting set of R. meliloti strains were tested for ability to fix nitrogen after establishment of symbiosis with alfalfa. I found two clusters of Tn5 insertions 6.3 kb and at least 5.0 kb long which upon nodulation of alfalfa cause R. meliloti to be incapable of nitrogen fixation (Fix('-)). This analysis proved that the region of the R. meliloti genome containing homology to the nif genes of K. pneumoniae in fact functions in symbiotic nitrogen fixation.; Complementation analysis between pairs of nif::Tn5 mutations allowed the assignment of three transcription units in the R. meliloti nif region. One operon containing the genes for nitrogenase was characterized in detail and contains the genes Rm nifH, Rm nifD, and Rm nifK transcribed in that order.; A set of random Tn5 mutagenized R. meliloti strains containing symbiotic mutations was physically screened by Southern hybridization to cloned R. meliloti nif genes and an endogenous insertion element which transposes at a frequency of 10('-2) to 10('-3) preferentially into the nif region of R. meliloti was detected.; A method to detect in vitro DNA protection from methylation by dimethyl sulfate (DMS) or nicking by DNaseI using crude cell extracts rather than purified proteins was developed and was used to show that the lac operator is protected from DMS methylation or DNaseI nicking by crude cell extracts prepared from lacI('q) cells but not by cell extracts prepared from lacI('-) cells.