Nitrogen fixation by Rhizobium leguminosarum PRE : a genetical and biochemical approach

Nitrogen fix ation by Rhizobium and Bradyrhizobium bacteria in symbiosis with their leguminous host plants forms an attractive alternative for the industrial production of nitrogenous fertilizers, both from an economic as well as an enviromnental point of view, and is the topic of many scientific research programs nowadays. Ultimate goals in many of these programs are improving the efficiency of nitrogen fix ation, the extension of the host range of the bacteria to important, non-leguminous crops and the transfer of the nitrogen fix ing ability from the bacterium to the plant. A thorough knowledge of the biochemical processes and the genetic determinants involved in both plant and bacterium, however, is a prerequisite for the achievement of any of the above mentioned goals.

In this thesis a genetic and biochemical analysis of R.leguminosarum PRE is described. The organization of the nif and fix genes on the sym plasmid, involved in the reduction of dinitrogen, was studied by the construction of extended physical maps of parts of this plasmid (chapter 2). Furthermore, the region upstream of the fix ABC operon was studied in detail. A novel fix gene ( fix W), located immediately upstream of fix A, was identified and the expression of this gene was studied (chapter 3). Four regions on the PRE sym plasmid were found to contain reiterations of (parts) of functional nif and fix genes.

To elucidate the route by which the enzyme nitrogenase is supplied with electrons, the membrane fraction of R.leguminosarum bacteroids was analyzed for NADH dehydrogenase activity and a bacteroid specific NADH dehydrogenase complex, DH1, was isolated (chapter 4). With the aid of specific antisera directed against the different subunits in this dehydrogenase complex, it was shown that only one subunit, with a molecular weight of 35 kD, is bacteroid specific. To investigate a possible role of complex DH1 in the electron transport to nitrogenase, an attempt was made to construct R.leguminosarum mutants disturbed in the synthesis of this 35K subunit (chapter 5). Therefore, this protein was isolated and the N-terminal amino acid sequence was determined. Based on this sequence oligodeoxynucleotide probes were synthesized and used to screen a cosmid library in order to identify the gene encoding the 35K subunit. Although this gene was not found, several other DNA fragments were cloned showing a high degree of homology to the deduced 35K-gene nucleotide sequence, which indicates that the experimental procedure followed eventually can lead to the isolation of this gene. In chapter 6 an analysis of proteins of the peribacteroid space (PBS) is described. This symbiotic compartment forms the interface between the Rhizobium bacteroid and the plant host, and the proteins of this space may have an important role in transport processes and in signal transduction between the two partners in the symbiosis. The bulk of the proteins (about 90%) was found to be excreted by the bacteroid into the PBS, whereas the remaining 10% probably is of plant origin. About one third of the PBS proteins appeared to occur also in the periplasmic space of free-living bacteria. Four bacteroid encoded PBS proteins were identified, which are not present in free-living R.leguminosarum bacteria and thus might play a role in nitrogen fix ation. These proteins might lead to the identification of novel symbiotic loci on the R.leguminosarum genome.