The objectives of this research program are to conduct molecular genetic analyses of the Rhizobium-Legume symbiosis, which, in world food production, is of great agronomic importance. The study of symbioses at the molecular level challenges us to understand how two different organisms intertwine their fundamental biochemical life processes and interregulate the genes responsible for these processes. To accomplish this, as genetic probes, translational, in-frame gene fusions will be constructed between Rhizobium N 2 fixation genes and either E. coli lacZ (encoding B-galactosidase), uidA (encoding B- glucuronidase), or V. fischerii lux (encoding bacterial luciferase) genes by perfect gene replacement. To understand how N 2 fixation is orchestrated during both symbiosis and free- living bacterial growth, using these gene-engineered Rhizobium strains, the regulation of N 2 fixation processes will be compared in culture and in planta. To understand how these processes are genetically controlled, the genes responsible for this regulation, will be isolated defined Rhizobium mutants in which these genes have been altered, will be constructed and the phenotypes resulting from such alterations will be tested. Because it offers a powerful method to manipulate genes in defined ways, the capacity to deliver to any bacterium defined DNA sequences via phage will be exploited. Rhizobium is a genus of gram-negative bacteria which can invade the roots of legumes and stimulate the development of root nodules. ln this symbiotic relationship, the bacteria and plant are able to convert atmospheric nitrogen (dinitrogen) to biologically useful forms by the process known as nitrogen fixation. The goal of this project is to understand the genetic regulatory events that bring about this symbiosis. Emphasis will be placed on isolating the Rhizobium genes that participate in nitrogen fixation and on identifying the mechanisms that regulate the expression of these genes. This is an economically important area of research as symbiotic nitrogen fixation represents an efficient alternative to nitrogen fertilizers.
