Robert Ludwig - US grants

In preliminary studies of the Rhzobium-legume symbiosia, we have promulgated a novel hypothesis: that Rhizobium sp. ORS571 conducts synergistic nicotinate oxidation and N2 fixation. We propose to study ORS571 nicotinate oxidation by the Vector-insertion mutanesis/cloning technique that we have developed. We also propose to study the biochemistry and physiology of nicotinate oxidation reactions. We propose to integrate the two approaches by using nicotinate oxidation Vector-insertion mutants obtained from the genetic studies as experimental subjects for the biochemical and physiological studies. We shall access: (a) the nicotinate catabolism genes and, (b) the genes integrating nicotinate catabolism and N2 fixation. We shall construct genetic and physical maps ORS571 nicotinate catabolism genes. We shall attempt to compare and contrast synergistic nicotinate oxidation and N2 fixation in free-living rhizobia and symbiotic "bacteroids". We also hope to assess how nicotinate-catabolizing, N2-fixing ORS571 cells actively assimilate ammonium, and whether ORS571 nicotinate oxidation genes are integrated with the regulation of general N-metabolism. In contrast to the free-living process, we hope to understand how fixed-N is charted during symbiosis in Sesbania rostrata and thus how the host plant controls a potential pathogen.

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.

1. We will use high-efficiency Arabidopsis whole-plant DNA transformation as a tool for gene rescue. By a "successive approximation" method, we will conduct large-scale, whole-plant transformation with a matrix- organized DNA library so as to be able to infer individual clones (library members) giving rise to transformants. 2. We will use the transformation-competent Arabidopsis genomic library in Agrobacterium as donor DNA to rescue: (a) the ahs1-1 gene conferring resistance to the sulfonylurea herbicide chlorsulfuron, (b) the DET3 gene which controls light regulated growth and development, and will collaborate on the rescue of TTG, er, bp and yi genes, all from the same, large-scale transformation experiment. 3. We will define a simple procedure for gene-targeting in Arabidopsis plants. To do so, we will screen phenotypically rescued mutants for perfect DNA replacements resulting from homologous, double-recombination events. 4. We will YAC-sort the transformation competent Arabidopsis genomic library so as to facilitate map-based gene cloning. This will cross- reference our Arabidopsis cosmid library with the Arabidopsis YAC libraries, whose clones are anchored to the genomic physical map. Gene-rescue will allow the expeditious isolation of Arabidopsis genes central to the control of plant growth and development. It will also allow efficient integration of Arabidopsis physical-genome and genetic maps.