Our proposal concerns the regulation of gene expression in a prokaryote, Rhizobium meliloti, that infects and establishes symbiosis with eukaryotic plant hosts such as alfalfa (Medicago sativa). We have previously established a role for plant inducers in combination with a bacterial regulatory apparatus including the gene activator protein NodD. An inducer-independent pathway involving two other activators, SyrM and NodD3, has been found, and we have shown it is under complex control. We have recently shown that the NodD and SyrM activators require one of two groESL genes to be active. In the next project period, we will pursue further the control of nodulation genes, both in definition of the circuits that control it, and in examining the biochemical mechanism of nod promoter-NodD activity. I. We will develop new genetic tools including transposons, vectors, conditional phage mutants and a restriction R. meliloti strain, to facilitate further global genetic searches for loci needed for nod promoter expression. We will complete analysis of the syrB repression of syrM. We will examine the genetic basis for the NodD-independent growth-promotive effect of the inducer luteolin. II. We present a model that guides the design of experiments on nod promoter expression. III. We will map the syrM start site and several other promoters, to increase our knowledge of native promoters. We will use mutagenesis and selection of artificial sites to define the requirements for NodD and SyrM binding. By deletion analysis, we will examine the role of the nod mRNA leader sequence. IV. We will use a series of mutant screens, including some that distinguish between intact promoters and half sites, to identify domains in NodD3 and NodD1 for DNA binding, for protein-protein cooperativity, and for interaction with polymerase. We will test independently for the presence of dimerization domains in NodD. Based on our defining an activator domain within the two NodD's we will ask whether the SyrM protein requires the same domain for activation of RNA polymerase. V. We will further purify and characterize RNA polymerase and will purify GroEL and GroES for further biochemical study. We will determine the role played by GroE in the activity of NodD and ask whether it has an effect in the interactions of NodD with RNA polymerase. We will assay the effect of inducers such as luteolin on the interactions of RNA polymerase, NodD, and GroEL. VI. We will look for the genetic circuit that causes the decrease of nod promoter expression in bacteroids. We will also test for sites that are important for this decrease, including any identified from the leader in part I.