Recent studies are consistent with a two-component model of sensory transduction and transcriptional regulation that applies to a number of biological responses in many eubacterial species. Functional domains appear to have evolved to make repeated use of existing protein-protein and protein-DNA interactions to create novel regulatory systems. Validating the presence of these functional domains can be achieved by either following regulatory phenotypes after rearranging existing domains in hybrid genes of by mapping second site reversions of numerous point mutations to demonstrate interactive domains. Experiments are designed to utilize C4-dicarboxylate transport (dct), nitrogen fixation (nif) and nitrogen assimilation (ntr) genes of Rhizobium to test the validity of the model; results will simultaneously reveal mechanisms of gene expression essential to biological nitrogen fixation. Previous work provides nif structural genes and appropriate promoter::reporter gene constructions, regulatory genes nifA, ntrB and ntrC, and the alternate sigma factor encoded by ntrA. Unpublished work provides the dctA structural gene and regulatory genes dctB and dctD. Hybrid genes, expression vectors for producing regulatory proteins, a dctA::lacZ fusion gene for reporting transcription and various mutant strains will be constructed. These tools will then facilitate phenotype screening to discover how similar polypeptide regions are used in a specific fashion to regulate numerous pathways in Rhizobium meliloti. Broad academic and practical interest in this study is anticipated because related gene products control plant tumor formation by Agrobacteria, sporulation by Bacillus and phosphate scavenging and chemotaxis in enterics; similar genes are also implicated in initiation and maintenance of infection by several gram negative pathogens by controlling expression of pillin genes, regulation of differentiation by Caulobacter, and control of expression of xylose degredation genes and carboxypeptidase G2 in Pseudomonas.
| Ledebur, H; Nixon, B T (1992) Tandem DctD-binding sites of the Rhizobium meliloti dctA upstream activating sequence are essential for optimal function despite a 50- to 100-fold difference in affinity for DctD. Mol Microbiol 6:3479-92 |
