Rhodobacter capsulatus is a Gram-negative photosynthetic bacterium which possesses the ability to adapt metabolically to a wide range of growth conditions. The capacity to convert from one mode to another is facilitated by complex regulatory systems which respond to changes in culture conditions. The long term goal of the proposed study is to understand these complex regulatory capabilities of R. capsulatus with particular emphasis on the genes for nitrogen fixation. The two major research objectives are: (1) To understand transcriptional regulation of nif genes in response to fixed nitrogen. (2) To determine the mechanism of transcriptional regulation of nif genes and other genes in response to oxygen. It is anticipated that the basic control mechanisms used by R. capsulatus can be added to other organisms with complex regulatory needs.
Specific aims i nclude: sequencing and mutagenesis of an ntrA-like gene which codes for a RNA polymerase sigma factor used for nitrogen regulated promoters. 2) Use ntrA-like mutants and mutants in four other nif regulatory genes (called nifR1 through R4) to analyze the regulatory cascades. 3) Testing the hypothesis that various ntrC- like DNA binding proteins (all requiring an ntrA-like sigma factor) modulate activation of different sets of similarly controlled genes. nifR1 from R. capsulatus is required for activation of all the nif genes and has been shown to be homologous to E. coli ntrC. 4) Isolation and characterization of nifR1 through R4- constitutive genes which are locked in their activating states. 5) Sequencing and characterization of nifR5, a gene which complements (i.e. restores) some nifc strains to normal regulation. 6) Since results suggest that DNA supercoiling plays a role in the aerobic repression of nif genes, R. capsulatus strains with varying degrees of DNA topology will be constructed to analyze nif and other anaerobic specific gene transcription. Anaerobic/aerobic gene regulation and complex regulatory cascades are universally employed by both procaryotes and eucaryotes. Basic knowledge of these mechanisms learned in genetically attractive systems such as R. capsulatus can certainly be applied to other bacteria, including anaerobic, aerobic and facultative pathogens. The functional significance of DNA supercoiling on in vivo gene expression is a question of fundamental importance in cells as diverse as bacterial pathogens to human carcinomas.
