MOLECULAR GENETICS OF BACILLUS SUBTILIS RNA POLYMERASE

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Stress responses are important components of pathogenetic mechanisms in Gram negative bacteria. Less is known regarding stress responses among Gram positive bacteria, a group containing many significant human pathogens. The long term objective of this proposal is to establish key features of the mechanism controlling stress-responsive transcription in Bacillus subtilis, a model Gram positive system. The general stress response of B. subtilis is governed by the alternative transcription factor sigma B, which associates with the catalytic core of RNA polymerase to direct transcription of over 40 stress genes. Activity of sigma B is controlled post-translationally by a signal transduction network comprising at least seven Rsb proteins encoded in the sig B operon. The Rsb network responds to two different classes of stress: (i) signals of energy stress, such as entry into stationary phase, and (il) signals of environmental stress, such as salt, heat, or ethanol shock. This network is based on the newly recognized "partner switching" mechanism which is distinct from the two- component systems commonly used for bacterial signal transduction. However, in both mechanisms, key protein contacts are controlled by reversible phosphorylation. The Rsb network consists of two linked partner switching modules, each containing a phosphatase, an antagonist protein, and a switch protein-kinase. The modules convey and integrate the two different classes of stress signals to which sigmaB responds. The upstream module is required to transmit environmental stress signals to the downstream module. The downstream module then integrates these upstream signals with energy stress signals and transmits them to sigma B via the RsbW anti-sigma factor, the switch protein-kinase of the downstream module. The specific aims address the following significant questions: (l) What are the critical aspects of the partner switching mechanism? (2) How do signals of environmental stress enter the Rsb network? and (3) How does sigma B associate with the RNA polymerase core enzyme? We will determine the activities and interactions of purified wild type and mutant Rsb proteins in vitro, and will establish the in vivo relevance of these activities and interactions by genetic analysis. Recent data suggests the existence of additional, undiscovered Rsb regulators, which we will identify by a genetic screen. We will also use a combined biochemical and genetic approach to identify residues in the RNA polymerase core subunits that are impOrtant for the a-core association. Our results should have direct relevance to stress response in other Gram positive bacteria, in which sigma B homologues and Rsb regulators have recently been discovered.