Synthesis of the two major porin protein of Escherichia coli, OmpF and OmpC, is controlled by complex, overlapping regulatory mechanisms that each respond to key environmental cues. Accordingly, the porin regulon provides a powerful experimental system for studying the integrated molecular mechanisms of bacterial adaptation. Knowledge of these mechanisms will reveal chinks in the bacterial armor that can be exploited in our never-ending fight against infectious disease. The OmpR/EnvZ two-component signal transduction system control transcription of both porin structural genes in reciprocal fashion in response to media osmolarity. Our genetic analysis has revealed structural motifs that are important for the multiple functions performed by each regulatory protein. In the case of OmpR, our data fit beautifully with the recently determined three-dimensional structure of the DNA-binding domain. However, important questions about the interactions between OmpR and DNA, and OmpR and RNA polymerase remain. We propose specific genetic experiments to address these issues. Our work with EnvZ has helped clarify the mechanistic difference between the opposed kinase and phosphatase activities of this prototypic sensor. Future experiments here will use genetic and physical methods to try and understand how this integral membrane protein senses media osmolarity. MicF, an antisense RNA, prevents translation of the mRNA for the larger porin, OmpF, when certain toxic molecules such as bile salts are present. We will use micF-lacZ fusions to investigate the mechanism that upregulates synthesis of this antisense RNA in response to growth temperature. In addition, RpoS, the stationary phase sigma factor, blocks transcription of ompF during an elaborately programmed effort to maximize changes for survival when the cell is faced with the unpredictable stresses that might occur following starvation. We have discovered two novel proteins, SprE and Crl, that function to control the stability and activity of RpoS respectively. Genetic and biochemical experiments are proposed to elucidate these novel regulatory mechanisms.
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