H-067. Determination of the Global Regulatory Role of CsrA in Escherichia coli

A. N. Edwards1, J. W. Mercante1, P. Babitzke2, T. Romeo1;
1Emory Univ., Atlanta, GA, 2Pennsylvania State Univ., College Park , PA.

Carbon storage regulator A (CsrA) is the key component of a global regulatory system of Escherichia coli K-12 and many other eubacteria. CsrA is a small, dimeric protein that binds to the leaders of target mRNA molecules, affecting their translation and/or stability. Thereby, CsrA activates certain processes associated with exponential phase growth while repressing several stationary phase functions. CsrA activity in the cell is antagonized by two small regulatory RNAs, CsrB and CsrC, which contain multiple CsrA binding sites that permit CsrA sequestration. A GGDEF-EAL domain protein, CsrD, acts as a specificity factor that targets CsrB/C RNAs for degradation by the endoribonuclease RNase E and influences CsrA-mediated regulation. In E. coli, CsrA activates glycolysis, motility, and acetate metabolism while repressing glycogen synthesis and catabolism, gluconeogensis, peptide uptake, and biofilm formation. Because CsrA appears to play a broad role in bacterial physiology, we hypothesized that the Csr regulon likely extends to other important pathways and cellular functions. To determine the RNAs that are the direct targets for CsrA binding, a recombinant protein (CsrA-His6) was purified, followed by extraction of bound RNA and subsequent cDNA synthesis. The identities of the resulting cDNAs were determined using 454 pyrosequencing technology. The results of this analysis indicated that CsrA binds to mRNAs corresponding to more than 600 genes that are important for many fundamental processes, including basic transcriptional and translational machinery of the cell, cell division, novel metabolic and transport systems, and several global regulatory systems. These data suggest that CsrA has a much greater impact on cellular physiology than was previously recognized. RNA binding and regulation by CsrA of a select number of the most important transcripts and processes identified in these studies is being confirmed using quantitative real-time RT-PCR, RNA gel shifts, immunoblotting, reporter fusions and other analyses.