(105d) The Regulatory Small RNA Spot42 Helps Optimize Carbon Source Utilization in Escherichia Coli through a Common Feedforward Loop

Regulatory small RNAs (sRNAs) represent a previously overlooked class of critical regulators in all three domains of life. In bacteria, sRNAs have been linked to diverse environmental responses, including changes in nutrient conditions, cell-cell signaling, and virulence. Most bacterial sRNAs regulate gene expression through limited base pairing interactions with target mRNAs. Base pairing modulates mRNA stability or translational efficiency, resulting in an increase or decrease in gene expression. In the model organism Escherichia coli, over 30 base pairing sRNAs have been identified to date. Despite an accumulating number of identified base pairing sRNAs, little is known how these sRNAs participate in regulatory networks to help cells respond to environmental signals. Understanding how sRNAs function within the regulatory architecture would help explain why RNA regulators are employed over protein regulators and suggest novel applications of RNA regulators in biotechnology and medicine. Toward this goal, we are characterizing how the base pairing sRNA Spot42 contributes to the optimal utilization of carbon sources in E. coli.

Spot42 was discovered over three decades as a highly abundant cellular RNA in the presence of glucose. Later studies revealed that the catabolite repressor protein (CRP), whose regulatory activity is inhibited following glucose import, represses the expression of Spot42. Most recently, Spot42 was shown to act as a base pairing sRNA by repressing the expression of the galactokinase gene galK. While galK is the only reported target, we hypothesized that Spot42 plays a broader role in cellular metabolism since most base pairing sRNAs target multiple genes.

We performed microarray analyses following Spot42 overexpression to identify additional genes regulated by Spot42. These analyses identified 16 genes, including galK, involved in central catabolism, redox balancing, and the catabolism of diverse non-preferred carbon sources such as fucose, xylose, and sorbitol. Results from Northern blotting, lacZ transcriptional fusions, and mutational analyses confirmed direct base pairing between Spot42 and at least half of the identified mRNAs. Overexpressing Spot42 limited growth on fucose, xylose, and sorbitol, suggesting that Spot42 functions in cellular metabolism to limit the uptake and catabolism of non-preferred carbon sources in the presence of glucose.

Most of the identified genes are transcriptionally activated by CRP. Since CRP also represses Spot42, these two regulators form a general motif called a feedforward loop. Feedforward loops are found throughout the regulatory architecture of many organisms and are predicted to alter the dynamics and strength of gene regulation. To examine the contributions of the CRP-Spot42 feedforward loop, we are measuring the steady-state levels and rates of transition following addition or removal of glucose. These results will elucidate how this feedforward loop contributes to carbon source utilization and whether inclusion of sRNA-based regulation alters the functionality of the loop. Overall, our study lends insights into the unique roles played by sRNAs in cellular regulation and suggests how sRNAs can be implemented in metabolic engineering and the construction of synthetic gene regulatory networks.