K-128. Functional Characterization of Small Proteins and Small RNAs in Escherichia coli by Signature-Tagged Mutagenesis

E. C. Hobbs, J. L. Astarita, G. Storz;
NIH, Bethesda, MD.

Background: Previous work from our lab has identified a number of small RNAs and small proteins (less than or equal to 50 amino acids in length) in Escherichia coli. The functions of the majority of these gene products are unknown. We are undertaking global approaches to identify phenotypes associated with deletion mutants of each of these small RNAs and small proteins. These phenotypes will serve as a springboard for employing standard biochemical, genetic, and cytological methods to discover the molecular and physiological functions served by each of these genes. Methods: We have employed the signature-tagged mutagenesis (STM) methodology described by Davis and colleagues (Nature Protocols, 2007; 2(11): 2958-74) to facilitate the rapid characterization of uniquely tagged deletion mutants. In brief, antibiotic resistance cassettes flanked by two unique 20-mer DNA sequences (“Up” and “Down” tags) were generated by PCR for each gene that was deleted. The tagged cassettes were then incorporated into the E. coli genome by homologous recombination at loci coding for small RNAs and proteins. Thus far we have generated uniquely tagged deletion mutants of 110 genes. All of these deletion mutants were mixed and subjected to a stress or mock treatment. The “Up” and “Down” tags were PCR-amplified using genomic DNA isolated from the stress- and mock-treated samples as templates. The numbers of each deletion mutant present after either treatment were measured by hybridizing these amplified tags to a microarray containing probes against the “Up” and “Down” tags and comparing the fluorescence intensities for each tag in the two samples. The microarray results were verified by directly competing the mutants in question against otherwise wild-type lacZ mutants under the appropriate stress condition. Results: We have shown that cells mutant for ssrA, micA, yqcG, ybaM, yobF and ryeF are sensitive to outer membrane stress while blr mutants are more resistant than wild-type cells. Conclusions: Directed STM is an effective method to rapidly associate phenotypes with deletion mutants of previously uncharacterized bacterial genes in a highly parallel manner.