Finding sRNA-associated phenotypes by competition assays: An example with Staphylococcus aureus

- DNA barcodes allow many mutants to be monitored in the same culture.
- Competition experiments using DNA barcodes uncover sRNA gene phenotypes.
- Multiplexing allows numerous conditions to be tested in one DNA-seq run.
- A DNA barcoding strategy was applied to Staphylococcus aureus.

Bacteria optimize their fitness in response to a changing environment by tight regulation of gene expression. Regulation can be controlled at both transcriptional and post-transcriptional levels via key players such as sigma factors, regulatory proteins and regulatory RNAs. The identification of phenotypes associated with gene deletions is the established method for finding gene functions but may require testing many conditions for each studied mutant. As regulatory RNAs often contribute to fine-tuning gene expression, phenotypes associated with their inactivation are often weak and difficult to detect. Nevertheless, minor phenotypes conferring modest advantages, may allow bacteria to emerge after some generations under selective pressure. A strategy employing DNA barcodes can be used to perform competition experiments between mutants and to monitor fitness associated with mutations in different growth conditions. We combined this strategy with deep sequencing to study regulatory RNAs in Staphylococcus aureus, a major opportunistic pathogen.