A method for discovery of transcription factors controlling <i>Brucella</i> sRNAs

In the genus <i>Brucella</i>, approximately 40 small regulatory RNAs (sRNAs) have been identified and validated via northern blot analysis, but the transcription factors controlling sRNA transcript levels are largely unknown. Here, we present a method for more rapidly discovering conditions and transcription factors controlling sRNA levels from transcriptomic data sets. Using this approach, we reanalyzed publicly available RNA-seq data sets and generated a large list of predicted regulatory relationships. Northern blot analyses confirmed several of these predictions, covering a wide range of potential transcription factors. Specific findings include that VjbR, a major quorum-sensing regulator of virulence in <i>Brucella</i>, controls levels of the MavR sRNA, and the alternative sigma factor RpoE1 is absolutely required for the expression of <i>bsr6</i> while also contributing to Bsr8 production during oxidative stress. We also found that RNase E is linked to transcript levels of Bsr7, and the transcriptional regulator PhyK modulates the expression of two <i>Brucella</i> sRNAs. Overall, the methods described here can be used in future transcriptomic studies to predict and validate transcriptional regulators and conditions that influence sRNA levels in bacteria.IMPORTANCERegulatory small RNAs control translation for numerous proteins linked to virulence in pathogenic bacteria, but have proven difficult to fully characterize. We developed a new method to discover transcription factors and conditions controlling small regulatory RNAs (sRNA) expression. This method can be applied to other bacteria and future <i>Brucella</i> transcriptomic studies to aid in understanding pathogenesis and virulence factors mediated by sRNAs.