(648a) Coupled Experimental and Bioinformatic Approach Elucidates Transcriptional Regulators of Small Non-Coding RNAs in Pathogens

Small non-coding RNAs (sRNAs) are powerful post-transcriptional regulators in bacteria that are rapidly produced in response to environmental stresses, including those experienced by pathogens during infection. As such, sRNAs have been found to regulate the stability and translation of mRNAs implicated in pathogenesis. However, the transcriptional regulation of sRNAs remain under-characterized due, in part, to the diversity of conditions that activate sRNA expression. The complex transcription of sRNAs suggests tight coordination of many cooperative and competitive DNA-binding proteins near the sRNA promoters. Recently, a technique called IPOD-HR, or in vivo protein occupancy display—high resolution, has allowed for the isolation of all DNA regions bound by proteins at a given time, agnostic to the identity of each protein that might be involved in transcriptional regulation.¹ IPOD-HR has been benchmarked in E. coli to efficiently capture known transcription factor networks.¹

To investigate the potential use of this technique in uncovering sRNA regulatory networks, we developed a coupled experimental and bioinformatic approach to predict transcriptional regulators for 91 sRNAs using a limited IPOD-HR dataset in E. coli. Along with differential sRNA expression from datamined RNA-Seq datasets covering >30 different stress conditions, we applied motif searching algorithms to identified DNA regions of protein occupancy near 91 sRNAs to propose >150 putative sRNA transcriptional DNA-binding proteins. We demonstrated the success of this method by biochemically confirming a transcriptional silencer, H-NS, of an elusive sRNA, RseX, in E. coli for which transcriptional expression remained undetected.²

In this talk, we will discuss our current work in expanding the IPOD-HR supported experimental and bioinformatic approach to uncover larger networks of sRNA transcription factors in relation to pathogenesis. Currently, we have collected new datasets in E. coli, under different nutritional conditions, and have gathered >50 putative transcription factors predicted to bind in sRNA promoters that we are examining experimentally. Notably, most of these sRNA promoters have numerous protein occupancy regions throughout growth, supporting the hypothesis of coordinated DNA-binding proteins controlling their complex induction. We are also concurrently extending these approaches to pathogenic bacteria, including the pathogenic gram-positive family, Mycobacteria. Overall, these high-throughput approaches are proving to be highly useful in our efforts to map regulatory networks of sRNAs in response to environmental stresses, including those of high relevance to pathogenesis.

¹ Freddolino PL, Amemiya HM, Goss TJ, Tavazoie S (2021) PLOS Biol 19(6):e3001306.

² Mihailovic MK, et al. (2021) Front Cell Infect Microbiol 11:696533.