Project Summary Functional profiling of microbial communities is critical to understanding their overall effects on host health. Most often, metagenomic shotgun sequencing of microbiome samples is used to assess total functional capacity. Yet, transcriptional responses may vary dramatically between organisms depending on the context, with potentially large effects. Many metabolic functions are only expressed after the organism acutely senses the presence of particular substrates in their environment. Pathogens may only express virulence factors after obtaining a critical quorum of pathogens. Overall, stress responses are critical for survival under changing abiotic and biotic conditions. Being able to comprehensively map out these pathways, which determine the resilience, plasticity, and patho-functions of the microbiome, requires sensitive, robust transcriptional –omics tools. Performing traditional RNAseq analyses on bacterial communities has been the predominant method to gain transcriptional information, but it is hampered by the need for technical workarounds and it provides incomplete information about the transcriptional landscape. Ribosomal RNA needs to be depleted prior to sequencing, it has a poor signal-to-noise ratio arising from varying RNA decay rates, and it is insensitive to the transcription of non-coding RNA that has secondary structure or post-transcriptional modifications. Alternatively, the position of RNA polymerase (RNAP) can be assessed, which provides a real-time readout of transcription. Although so-called nascent transcript sequencing has been performed in E. coli, revealing transcriptional pause sites and other phenomenon elusive when using RNAseq alone, these protocols rely on immunoprecipitation of RNAP and are therefore unsuitable for complex microbial communities where RNAP may be quite diverse and require species-specific antibodies. As a solution, Precision Run-On and SEQuencing (PRO-seq), a method originally created for examining transcription in eukaryotes, may provide an unbiased method to examine transcriptional dynamics on cultured bacteria or in complex microbial communities, such as the human microbiome. Our goal is to test the feasibility of PRO-seq when applied to prokaryotes and to evaluate its ability to capture transcriptional dynamics associated with canonical stress response pathways (heat-shock, oxygen exposure and DNA damage), using a set of quantitative metrics. We aim to validate, and if necessary, modify the protocol so it can be used robustly across species. We plan to develop a computational approach to test the full breadth of transcriptional phenomena that can be observed using this method, such as transcriptional pausing, bidirectional transcription, differences in RNAP function apparent across species, and RNA decay rates, among other aspects. If successful, we expect that PRO-seq will be adopted to study the responses of human-associated microbiota to host diet, inflammatory signals, xen...