Zhu, D. X. | bioRxiv Science

bioRxiv2026

Distribution of prophage-encoded Pas sRNAs across pathogenic Escherichia coli

Numerous base pairing small RNAs (sRNAs), which are an integral part of regulatory networks in bacteria, are encoded on mobile genetic elements (MGEs) in pathogenic strains of Escherichia coli. These sRNAs help coordinate the expression of MGE-encoded virulence factors with core genome-encoded cellular pathways. To investigate the evolution of MGE-encoded sRNAs, we queried public databases to characterize the distribution of PasA, PasB, PasC, PasD1, and PasD2, five prophage-encoded sRNAs discovered in enteropathogenic E. coli. We find that while the Pas sRNAs are largely restricted to pathogenic lineages of Escherichia and Shigella, they exhibit diversity in sequence, genomic presence, and copy number across strains. Based on phylogenetic analysis, the Pas sRNAs originate from multiple ancestral lineages and associate with specific E. coli pathovars, consistent with horizontal acquisition followed by retention. Syntenic analysis suggests a phage origin for the Pas sRNAs, likely from Shiga-toxin encoding phages, but the sRNAs appear to have diverged substantially following their integration into bacterial chromosomes. Comparative and structural analyses further suggest that the PasA and PasC sRNAs share a common ancestor as is the case for PasD and STnc100, another prophage-encoded sRNA. These findings add to our understanding of how accessory genome-encoded sRNAs emerge and evolve.

evolutionary biology↗

bioRxiv2023

Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) employ transcription factors to adapt their physiology to the diverse environments within their host. CarD is a conserved bacterial transcription factor that is essential for viability in Mtb. Unlike classical transcription factors that recognize promoters by binding to specific DNA sequence motifs, CarD binds directly to the RNA polymerase (RNAP) to stabilize the open complex intermediate (RPo) during transcription initiation. We previously showed using RNA-sequencing that CarD is capable of both activating and repressing transcription in vivo. However, it is unknown how CarD achieves promoter specific regulatory outcomes in Mtb despite binding indiscriminate of DNA sequence. We propose a model where CarDs regulatory outcome depends on the promoters basal RPo stability and test this model using in vitro transcription from a panel of promoters with varying levels of RPo stability. We show that CarD directly activates full-length transcript production from the Mtb ribosomal RNA promoter rrnAP3 (AP3) and that the degree of transcription activation by CarD is negatively correlated with RPo stability. Using targeted mutations in the extended -10 and discriminator region of AP3, we show that CarD directly represses transcription from promoters that form relatively stable RPo. DNA supercoiling also influenced RPo stability and affected the direction of CarD regulation, indicating that the outcome of CarD activity can be regulated by factors beyond promoter sequence. Our results provide experimental evidence for how RNAP-binding transcription factors like CarD can exert specific regulatory outcomes based on the kinetic properties of a promoter.

microbiology↗