In recent years, there has been increased awareness of the importance of post-transcriptional regulation of gene expression by regulatory RNAs in all three domains of life: bacteria, archaea, and eukaryotes. The major group of regulatory RNAs in bacteria is comprised of short, 50-400 nt long, RNA molecules denoted small RNAs (sRNAs). sRNAs affect many aspects of cell physiology, including pathogenicity.

Most of the sRNAs act in trans by base-pairing with their RNA targets upon binding the RNA chaperone Hfq and affect the target stability/and or translation. For a long time, a major unresolved challenge was the identification of the RNA pairs on Hfq. For this reason, we developed a broadly applicable method, named RIL-seq (RNA Interaction by Ligation and sequencing), for a transcriptome-wide identification of mRNA targets of sRNAs. In RIL-seq, RNAs are UV-crosslinked to an RNA-binding protein, and the protein is purified with its bound RNAs. Proximal RNA ends are ligated, yielding RNA chimeras. These chimeras are sequenced and filtered for statistically significant over-represented chimeras, generating a reliable dataset of RNA pairs.

Application of RIL-seq to Escherichia coli Hfq revealed thousands of novel interactions, significant re-wiring of the network upon changes in cellular conditions, and the fact that sRNAs are encoded in all regions of the genome. Application of RIL-seq to another RNA chaperone in E. coli, the understudied ProQ, demonstrated that the Hfq and ProQ interactomes overlap, resulting in competition for some RNA pairs.

The goal of the Melamed lab is to understand the roles played by sRNAs during infection and in the responses to the environment, bacteriophage (phage) and other bacteria. Understanding the role sRNAs play in these relationships can be valuable for coping with different pathogens.

Phages have a great impact on bacterial populations. Yet, the impact of phages on post-transcription regulation in bacteria and the impact of regulation at the post-transcriptional level on phage infection is understudied. The possibility of cross-regulation between bacteria and phages at the RNA level, where RNA from one partner is regulating gene expression in the other partner, is an intriguing route for bacteria-phage communication. We study the sRNA-RNA network during phage infection and decode how bacteria- and phage-encoded regulatory RNAs participate in and control the interactions of bacteria with the phages.

Model of cross-regulation between bacteria and phages at the RNA level upon infection of E. coli with phage lambda. Light gray icons represent Hfq and dark gray icons represent ProQ.

In their natural habitats, bacteria reside in communities with other bacterial species, which can result in conditions that differ greatly from a pure culture and lead to cooperative or competitive relationships between the bacteria. We investigate the RNA-RNA interaction network in different bacterial species in a community and study the role sRNAs play in these relationships.

Pseudomonas aeruginosa (on the right) and Staphylococcus aureus (on the left) that were grown in co-culture.

RIL-seq analysis revealed a new class of sRNAs, four UTR-derived sRNAs whose expression is controlled by the flagella sigma factor (σF). These sRNAs are conserved among pathogenic species and we decipher the mode of action of the σF-dependent sRNAs and uncover their effect on flagella synthesis, bacterial physiology and pathogenicity.

Swarming of two E. coli strains on a low concentration agar plate, one emits a red fluorescence signal and the other one emits a green fluorescence signal.