Poster Presentation 49th Lorne Conference on Protein Structure and Function 2024

Transcriptional regulation study by RIL-seq revealed potential fine-tuning events on the outer membrane through the actions of the sRNAs. (#429)

Kwok-Jian Goh 1 2 3 , Yael Altuvia 4 , Liron Argaman 4 , Yair Raz 4 , Amir Bar 4 , Chris Stubenrauch 3 , Francesca Short 3 , Trevor Lithgow 3 , Hanah Margalit 4 , Yunn-Hwen Gan 1 2
  1. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  2. Infectious Diseases Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
  3. Department of Microbiology, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
  4. Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

The Gram-negative bacterium Klebsiella pneumoniae (Kp) is regarded by the CDC and WHO as an urgent threat because of resistance towards antibiotics as well as hypervirulent phenotypes that now allows for Kp to infect healthy individuals. We are seeking a better understanding of their outer membrane biology including the associated protective layer made of K-type capsule, as well as arrays of “major” porins that facilitate the passage of small molecules, including some of the antibiotics, across the outer membrane. Accumulating evidence is starting to hint that rapid response in protein expression and turnover of the outer membrane elements are crucial for Kp to survive and even thrive in unfavourable environments. We are investigating the potential role for small RNAs (sRNAs) in fine-tuning outer membrane protein remodelling in response to environmental change, including the possibility that sRNAs might be major post-transcriptional regulators of the steady-state levels of porins. Here, RNA Interaction by Ligation and sequencing (RIL-seq) is applied to investigate Kp for the first time in attempt to decipher its post-transcriptional regulatory networks that involves Hfq-dependent RNA-RNA interactions. The RRI network appeared to be dominated by sRNAs, demonstrating the importance of sRNA regulation. We have found that the capsule locus is a target hub where multiple sRNAs interact with genes within the locus. Interestingly, the capsule production and hypermucoviscosity were suppressed when sRNA OmrB was overexpressed, and the capsule regulator KvrA was likely one of the major targets of OmrB that led to the phenotypes. Further, investigation of the four major porins in Kp (OmpK35, OmpK36, OmpK37 and OmpK38), three showed evidence of interaction with various sRNAs. Using biochemical assays, we are mapping the post-transcriptional regulation and post-translational control that determine the steady-state levels of the major porins in Kp.