Identification and characterization of putative Mn2+ binding domain in the small RNA AbsR28 and its role in the regulation of type VI secretion system in Acinetobacter baumannii

Abstract

Small RNAs (sRNAs) play a crucial role in bacterial physiology and pathogenesis as they provide a means for precise and quick regulation of genes in response to several environmental factors. The sRNAs binds to their cognate mRNA and results in their silencing or upregulation. It has been experimentally observed that the activity of the sRNA can be controlled by the presence of certain ions or nutrient molecules in the extracellular media. Acinetobacter baumannii is a Gram-negative pathogen responsible for wound infection, urinary tract infection and hospital acquired pneumonia in immunocompromised patients. In a recent study by the lab, a cross talk between the Mn transporter mntH and type six secretion system (T6SS) was observed and this cross talk is mediated by AbsR28 in Mn2+-dependent manner through an unknown molecular mechanism (Bhowmik, S. and Pathania, R., 2020; unpublished data). Multiple sequence alignment with sequences from a characterised Mn2+ binding riboswitch family yybP-yKoY revealed that the sRNA, AbsR28 had regions that shared similarity with the sequence domains identified to play a role in the in Mn2+ binding. Megaprimer PCR was performed to mutate the sRNA and study the importance of the conserved domains. Loss of binding properties of sRNA upon mutation determined in vitro by interaction studies such as isothermal titration calorimetry (ITC), electrophoretic mobility shift assay (EMSA) or equilibrium dialysis would suggest that the domain is essential for its binding properties. The further evaluation of its virulence and ability to repress T6SS by in vivo pulse expression analysis would help determine the importance of Mn2+ binding domain to AbsR28 mediated regulation.