Shaibah, A. | bioRxiv Science

bioRxiv2025

Development of two compatible plasmids to assess sRNA-mediated post-transcriptional regulation in Acinetobacter baumannii

Post-transcriptional regulation can be mediated by small, regulatory RNAs in bacteria, which can act by base-pairing to a target messenger RNA. The discovery and mechanistic validation of base-pairing sRNAs in multidrug resistant Acinetobacter baumannii has been hampered by the lack of genetic tools to assess RNA-RNA interactions. Here, we created two compatible plasmids for A. baumannii, which addresses this need. The newly designed plasmids validated the known Aar sRNA-carO mRNA, and a new interaction of sRNA44 and the mRNA of the biofilm-associated protein Bap. The new plasmid system should accelerate the mechanistic characterisation of small, regulatory RNAs in A. baumannii. IMPACT STATEMENTMulti-drug resistance of pathogenic microorganisms is one of the greatest challenges for modern medicine. Carbapenem-resistant Acinetobacter baumannii are considered a highly critical organism, yet we are only beginning to understand its physiology and mechanisms of gene regulation. Post-transcriptional regulation by base-pairing, small RNAs is an understudied area, partly because of the lack of genetic tools to investigate them. In this study, we developed a 2-plasmid system to assess sRNA-mRNA interactions, which will greatly accelerate the discovery and validation of small, regulatory RNAs and their target molecules. DATA SUMMARYPlasmid sequences of pAMCK14-sRNA44 and pAMCK18-Bap have been made available in GenBank of National Center for Biotechnology Information (accession numbers PV916437 and PV916438).

microbiology↗

bioRxiv2023

Global analysis of the RNA-RNA interactome in Acinetobacter baumannii AB5075 uncovers a small regulatory RNA repressing the virulence-related outer membrane protein CarO

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that infects critically ill patients. The emergence of antimicrobial resistant A. baumannii has exacerbated the need to functionally characterise environmental adaptation, antibiotic resistance and pathogenicity of this organism and their genetic regulators to inform intervention strategies. Critical to rapid adaptation to changing environments in bacteria are small regulatory RNAs (sRNAs), however, the role that sRNAs play in the biology of A. baumannii is poorly understood. To assess the regulatory function of sRNAs and to uncover their RNA interaction partners in A. baumannii, we employed an RNA proximity ligation and sequencing method (Hi-GRIL-seq) in three different environmental conditions. We found that 40 sRNA candidates were ligated to sRNA-RNA chimeric sequencing reads, suggesting that sRNA-mediated gene regulation is pervasive in A. baumannii and that sRNAs act as direct regulators of mRNA molecules through antisense base-pairing. In-depth characterisation uncovered the sRNA Aar to be a post-transcriptional regulator of four mRNA targets including that of the outer membrane protein CarO and the siderophore receptor BfnH. We show that Aar initiates base-pairing with these mRNA molecules using a conserved seed region of nine nucleotides, sequestering the ribosome binding sites and inhibiting translation. Aar is differentially expressed in response to multiple stress stimuli suggesting a role in fine-tuning translation of the Aar-target molecules in A. baumannii under hostile conditions. Together, our study provides mechanistic insights into sRNA-mediated gene expression control in A. baumannii and represents a valuable resource for future RNA-centric research endeavours in this ESKAPE pathogen.

microbiology↗