Small Noncoding RNA AbcR1 Addressing Multiple Target mRNAs From Transcriptional Factor and Two-Component Response Regulator of Brucella melitensis

Abstract

Background: AbcR, as a small noncoding RNA (sRNA), is a typical example of a molecule acting on transencoded target mRNAs. It is a principal element of bacterial gene regulation, required for wild-type virulence in α-proteobacteria. Objectives: The present study was conducted to determine the interaction between sRNA AbcR1 and target mRNA in Brucella melitensis. Methods: Using the bioinformatics method, 7 targets were selected from the transcriptional factor and Two-component response regulators of B. melitensis. The interaction between AbcR1 and target mRNA was experimentally validated. Results: The Two-plasmid superfolder green fluorescent protein (GFP) reporter system revealed that AbcR1 regulates the expression of target mRNA to perform regulatory functions. We confirmed this interaction with Western blot and quantitative real-time polymerase chain reaction (RT-PCR) analyses. Significant up/downregulation was observed, which showed that the conserved seed region of AbcR1 is responsible for regulating all the selected target mRNAs. The target mRNAs showed significant up/downregulation with an AbcR1 mutant. Point mutation in the seed regions of targets resulted in the up/downregulation of the target mRNA. Furthermore, we constructed a novel shuttle expression plasmid for gene expression, enabling stable replication in B. melitensis. We transformed the plasmids in B. melitensis 16M competent cells and observed significant up/downregulation of target mRNAs. Conclusion: The present findings suggest that the conserved seed region of AbcR1 is responsible for regulating multiple target mRNAs. Transcriptional and Two-component response regulators of B. melitensis were direct targets of sRNA AbcR1.