Analysis of tRNACys processing under salt stress in Bacillus subtilis spore outgrowth using RNA sequencing data

Abstract

Background: In spore-forming bacteria, the molecular mechanisms of accumulation of transfer RNA (tRNA) during sporulation must be a priority as tRNAs play an essential role in protein synthesis during spore germination and outgrowth. However, tRNA processing has not been extensively studied in these conditions, and knowledge of these mechanisms is important to understand long-term stress survival.    Methods:To gain further insight into tRNA processing during spore germination and outgrowth, the expression of the single copy tRNACys gene was analyzed in the presence and absence of 1.2 M NaCl in Bacillus subtilis using RNA-Seq data obtained from the Gene Expression Omnibus (GEO) database. The CLC Genomics work bench 12.0.2 (CLC Bio, Aarhus, Denmark, https://www.qiagenbioinformatics.com/) was used to analyze reads from the tRNACys gene.  Results:The results show that spores store different populations of tRNACys-related molecules.  One such population, representing 60% of total tRNACys, was composed of tRNACys fragments.  Half of these fragments (3´-tRF) possessed CC, CCA or incorrect additions at the 3´end. tRNACys with correct CCA addition at the 3´end represented 23% of total tRNACys, while with CC addition represented 9% of the total and with incorrect addition represented 7%. While an accumulation of tRNACys precursors was induced by upregulation of the rrnD operon under the control of  σA -dependent promoters under both conditions investigated, salt stress produced only a modest effect on tRNACys expression and the accumulation of tRNACys related species. Conclusions:The results demonstrate that tRNACys molecules resident in spores undergo dynamic processing to produce functional molecules that may play an essential role during protein synthesis.