Analysis of tRNA Cys 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 tRNA ^Cys 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 tRNA ^Cys gene. Results:The results show that spores store different populations of tRNA ^Cys-related molecules. One such population, representing 60% of total tRNA ^Cys, was composed of tRNA ^Cys fragments. Half of these fragments (3´-tRF) possessed CC, CCA or incorrect additions at the 3´end. tRNA ^Cys with correct CCA addition at the 3´end represented 23% of total tRNA ^Cys, while with CC addition represented 9% of the total and with incorrect addition represented 7%. While an accumulation of tRNA ^Cys precursors was induced by upregulation of the rrnD operon under the control of σ ^A -dependent promoters under both conditions investigated, salt stress produced only a modest effect on tRNA ^Cys expression and the accumulation of tRNA ^Cys related species. Conclusions:The results demonstrate that tRNA ^Cys molecules resident in spores undergo dynamic processing to produce functional molecules that may play an essential role during protein synthesis.

Keywords: B. subtilis spore germination; RNA-Seq data; outgrowth; salt stress; tRNACys processing.