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.

Figure 1.. Overview of subpopulations of tRNA ^Cys-related molecules in B. subtilis dormant spores.

Reads categorized were: I) precursors, II) immature tRNA, III) mature tRNA with 3’ CCA ends, IV) immature tRNA with 3’ CC ends, V) 3’-tRF fragments detected with 3’ CCA ends, VI) 3’-tRF fragments detected with 3’ CC ends, VII) 5’-tRF or internal fragments, VIII) complete tRNA with incorrect 3´ tails added and IX) 3´-tRF fragments with incorrect 3´ tails added. The numbers after each category indicate the percentage of total tRNA ^Cys represented by that species. The table at the right shows the tails associated with the various tRNA ^Cys species in green.

Figure 2.. Distribution of subpopulations of tRNA ^Cys molecules in dormant spores (T0) and during the outgrowth process 30, 60 and 90 minutes after the initiation of germination in the absence and presence of 1.2 M of NaCl.

We show the last ten nucleotides of tRNA ^Cys, with correct or incorrect additions at the 3´end. Fragments (total 5´-tRF and internal fragments), 3´tRF and precursors are indicated at the left side. The average of mapped reads for each subpopulation is indicated by a number and by the bar graph at the right side and its presence at each sampled time by a colored bar in the graph.

Figure 3.. Overview of subpopulations of tRNA ^Cys molecules in B. subtilis dormant spores and during the outgrowth phase in the presence and absence of 1.2 M NaCl.

Average of reads mapped for each category were plotted. Error lines represent standard errors of means.