Cappable-Seq and Direct RNA Sequencing Reveals Novel insights into the Transcriptome of Listeria monocytogenes

Abstract

Abstract Background Listeria monocytogenes is a foodborne pathogen that can survive various stresses. To inactivate Listeria monocytogenes, food processing facilities use high energy methods, such as high-pressure processing (HPP). In this study, we explored the transcriptional units of barotolerant L. monocytogenes RO15 using Cappable-seq and direct RNA sequencing, two novel techniques. Results We detected 1641 transcription start sites (TSSs) in L. monocytogenes RO15, including six HPP-specific TSSs, showing that HPP influences the TSS selection. In addition, we predicted small RNAs (sRNAs) candidates and examined promoter motifs, which revealed new regulatory elements that control gene expression. By integrating short and long RNA-seq reads, we predicted the operon structure of L. monocytogenes RO15 and found 658 operons, comprising 71% of all the genes. The largest operons were mainly located in prophage regions. Moreover, we identified A-to-I RNA editing events in L. monocytogenes for the first time. HPP treatment statistically significantly (p < 0.05) increased the A-to-I editing of several genes including hpf and mdxE suggesting a role in the stress response. We predicted m6A RNA modifications in L. monocytogenes RO15 using direct RNA sequencing reads. This is the first report of m6A RNA modifications in L. monocytogenes by using direct RNA sequencing. Conclusions This study provides novel insights into the transcriptome complexity and diversity, stress response strategies, and post-transcriptional modifications of L. monocytogenes. Our results uncover the genomic mechanisms of adaptation of L. monocytogenes to HPP and indicate potential targets for developing new strategies to control this pathogen. However, further studies are needed to validate the functional roles of the identified sRNAs, RNA editing events, and RNA modifications in L. monocytogenes.