RNA editing, RNA modifications, and transcriptional units in Listeria monocytogenes

Abstract Background: The Gram-positive foodborne pathogen Listeria monocytogenes can resist several stress conditions, and therefore, food processing facilities employ methods with high energy demand, such as high pressure processing (HPP), to eliminate L. monocytogenes. Detection of novel genomic and transcriptomic functions may enable food processing methods to better target pathogens. In this study, we aimed to reveal organization of the transcriptional units of barotolerant L. monocytogenes strain RO15 using novel sequencing methods such as Cappable-seq and direct RNA sequencing. The identified transcriptional units allowed prediction of transcription terminator, promoter, and operon structures. Moreover, RNA modification of the whole transcriptome by direct RNA-seq in L. monocytogenes strain RO15 and ScottA revealed post-transcriptional mechanisms in L. monocytogenes. RNA editing was investigated in both strain RO15 and ScottA to elucidate functions of RNA editing during HPP.Results: We observed 1641 transcription start sites (TSSs) in L. monocytogenes strain RO15 based on Cappable-seq. Comparison of HPP-treated and control samples indicated that some prophage genes might alter their TSSs used under different conditions. Short Illumina RNA-seq reads indicate that RNA editing (A to I) occurs in L. monocytogenes on several genes. Some of the target genes, such as hpf, RNA editing happened only after HPP, indicating that RNA editing might be an important mechanism in L. monocytogenes when recovering óf the HPP injury. TACG was the most common motif of RNA editing in both strains (RO15 and ScottA). We observed that RNA editing sites cannot be identified by long Nanopore direct RNA-seq reads with the current methodologies. The whole transcriptome RNA modification analysis showed that thousands of bases were modified in both strains. Motif analysis of the RNA modification sites detected via direct RNA sequencing indicated that a GATGA-like motif is used in L. monocytogenes during RNA modification. Lastly, we were able to construct the whole transcriptome annotation using the long reads, which revealed operon structures in both L. monocytogenes RO15 and ScottA.Conclusion: Here, we employed two novel sequencing methods, Cappable-seq and direct RNA sequencing, for L. monocytogenes. The results enhanced our understanding of transcriptional units of L. monocytogenes. We revealed that RNA editing is used in L. monocytogenes during HPP recovery, which brings a new perspective to developing novel methods to eliminate L. monocytogenes during food processes. Whole transcriptome RNA modification analysis was performed for the first time in bacteria, potentially providing a new basis for studying novel post-transcriptional functionalities in bacteria.