RNA-sequencing of L. monocytogenes under PrfA-inducing conditions refines the PrfA regulon and reveals a putative novel PrfA-dependent gene and its associated PrfA box

Purpose: The objective of the present study was to refine the PrfA regulatory network using RNA-seq to characterize the L. monocytogenes transcriptome in a wt, ?prfA, and prfA* under PrfA-inducing conditions. Methods: Sequence reads were aligned to the L. monocytogenes 10403S genome using the BWA-MEM algorithm, version 0.7.3a. The data for coverage per base on the sense and antisense strands were analyzed separately using SAMtools. Differential expression of genes in each strain comparison (wt/?prfA, wt/prfA*, and prfA*/?prfA) were analyzed using the baySeq package for R version 2.2.0. Transcripts were considered differentially expressed if the FDR (False Discovery Rate) was = 0.05, the FC (Fold Change) was = 2.0 for up-regulated transcripts and = 0.5 for down-regulated transcripts. Results: Our data indicate that we (i) confirmed transcription of known PrfA-dependent genes and (ii) identified a novel PrfA box and its putative ??????A-dependent promoter region. Conclusions: Combined with many studies that have investigated the PrfA regulon and virulence in L. monocytogenes, we have shown tha PrfA directly regulates a small subset of genes; however, using RNA-seq and bioinformatics we were able to identify a novel PrfA-dependent gene and its putative PrfA box. This finding highlights the importance of using advanced approaches to analyze RNA-seq data, which can potentially reveal previously unidentified genes regulated under specific conditions. Overall design: RNA-sequencing characterization of the PrfA regulon by comparing the transcript level between (i) wt (10403S) and prfA*, (ii) wt and ?prfA, and (iii) prfA* and ?prfA in triplicate, NextSeq 500