An advanced bioinformatics approach for analyzing RNA-seq data reveals sigma H-dependent regulation of competence genes in Listeria monocytogenes

Abstract

Background: Alternative σ factors are important transcriptional regulators in bacteria. While σ(B) has been shown to control a large regulon and play important roles in stress response and virulence in the pathogen Listeria monocytogenes, the function of σ(H) has not yet been well defined in Listeria, even though σ(H) controls a large regulon in the closely related non-pathogenic Bacillus subtilis.

Results: Using RNA-seq characterization of a L. monocytogenes strain with deletions of all 4 genes encoding alternative σ factors (ΔBCHL), which was further modified to overexpress sigH (ΔBCHL::P rha -sigH), we identified 6 transcription units (TUs) that are transcribed from σ(H)-dependent promoters. Five of these TUs had not been previously identified. Identification of these promoters was facilitated by use of a bio-informatics approach that compared normalized RNA-seq coverage (NRC), between ΔBCHL::P rha -sigH and a ΔBCHL control, using sliding windows of 51 nt along the whole genome rather than comparing NRC calculated only for whole genes. Interestingly, we found that three operons that encode competence genes (comGABCDEFG, comEABC, coiA) are transcribed from σ(H)-dependent promoters. While these promoters were highly conserved in L. monocytogenes, none of them were found in all Listeria spp. and coiA and its σ(H)-dependent promoter were only found in L. monocytogenes.

Conclusions: Our data indicate that a number of L. monocytogenes competence genes are regulated by σ(H). This σ(H)-dependent regulation of competence related genes is conserved in the pathogen L. monocytogenes, but not in other non-pathogenic Listeria strains. Combined with prior data that indicated a role of σ(H) in virulence in a mouse model, this suggests a possible novel role of σ(H)-dependent competence genes in L. monocytogenes virulence. Development and implementation of a sliding window approach to identify differential transcription using RNA-seq data, not only allowed for identification of σ(H)-dependent promoters, but also provides a general approach for sensitive identification of differentially transcribed promoters and genes, particularly for genes that are transcribed from multiple promoter elements only some of which show differential transcription.

Fig. 1

Schematic of σ^H-dependent transcriptional units identified here. a LMRG_00908-dnaG-rpoD operon; b comEABC operon; c coiA, d lytG; e comGABCDEFG operon; f LMRG_01010-LMRG_01005 operon. Lines show average RNAseq coverage for a sliding window of 100 nt. Blue lines indicate RNA-seq coverage for the three replicates experiments with the 10403S::ΔBCHL P _rha -sigH strain (which over expresses sigH), while green lines indicate RNA-seq coverage for the three replicates experiments with the 10403S::ΔBCHL P _rha strain (which does not contain sigH). Maximum average coverages are shown on the left side of each panel. Black arrows indicate the direction in which the RNA-seq reads were mapped to the chromosome. Genes significantly differentially expressed by the standard approach are labeled with a * next to the gene name. Position of significant fragments is shown as dotted lines underneath the genes with their maximum sliding window fold change shown underneath. Stem loop symbols indicate transcriptional terminators. Genes colored in gray are part of the operons found to be significantly differentially expressed by the sliding window approach and are drawn to scale. Genes colored in magenta are not part of the significant operon and are not drawn to scale. Promoters are indicated by stemmed arrows. A window size of 100 nt was used for the smoothing method. Values on the graph represent the center of these 100 nt windows

Fig. 2

Sequence logos for σ^H-dependent promoters. a Sequence logos for the six σ^H-dependent promoters found in this study based on alignment of 24 L. monocytogenes strains. −35 and −10 regions are shown. b Sequence logo of the consensus sequence of σ^H-dependent promoters based on alignment of sequences from [9]

Fig. 3

Alignment of σ^H-dependent promoters found in Listeria species. L. monocytogenes strains 10403S (lineage II), F2365 (lineage I) and HCC23 (lineage III) are used for comparison. −35 and −10 regions are shown