The architecture and ppGpp-dependent expression of the primary transcriptome of Salmonella Typhimurium during invasion gene expression

Abstract

Background: Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium (S. Typhimurium) requires expression of the extracellular virulence gene expression programme (ST(EX)), activation of which is dependent on the signalling molecule guanosine tetraphosphate (ppGpp). Recently, next-generation transcriptomics (RNA-seq) has revealed the unexpected complexity of bacterial transcriptomes and in this report we use differential RNA sequencing (dRNA-seq) to define the high-resolution transcriptomic architecture of wild-type S. Typhimurium and a ppGpp null strain under growth conditions which model ST(EX). In doing so we show that ppGpp plays a much wider role in regulating the S. Typhimurium ST(EX) primary transcriptome than previously recognised.

Results: Here we report the precise mapping of transcriptional start sites (TSSs) for 78% of the S. Typhimurium open reading frames (ORFs). The TSS mapping enabled a genome-wide promoter analysis resulting in the prediction of 169 alternative sigma factor binding sites, and the prediction of the structure of 625 operons. We also report the discovery of 55 new candidate small RNAs (sRNAs) and 302 candidate antisense RNAs (asRNAs). We discovered 32 ppGpp-dependent alternative TSSs and determined the extent and level of ppGpp-dependent coding and non-coding transcription. We found that 34% and 20% of coding and non-coding RNA transcription respectively was ppGpp-dependent under these growth conditions, adding a further dimension to the role of this remarkable small regulatory molecule in enabling rapid adaptation to the infective environment.

Conclusions: The transcriptional architecture of S. Typhimurium and finer definition of the key role ppGpp plays in regulating Salmonella coding and non-coding transcription should promote the understanding of gene regulation in this important food borne pathogen and act as a resource for future research.

Figure 1

Annotation of TSSs. (A) TSSs were defined as primary (P), secondary (S) or internal (I). Primary TSSs were identified by higher read counts relative to secondary TSSs. Internal TSSs were located within the coding region (CDR) of a gene where a TSS was annotated for the gene immediately upstream. Primary and secondary internal TSSs (P,I and S,I respectively) were located within the CDR of a gene where there were no TSSs annotated for the gene immediately downstream. (B) Pie chart showing percentage distribution of TSSs within the S. Typhimurium transcriptome.

Figure 2

Comparison of published, experimentally-mapped S. Typhimurium TSSs and dRNA-seq mapped TSSs. The positions of 85% and 92% of the dRNA-seq identified TSSs were located within 2 nt and 10 nt of the experimentally determined TSSs respectively.

Figure 3

Conserved motifs identified from promoter regions. A MEME analysis of 2695 promoters from chromosomal and SLP1-3 SL1344 genes. (A) Conserved -10 motif in 1932 promoters. (B) Conserved -35 motif in 365 promoters. (C) Conserved motif (designated motif 1) in 264 promoters. A MEME analysis of ncRNA promoters revealed (D) conserved -10 and -35 motifs in all of the known and predicted sRNAs (100) and (E) a conserved -10 motif in 280 sites from 302 asRNA promoters. (F) Discriminator region identified from a MEME analysis of 52 ppGpp-repressed promoters (greater than 4-fold).

Figure 4

Length distribution of 5' leader sequences. The frequency of individual 5' Leader lengths was based on an analysis of 1942 primary and secondary TSSs (additional file 2: Table S1).

Figure 5

Promoter architecture of the S. Typhimurium hfq gene reveals a ppGpp-dependent TSS. Enriched (+) and non-enriched (-) cDNAs of S. Typhimurium wild-type (black) or ΔrelAΔspoT (red) strains mapped onto the miaA-hfq locus. The TSSs are marked by black arrows (P1 - 4625091, P2 - 4624672, P3 - 4624269). The Y axis in each lane represents 0-150 mapped reads per genome position. The genome co-ordinates are shown across the bottom. Note that hfq is operonic with the downstream genes, hflXKC.

Figure 6

The S. Typhimurium pykF gene is expressed from alternative ppGpp-dependent and independent promoters. Enriched (+) and non-enriched (-) cDNAs of S. Typhimurium wild-type (black) or ΔrelAΔspoT (red) strains mapped onto the pykF gene (encoding pyruvate kinase). The Y axis in each lane represents 0-80 mapped reads per genome position. The genome co-ordinates are shown across the top. Enriched reads show the presence of alternative TSSs in the wild-type strain (black arrow) and the ΔrelAΔspoT strain (red arrow). A possible third TSS showing equal read numbers in both wild-type and ΔrelAΔspoT strains is indicated by a shorter black arrow.

Figure 7

Promoter architecture and ppGpp-dependency of the S. Typhimurium SPI1 pathogenicity island. Enriched (+) and non-enriched (-) cDNAs of S. Typhimurium wild-type (black) or ΔrelAΔspoT (red) strains mapped onto SPI1. The Y axis in each lane represents 0-50 mapped reads per genome position. Grey boxes represent individual SPI1 genes and operonic transcripts are indicated by black arrows and labelled according to the first gene of the operon. The relevant genome co-ordinates span the centre of the figure.

Figure 8

Functional category analysis of ppGpp-dependent genes. Functional categories were compiled from the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) and The Comprehensive Microbial Resource (CMR) at the J. Craig Ventner Institute (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi) and a manual inspection based on the published literature. Open and shaded bars represent ppGpp-repressed and activated genes respectively. The total number of ORFs present in each category is indicated in parentheses after the category designation.