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

Ramachandran VK, Shearer N, Jacob JJ, Sharma CM, Thompson A - BMC Genomics (2012)

Bottom Line: 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 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.The transcriptional architecture of S.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Food Research, Norwich, UK, University of Würzburg, Josef-Schneider-Str, 2/Bau D15, 97080 Würzburg, Germany.

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 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.

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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).
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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).

Mentions: TSS mapping of S. Typhimurium wild-type and ΔrelAΔspoT strains revealed considerable variation in the length of mRNA 5' leader regions ranging from 0-933 nt with a peak at 26 nt and median of 58 nt (Figure 4). We discovered that 735 genes were synthesised from one or more mRNAs containing 5' leaders > 100 nt in length, suggesting that regulatory mechanisms associated with long 5' leaders are widely used by S. Typhimurium. As has been reported for E. coli [47], no global link between the length of the 5' leader and the functional category of the encoded protein was observed (results not shown). However, some of the longest S. Typhimurium 5' leaders were associated with genes involved in global and virulence gene regulation, including hfq (887 nt), lrhA (712 nt), invF (642 nt), rpoS (566 nt) and hilD (551 nt). One of the longest 5' leaders (887 nt) was transcribed from one of the three promoters regulating the expression of the RNA chaperone hfq (Figure 5). The E. coli hfq gene is also transcribed from 3 promoters and the TSSs identified by primer extension exactly match our dRNA-seq predicted TSSs in S. Typhimurium [48]. The distal hfq promoter directing the longest 5' leader is σ32 dependent in E. coli and a clear σ32 consensus sequence was found in the corresponding S. Typhimurium promoter. Although the role of the leader in regulating gene expression has not yet been defined we found that this promoter was repressed by ppGpp (Figure 5).


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

Ramachandran VK, Shearer N, Jacob JJ, Sharma CM, Thompson A - BMC Genomics (2012)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293720&req=5

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).
Mentions: TSS mapping of S. Typhimurium wild-type and ΔrelAΔspoT strains revealed considerable variation in the length of mRNA 5' leader regions ranging from 0-933 nt with a peak at 26 nt and median of 58 nt (Figure 4). We discovered that 735 genes were synthesised from one or more mRNAs containing 5' leaders > 100 nt in length, suggesting that regulatory mechanisms associated with long 5' leaders are widely used by S. Typhimurium. As has been reported for E. coli [47], no global link between the length of the 5' leader and the functional category of the encoded protein was observed (results not shown). However, some of the longest S. Typhimurium 5' leaders were associated with genes involved in global and virulence gene regulation, including hfq (887 nt), lrhA (712 nt), invF (642 nt), rpoS (566 nt) and hilD (551 nt). One of the longest 5' leaders (887 nt) was transcribed from one of the three promoters regulating the expression of the RNA chaperone hfq (Figure 5). The E. coli hfq gene is also transcribed from 3 promoters and the TSSs identified by primer extension exactly match our dRNA-seq predicted TSSs in S. Typhimurium [48]. The distal hfq promoter directing the longest 5' leader is σ32 dependent in E. coli and a clear σ32 consensus sequence was found in the corresponding S. Typhimurium promoter. Although the role of the leader in regulating gene expression has not yet been defined we found that this promoter was repressed by ppGpp (Figure 5).

Bottom Line: 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 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.The transcriptional architecture of S.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Food Research, Norwich, UK, University of Würzburg, Josef-Schneider-Str, 2/Bau D15, 97080 Würzburg, Germany.

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 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.

Show MeSH
Related in: MedlinePlus