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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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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.
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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.

Mentions: In order to determine the roles of ppGpp-dependent genes we performed a functional category analysis (Figure 8). We assigned the ppGpp-dependent genes into 25 functional categories. The largest ppGpp-repressed functional categories contained genes related to fatty acid and lipid metabolism, including peptidoglycan metabolism which play a role in the alterations to cell wall structure that occur at the late-log phase of growth. As well as the expected ppGpp-repression of translation related genes, we also observed repression of genes within the categories of pyrimidine and purine metabolism, and DNA/RNA interactions, replication and metabolism. These ppGpp-dependent processes are likely related to adaptation to the decreased growth rate that occurs at late-log phase. We also note that 28 transcriptional regulators were ppGpp-repressed suggesting that some ppGpp-dependent repression may occur via indirect mechanisms. It has been shown that ppGpp-repressed ribosomal RNA genes contain GC-rich discriminator regions located between the TSS and -10 regions that play a role in destabilisation of the RNAP-promoter complex [62,63]. A MEME analysis revealed that 66% of the genes that were ppGpp-repressed by greater than 16-fold contained a conserved 6 nt long GC rich discriminator regions and a Weblogo analysis (http://weblogo.berkeley.edu/) showed a tendency towards C rather than G residues in all 6 positions (Figure 3F). The remaining 34% of the highly ppGpp-repressed genes did not contain GC rich discriminator regions and may therefore be indirectly regulated.


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)

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

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

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.
Mentions: In order to determine the roles of ppGpp-dependent genes we performed a functional category analysis (Figure 8). We assigned the ppGpp-dependent genes into 25 functional categories. The largest ppGpp-repressed functional categories contained genes related to fatty acid and lipid metabolism, including peptidoglycan metabolism which play a role in the alterations to cell wall structure that occur at the late-log phase of growth. As well as the expected ppGpp-repression of translation related genes, we also observed repression of genes within the categories of pyrimidine and purine metabolism, and DNA/RNA interactions, replication and metabolism. These ppGpp-dependent processes are likely related to adaptation to the decreased growth rate that occurs at late-log phase. We also note that 28 transcriptional regulators were ppGpp-repressed suggesting that some ppGpp-dependent repression may occur via indirect mechanisms. It has been shown that ppGpp-repressed ribosomal RNA genes contain GC-rich discriminator regions located between the TSS and -10 regions that play a role in destabilisation of the RNAP-promoter complex [62,63]. A MEME analysis revealed that 66% of the genes that were ppGpp-repressed by greater than 16-fold contained a conserved 6 nt long GC rich discriminator regions and a Weblogo analysis (http://weblogo.berkeley.edu/) showed a tendency towards C rather than G residues in all 6 positions (Figure 3F). The remaining 34% of the highly ppGpp-repressed genes did not contain GC rich discriminator regions and may therefore be indirectly regulated.

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