Dataset Information

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

ABSTRACT: 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.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.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.

SUBMITTER: Ramachandran VK

PROVIDER: S-EPMC3293720 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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

Ramachandran Vinoy K VK Shearer Neil N Jacob Jobin J JJ Sharma Cynthia M CM Thompson Arthur A

BMC genomics 20120117

<h4>Background</h4>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 hi ...[more]

PMID: 22251276

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Project description:Salmonella enterica is one of the most common foodborne illnesses in the United States and worldwide, with nearly one-third of the cases attributed to contaminated eggs and poultry products. Vaccination has proven to be an effective strategy to reduce Salmonella load in poultry. The Salmonella Typhimurium Δcrp-cya (MeganVac1) strain is the most commonly used vaccine in the United States; however, the mechanisms of virulence attenuation and host response to this vaccine strain are poorly understood. Here, we profiled the invasion and intracellular survival phenotypes of Δcrp-cya and its derivatives (lacking key genes required for intra-macrophage survival) in HD11 macrophages and the transcriptome response in primary chicken macrophages using RNA-seq. Compared to the parent strain UK1, all the mutant strains were highly defective in metabolizing carbon sources related to the TCA cycle and had greater doubling times in macrophage-simulating conditions. Compared to UK1, the majority of the mutants were attenuated for invasion and intra-macrophage survival. Compared to Δcrp-cya, while derivatives lacking phoPQ, ompR-envZ, feoABC and sifA were highly attenuated for invasion and intracellular survival within macrophages, derivatives lacking ssrAB, SPI13, SPI2, mgtRBC, sitABCD, sopF, sseJ and sspH2 showed increased ability to invade and survive within macrophages. Transcriptome analyses of macrophages infected with UK1, Δcrp-cya and its derivatives lacking phoPQ, sifA and sopF demonstrated that, compared to uninfected macrophages, 138, 148, 153, 155 and 142 genes were differentially expressed in these strains, respectively. Similar changes in gene expression were observed in macrophages infected with these strains; the upregulated genes belonged to innate immune response and host defense and the downregulated genes belonged to various metabolic pathways. Together, these data provide novel insights on the relative phenotypes and early response of macrophages to the vaccine strain and its derivatives. The Δcrp-cya derivatives could facilitate development of next-generation vaccines with improved safety.

Project description:BACKGROUND:The molecular mechanisms underlying bacterial cell death due to stresses or bactericidal antibiotics are complex and remain puzzling. Due to the current crisis of antibiotic resistance, development of effective antibiotics is urgently required. Previously, it has been shown that iron is required for effective killing of bacterial cells by numerous bactericidal antibiotics. RESULTS:We investigated the death or growth inhibition of S. Typhimurium under iron-restricted conditions, following disruption of essential genes, by transposon mutagenesis using transposon sequencing (Tn-seq). Our high-resolution Tn-seq analysis revealed that transposon mutants of S. Typhimurium with insertions in essential genes escaped immediate killing or growth inhibition under iron-restricted conditions for approximately one-third of all previously known essential genes. Based on this result, we classified all essential genes into two categories, iron-dependent essential genes, for which the insertion mutants can grow slowly if iron is restricted, and iron-independent essential genes, for which the mutants become nonviable regardless of iron concentration. The iron-dependency of the iron-dependent essential genes was further validated by the fact that the relative abundance of these essential gene mutants increased further with more severe iron restrictions. Our unexpected observation can be explained well by the common killing mechanisms of bactericidal antibiotics via production of reactive oxygen species (ROS). In this model, iron restriction would inhibit production of ROS, leading to reduced killing activity following blocking of essential gene functions. Interestingly, the targets of most antibiotics currently in use clinically are iron-dependent essential genes. CONCLUSIONS:Our result suggests that targeting iron-independent essential genes may be a better strategy for future antibiotic development, because blocking their essential gene functions would lead to immediate cell death regardless of the iron concentration. This work expands our knowledge on the role of iron to a broad range of essential functions and pathways, providing novel insights for development of more effective antibiotics.

Dataset Information

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

Publications

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

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