More than 50 y of research have provided great insight into the physiology, metabolism, and molecular biology of Salmonella enterica serovar Typhimurium ( S . Typhimurium), but important gaps in our knowledge remain. It is clear that a precise choreography of gene expression is required for Salmonella infection, but basic genetic information such as the global locations of transcription start sites (TSSs) has been lacking. We combined three RNA-sequencing techniques and two sequencing platforms to generate a robust picture of transcription in S . Typhimurium. Differential RNA sequencing identified 1,873 TSSs on the chromosome of S . Typhimurium SL1344 and 13% of these TSSs initiated antisense transcripts. Unique findings include the TSSs of the virulence regulators phoP , slyA , and invF . Chromatin immunoprecipitation revealed that RNA polymerase was bound to 70% of the TSSs, and two-thirds of these TSSs were associated with σ ⁷⁰ (including phoP , slyA , and invF ) from which we identified the −10 and −35 motifs of σ ⁷⁰ -dependent S . Typhimurium gene promoters. Overall, we corrected the location of important genes and discovered 18 times more promoters than identified previously. S . Typhimurium expresses 140 small regulatory RNAs (sRNAs) at early stationary phase, including 60 newly identified sRNAs. Almost half of the experimentally verified sRNAs were found to be unique to the Salmonella genus, and <20% were found throughout the Enterobacteriaceae. This description of the transcriptional map of SL1344 advances our understanding of S . Typhimurium, arguably the most important bacterial infection model.

Salmonella enterica serovar Typhimurium is arguably the world’s best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection. The burden of Salmonellosis remains unacceptably high throughout the world and control measures have had limited success. Because Salmonella bacteria can be transmitted from the wider environment to animals and humans, the bacteria encounter diverse environments that include food, water, plant surfaces and the extracellular and intracellular phases of infection of eukaryotic hosts. An intricate transcriptional network has evolved to respond to a variety of environmental signals and control the “right time/ right place” expression of virulence genes. To understand how transcription is rewired during intracellular infection, we determined the primary transcriptome of Salmonella enterica serovar Typhimurium within murine macrophages. We report the coding genes, sRNAs and transcriptional start sites that are expressed within macrophages at 8 hours after infection, and use these to infer gene function. We identified gene promoters that are specifically expressed within macrophages and could drive the intracellular delivery of antigens by S. Typhimurium vaccine strains. These data contribute to our understanding of the mechanisms used by Salmonella to regulate virulence gene expression whilst replicating inside mammalian cells.

We know a great deal about the genes used by the model pathogen Salmonella enterica serovar Typhimurium to cause disease, but less about global gene regulation. New tools for studying transcripts at the single nucleotide level now offer an unparalleled opportunity to understand the bacterial transcriptome, and expression of the small RNAs (sRNA) and coding genes responsible for the establishment of infection. Here, we define the transcriptomes of 18 mutants lacking virulence-related global regulatory systems that modulate the expression of the SPI1 and SPI2 Type 3 secretion systems of S. Typhimurium strain 4/74. Using infection-relevant growth conditions, we identified a total of 1257 coding genes that are controlled by one or more regulatory system, including a sub-class of genes that reflect a new level of cross-talk between SPI1 and SPI2. We directly compared the roles played by the major transcriptional regulators in the expression of sRNAs, and discovered that the RpoS (σ³⁸) sigma factor modulates the expression of 23% of sRNAs, many more than other regulatory systems. The impact of the RNA chaperone Hfq upon the steady state levels of 280 sRNA transcripts is described, and we found 13 sRNAs that are co-regulated with SPI1 and SPI2 virulence genes. We report the first example of an sRNA, STnc1480, that is subject to silencing by H-NS and subsequent counter-silencing by PhoP and SlyA. The data for these 18 regulatory systems is now available to the bacterial research community in a user-friendly online resource, SalComRegulon. The transcriptional networks and the functions of small regulatory RNAs of Salmonella enterica serovar Typhimurium are being studied intensively. S. Typhimurium is becoming the ideal model pathogen for linking transcriptional and post-transcriptional gene regulation to bacterial virulence. Here, we systematically defined the regulatory factors responsible for controlling the expression of S. Typhimurium coding genes and sRNAs under infection-relevant growth conditions. As well as confirming published regulatory inputs for Salmonella pathogenicity islands, such as the positive role played by Fur in the expression of SPI1, we report, for the first time, the global impact of the FliZ, HilE and PhoB/R transcription factors and identify 124 sRNAs that belong to virulence-associated regulons. We found a subset of genes of known and unknown function that are regulated by both HilD and SsrB, highlighting the cross-talk mechanisms that control Salmonella virulence. An integrative analysis of the regulatory datasets revealed 5 coding genes of unknown function that may play novel roles in virulence. We hope that the SalComRegulon resource will be a dynamic database that will be constantly updated to inspire new hypothesis-driven experimentation, and will contribute to the construction of a comprehensive transcriptional network for S. Typhimurium.

The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase. Selection for restored export allowed for the isolation of Tat translocases possessing single mutations in either the amino-terminal domain of TatB or the first cytosolic domain of TatC. The mutant Tat translocases still efficiently accepted the unaltered precursor protein, indicating that the substrate specificity of the translocases was not strictly changed; rather, the translocases showed an increased tolerance toward variations of the amino acids occupying the positions of the twin arginine residues in the consensus motif of a Tat signal peptide.

Significance: Invasive nontyphoidal Salmonella disease is a major and previously neglected tropical disease responsible for an estimated ∼390,000 deaths per year in Africa, largely caused by a variant of Salmonella Typhimurium called ST313. Despite the availability of >100,000 Salmonella genomes, it has proven challenging to associate individual SNPs with pathogenic traits of this dangerous bacterium. Here, we used a transcriptomic strategy to identify a single-nucleotide change in a promoter region responsible for crucial phenotypic differences of African S. Typhimurium. Our findings show that a noncoding nucleotide of the bacterial genome can have a profound effect upon the pathogenesis of infectious disease.