Bacterial transcriptional networks consist of hundreds of transcription factors and thousands of promoters. However, the true complexity of transcription in a bacterial pathogen and the effect of the environments encountered during infection remain to be established. We present a simplified approach for global promoter identification in bacteria using RNA-seq-based transcriptomic analyses of 22 distinct infection-relevant environmental conditions. Individual RNA samples were combined to identify most of the 3,838 Salmonella enterica serovar Typhimurium promoters in just two RNA-seq runs. Individual in vitro conditions stimulated characteristic transcriptional signatures, and the suite of 22 conditions induced transcription of 86% of all S. Typhimurium genes. We highlight the environmental conditions that induce the Salmonella pathogenicity islands and present a small RNA expression landscape of 280 sRNAs. This publicly available compendium of environmentally controlled expression of every transcriptional feature of S. Typhimurium constitutes a useful resource for the bacterial research community.

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 RNAsequencing 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. transcriptional mapping | noncoding RNA | posttranscriptional regulation | pathogenicity | genome sequence

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.

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

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.

Knockout mutation of STM4432 resulted in a growth-deficient phenotype of Salmonella enterica serovar Typhimurium in the presence of myo-inositol (MI) as the sole carbon source. STM4432 is part of a 22.6-kb genomic island which spans STM4417 to STM4436 (genomic island 4417/4436) and is responsible for MI degradation. Genome comparison revealed the presence of this island in only six Salmonella strains and a high variability of the iol gene organization in gram-negative bacteria. Upon nonpolar deletion of 11 island loci, the genes involved in six enzymatic steps of the MI pathway were identified. The generation time of S. enterica serovar Typhimurium in minimal medium with MI decreases with higher concentrations of this polyol. Reverse transcriptase PCR showed five separate transcriptional units encompassing the genes iolA-iolB, iolE-iolG1, iolC1-iolC2, iolD1-iolD2-iolG2, and iolI2-iolH. Luciferase reporter assays revealed a strong induction of their promoters in the presence of MI but not glucose. The main regulator, IolR, was identified due to a reduced lag phase of a strain mutated in STM4417 (iolR). Deletion of iolR resulted in stimulation of the iol operons, indicating its negative effect on the iol genes of S. enterica serovar Typhimurium in rich medium at a transcriptional level. Bandshift assays demonstrated the binding of this putative repressor to promoter sequences of iolA, iolC1, and iolD1. Binding of IolR to its own promoter and induced iolR expression in an IolR-negative background demonstrate that its transcription is autoregulated. This is the first characterization of MI degradation in a gram-negative bacterium, revealing a complex transcriptional organization and regulation of the S. enterica serovar Typhimurium iol genes.More than 60 carbon sources are known to be utilizable by Salmonella enterica serovar Typhimurium (10). Among them is myo-inositol (MI), a substrate that is ubiquitous in soil and plants, where it appears as a free form or as phospholipid derivatives. Old reported that MI utilization by S. enterica serovar Typhimurium strains is temperature dependent and that 95% of all strains investigated fermented MI at 25°C, although they had been designated inositol nonfermenting at 37°C (23).In addition to S. enterica serovar Typhimurium, growth of gram-negative bacteria on MI has been demonstrated so far for representatives of the genera Serratia and Klebsiella (18) and for Rhizobium leguminosarum (24). The enzymatic steps of the MI degradation were partially analyzed in Aerobacter (reclassified as Klebsiella) aerogenes (3). The genetics and biochemistry of bacterial MI utilization have been described in most detail for Bacillus subtilis (31,33,35). In this organism, the iol divergon, comprising the operons iolABCDEFGHIJ and iolRS, and the gene iolT, located elsewhere on the chromosome, were shown to be responsible for MI degradation that finally results in an equimolar mixture of dihydroxyacetone phosphate, acetyl coenzyme A, and CO 2 (33). Two transporters belonging to the major facilit...

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 aminoterminal 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.

SignificanceInvasive 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.