Abstract

Salmonella Typhi and Typhimurium diverged only ∼50 000 years ago, yet have very different host ranges and pathogenicity. Despite the availability of multiple whole-genome sequences, the genetic differences that have driven these changes in phenotype are only beginning to be understood. In this study, we use transposon-directed insertion-site sequencing to probe differences in gene requirements for competitive growth in rich media between these two closely related serovars. We identify a conserved core of 281 genes that are required for growth in both serovars, 228 of which are essential in Escherichia coli . We are able to identify active prophage elements through the requirement for their repressors. We also find distinct differences in requirements for genes involved in cell surface structure biogenesis and iron utilization. Finally, we demonstrate that transposon-directed insertion-site sequencing is not only applicable to the protein-coding content of the cell but also has sufficient resolution to generate hypotheses regarding the functions of non-coding RNAs (ncRNAs) as well. We are able to assign probable functions to a number of cis -regulatory ncRNA elements, as well as to infer likely differences in trans -acting ncRNA regulatory networks.

INTRODUCTION

Salmonella enterica subspecies enterica serovars Typhi ( S. Typhi) and Typhimurium ( S. Typhimurium) are important human pathogens with distinctly different lifestyles. S . Typhi is host-restricted to humans and causes typhoid fever. This potentially fatal systemic illness affects at least 21 million people annually, primarily in developing countries ( 1–3 ) and is capable of colonizing the gall bladder creating asymptomatic carriers; such individuals are the primary source of this human restricted infection, exemplified by the case of ‘Typhoid Mary’ ( 4 ). S. Typhimurium, conversely, is a generalist, infecting a wide range of mammals and birds in addition to being a leading cause of foodborne gastroenteritis in human populations. Control of S. Typhimurium infection in livestock destined for the human food chain is of great economic importance, particularly in swine and cattle ( 5 , 6 ). Additionally, S. Typhimurium causes an invasive disease in mice, which has been used extensively as a model for pathogenicity in general and human typhoid fever specifically ( 7 ).

Despite this long history of investigation, the genomic factors that contribute to these differences in lifestyle remain unclear. More than 85% of predicted coding sequences are conserved between the two serovars in sequenced genomes of multiple strains ( 8–11 ). The horizontal acquisition of both plasmids and pathogenicity islands during the evolution of the salmonellae is believed to have impacted upon their disease potential. A 100 kb plasmid, encoding the spv ( Salmonella plasmid virulence) genes, is found in some S. Typhimurium strains and contributes significantly towards systemic infection in animal models ( 12 , 13 ). S. Typhi is known to have harboured IncHI1 plasmids conferring antibiotic resistance since the 1970’s ( 14 ), and there is evidence that these strains present a higher bacterial load in the blood during human infection ( 15 ). Similar plasmids have been isolated from S. Typhimurium ( 16–18 ). Salmonella pathogenicity islands (SPI)-1 and -2 are common to both serovars and are required for invasion of epithelial cells [reviewed in ( 19 )] and survival inside macrophages respectively ( 20 , 21 ). S. Typhi additionally incorporates SPI-7 and SPI-10, which contain the Vi surface antigen and a number of other putative virulence factors ( 22–24 ).

Acquisition of virulence determinants is not the sole explanation for the differing disease phenotypes displayed in humans by S. Typhimurium and S. Typhi; genome degradation is an important feature of the S. Typhi genome, in common with other host-restricted serovars such as S. Paratyphi A (humans) and S. Gallinarum (chickens). In each of these serovars, pseudogenes account for 4–7% of the genome ( 9 , 25–27 ). Loss of function has occurred in a number of S. Typhi genes that have been shown to encode intestinal colonization and persistence determinants in S. Typhimurium ( 28 ). Numerous sugar transport and degradation pathways have also been interrupted ( 9 ) but remain intact in S. Typhimurium, potentially underlying the restricted host niche occupied by S. Typhi.

In addition to its history as a model organism for pathogenicity, S. Typhimurium has recently served as a model organism for the elucidation of non-coding RNA (ncRNA) function ( 29 ). These include cis -acting switches, such as RNA-based temperature and magnesium ion sensors ( 30 , 31 ), together with a host of predicted metabolite-sensing riboswitches. Additionally, a large number of trans -acting small RNAs (sRNAs) have been identified within the S. Typhimurium genome ( 32 ), some with known roles in virulence ( 33 ). These sRNAs generally control a regulon of mRNA transcripts through an antisense binding mechanism mediated by the protein Hfq in response to stress. The functions of these molecules have generally been explored in either S. Typhimurium or Escherichia coli , and it is unknown how stable these functions and regulons are over evolutionary time ( 34 ).

Transposon mutagenesis has previously been used to assess the requirement of particular genes for cellular viability. The advent of next-generation sequencing has allowed simultaneous identification of all transposon insertion sites within libraries of up to 1 million independent mutants ( 35–38 ), enabling us to answer the basic question of which genes are required for in vitro growth with extremely fine resolution. By using transposon mutant libraries of this density, which in S. Typhi represents on average >80 unique insertions per gene ( 35 ), shorter regions of the genome can be interrogated, including ncRNAs ( 38 ). In addition, once these libraries exist, they can be screened through various selective conditions to further reveal which functions are required for growth/survival.

Using Illumina-based transposon-directed insertion-site sequencing [TraDIS ( 35 )] with large mutant libraries of both S. Typhimurium and S. Typhi, we investigated whether these Salmonellae require the same protein-coding and ncRNA gene sets for competitive growth under laboratory conditions, and whether there are differences that reflect intrinsic differences in the pathogenic niches these bacteria inhabit.

MATERIALS AND METHODS

Strains

S. Typhimurium strain SL3261 was used to generate the large transposon mutant library, and contains a deletion relative to the parent strain SL1344. The 2166 bp deletion ranges from 153 bp within aroA (normally 1284 bp) to the last 42 bp of cmk , forming two pseudogenes and deleting the intervening gene SL0916 completely. For comparison, we used our previously generated S. Typhi Ty2 transposon library ( 35 ).

Annotation

For S. Typhimurium strain SL3261, we used feature annotations drawn from the SL1344 genome (EMBL-Bank accession FQ312003.1), ignoring the deleted aroA , ycaL and cmk genes. We re-analysed our S. Typhi Ty2 transposon library with features drawn from an updated genome annotation (EMBL-Bank accession AE014613.1). We supplemented the EMBL-Bank annotations with ncRNA annotations drawn from Rfam 10.1 ( 39 ), Sittka et al. ( 40 ), Chinni et al. ( 41 ), Raghavan et al. ( 42 ) and Kröger et al. ( 32 ). Selected protein-coding gene annotations were supplemented using the HMMER webserver ( 43 ) and Pfam ( 44 ).

Creation of S. Typhimurium transposon mutant library

S. Typhimurium was mutagenized using a Tn5-derived transposon as described previously ( 35 ). Briefly, the transposon was combined with the EZ-Tn5 transposase (Epicenter, Madison, USA) and electroporated into S. Typhimurium. Transformants were selected by plating on LB agar containing 15 μg/ml kanamycin and harvested directly from the plates following overnight incubation. A typical electroporation experiment generated a batch of between 50 000 and 150 000 individual mutants. Ten batches were pooled together to create a mutant library comprising ∼930 000 transposon mutants.

DNA manipulations and sequencing

Genomic DNA was extracted from the library pool samples using tip-100 g columns and the genomic DNA buffer set from Qiagen (Crawley, UK). DNA was prepared for nucleotide sequencing as described previously ( 35 ). Before sequencing, a 22-cycle PCR was performed as previously described ( 35 ). Sequencing took place on a single end Illumina flowcell using an Illumina GAII sequencer, for 36 cycles of sequencing, using a custom sequencing primer and 2× Hybridization Buffer ( 35 ). The custom primer was designed such that the first 10 bp of each read was transposon sequence.

Sequence analysis

The Illumina FASTQ sequence files were parsed for 100% identity to the 5′ 10 bp of the transposon (TAAGAGACAG). Sequence reads, which matched, were stripped of the transposon tag and subsequently mapped to the S. Typhimurium SL1344 or S. Typhi Ty2 chromosomes using Maq version maq-0.6.8 ( 45 ). Approximately 12 million sequence reads were generated from the sequencing run, which used two lanes on the Illumina flowcell. Precise insertion sites were determined using the output from the Maq mapview command, which gives the first nucleotide position to which each read mapped. The number and frequency of insertions mapping to each nucleotide in the appropriate genome was then determined.

Statistical analysis of required genes

The number of insertion sites for any gene is dependent on its length; therefore, the values were made comparable by dividing the number of insertion sites by the gene length, giving an ‘insertion index’ for each gene. As before ( 35 ), the distribution of insertion indices was bimodal, corresponding to the required (mode at 0) and non-required models. We fitted gamma distributions for the two modes using the R MASS library ( http://www.r-project.org ). Log 2 -likelihood ratios (LLR) were calculated between the required and non-required models, and we called a gene required if it had an LLR of less than −2, indicating it was at least four times more likely according to the required model than the non-required model. ‘Non-required’ genes were assigned for an LLR of >2. Genes falling between the two thresholds were considered ‘ambiguous’ for the purpose of this analysis. This procedure lead to genes being called as required in S. Typhimurium when their insertion index was <0.020 and ambiguous between 0.020 and 0.027. The equivalent cut-offs for the S . Typhi library are 0.0147 and 0.0186, respectively.

We calculated a P -value for the observed number of insertion sites per gene using a Poisson approximation with rate R = N/G where N is the number of unique insert sites (549 086) and G is the number of bases in the genome (4 878 012). The P -value for at least X consecutive bases without an insert site is e (-RX) , giving a 5% cut-off at 27 bp and a 1% cut-off at 41 bp.

For every gene g with n g,A reads observed in S. Typhi and n g,B reads observed in S. Typhimurium, we calculated the log 2 fold change ratio S g,A,B = log 2 [ (n g,A + 100)/(n g,B + 100) ] . The correction of 100 reads smoothes out the high scores for genes with very low numbers of observed reads. We fitted a normal model to the mode +/− 2 sample standard deviations of the distribution of S A,B and calculated P -values for each gene according to the fit. We considered genes with a P -value of ≤0.05 under the normal model to be uniquely required by one serovar.

RESULTS AND DISCUSSION

TraDIS assay of every Salmonella Typhimurium protein-coding gene

Approximately 930 000 mutants of S. Typhimurium were generated using a Tn5 -derived transposon. In all, 549 086 unique insertion sites were recovered from the mutant library using short-read sequencing with transposon-specific primers. This is a substantially higher density than the 371 775 insertions recovered from S . Typhi previously ( 35 ). The S. Typhimurium library contains an average of one insertion every 9 bp or >100 unique inserts per gene ( Figure 1 ). The large number of unique insertion sites allowed every gene to be assayed; assuming random insertion across the genome, a region of 41 bp without an insertion was statistically significant ( P < 0.01). As previously noted in S . Typhi, the distribution of length-normalized insertions per gene is bimodal (see Supplementary Figure S1 ), with one mode at 0. We interpret genes falling in to the distribution around this mode as being required for competitive growth within a mixed population under laboratory conditions (hereafter ‘required’). Of these, 57 contained no insertions whatsoever and were mostly involved in core cellular processes (see Table 1 , Supplementary Data Set ).

Figure 1.

Genome-wide transposon mutagenesis of S. Typhimurium. Circular plot showing gene content, distribution of required genes and insertion density along the S. Typhimurium chromosome. The outer scale is marked in megabases. Circular tracks range from 1 (outer track) to 6 (inner track). Track 1, all forward-strand genes (colour-coded according to function: dark blue, pathogenicity/adaptation; black, energy metabolism; red, information transfer; dark green, membranes/surface structures; cyan, degradation of macromolecules; purple, degradation of small molecules; yellow, central/intermediary metabolism; light blue, regulators; pink, phage/IS elements; orange, conserved hypothetical; pale green, unknown function; brown, pseudogenes.); track 2, all reverse-strand genes (colour-coded as for forward-strand genes); track 3, S. Typhimurium required genes (red); track 4, 56 genes required by S. Typhimurium, but not by S. Typhi (dark blue, see also Table 1 ); track 5, transposon insertion density; track 6, GC bias [(G − C)/(G + C)]), khaki indicates values >1; purple <1.

Figure 1.

Genome-wide transposon mutagenesis of S. Typhimurium. Circular plot showing gene content, distribution of required genes and insertion density along the S. Typhimurium chromosome. The outer scale is marked in megabases. Circular tracks range from 1 (outer track) to 6 (inner track). Track 1, all forward-strand genes (colour-coded according to function: dark blue, pathogenicity/adaptation; black, energy metabolism; red, information transfer; dark green, membranes/surface structures; cyan, degradation of macromolecules; purple, degradation of small molecules; yellow, central/intermediary metabolism; light blue, regulators; pink, phage/IS elements; orange, conserved hypothetical; pale green, unknown function; brown, pseudogenes.); track 2, all reverse-strand genes (colour-coded as for forward-strand genes); track 3, S. Typhimurium required genes (red); track 4, 56 genes required by S. Typhimurium, but not by S. Typhi (dark blue, see also Table 1 ); track 5, transposon insertion density; track 6, GC bias [(G − C)/(G + C)]), khaki indicates values >1; purple <1.

Table 1.

Core gene functions in S. Typhimurium

Biological process Sub-process Required genes Non-required genes 
Cell division  ftsALKQWYZ , minE, mukB , SL2391 ftsHJNX* , minCD, sdiA, cedA, sulA  
DNA replication Polymerases I, II and III dnaENQX, holAB polA B, holC D E  
Supercoiling gyrAB, parCE  
Primosome-associated dnaBCGT, priA, ssb priB *C, rep 
Transcription RNA polymerase rpoABC  
Sigma, elongation, anti- and termination factors nusBG, rpoDH, rho nusA, rpoE NS  
Translation tRNA-synthetases alaS, argS, asnS, aspS, cysS, glnS, gltX, glyQS, hisS, ileS, leuS, lysS, metG, pheST, proS, serS, thrS, tyrS, valS ,  trpS, trpS2 
Ribosome components rplBCDEFJKLMNOPQRSTUVWXY, rpmABCDHI, rpsABCDEFGHIJKLMNPQST  rplAI, rpmEE2, rpmFG J J2, rps OR * U *V  
Initiation, elongation and peptide chain release factors fusA, infABC, prfAB, tsf, yrdC efp, prfCH, selB, tuf 
Biosynthetic pathways    
Peptidoglycan  murABCDEFGI ,  ddl, ddlA 
Fatty acids  accABCD, fabABDGHIZ – 
Biological process Sub-process Required genes Non-required genes 
Cell division  ftsALKQWYZ , minE, mukB , SL2391 ftsHJNX* , minCD, sdiA, cedA, sulA  
DNA replication Polymerases I, II and III dnaENQX, holAB polA B, holC D E  
Supercoiling gyrAB, parCE  
Primosome-associated dnaBCGT, priA, ssb priB *C, rep 
Transcription RNA polymerase rpoABC  
Sigma, elongation, anti- and termination factors nusBG, rpoDH, rho nusA, rpoE NS  
Translation tRNA-synthetases alaS, argS, asnS, aspS, cysS, glnS, gltX, glyQS, hisS, ileS, leuS, lysS, metG, pheST, proS, serS, thrS, tyrS, valS ,  trpS, trpS2 
Ribosome components rplBCDEFJKLMNOPQRSTUVWXY, rpmABCDHI, rpsABCDEFGHIJKLMNPQST  rplAI, rpmEE2, rpmFG J J2, rps OR * U *V  
Initiation, elongation and peptide chain release factors fusA, infABC, prfAB, tsf, yrdC efp, prfCH, selB, tuf 
Biosynthetic pathways    
Peptidoglycan  murABCDEFGI ,  ddl, ddlA 
Fatty acids  accABCD, fabABDGHIZ – 

Protein-coding genes providing fundamental biological functions in S. Typhimurium. Genes in bold are required in S . Typhi (LLR between required and non-required models less than −2; see ‘Materials and Methods’ section). Asterisk indicates genes ambiguous in S. Typhimurium, having a LLR between −2 and 2.

There was a bias in the frequency of transposon insertion towards the origin of replication. This likely occurred as the bacteria were in exponential growth phase immediately before transformation with the transposon. In this phase of growth, multiple replication forks would have been initiated, meaning genes closer to the origin were in greater copy number and hence more likely to be a target for insertion. We also observed a bias for transposon insertions in A + T rich regions, as was previously observed in the construction of an S . Typhi mutant library ( 35 ). However, the insertion density achieved is sufficient to discriminate between required and non-required genes easily. As was first seen in S . Typhi ( 35 ), we observed transposon insertions into genes upstream of required genes in the same operon, suggesting that most insertions do not have polar effects leading to the inactivation of downstream genes.

Analysis of the S. Typhimurium mutant library allowed us to identify 353 coding sequences required for growth under laboratory conditions, and 4112 non-required coding sequences (see Supplementary Data Set ). We were unable to assign 65 genes to either the required or non-required category. Sixty of these genes, which we will refer to as ‘ambiguous’, had LLRs between −2 and 2. The final five unassigned genes had lengths <60 bases, and they were removed from the analysis. All other genes contained enough insertions or were of sufficient length to generate credible LLR scores. Thus, every gene was assayed, and we were able to draw conclusions for 98.7% of the coding genome in a single sequencing run ( Figure 1 ).

Cross-species comparison of genes required for growth

Gene essentiality has previously been assayed in Salmonella using insertion-duplication mutagenesis ( 46 ). Knuth et al. estimated 490 genes are essential to growth in clonal populations, though 36 of these have subsequently been successfully deleted ( 47 ). Although TraDIS assays gene requirements after a brief period of competitive growth on rich media, we identify a smaller required set than Knuth et al. of ∼350 genes in each serovar, closer to current estimates of ∼300 essential genes in E. coli ( 48 ).

To demonstrate that TraDIS does identify genes known to have strong effects on growth, as well as to test our predictive power for determining gene essentiality, we compared our required gene sets in S. Typhimurium and S . Typhi to essential genes determined by systematic single-gene knockouts in the E. coli K-12 Keio collection ( 48 ). We identified orthologous genes in the three data sets by best reciprocal FASTA hits exhibiting >30% sequence identity for the amino acid sequences. Required orthologous genes identified in this manner share a significantly higher average percentage sequence identity with their E. coli counterparts than expected for a random set of orthologues, at ∼94% identity as compared with ∼87% for all orthologous genes. In 100 000 randomly chosen gene sets of the same size as our required set, we did not find a single set where the average shared identity exceeded 90%, indicating that required genes identified by TraDIS are more highly conserved at the nucleotide level than other orthologous protein-coding sequences.

Baba et al. ( 48 ) have defined an essentiality score for each gene in E. coli based on evidence from four experimental techniques for determining gene essentiality: targeted knockouts using λ-red mediated homologous recombination ( 48 ), genetic footprinting ( 49 , 50 ), large-scale chromosomal deletions ( 51 ) and transposon mutagenesis ( 52 ). Scores range from −4 to 3, with negative scores indicating evidence for non-essentiality and positive scores indicating evidence for essentiality. Comparing the overlap between essential gene sets in E. coli , S. Typhi and S. Typhimurium, we find a set of 228 E. coli genes, which have a Keio essentiality score of at least 0.5 (i.e. there is evidence for gene essentiality; see Figure 2 ) that have TraDIS-predicted required orthologues in both S . Typhi and S. Typhimurium, constituting ∼85% of E. coli genes with evidence for essentiality indicating that gene requirements are largely conserved between these genera. Including orthologous genes that are only predicted to be essential by TraDIS in S . Typhi or S . Typhimurium raises this figure to nearly 93%. The majority of shared required genes between all three bacteria are responsible for fundamental cell processes, including cell division, transcription and translation. A number of key metabolic pathways are also represented, such as fatty acid and peptidoglycan biosynthesis ( Table 1 ). A recent study in the alphaproteobacteria Caulobacter crescentus reported 210 shared essential genes with E. coli , despite C. crescentus sharing less than a third as many orthologous genes with E. coli as Salmonella serovars ( 38 ). This suggests the existence of a shared core of ∼200 essential proteobacterial genes, with the comparatively rapid turnover of 150–250 ‘non-core’ lineage-specific essential genes.

Figure 2.

Comparison of required genes. Venn diagrams showing ( A ) the overlap of all genes (outer circles, light colours) and required genes (inner circles, dark colours) between S. Typhimurium and S . Typhi (excluding genes required in one serovar only, which do not have significantly different read-counts). Black numbers refer to all genes, white numbers to required genes. ( B ) the overlap of all required genes between S. Typhimurium (blue), S . Typhi (green) and E. coli (purple). White numbers refer to genes with Keio essentiality scores ≥0.5.

Figure 2.

Comparison of required genes. Venn diagrams showing ( A ) the overlap of all genes (outer circles, light colours) and required genes (inner circles, dark colours) between S. Typhimurium and S . Typhi (excluding genes required in one serovar only, which do not have significantly different read-counts). Black numbers refer to all genes, white numbers to required genes. ( B ) the overlap of all required genes between S. Typhimurium (blue), S . Typhi (green) and E. coli (purple). White numbers refer to genes with Keio essentiality scores ≥0.5.

If we make the simplistic assumption that gene essentiality should be conserved between E. coli and Salmonella , we can use the overlap of our predictions with the Keio essential genes to provide an estimate of our TraDIS libraries’ accuracy for predicting that a gene will be required in a clonal population. Of the 2632 orthologous E. coli genes, which have a Keio essentiality score of less than −0.5 (i.e. there is evidence for gene non-essentiality), only 33 are predicted to be required by TraDIS in both Salmonella serovars. S . Typhi contains the largest number of genes predicted by TraDIS to be required with E. coli orthologues with negative Keio essentiality scores. However, even if we assume these are all incorrect predictions of gene essentiality, this still gives a gene-wise false positive rate (FPR) of ∼2.7% (81 of 2981 orthologues) and a positive predictive value (PPV) of ∼75% (247 with essentiality scores ≥0.5 of 328 predictions with some Keio essentiality score). Under these same criteria, the S. Typhimurium data set has a lower gene-wise FPR of ∼1.6% (51 of 3122 orthologues) and a higher PPV of ∼82% (234 of 285 predictions as before), as we would expect given the library’s higher insertion density. In reality, these FPRs and PPVs are only estimates; genes that are not essential in E. coli may become essential in the different genomic context of Salmonella serovars and vice versa, particularly in the case of S . Typhi where wide-spread pseudogene formation has eliminated potentially redundant pathways ( 26 , 27 ). Additionally, TraDIS will naturally over-predict essentiality in comparison with targeted knockouts, as our library creation protocol necessarily contains a short period of competitive growth between mutants during the recovery from electro-transformation and selection. As a consequence, genes that cause major growth defects, but not necessarily a complete lack of viability in clonal populations, may be reported as ‘required’.

Serovar-specific genes required for growth

Many of the required genes present in only one serovar encoded phage repressors, for instance the cI proteins of Fels-2/SopE and ST35 (see Supplementary Tables S2 and S3 ). Repressors maintain the lysogenic state of prophage, preventing transcription of early lytic genes ( 53 ). Transposon insertions into these genes will relieve this repression and trigger the lytic cycle, resulting in cell death, and consequently mutants are not represented in the sequenced library. This again broadens the definition of ‘required’ genes; such repressors may not be required for cellular viability in the traditional sense, but once present in these particular genomes, their maintenance is required for continued viability as long as the rest of the phage remains intact.

Serovars Typhimurium and Typhi both contain eight apparent large phage-derived genomic regions ( 54 , 55 ). We were able to identify required repressors in all the intact lambdoid, P2-like and P22-like prophage in both genomes, including Gifsy-1, Gifsy-2 and Fels-2/SopE (see Supplementary Tables S2 and S3 ). With the exception of the SLP203 P22-like prophage in S. Typhimurium, all of these repressors lack the peptidase domain of the classical lambda repressor gene cI. This implies that the default anti-repression mechanism of Salmonella prophage may be more similar to a trans -acting mechanism recently discovered in Gifsy phage ( 56 ) than to the phage lambda repressor’s RecA-induced self-cleavage mechanism. We are also able to confirm that most phage remnants and fusions contained no active repressors, with the exception of the SLP281 degenerate P2-like prophage in S. Typhimurium. This degenerate prophage contains both intact replication and integration genes, but appears to lack tail and head proteins, suggesting it may depend on another phage for production of viral particles. Both genomes also encode P4-like satellite prophage, which rely on ‘helper’ phage for lytic functions and use a complex antisense-RNA based regulation mechanism for decision pathways regarding cell fate ( 57 ) using structural homologs of the IsrK ( 58 ) and C4 ncRNAs ( 59 ), known as seqA and CI RNA in the P4 literature, respectively. Although the mechanism of P4 lysogenic maintenance is not known, the IsrK-like ncRNAs of two potentially active P4-like prophage in S . Typhi are required under TraDIS. This sequence element has previously been shown to be essential for the establishment of the P4 lysogenic state ( 60 ), and we predict based on our observations that it may be necessary for lysogenic maintenance as well. The fact that some lambdoid prophage in S. Typhimurium encode non-coding genes structurally similar to the IsrK-C4 immunity system of P4 raises the possibility that these systems may be acting as a defense mechanism of sorts, protecting the prophage from predatory satellite phage capable of co-opting its lytic genes.

In addition to repressors, 4 prophage cargo genes in S . Typhimurium and one in S . Typhi are required (See Tables 2 and 3 ; Supplementary Tables S2 and S3 ). The S. Typhimurium prophage cargo genes encode a PhoPQ-regulated protein, a protein predicted to be involved in natural transformation, an endodeoxyribonuclease and a hypothetical protein. The S . Typhi prophage cargo gene encodes a protein containing the DNA-binding HIRAN domain ( 62 ) believed to be involved in the repair of damaged DNA. These warrant further investigation, as they are genes that have been recently acquired and become necessary for survival in rich media.

Table 2.

Genes uniquely required in S. Typhimurium

Ty inserts Ty reads SL inserts SL reads SL ID SL gene length Ty ID Ty gene length Name Function 
No orthologue in S. Typhi  
  18 123 SL0742 1269    putative cation transporter 
  80 SL0830 516    conserved hypothetical protein 
  21 SL0831 855    putative electron transfer flavoprotein (beta subunit) 
  SL0950 323    predicted bacteriophage protein, potential phage repressor Gifsy-2 
  11 75 SL1179 789   envF lipoprotein 
  18 SL1480 249    antitoxin Phd_YefM, type II toxin-antitoxin system 
  32 SL1527 264   ydcX putative inner membrane protein 
  SL1560 717    putative membrane protein 
  50 SL1601 859    putative transcriptional regulator (pseudogene) 
  36 SL1799 201    bacteriophage encoded pagK (phoPQ-activated protein) 
  22 SL1830A 434    conserved hypothetical protein (pseudogene) 
  27 SL1967 677    predicted bacteriophage protein, potential phage repressor SLP203 
  15 SL2045A 63   yoeI short ORF 
  17 107 SL2066 900   rfbJ CDP-abequose synthase 
  34 SL2549 209    endodeoxyribonuclease 
  149 SL2593 449    putative DNA-binding protein, potential phage repressor Gifsy-1 SLP272 
  SL2633 846    putative repressor protein, phage SLP281 
   21 SL2695 978   smf putative competence protein 
  39 SL4132 291    hypothetical protein 
  45 SL4354A 303    conserved hypothetical protein 
Present in S. Typhi but required only in S. Typhimurium * 
    36 474 26 SL0032 441 t0033 306  putative transcriptional regulator 
    71 349 11 48 SL0623 642 t2232 576 lipB lipoate-protein ligase B 
    151 3546 10 64 SL0702 897 t2156 894  putative glycosyl transferase 
    194 3007 61 SL0703 1134 t2155 1134  galactosyltransferase 
    231 3499 15 67 SL0706 1779 t2152 1780  putative glycosyltransferase, cell wall biogenesis 
    84 1041 SL0707 834 t2151 834  putative glycosyltransferase, cell wall biogenesis 
    49 367 14 70 SL0722 1569 t2136 1569 cydA cytochrome d ubiquinol oxidase subunit I 
    74 1613 22 SL1069 693 t1789 693  putative secreted protein 
    20 199 SL1203 150 t1146 156  hypothetical protein 
    20 290 SL1264 315 t1209 315  putative membrane protein 
    84 384 26 SL1327 402 t1261 384 spiC putative pathogenicity island 2 secreted effector protein 
    66 769 35 SL1331 270 t1265 327 sseA T3SS chaperone 
    36 307 SL1341 228 t1275 228 ssaH putative pathogenicity island protein 
    47 407 SL1342 249 t1276 249 ssaI putative pathogenicity island protein 
    144 3197 14 SL1343 750 t1277 750 ssaJ putative pathogenicity island lipoprotein 
    63 847 26 SL1354 267 t1288 267 ssaS putative type III secretion protein 
    73 762 44 SL1355 780 t1289 780 ssaT putative type III secretion protein 
    30 226 12 48 SL1386 693 t1322 693 rnfE/ydgQ Electron transport complex protein rnfE 
    265 3337 29 165 SL1473 1557 t1463 1557 pqaA PhoPQ-activated protein 
    85 765 35 SL1532 951 t1511 951 sifB putative virulence effector protein 
    22 156 16 174 SL1561 1227  t1534 a 141 sseJ Salmonella translocated effector protein (SseJ) 
    119 1639 10 44 SL1563 762 t1536 762  putative periplasmic amino acid-binding protein 
    107 2440 44 SL1564 648 t1537 648  putative ABC amino acid transporter permease 
    183 1646 20 118 SL1628 1355 t1612 1364  hypothetical protein 
    23 177 SL1659 183 t1640 183 yciG conserved hypothetical protein 
    78 617 16 104 SL1684 1014 t1664 1014 Hnr putative regulatory protein 
    37 277 25 SL1785 396 t1022 396  conserved hypothetical protein 
    166 2823 27 SL1793 915 t1016 915 pagO inner membrane protein 
    28 311 22 SL1794 159 t1015 159  putative inner membrane protein 
    23 155 SL1823 972 t0988 972 msbB lipid A acyltransferase 
    60 402 11 58 SL2064 1002 t0786 1002 rfbV putative glycosyl transferase 
    87 524 59 SL2065 1293 t0785 1299 rfbX putative O-antigen transporter 
    66 559 13 74 SL2069 774 t0780 774 rfbF glucose-1-phosphate cytidylyltransferase 
    41 204 14 SL3828 1830 t3658 1830 glmS glucosamine-fructose-6-phosphate aminotransferase 
    27 288 23 SL4250 288 t4220 288  putative GerE family regulatory protein 
    148 2633 16 89 SL4251 876 t4221 876  araC family regulatory protein 
Ty inserts Ty reads SL inserts SL reads SL ID SL gene length Ty ID Ty gene length Name Function 
No orthologue in S. Typhi  
  18 123 SL0742 1269    putative cation transporter 
  80 SL0830 516    conserved hypothetical protein 
  21 SL0831 855    putative electron transfer flavoprotein (beta subunit) 
  SL0950 323    predicted bacteriophage protein, potential phage repressor Gifsy-2 
  11 75 SL1179 789   envF lipoprotein 
  18 SL1480 249    antitoxin Phd_YefM, type II toxin-antitoxin system 
  32 SL1527 264   ydcX putative inner membrane protein 
  SL1560 717    putative membrane protein 
  50 SL1601 859    putative transcriptional regulator (pseudogene) 
  36 SL1799 201    bacteriophage encoded pagK (phoPQ-activated protein) 
  22 SL1830A 434    conserved hypothetical protein (pseudogene) 
  27 SL1967 677    predicted bacteriophage protein, potential phage repressor SLP203 
  15 SL2045A 63   yoeI short ORF 
  17 107 SL2066 900   rfbJ CDP-abequose synthase 
  34 SL2549 209    endodeoxyribonuclease 
  149 SL2593 449    putative DNA-binding protein, potential phage repressor Gifsy-1 SLP272 
  SL2633 846    putative repressor protein, phage SLP281 
   21 SL2695 978   smf putative competence protein 
  39 SL4132 291    hypothetical protein 
  45 SL4354A 303    conserved hypothetical protein 
Present in S. Typhi but required only in S. Typhimurium * 
    36 474 26 SL0032 441 t0033 306  putative transcriptional regulator 
    71 349 11 48 SL0623 642 t2232 576 lipB lipoate-protein ligase B 
    151 3546 10 64 SL0702 897 t2156 894  putative glycosyl transferase 
    194 3007 61 SL0703 1134 t2155 1134  galactosyltransferase 
    231 3499 15 67 SL0706 1779 t2152 1780  putative glycosyltransferase, cell wall biogenesis 
    84 1041 SL0707 834 t2151 834  putative glycosyltransferase, cell wall biogenesis 
    49 367 14 70 SL0722 1569 t2136 1569 cydA cytochrome d ubiquinol oxidase subunit I 
    74 1613 22 SL1069 693 t1789 693  putative secreted protein 
    20 199 SL1203 150 t1146 156  hypothetical protein 
    20 290 SL1264 315 t1209 315  putative membrane protein 
    84 384 26 SL1327 402 t1261 384 spiC putative pathogenicity island 2 secreted effector protein 
    66 769 35 SL1331 270 t1265 327 sseA T3SS chaperone 
    36 307 SL1341 228 t1275 228 ssaH putative pathogenicity island protein 
    47 407 SL1342 249 t1276 249 ssaI putative pathogenicity island protein 
    144 3197 14 SL1343 750 t1277 750 ssaJ putative pathogenicity island lipoprotein 
    63 847 26 SL1354 267 t1288 267 ssaS putative type III secretion protein 
    73 762 44 SL1355 780 t1289 780 ssaT putative type III secretion protein 
    30 226 12 48 SL1386 693 t1322 693 rnfE/ydgQ Electron transport complex protein rnfE 
    265 3337 29 165 SL1473 1557 t1463 1557 pqaA PhoPQ-activated protein 
    85 765 35 SL1532 951 t1511 951 sifB putative virulence effector protein 
    22 156 16 174 SL1561 1227  t1534 a 141 sseJ Salmonella translocated effector protein (SseJ) 
    119 1639 10 44 SL1563 762 t1536 762  putative periplasmic amino acid-binding protein 
    107 2440 44 SL1564 648 t1537 648  putative ABC amino acid transporter permease 
    183 1646 20 118 SL1628 1355 t1612 1364  hypothetical protein 
    23 177 SL1659 183 t1640 183 yciG conserved hypothetical protein 
    78 617 16 104 SL1684 1014 t1664 1014 Hnr putative regulatory protein 
    37 277 25 SL1785 396 t1022 396  conserved hypothetical protein 
    166 2823 27 SL1793 915 t1016 915 pagO inner membrane protein 
    28 311 22 SL1794 159 t1015 159  putative inner membrane protein 
    23 155 SL1823 972 t0988 972 msbB lipid A acyltransferase 
    60 402 11 58 SL2064 1002 t0786 1002 rfbV putative glycosyl transferase 
    87 524 59 SL2065 1293 t0785 1299 rfbX putative O-antigen transporter 
    66 559 13 74 SL2069 774 t0780 774 rfbF glucose-1-phosphate cytidylyltransferase 
    41 204 14 SL3828 1830 t3658 1830 glmS glucosamine-fructose-6-phosphate aminotransferase 
    27 288 23 SL4250 288 t4220 288  putative GerE family regulatory protein 
    148 2633 16 89 SL4251 876 t4221 876  araC family regulatory protein 

Genes determined to be uniquely required in S. Typhimurium. SL, S. Typhimurium; Ty, S . Typhi; inserts refer to the number of unique insertion sites within a gene; reads refer to the number of sequence reads over all insertions sites within a gene. Shaded rows indicate genes shown to be H-NS repressed in ( 61 ).

* P -value (associated with log 2 read ratio) < 0.05.

asseJ is a pseudogene in S . Typhi.

Table 3.

Genes uniquely required in S. Typhi

SL inserts SL reads Ty inserts Ty reads Ty ID Ty gene length SL ID SL gene length Name Function 
No orthologue in S. Tm  
  t1332 132   malY pseudogene 
  32 t1920 405    possible repressor protein, prophage 10/Gifsy-2 
  12 t3157 165    conserved hypothetical protein 
  12 t3166 228    spurious ORF annotation overlapping the RnaseP/M1 RNA 
  196 t3402 570   cI repressor protein, cs 73 prophage 
  58 t3415 741    HIRAN-domain family gene, potential DNA repair 
  t4531 150    hypothetical secreted protein 
Present in S. Typhimurium but required only in S. Typhi * 
    199 1792 18 59 t0095 1287 SL0093 1287 surA survival protein SurA precursor 
    45 498 22 t0123 459 SL0119 459 yabB/mraZ conserved hypothetical protein 
    120 589 11 32 t0203 1281 SL0203 1281 hemL glutamate-1-semialdehyde 2,1-aminomutase 
    123 982 25 t0224 1353 SL0224 1353 yaeL/rseP Zinc metallopeptidase 
    67 452 14 t0270 576 SL2604 576 rpoE RNA polymerase sigma-E factor 
    140 760 t0587 2286 SL2246 2286 nrdA ribonucleoside-diphosphate reductase 1 alpha chain 
    113 641 15 42 t2140 2802 SL0718 2802 sucA 2-oxoglutarate dehydrogenase E1 component 
    116 753 13 36 t2177 1641 SL0680 1641 pgm phosphoglucomutase 
    80 542 15 t2276 1008 SL0580 1008 fepD ferric enterobactin transport protein FepD 
    93 591 t2277 990 SL0579 990 fepG ferric enterobactin transport protein FepG 
    64 508 t2278 795 SL0578 795 fepC ferric enterobactin transport ATP-binding protein FepC 
    201 1129 12 116 t2410 2355 SL0444 2355 lon Lon protease 
    95 518 20 t2730 1062 SL2809 1062  recA a recA protein 
    135 719 16 39 t2996 1992 SL3052 1947 tktA transketolase 
    76 358 t3120 1434 SL3173 1434 rfaE ADP-heptose synthase 
    213 1976 50 t3265 1071 SL3321 1071 degS serine protease 
    43 448 10 t3326 606 SL3925 606 yigP conserved hypothetical protein 
    124 571 17 36 t3384 2025 SL3872 2025 rep ATP-dependent DNA helicase 
    175 1208 21 t3621 2787 SL3947 2787 polA DNA polymerase I 
    117 797 13 t3808 1047 SL3677 1047 waaF ADP-heptose-LPS heptosyltransferase II 
    176 1628 14 59 t4153 1080 SL4183 1080 alr alanine racemase 
    140 1127 10 38 t4411 951 SL4294 951 miaA tRNA delta-2-isopentenylpyrophosphate transferase 
SL inserts SL reads Ty inserts Ty reads Ty ID Ty gene length SL ID SL gene length Name Function 
No orthologue in S. Tm  
  t1332 132   malY pseudogene 
  32 t1920 405    possible repressor protein, prophage 10/Gifsy-2 
  12 t3157 165    conserved hypothetical protein 
  12 t3166 228    spurious ORF annotation overlapping the RnaseP/M1 RNA 
  196 t3402 570   cI repressor protein, cs 73 prophage 
  58 t3415 741    HIRAN-domain family gene, potential DNA repair 
  t4531 150    hypothetical secreted protein 
Present in S. Typhimurium but required only in S. Typhi * 
    199 1792 18 59 t0095 1287 SL0093 1287 surA survival protein SurA precursor 
    45 498 22 t0123 459 SL0119 459 yabB/mraZ conserved hypothetical protein 
    120 589 11 32 t0203 1281 SL0203 1281 hemL glutamate-1-semialdehyde 2,1-aminomutase 
    123 982 25 t0224 1353 SL0224 1353 yaeL/rseP Zinc metallopeptidase 
    67 452 14 t0270 576 SL2604 576 rpoE RNA polymerase sigma-E factor 
    140 760 t0587 2286 SL2246 2286 nrdA ribonucleoside-diphosphate reductase 1 alpha chain 
    113 641 15 42 t2140 2802 SL0718 2802 sucA 2-oxoglutarate dehydrogenase E1 component 
    116 753 13 36 t2177 1641 SL0680 1641 pgm phosphoglucomutase 
    80 542 15 t2276 1008 SL0580 1008 fepD ferric enterobactin transport protein FepD 
    93 591 t2277 990 SL0579 990 fepG ferric enterobactin transport protein FepG 
    64 508 t2278 795 SL0578 795 fepC ferric enterobactin transport ATP-binding protein FepC 
    201 1129 12 116 t2410 2355 SL0444 2355 lon Lon protease 
    95 518 20 t2730 1062 SL2809 1062  recA a recA protein 
    135 719 16 39 t2996 1992 SL3052 1947 tktA transketolase 
    76 358 t3120 1434 SL3173 1434 rfaE ADP-heptose synthase 
    213 1976 50 t3265 1071 SL3321 1071 degS serine protease 
    43 448 10 t3326 606 SL3925 606 yigP conserved hypothetical protein 
    124 571 17 36 t3384 2025 SL3872 2025 rep ATP-dependent DNA helicase 
    175 1208 21 t3621 2787 SL3947 2787 polA DNA polymerase I 
    117 797 13 t3808 1047 SL3677 1047 waaF ADP-heptose-LPS heptosyltransferase II 
    176 1628 14 59 t4153 1080 SL4183 1080 alr alanine racemase 
    140 1127 10 38 t4411 951 SL4294 951 miaA tRNA delta-2-isopentenylpyrophosphate transferase 

Genes determined to be uniquely required in S . Typhi. SL, S. Typhimurium; Ty, S . Typhi; inserts refer to the number of unique insertion sites within a gene; reads refer to the number of sequence reads over all insertions sites within a gene.

* P -value (associated with log 2 read ratio) < 0.05.

a The assignment of recA as a required gene has been described previously ( 35 ), but briefly is believed to be due to the presence of the priC pseudogene in Typhi.

To compare differences between requirements for orthologous genes in both serovars, we calculated log-fold read ratios to eliminate genes, which were classified differently in S. Typhi and S. Typhimurium but did not have significantly different read densities (see ‘Materials and Methods’ section.) Even after this correction, 36 S. Typhimurium genes had a significantly lower frequency of transposon insertion compared with the equivalent genes in S. Typhi ( P < 0.05), including four encoding hypothetical proteins ( Table 2 ). This indicates that these gene products play a vital role in S. Typhimurium, but not in S. Typhi when grown under laboratory conditions.

A major difference between the two serovars is in the requirement for genes involved in cell wall biosynthesis (see Figure 3 ). A set of four genes (SL0702, SL0703, SL0706 and SL0707) in an operonic structure putatively involved in cell wall biogenesis is required in S. Typhimurium, but not in S. Typhi. The protein encoded by SL0706 is a pseudogene in S. Typhi (Ty2 unique ID: t2152) owing to a 1 bp deletion at codon 62 that causes a frameshift ( Figure 4 a). This operon contains an additional two pseudogenes in S. Typhi (t2154 and t2150), as well as a single different pseudogene (SL0700) in S. Typhimurium, indicating that this difference in gene requirements reflects the evolutionary adaptation of these serovars to their respective niches. Similarly, four genes ( rfbV , rfbX , rfbJ and rfbF ) within an O-antigen biosynthetic operon are required by S. Typhimurium, but not S. Typhi. There appears to have been a shuffling of O-antigen biosynthetic genes since the divergence between the two serovars, and rfbJ , encoding a CDP-abequose synthase, has been lost from S. Typhi altogether. These broader requirements for cell wall-associated biosynthetic and transporter genes suggest that surface structure biogenesis is of greater importance in S. Typhimurium.

Figure 3.

Comparison of cell surface operon structure and requirements. Diagram illustrating cell surface operons with different requirement patterns in S. Typhimurium and S. Typhi. The top figure is of an uncharacterized operon putatively involved in cell wall biogenesis, whereas the bottom figure shows a portion of the rfb operon involved in O-antigen biosynthesis. Plots along the top and bottom of each figure show insertions in S. Typhimurium and S. Typhi, respectively, with read depth on the y -axis with a maximum cut-off of 100 reads. Genes in blue are required in S. Typhimurium, genes in white are pseudogenes; others are in grey. Grey rectangles represent BLAST hits between orthologous genes, with percentage nucleotide identity coloured on the scale to the right of each figure.

Figure 3.

Comparison of cell surface operon structure and requirements. Diagram illustrating cell surface operons with different requirement patterns in S. Typhimurium and S. Typhi. The top figure is of an uncharacterized operon putatively involved in cell wall biogenesis, whereas the bottom figure shows a portion of the rfb operon involved in O-antigen biosynthesis. Plots along the top and bottom of each figure show insertions in S. Typhimurium and S. Typhi, respectively, with read depth on the y -axis with a maximum cut-off of 100 reads. Genes in blue are required in S. Typhimurium, genes in white are pseudogenes; others are in grey. Grey rectangles represent BLAST hits between orthologous genes, with percentage nucleotide identity coloured on the scale to the right of each figure.

Figure 4.

Proposed differences in sRNA utilization. Diagram illustrating inferred required sRNA regulatory networks under TraDIS. Molecules required in S. Typhi are in black and in S. Typhimurium are in white. RseA, in black/grey check, is ambiguous in S. Typhi. Non-required molecules are in grey. Diamonds indicate sRNAs, circles regulatory proteins, ovals proteases, oblong shapes are membrane-anchored proteins, and rounded squares are outer membrane porins.

Figure 4.

Proposed differences in sRNA utilization. Diagram illustrating inferred required sRNA regulatory networks under TraDIS. Molecules required in S. Typhi are in black and in S. Typhimurium are in white. RseA, in black/grey check, is ambiguous in S. Typhi. Non-required molecules are in grey. Diamonds indicate sRNAs, circles regulatory proteins, ovals proteases, oblong shapes are membrane-anchored proteins, and rounded squares are outer membrane porins.

We also identified seven genes from the shared pathogenicity island SPI-2 that appear to contain few or no transposon insertions only in S. Typhimurium under laboratory conditions. These genes ( spiC , sseA and ssaHIJT ) are thought to encode components of the SPI-2 type III secretion system apparatus (T3SS) ( 63 ). In addition, the effector genes sseJ and sifB , whose products are secreted through the SPI-2-encoded T3SS ( 64 , 65 ), also fell into the ‘required’ category in S. Typhimurium alone. All of these genes display high A + T nucleotide sequence and have been previously shown (in S. Typhimurium) to be strongly bound by the nucleoid-associated protein H-NS, encoded by hns ( 61 , 66 ). Therefore, rather than being ‘required’, it is instead possible that access for the transposon was sufficiently restricted that very few insertions occurred at these sites. In further support of this hypothesis, a comparison of the binding pattern of H-NS detected in studies using S. Typhimurium LT2 with the TraDIS results from the SPI-2 locus indicated that high regions of H-NS enrichment correlated well with both the ssa genes described here and with sseJ ( 61 , 66 ) (see Supplementary Figure S1 ). An earlier study also suggests that high-density DNA-binding proteins can block Mu, Tn 5 and Tn 10 insertion ( 67 ); however, a genome-wide study of the effects of H-NS binding on transposition would be necessary to confirm this effect.

Indeed, the generation of null S. Typhimurium mutants in sseJ and sifB , as well as many others generated at the SPI-2 locus suggest that these genes are not truly a requirement for growth in this serovar ( 65 , 68–70 ). Although this is a reminder that the interpretation of gene requirement needs to be made with care, the effect of H-NS on transposon insertion is not genome-wide. If this were the case, there would be an under-representation of transposon mutants in high A + T regions (known for H-NS binding), which is not what we observed. In total, only 21 required genes fall into the ‘ hns -repressed’ category described in Navarre et al. ( 61 ) (see Table 2 ; Supplementary Table S1 ); the remainder (almost 400) contained sufficient transposon insertions to conclude they were non-required. In addition, all SPI-1 genes that encode another T3SS and are of high A + T content were also found to be non-required. This phenomenon was not observed in S. Typhi, possibly because the strain used harbours the pHCM1 plasmid, which encodes the H-NS-like protein sfh and has been shown to affect H-NS binding ( 71 , 72 ).

Twenty-two S. Typhi genes had a significantly lower frequency of transposon insertion compared with orthologues in S. Typhimurium ( P < 0.05), indicating that they are required only in S . Typhi for growth under laboratory conditions ( Table 3 ), including the fepBDGC operon. This indicates a requirement for ferric [Fe(III)] rather than ferrous [Fe(II)] iron. This can be explained by the presence of Fe(III) in the bloodstream, where S. Typhi can be found during typhoid fever ( 15 ). These genes function to recover the ferric chelator enterobactin from the periplasm, acting with a number of proteins known to aid the passage of this siderophore through the outer membrane ( 73 ). It has long been noted that aroA mutants of S. Typhi, deficient in their ability to synthesize enterobactin, exhibit severe growth defects on complex media, whereas similar mutants of S. Typhimurium grow normally under the same conditions ( 74 ), though the mechanism has not been clear. Our results suggest that this difference in growth of aroA mutants is caused by a requirement for iron uptake through the fep system in S. Typhi. During host adaptation, S. Typhi has accumulated pseudogenes in many iron transport and response systems ( 27 ), presumably because they are not necessary for survival in the niche S. Typhi occupies in the human host, which may have led to this dependence on fep genes. In contrast, S. Typhimurium generally causes intestinal rather than systemic infection and is able to use a wider range of iron sources, including Fe(II), a soluble form of iron present under anaerobic conditions such as those found in the intestine ( 75 ).

TraDIS provides resolution sufficient to evaluate ncRNA contributions to fitness

Under a Poisson approximation to the transposon insertion process, a region of 41 (in S. Typhimurium) or 60 bases (in S . Typhi) has only a 1% probability of not containing an insertion by chance. NcRNAs tend to be considerably shorter than their protein-coding counterparts, but this gives us sufficient resolution to assay most of the non-coding complement of the Salmonella genome. As a proof of principle, we performed an analysis of the best-understood class of small ncRNAs, the tRNAs. Francis Crick hypothesized that a single tRNA could recognize more than one codon through wobble recognition ( 76 ), where a non-canonical G-U base pair is formed between the first (wobble) position of the anticodon and the third nucleotide in the codon. As a result, some codons are covered by multiple tRNAs, whereas others are covered non-redundantly by a single tRNA. We expect that singleton wobble-capable tRNAs, i.e. wobble tRNAs which recognize a codon uniquely, will be required. In addition, we inferred the requirement for other tRNAs through the non-redundant coverage of their codons and used this to benchmark our ability to use TraDIS to reliably interrogate short genomic intervals.

The S . Typhi and S. Typhimurium genomes encode 78 and 85 (plus one pseudogene) tRNAs, respectively, with 40 anticodons, as identified by tRNAscan-SE ( 77 ). In S . Typhi, 10 of 11 singleton wobble tRNAs are predicted to be required or ambiguous, compared with 16 tRNAs below the ambiguous LLR cut-off overall (significant enrichment at the 0.05 level, two-tailed Fisher’s exact test P -value: 6.4e-08). Similarly in S. Typhimurium, 9 of 11 singleton wobble tRNAs are required or ambiguous compared with 15 required or ambiguous tRNAs overall, again showing a significant enrichment of required tRNAs in this subset (Fisher’s exact test P -value: 5.2e-07). The one singleton wobble tRNA, which is consistently not required in both serovars is the tRNA-Pro(GGG), which occurs within a four-member codon family. It has previously been shown in S. Typhimurium that tRNA-Pro(UGG) can read all four proline codons in vivo owing to a cmo 5 U34 modification to the anticodon, obviating the need for a functional tRNA-Pro(GGG) ( 78 ) and making this tRNA non-required. The other non-required singleton wobble tRNA in S. Typhimurium, tRNA-Leu(GAG), is similarly a member of a four-member codon family. We predict tRNA-Leu(TAG) may also be capable of recognizing all four leucine codons in this serovar; such a leucine ‘four-way wobble’ has been previously inferred in at least one other bacterial species ( 79 , 80 ).

Of the six required non-wobble tRNAs in each serovar, four are shared. These include two non-wobble singleton tRNAs covering codons uniquely, as well as a tRNA with the ATG anticodon, which is post-transcriptionally modified by the required protein mesJ/tilS to recognize the isoleucine codon ATA ( 80 ). An additional two required tRNAs in both serovars, one shared and one with a differing anticodon, contain Gln anticodons and are part of a polycistronic tRNA operon containing other required tRNAs. This operon is conserved in E. coli with the exception of an additional tRNA-Gln at the 3′ end that has been lost in the Salmonella lineage. It is possible that transposon insertions early in the operon may interfere with processing of the polycistronic transcript in to mature tRNAs. Finally, we do not observe insertions in a tRNA-Met and a tRNA-Val in S . Typhi and S. Typhimurium, respectively.

Using this analysis of the tRNAs, we estimate a worst-case PPV for these short molecules (∼76 bases) at 81%, in line with our previous estimates for conserved protein-coding genes, and a FPR of <4%, higher than for protein-coding genes but still well within the typical tolerance of high-throughput experiments. This assumes that the ‘required’ operonic tRNA-Glns and the serovar-specific tRNA-Met and tRNA-Val are all false positives; it is not clear that this is in fact the case.

Surveying the shared required ncRNA content of both serovars (see Table 4 ), we find that the RNA components of the signal recognition particle (SRP) and RNaseP, two universally conserved ncRNAs, are required as expected. The SRP is an essential component of the cellular secretion machinery, whereas RnaseP is necessary for the maturation of tRNAs. We also find a number of required known and potential cis -regulatory molecules associated with genes required for growth under laboratory conditions in both serovars. The RFN riboswitch controls ribB , a 3,4-dihydroxy-2-butanone 4-phosphate synthase involved in riboflavin biosynthesis, in response to flavin mononucleotide concentrations ( 83 ). Additionally, we are able to assign putative functions to a number of previously uncharacterized required non-coding transcripts through their 5′ association with required genes. SroE, a 90 nt molecule discovered in an early sRNA screen ( 84 ), is consistently located at the 5′ end of the required hisS gene across its phylogenic distribution in the Enterobacteriaceae. Given this consistent association and the function of HisS as a histidyl-tRNA synthetase, we hypothesize that this region may act in a manner similar to a T-box leader, inducing or repressing expression in response to tRNA-His levels. The thrU leader sequence, recently discovered in a deep-sequencing screen of E. coli ( 42 ), appears to regulate a polycistronic operon of required singleton wobble tRNAs. Three additional required cis -regulatory elements, t44, S15 and StyR-8, are associated with required ribosomal proteins, highlighting the central role ncRNA elements play in regulating fundamental cellular processes.

Table 4.

Candidate required ncRNAs greater than 60 nt in length, excluding rRNA and tRNA

Element name Rfam accession Function Hfq-binding Downstream protein-coding gene(s) Downstream gene required References 
Required or ambiguous in both S. Typhi and S. Typhimurium  
    SRP RF00169 RNA component of the signal recognition particle     ( 81 )  
    RNase P RF00010 RNA component of RNase P  ybaZ  ( 82 )  
    RFN RF00050 FMN-sensing riboswitch controlling the ribB gene  ribB  ( 83 )  
    SroE RF00371 Putative cis-regulatory element controlling the hisS gene  hisS  ( 84 )  
    ThrU Leader NA Putative cis-regulatory element controlling the ThrU tRNA operon     ( 42 )  
    t44 RF00127 Cis-regulatory element controlling the ribosomal rpsB gene  rpsB  ( 85-87 )  
    S15 b RF00114 Translational regulator of the ribosomal S15 protein  rpsO  ( 88 )  
    StyR-8 NA Putative cis-regulatory element controlling the ribosomal rpmB gene  rpmB  ( 41 )  
    MicA RF00078 sRNA involved in cellular response to extracytoplasmic stress luxS  ( 29 )  
    DsrA b RF00014 sRNA regulator of H-NS mngB  ( 89 )  
    STnc10 NA Putative sRNA  nhaA  ( 40 )  
    STnc60 b NA Putative sRNA     ( 40 )  
    STnc840 NA Verified sRNA derived from 3' UTR of the flgL gene    ( 90 )  
    IS0420 a,b NA Putative ncRNA  rmf  ( 42 , 91 )  
    RGO0 b NA Putative sRNA identified in E. coli     ( 42 )  
Required or ambiguous in S. Typhimurium only  
    rne5 RF00040 RNase E autoregulatory 5' element  rne  ( 92 )  
    RydC RF00505 sRNA regulator of the yejABEF ABC transporter    ( 93 )  
    RydB RF00118 Putative ncRNA     ( 94 )  
    STnc510 NA Putative sRNA  pagD/pagC Y/N  ( 40 )  
    STnc460 b NA Putative sRNA     ( 40 )  
    STnc170 NA Putative sRNA  SL1458  ( 40 )  
    STnc130 NA Putative sRNA  dmsA  ( 40 )  
    RseX RF01401 sRNA regulator of OmpA and OmpC    ( 95 )  
    IsrJ RF01393 sRNA regulator of SPI-1 effector protein secretion     ( 40 , 58 )  
    IsrI RF01392 Island-encoded Hfq-binding sRNA SL1028  ( 40 , 58 , 90 )  
Required or ambiguous in S. Typhi only  
    RybB RF00110 sRNA involved in cellular response to extracytoplasmic stress    ( 29 )  
    tk5 a NA Putative ncRNA     ( 42 , 96 )  
    STnc750 NA Verified sRNA SpeB  ( 32 , 90 )  
    StyR-44 a RF01830  Putative multicopy (2/6 copies required in S. Typhi) ncRNA associated with ribosomal RNA operon   23S rRNA  ( 41 )  
    tp2 NA Putative ncRNA  aceE  ( 42 , 96 )  
    RdlD RF01813  RdlD RNA anti-toxin, 1/2 copies required in S. Typhi      ( 97 )  
    STnc120 a NA Putative sRNA     ( 40 )  
    tp28 a NA Putative ncRNA  fur  ( 42 , 96 )  
    Phe Leader a RF01859 Phenylalanine peptide leader sequence associated with the required PheST operon  PheS  ( 98 )  
    RimP Leader RF01770 Putative cis-regulator of the rimP-nusA-infB operon  rimP  ( 99 )  
    GlmY RF00128 Trans-acting regulator of the GlmS gene     ( 100 )  
Element name Rfam accession Function Hfq-binding Downstream protein-coding gene(s) Downstream gene required References 
Required or ambiguous in both S. Typhi and S. Typhimurium  
    SRP RF00169 RNA component of the signal recognition particle     ( 81 )  
    RNase P RF00010 RNA component of RNase P  ybaZ  ( 82 )  
    RFN RF00050 FMN-sensing riboswitch controlling the ribB gene  ribB  ( 83 )  
    SroE RF00371 Putative cis-regulatory element controlling the hisS gene  hisS  ( 84 )  
    ThrU Leader NA Putative cis-regulatory element controlling the ThrU tRNA operon     ( 42 )  
    t44 RF00127 Cis-regulatory element controlling the ribosomal rpsB gene  rpsB  ( 85-87 )  
    S15 b RF00114 Translational regulator of the ribosomal S15 protein  rpsO  ( 88 )  
    StyR-8 NA Putative cis-regulatory element controlling the ribosomal rpmB gene  rpmB  ( 41 )  
    MicA RF00078 sRNA involved in cellular response to extracytoplasmic stress luxS  ( 29 )  
    DsrA b RF00014 sRNA regulator of H-NS mngB  ( 89 )  
    STnc10 NA Putative sRNA  nhaA  ( 40 )  
    STnc60 b NA Putative sRNA     ( 40 )  
    STnc840 NA Verified sRNA derived from 3' UTR of the flgL gene    ( 90 )  
    IS0420 a,b NA Putative ncRNA  rmf  ( 42 , 91 )  
    RGO0 b NA Putative sRNA identified in E. coli     ( 42 )  
Required or ambiguous in S. Typhimurium only  
    rne5 RF00040 RNase E autoregulatory 5' element  rne  ( 92 )  
    RydC RF00505 sRNA regulator of the yejABEF ABC transporter    ( 93 )  
    RydB RF00118 Putative ncRNA     ( 94 )  
    STnc510 NA Putative sRNA  pagD/pagC Y/N  ( 40 )  
    STnc460 b NA Putative sRNA     ( 40 )  
    STnc170 NA Putative sRNA  SL1458  ( 40 )  
    STnc130 NA Putative sRNA  dmsA  ( 40 )  
    RseX RF01401 sRNA regulator of OmpA and OmpC    ( 95 )  
    IsrJ RF01393 sRNA regulator of SPI-1 effector protein secretion     ( 40 , 58 )  
    IsrI RF01392 Island-encoded Hfq-binding sRNA SL1028  ( 40 , 58 , 90 )  
Required or ambiguous in S. Typhi only  
    RybB RF00110 sRNA involved in cellular response to extracytoplasmic stress    ( 29 )  
    tk5 a NA Putative ncRNA     ( 42 , 96 )  
    STnc750 NA Verified sRNA SpeB  ( 32 , 90 )  
    StyR-44 a RF01830  Putative multicopy (2/6 copies required in S. Typhi) ncRNA associated with ribosomal RNA operon   23S rRNA  ( 41 )  
    tp2 NA Putative ncRNA  aceE  ( 42 , 96 )  
    RdlD RF01813  RdlD RNA anti-toxin, 1/2 copies required in S. Typhi      ( 97 )  
    STnc120 a NA Putative sRNA     ( 40 )  
    tp28 a NA Putative ncRNA  fur  ( 42 , 96 )  
    Phe Leader a RF01859 Phenylalanine peptide leader sequence associated with the required PheST operon  PheS  ( 98 )  
    RimP Leader RF01770 Putative cis-regulator of the rimP-nusA-infB operon  rimP  ( 99 )  
    GlmY RF00128 Trans-acting regulator of the GlmS gene     ( 100 )  

Known and putative non-coding elements classified as required or ambiguous in this screen. Required ncRNAs have an LLR between required and non-required models of less than −2; see ‘Materials and Methods’ section.

a,b ncRNAs that are amibiguous (LLR between −2 and 2) in S . Typhi( a ) or in S. Typhimurium( b ). Hfq-binding annotations are taken from ( 90 ). The downstream protein-coding genes columns report annotated CDS or ribosomal RNA start sites within 100 bases of each candidate required non-coding element on either strand and whether these downstream sequences are also classified as required.

The sRNAs required for competitive growth

Inferring functions for potential trans -acting ncRNA molecules, such as anti-sense binding sRNAs, from requirement patterns alone is more difficult than for cis -acting elements, as we cannot rely on adjacent genes to provide any information. It is also important to keep in mind that TraDIS assays requirements after a brief competition within a large library of mutants on permissive media. This may be particularly important when surveying the bacterial sRNAs, which are known to participate in responses to stress ( 29 ).

This is demonstrated by two sRNAs involved in the σ E -mediated extracytoplamic stress response, RybB and RseX, both of which can be successfully knocked out in S. Typhimurium ( 101 ). In S . Typhi, rpoE is required, as it also is in E. coli ( 48 , 102 ). However, in S. Typhimurium, rpoE tolerates a heavy insertion load, implying that σ E mutants are not disadvantaged in competitive growth. In S. Typhimurium, the sRNA RseX is required. Overexpression of RseX has previously been shown to compensate for σ E essentiality in E. coli by degrading ompA and ompC transcripts ( 95 ). This suggests that RseX may also be short-circuiting the σ E stress response network in S. Typhimurium ( Figure 4 ). To our knowledge, this is the first evidence of a native (i.e. not experimentally induced) activity of RseX.

S . Typhi on the other hand requires σ E along with its activating proteases RseP and DegS and anchoring protein RseA, as well as the σ E -dependent sRNA RybB, which also regulates OmpA and OmpC in S. Typhimurium, along with a host of other OMPs ( 103 ). It is unclear why the σ E response is required in S . Typhi, but not S. Typhimurium, though it may partially be due to the major differences in the cell wall and outer membrane between the two serovars. In addition, there are significant differences in the OMP content of the S . Typhi and S. Typhimurium membranes that may be driving alternative mechanisms for coping with membrane stress. For instance, S . Typhi completely lacks OmpD, a major component of the S . Typhimurium outer membrane ( 104 ) and a known target of RybB ( 29 ).

Two additional sRNAs involved in stress response are also required by both S . Typhi and S. Typhimurium. The first, MicA, is known to regulate ompA and the lamB porin-coding gene in S. Typhimurium ( 105 ), contributing to the extracytoplasmic stress response. The second, DsrA, has been shown to negatively regulate the nucleoid-forming protein H-NS and enhance translation of the stationary-phase alternative sigma factor σ S in E. coli ( 89 ), though its regulation of σ S does not appear to be conserved in S. Typhimurium ( 106 ). Both have been previously deleted in S. Typhimurium and thus are not essential. H-NS knockouts have previously been shown to have severe growth defects in S. Typhimurium that can be rescued by compensatory mutations in either the phoPQ two-component system or rpoS , implying that the lack of H-NS is allowing normally silenced detrimental regions to be transcribed ( 61 ). As MicA has recently been shown to negatively regulate phoPQ expression in E. coli ( 107 ), it is tempting to speculate that MicA may be moderating the effects of DsrA-induced H-NS repression; however, it is currently unclear whether sRNA regulons are sufficiently conserved between E. coli and S. enterica to justify this hypothesis.

CONCLUSION

The extremely high resolution of TraDIS has allowed us to assay gene requirements in two very closely related Salmonellae with different host ranges. We found, under laboratory conditions, that 58 genes present in both serovars were required in only one, suggesting that identical gene products do not necessarily have the same phenotypic effects in the two different serovar backgrounds. Many of these genes occur in genomic regions or metabolic systems, which contain pseudogenes and/or have undergone reorganization since the divergence of S . Typhi and S. Typhimurium, demonstrating the complementarity of TraDIS and phylogenetic analysis. These changes may, in part, explain differences observed in the pathogenicity and host specificity of these two serovars. In particular, S. Typhimurium showed a requirement for cell surface structure biosynthesis genes; this may be partially explained by the fact that S . Typhi expresses the Vi-antigen, which masks the cell surface, though these genes are not required for survival in our assay. S . Typhi on the other hand has a requirement for iron uptake through the fep system, which enables ferric enterobactin transport. This dependence on enterobactin suggests that S . Typhi is highly adapted to the iron-scarce environments it encounters during systemic infections. Furthermore, this appears to represent a single point of failure in the S . Typhi iron utilization pathways and may present an attractive target for narrow-spectrum antibiotics.

Of the ∼4500 protein-coding genes present in each serovar, only ∼350 were sufficiently depleted in transposon insertions to be classified as required for growth in rich media. This means that >92% of the coding genome has sufficient insertion density to be queried in future assays. Dense transposon mutagenesis libraries have been used to assay gene requirements under conditions relevant for infection, including S . Typhi survival in bile ( 35 ), Mycobacterium tuberculosis catabolism of cholesterol ( 108 ), drug resistance in Pseudomonas aeruginosa ( 109 ) and Haemophilus influenzae survival in the lung ( 110 ). We expect that parallel experiments querying gene requirements under the same conditions in both serovars examined in this study will yield further insights in to the differences in the infective process between Typhi and Typhimurium and ultimately the pathways that underlie host-adaptation.

Both serovars possess substantial complements of horizontally acquired DNA. We have been able to use TraDIS to assay these recently acquired sequences. In particular, we have been able to identify, on a chromosome wide scale, active prophage through the requirement for their repressors. The P4 phage uses an RNA-based system to make decisions regarding cell fate, and structurally similar systems are used by P1, P7 and N15 phage ( 111 , 112 ). C4-like transcripts have been regarded as the primary repressor of lytic functions, though the IsrK-like sequence is known to be essential to the establishment of lysogeny in P4 and is transcribed in at least two phage types ( 60 , 112 ). Our observations in S . Typhi suggest an important role for the IsrK-like sequence in maintenance of the lysogenic state in P4-like phage, though the mechanism remains unclear.

Recent advances in high-throughput sequencing have greatly enhanced our ability to detect novel transcripts, such as ncRNAs and short open reading frames (sORFs). Our ability to identify these transcripts now far out-strips our ability to experimentally characterize these sequences. There have been previous efforts at high-throughput characterization of bacterial sRNAs and sORFs in enteric bacteria; however, these have relied on labour-intensive directed knockout libraries ( 47 , 113 ). Here, we have demonstrated that TraDIS has sufficient resolution to reliably query genomic regions as short as 60 bases, in agreement with a recent high-throughput transposon mutagenesis study in the alphaproteobacteria Caulobacter crescentus ( 38 ). Our method has the major advantage that library construction does not rely on genome annotation, and newly discovered elements can be surveyed with no further laboratory work.

We have been able to assign putative functions to a number of ncRNAs using TraDIS though consideration of their genomic and experimental context. In addition, ncRNA characterization generally is done in model organisms like E. coli or S. Typhimurium, and it is unclear how stable ncRNA regulatory networks are over evolutionary time. By assaying two serovars of Salmonella with the same method under the same conditions, we have seen hints that there may be differences in sRNA regulatory networks between S . Typhi and S. Typhimurium. In particular, we have found that under the same experimental conditions, S . Typhi appears to rely on the σ E stress response pathway, whereas S. Typhimurium does not; it is tempting to speculate that this difference in stress response is mediated by the observed difference in requirement for two sRNAs, RybB and RseX. We believe that this combination of high-throughput transposon mutagenesis with a careful consideration of the systems context of individual genes provides a powerful tool for the generation of functional hypotheses. We anticipate that the construction of TraDIS libraries in additional organisms, as well as the passing of these libraries through relevant experimental conditions, will provide further insights into the function of bacterial ncRNAs in addition to the protein-coding gene complement.

ACCESSION NUMBERS

ERA000097, ERA000217.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online: Supplementary Tables 1–3, Supplementary Figures 1–2 and Supplementary Data Set.

FUNDING

Wellcome Trust [WT076964, WT079643 and WT098051]; Medical Research Council. Funding for open access charge: Wellcome Trust.

Conflict of interest statement. None declared.

ACKNOWLEDGEMENTS

The authors would like to thank Leopold Parts for assistance with the statistical analysis of required genes under laboratory conditions, Derek Pickard for discussions of phage biology and Amy Cain for comments on the manuscript. The S. Typhimurium nucleotide sequencing data have been deposited in the European Short Read Archive under accession number ERA000217. The S. Typhi data can be accessed at ERA000097.

REFERENCES

1
Crump
JA
Luby
SP
Mintz
ED
The global burden of typhoid fever
Bull World Health Organ.
 , 
2004
, vol. 
82
 (pg. 
346
-
353
)
2
Bhutta
ZA
Threlfall
J
Addressing the global disease burden of typhoid fever
JAMA
 , 
2009
, vol. 
302
 (pg. 
898
-
899
)
3
Kothari
A
Pruthi
A
Chugh
TD
The burden of enteric fever
J. Infect. Dev. Ctries.
 , 
2008
, vol. 
2
 (pg. 
253
-
259
)
4
Soper
GA
The curious career of typhoid mary
Bull N.Y. Acad. Med.
 , 
1939
, vol. 
15
 (pg. 
698
-
712
)
5
 
CDC. (2009) Salmonella surveillance: Annual summary
6
Majowicz
SE
Musto
J
Scallan
E
Angulo
FJ
Kirk
M
O'Brien
SJ
Jones
TF
Fazil
A
Hoekstra
RM
The global burden of nontyphoidal Salmonella gastroenteritis
Clin. Infect. Dis.
 , 
2010
, vol. 
50
 (pg. 
882
-
889
)
7
Santos
RL
Zhang
S
Tsolis
RM
Kingsley
RA
Adams
LG
Baumler
AJ
Animal models of Salmonella infections: enteritis versus typhoid fever
Microbes Infect.
 , 
2001
, vol. 
3
 (pg. 
1335
-
1344
)
8
McClelland
M
Sanderson
KE
Spieth
J
Clifton
SW
Latreille
P
Courtney
L
Porwollik
S
Ali
J
Dante
M
Du
F
, et al.  . 
Complete genome sequence of Salmonella enterica serovar Typhimurium LT2
Nature
 , 
2001
, vol. 
413
 (pg. 
852
-
856
)
9
Parkhill
J
Dougan
G
James
KD
Thomson
NR
Pickard
D
Wain
J
Churcher
C
Mungall
KL
Bentley
SD
Holden
MT
, et al.  . 
Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18
Nature
 , 
2001
, vol. 
413
 (pg. 
848
-
852
)
10
Holt
KE
Parkhill
J
Mazzoni
CJ
Roumagnac
P
Weill
FX
Goodhead
I
Rance
R
Baker
S
Maskell
DJ
Wain
J
, et al.  . 
High-throughput sequencing provides insights into genome variation and evolution in Salmonella Typhi
Nat. Genet.
 , 
2008
, vol. 
40
 (pg. 
987
-
993
)
11
Deng
W
Liou
SR
Plunkett
G
3rd
Mayhew
GF
Rose
DJ
Burland
V
Kodoyianni
V
Schwartz
DC
Blattner
FR
Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18
J. Bacteriol.
 , 
2003
, vol. 
185
 (pg. 
2330
-
2337
)
12
Gulig
PA
Curtiss
R
3rd
Plasmid-associated virulence of Salmonella Typhimurium
Infect. Immun.
 , 
1987
, vol. 
55
 (pg. 
2891
-
2901
)
13
Gulig
PA
Doyle
TJ
The Salmonella Typhimurium virulence plasmid increases the growth rate of salmonellae in mice
Infect. Immun.
 , 
1993
, vol. 
61
 (pg. 
504
-
511
)
14
Phan
MD
Kidgell
C
Nair
S
Holt
KE
Turner
AK
Hinds
J
Butcher
P
Cooke
FJ
Thomson
NR
Titball
R
, et al.  . 
Variation in Salmonella enterica serovar Typhi IncHI1 plasmids during the global spread of resistant typhoid fever
Antimicrob. Agents Chemother.
 , 
2009
, vol. 
53
 (pg. 
716
-
727
)
15
Wain
J
Diep
TS
Ho
VA
Walsh
AM
Nguyen
TT
Parry
CM
White
NJ
Quantitation of bacteria in blood of typhoid fever patients and relationship between counts and clinical features, transmissibility, and antibiotic resistance
J. Clin. Microbiol.
 , 
1998
, vol. 
36
 (pg. 
1683
-
1687
)
16
Datta
N
Transmissible drug resistance in an epidemic strain of Salmonella Typhimurium
J. Hyg. (Lond.)
 , 
1962
, vol. 
60
 (pg. 
301
-
310
)
17
Holt
KE
Thomson
NR
Wain
J
Phan
MD
Nair
S
Hasan
R
Bhutta
ZA
Quail
MA
Norbertczak
H
Walker
D
, et al.  . 
Multidrug-resistant Salmonella enterica serovar Paratyphi A harbors IncHI1 plasmids similar to those found in serovar Typhi
J. Bacteriol.
 , 
2007
, vol. 
189
 (pg. 
4257
-
4264
)
18
Cain
AK
Hall
RM
Evolution of a multiple antibiotic resistance region in IncHI1 plasmids: reshaping resistance regions in situ
J. Antimicrob. Chemother.
 , 
2012
, vol. 
67
 (pg. 
2848
-
2853
)
19
Darwin
KH
Miller
VL
Molecular basis of the interaction of Salmonella with the intestinal mucosa
Clin. Microbiol. Rev.
 , 
1999
, vol. 
12
 (pg. 
405
-
428
)
20
Ochman
H
Soncini
FC
Solomon
F
Groisman
EA
Identification of a pathogenicity island required for Salmonella survival in host cells
Proc. Natl Acad. Sci. USA
 , 
1996
, vol. 
93
 (pg. 
7800
-
7804
)
21
Shea
JE
Hensel
M
Gleeson
C
Holden
DW
Identification of a virulence locus encoding a second type III secretion system in Salmonella Typhimurium
Proc. Natl Acad. Sci. USA
 , 
1996
, vol. 
93
 (pg. 
2593
-
2597
)
22
Pickard
D
Wain
J
Baker
S
Line
A
Chohan
S
Fookes
M
Barron
A
Gaora
PO
Chabalgoity
JA
Thanky
N
, et al.  . 
Composition, acquisition, and distribution of the Vi exopolysaccharide-encoding Salmonella enterica pathogenicity island SPI-7
J. Bacteriol.
 , 
2003
, vol. 
185
 (pg. 
5055
-
5065
)
23
Seth-Smith
HM
SPI-7: Salmonella 's Vi-encoding Pathogenicity Island
J. Infect. Dev. Ctries.
 , 
2008
, vol. 
2
 (pg. 
267
-
271
)
24
Townsend
SM
Kramer
NE
Edwards
R
Baker
S
Hamlin
N
Simmonds
M
Stevens
K
Maloy
S
Parkhill
J
Dougan
G
, et al.  . 
Salmonella enterica serovar Typhi possesses a unique repertoire of fimbrial gene sequences
Infect. Immun.
 , 
2001
, vol. 
69
 (pg. 
2894
-
2901
)
25
Thomson
NR
Clayton
DJ
Windhorst
D
Vernikos
G
Davidson
S
Churcher
C
Quail
MA
Stevens
M
Jones
MA
Watson
M
, et al.  . 
Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways
Genome Res.
 , 
2008
, vol. 
18
 (pg. 
1624
-
1637
)
26
Holt
KE
Thomson
NR
Wain
J
Langridge
GC
Hasan
R
Bhutta
ZA
Quail
MA
Norbertczak
H
Walker
D
Simmonds
M
, et al.  . 
Pseudogene accumulation in the evolutionary histories of Salmonella enterica serovars Paratyphi A and Typhi
BMC Genomics
 , 
2009
, vol. 
10
 pg. 
36
 
27
McClelland
M
Sanderson
KE
Clifton
SW
Latreille
P
Porwollik
S
Sabo
A
Meyer
R
Bieri
T
Ozersky
P
McLellan
M
, et al.  . 
Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid
Nat. Genet.
 , 
2004
, vol. 
36
 (pg. 
1268
-
1274
)
28
Kingsley
RA
Humphries
AD
Weening
EH
De Zoete
MR
Winter
S
Papaconstantinopoulou
A
Dougan
G
Baumler
AJ
Molecular and phenotypic analysis of the CS54 island of Salmonella enterica serotype Typhimurium: identification of intestinal colonization and persistence determinants
Infect. Immun.
 , 
2003
, vol. 
71
 (pg. 
629
-
640
)
29
Vogel
J
A rough guide to the non-coding RNA world of Salmonella
Mol. Microbiol.
 , 
2009
, vol. 
71
 (pg. 
1
-
11
)
30
Waldminghaus
T
Heidrich
N
Brantl
S
Narberhaus
F
FourU: a novel type of RNA thermometer in Salmonella
Mol. Microbiol.
 , 
2007
, vol. 
65
 (pg. 
413
-
424
)
31
Cromie
MJ
Shi
Y
Latifi
T
Groisman
EA
An RNA sensor for intracellular Mg(2+)
Cell
 , 
2006
, vol. 
125
 (pg. 
71
-
84
)
32
Kroger
C
Dillon
SC
Cameron
AD
Papenfort
K
Sivasankaran
SK
Hokamp
K
Chao
Y
Sittka
A
Hebrard
M
Handler
K
, et al.  . 
The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium
Proc. Natl Acad. Sci. USA
 , 
2012
, vol. 
109
 (pg. 
E1277
-
E1286
)
33
Hebrard
M
Kroger
C
Srikumar
S
Colgan
A
Handler
K
Hinton
J
sRNAs and the virulence of Salmonella enterica serovar Typhimurium
RNA Biol.
 , 
2012
, vol. 
9
 (pg. 
437
-
445
)
34
Richter
AS
Backofen
R
Accessibility and conservation: General features of bacterial small RNA-mRNA interactions?
RNA Biol.
 , 
2012
, vol. 
9
 (pg. 
954
-
965
)
35
Langridge
GC
Phan
MD
Turner
DJ
Perkins
TT
Parts
L
Haase
J
Charles
I
Maskell
DJ
Peters
SE
Dougan
G
, et al.  . 
Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants
Genome Res.
 , 
2009
, vol. 
19
 (pg. 
2308
-
2316
)
36
Goodman
AL
McNulty
NP
Zhao
Y
Leip
D
Mitra
RD
Lozupone
CA
Knight
R
Gordon
JI
Identifying genetic determinants needed to establish a human gut symbiont in its habitat
Cell Host Microbe.
 , 
2009
, vol. 
6
 (pg. 
279
-
289
)
37
van Opijnen
T
Bodi
KL
Camilli
A
Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms
Nat. Methods.
 , 
2009
, vol. 
6
 (pg. 
767
-
772
)
38
Christen
B
Abeliuk
E
Collier
JM
Kalogeraki
VS
Passarelli
B
Coller
JA
Fero
MJ
McAdams
HH
Shapiro
L
The essential genome of a bacterium
Mol. Syst. Biol.
 , 
2011
, vol. 
7
 pg. 
528
 
39
Burge
SW
Daub
J
Eberhardt
R
Tate
J
Barquist
L
Nawrocki
EP
Eddy
SR
Gardner
PP
Bateman
A
Rfam 11.0: 10 years of RNA families
Nucleic Acids Res.
 , 
2013
, vol. 
41
 (pg. 
D226
-
D232
)
40
Sittka
A
Sharma
CM
Rolle
K
Vogel
J
Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes
RNA Biol.
 , 
2009
, vol. 
6
 (pg. 
266
-
275
)
41
Chinni
SV
Raabe
CA
Zakaria
R
Randau
G
Hoe
CH
Zemann
A
Brosius
J
Tang
TH
Rozhdestvensky
TS
Experimental identification and characterization of 97 novel npcRNA candidates in Salmonella enterica serovar Typhi
Nucleic Acids Res.
 , 
2010
, vol. 
38
 (pg. 
5893
-
5908
)
42
Raghavan
R
Groisman
EA
Ochman
H
Genome-wide detection of novel regulatory RNAs in E. coli
Genome Res.
 , 
2011
, vol. 
21
 (pg. 
1487
-
1497
)
43
Finn
RD
Clements
J
Eddy
SR
HMMER web server: interactive sequence similarity searching
Nucleic Acids Res.
 , 
2011
, vol. 
39
 (pg. 
W29
-
W37
)
44
Punta
M
Coggill
PC
Eberhardt
RY
Mistry
J
Tate
J
Boursnell
C
Pang
N
Forslund
K
Ceric
G
Clements
J
, et al.  . 
The Pfam protein families database
Nucleic Acids Res.
 , 
2012
, vol. 
40
 (pg. 
D290
-
D301
)
45
Li
H
Ruan
J
Durbin
R
Mapping short DNA sequencing reads and calling variants using mapping quality scores
Genome Res.
 , 
2008
, vol. 
18
 (pg. 
1851
-
1858
)
46
Knuth
K
Niesalla
H
Hueck
CJ
Fuchs
TM
Large-scale identification of essential Salmonella genes by trapping lethal insertions
Mol. Microbiol.
 , 
2004
, vol. 
51
 (pg. 
1729
-
1744
)
47
Santiviago
CA
Reynolds
MM
Porwollik
S
Choi
SH
Long
F
Andrews-Polymenis
HL
McClelland
M
Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice
PLoS Pathog.
 , 
2009
, vol. 
5
 pg. 
e1000477
 
48
Baba
T
Ara
T
Hasegawa
M
Takai
Y
Okumura
Y
Baba
M
Datsenko
KA
Tomita
M
Wanner
BL
Mori
H
Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection
Mol. Syst. Biol.
 , 
2006
, vol. 
2
  
2006.0008
49
Gerdes
SY
Scholle
MD
Campbell
JW
Balazsi
G
Ravasz
E
Daugherty
MD
Somera
AL
Kyrpides
NC
Anderson
I
Gelfand
MS
, et al.  . 
Experimental determination and system level analysis of essential genes in Escherichia coli MG1655
J. Bacteriol.
 , 
2003
, vol. 
185
 (pg. 
5673
-
5684
)
50
Tong
X
Campbell
JW
Balazsi
G
Kay
KA
Wanner
BL
Gerdes
SY
Oltvai
ZN
Genome-scale identification of conditionally essential genes in E
coli by DNA microarrays. Biochem. Biophys. Res. Commun.
 , 
2004
, vol. 
322
 (pg. 
347
-
354
)
51
Hashimoto
M
Ichimura
T
Mizoguchi
H
Tanaka
K
Fujimitsu
K
Keyamura
K
Ote
T
Yamakawa
T
Yamazaki
Y
Mori
H
, et al.  . 
Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome
Mol. Microbiol.
 , 
2005
, vol. 
55
 (pg. 
137
-
149
)
52
Kang
Y
Durfee
T
Glasner
JD
Qiu
Y
Frisch
D
Winterberg
KM
Blattner
FR
Systematic mutagenesis of the Escherichia coli genome
J. Bacteriol.
 , 
2004
, vol. 
186
 (pg. 
4921
-
4930
)
53
Echols
H
Green
L
Establishment and maintenance of repression by bacteriophage lambda: the role of the cI, cII, and c3 proteins
Proc. Natl Acad. Sci. USA
 , 
1971
, vol. 
68
 (pg. 
2190
-
2194
)
54
Thomson
N
Baker
S
Pickard
D
Fookes
M
Anjum
M
Hamlin
N
Wain
J
House
D
Bhutta
Z
Chan
K
, et al.  . 
The role of prophage-like elements in the diversity of Salmonella enterica serovars
J. Mol. Biol.
 , 
2004
, vol. 
339
 (pg. 
279
-
300
)
55
Kropinski
AM
Sulakvelidze
A
Konczy
P
Poppe
C
Salmonella phages and prophages–genomics and practical aspects
Methods Mol. Biol.
 , 
2007
, vol. 
394
 (pg. 
133
-
175
)
56
Lemire
S
Figueroa-Bossi
N
Bossi
L
Bacteriophage crosstalk: coordination of prophage induction by trans-acting antirepressors
PLoS Genet.
 , 
2011
, vol. 
7
 pg. 
e1002149
 
57
Briani
F
Deho
G
Forti
F
Ghisotti
D
The plasmid status of satellite bacteriophage P4
Plasmid
 , 
2001
, vol. 
45
 (pg. 
1
-
17
)
58
Padalon-Brauch
G
Hershberg
R
Elgrably-Weiss
M
Baruch
K
Rosenshine
I
Margalit
H
Altuvia
S
Small RNAs encoded within genetic islands of Salmonella Typhimurium show host-induced expression and role in virulence
Nucleic Acids Res.
 , 
2008
, vol. 
36
 (pg. 
1913
-
1927
)
59
Forti
F
Dragoni
I
Briani
F
Deho
G
Ghisotti
D
Characterization of the small antisense CI RNA that regulates bacteriophage P4 immunity
J. Mol. Biol.
 , 
2002
, vol. 
315
 (pg. 
541
-
549
)
60
Sabbattini
P
Forti
F
Ghisotti
D
Deho
G
Control of transcription termination by an RNA factor in bacteriophage P4 immunity: identification of the target sites
J. Bacteriol.
 , 
1995
, vol. 
177
 (pg. 
1425
-
1434
)
61
Navarre
WW
Porwollik
S
Wang
Y
McClelland
M
Rosen
H
Libby
SJ
Fang
FC
Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella
Science
 , 
2006
, vol. 
313
 (pg. 
236
-
238
)
62
Iyer
LM
Babu
MM
Aravind
L
The HIRAN domain and recruitment of chromatin remodeling and repair activities to damaged DNA
Cell Cycle
 , 
2006
, vol. 
5
 (pg. 
775
-
782
)
63
Kuhle
V
Jackel
D
Hensel
M
Effector proteins encoded by Salmonella pathogenicity island 2 interfere with the microtubule cytoskeleton after translocation into host cells
Traffic
 , 
2004
, vol. 
5
 (pg. 
356
-
370
)
64
Miao
EA
Miller
SI
A conserved amino acid sequence directing intracellular type III secretion by Salmonella Typhimurium
Proc. Natl Acad. Sci. USA
 , 
2000
, vol. 
97
 (pg. 
7539
-
7544
)
65
Freeman
JA
Ohl
ME
Miller
SI
The Salmonella enterica serovar Typhimurium translocated effectors SseJ and SifB are targeted to the Salmonella -containing vacuole
Infect. Immun.
 , 
2003
, vol. 
71
 (pg. 
418
-
427
)
66
Lucchini
S
Rowley
G
Goldberg
MD
Hurd
D
Harrison
M
Hinton
JC
H-NS mediates the silencing of laterally acquired genes in bacteria
PLoS Pathog.
 , 
2006
, vol. 
2
 pg. 
e81
 
67
Manna
D
Porwollik
S
McClelland
M
Tan
R
Higgins
NP
Microarray analysis of Mu transposition in Salmonella enterica , serovar Typhimurium: transposon exclusion by high-density DNA binding proteins
Mol. Microbiol.
 , 
2007
, vol. 
66
 (pg. 
315
-
328
)
68
Hensel
M
Shea
JE
Raupach
B
Monack
D
Falkow
S
Gleeson
C
Kubo
T
Holden
DW
Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2
Mol. Microbiol.
 , 
1997
, vol. 
24
 (pg. 
155
-
167
)
69
Hensel
M
Shea
JE
Waterman
SR
Mundy
R
Nikolaus
T
Banks
G
Vazquez-Torres
A
Gleeson
C
Fang
FC
Holden
DW
Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages
Mol. Microbiol.
 , 
1998
, vol. 
30
 (pg. 
163
-
174
)
70
Ohlson
MB
Fluhr
K
Birmingham
CL
Brumell
JH
Miller
SI
SseJ deacylase activity by Salmonella enterica serovar Typhimurium promotes virulence in mice
Infect. Immun.
 , 
2005
, vol. 
73
 (pg. 
6249
-
6259
)
71
Doyle
M
Fookes
M
Ivens
A
Mangan
MW
Wain
J
Dorman
CJ
An H-NS-like stealth protein aids horizontal DNA transmission in bacteria
Science
 , 
2007
, vol. 
315
 (pg. 
251
-
252
)
72
Dillon
SC
Cameron
AD
Hokamp
K
Lucchini
S
Hinton
JC
Dorman
CJ
Genome-wide analysis of the H-NS and Sfh regulatory networks in Salmonella Typhimurium identifies a plasmid-encoded transcription silencing mechanism
Mol. Microbiol.
 , 
2010
, vol. 
76
 (pg. 
1250
-
1265
)
73
Rabsch
W
Voigt
W
Reissbrodt
R
Tsolis
RM
Baumler
AJ
Salmonella Typhimurium IroN and FepA proteins mediate uptake of enterobactin but differ in their specificity for other siderophores
J. Bacteriol.
 , 
1999
, vol. 
181
 (pg. 
3610
-
3612
)
74
Edwards
MF
Stocker
BA
Construction of delta aroA his delta pur strains of Salmonella Typhi
J. Bacteriol.
 , 
1988
, vol. 
170
 (pg. 
3991
-
3995
)
75
Tsolis
RM
Baumler
AJ
Heffron
F
Stojiljkovic
I
Contribution of TonB- and Feo-mediated iron uptake to growth of Salmonella Typhimurium in the mouse
Infect. Immun.
 , 
1996
, vol. 
64
 (pg. 
4549
-
4556
)
76
Crick
FH
Codon–anticodon pairing: the wobble hypothesis
J. Mol. Biol.
 , 
1966
, vol. 
19
 (pg. 
548
-
555
)
77
Lowe
TM
Eddy
SR
tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence
Nucleic. Acids. Res.
 , 
1997
, vol. 
25
 (pg. 
955
-
964
)
78
Nasvall
SJ
Chen
P
Bjork
GR
The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo
RNA
 , 
2004
, vol. 
10
 (pg. 
1662
-
1673
)
79
Osawa
S
Jukes
TH
Watanabe
K
Muto
A
Recent evidence for evolution of the genetic code
Microbiol. Rev.
 , 
1992
, vol. 
56
 (pg. 
229
-
264
)
80
Marck
C
Grosjean
H
tRNomics: analysis of tRNA genes from 50 genomes of Eukarya, Archaea, and Bacteria reveals anticodon-sparing strategies and domain-specific features
RNA
 , 
2002
, vol. 
8
 (pg. 
1189
-
1232
)
81
Rosenblad
MA
Larsen
N
Samuelsson
T
Zwieb
C
Kinship in the SRP RNA family
RNA Biol.
 , 
2009
, vol. 
6
 (pg. 
508
-
516
)
82
Frank
DN
Pace
NR
Ribonuclease P: unity and diversity in a tRNA processing ribozyme
Annu. Rev. Biochem.
 , 
1998
, vol. 
67
 (pg. 
153
-
180
)
83
Winkler
W
Nahvi
A
Breaker
RR
Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression
Nature
 , 
2002
, vol. 
419
 (pg. 
952
-
956
)
84
Vogel
J
Bartels
V
Tang
TH
Churakov
G
Slagter-Jager
JG
Huttenhofer
A
Wagner
EG
RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria
Nucleic Acids Res.
 , 
2003
, vol. 
31
 (pg. 
6435
-
6443
)
85
Tjaden
B
Saxena
RM
Stolyar
S
Haynor
DR
Kolker
E
Rosenow
C
Transcriptome analysis of Escherichia coli using high-density oligonucleotide probe arrays
Nucleic Acids Res.
 , 
2002
, vol. 
30
 (pg. 
3732
-
3738
)
86
Aseev
LV
Levandovskaya
AA
Tchufistova
LS
Scaptsova
NV
Boni
IV
A new regulatory circuit in ribosomal protein operons: S2-mediated control of the rpsB-tsf expression in vivo
RNA
 , 
2008
, vol. 
14
 (pg. 
1882
-
1894
)
87
Meyer
MM
Ames
TD
Smith
DP
Weinberg
Z
Schwalbach
MS
Giovannoni
SJ
Breaker
RR
Identification of candidate structured RNAs in the marine organism ‘Candidatus Pelagibacter ubique '
BMC Genomics
 , 
2009
, vol. 
10
 pg. 
268
 
88
Benard
L
Philippe
C
Ehresmann
B
Ehresmann
C
Portier
C
Pseudoknot and translational control in the expression of the S15 ribosomal protein
Biochimie
 , 
1996
, vol. 
78
 (pg. 
568
-
576
)
89
Lease
RA
Cusick
ME
Belfort
M
Riboregulation in Escherichia coli : DsrA RNA acts by RNA:RNA interactions at multiple loci
Proc. Natl Acad. Sci. USA
 , 
1998
, vol. 
95
 (pg. 
12456
-
12461
)
90
Chao
Y
Papenfort
K
Reinhardt
R
Sharma
CM
Vogel
J
An atlas of Hfq-bound transcripts reveals 3′ UTRs as a genomic reservoir of regulatory small RNAs
EMBO J.
 , 
2012
, vol. 
31
 (pg. 
4005
-
4019
)
91
Chen
S
Lesnik
EA
Hall
TA
Sampath
R
Griffey
RH
Ecker
DJ
Blyn
LB
A bioinformatics based approach to discover small RNA genes in the Escherichia coli genome
Biosystems
 , 
2002
, vol. 
65
 (pg. 
157
-
177
)
92
Diwa
A
Bricker
AL
Jain
C
Belasco
JG
An evolutionarily conserved RNA stem-loop functions as a sensor that directs feedback regulation of RNase E gene expression
Genes Dev.
 , 
2000
, vol. 
14
 (pg. 
1249
-
1260
)
93
Antal
M
Bordeau
V
Douchin
V
Felden
B
A small bacterial RNA regulates a putative ABC transporter
J. Biol. Chem.
 , 
2005
, vol. 
280
 (pg. 
7901
-
7908
)
94
Wassarman
KM
Repoila
F
Rosenow
C
Storz
G
Gottesman
S
Identification of novel small RNAs using comparative genomics and microarrays
Genes Dev.
 , 
2001
, vol. 
15
 (pg. 
1637
-
1651
)
95
Douchin
V
Bohn
C
Bouloc
P
Down-regulation of porins by a small RNA bypasses the essentiality of the regulated intramembrane proteolysis protease RseP in Escherichia coli
J. Biol. Chem.
 , 
2006
, vol. 
281
 (pg. 
12253
-
12259
)
96
Rivas
E
Eddy
SR
Noncoding RNA gene detection using comparative sequence analysis
BMC Bioinformatics
 , 
2001
, vol. 
2
 pg. 
8
 
97
Kawano
M
Oshima
T
Kasai
H
Mori
H
Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35-amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli
Mol. Microbiol.
 , 
2002
, vol. 
45
 (pg. 
333
-
349
)
98
Zurawski
G
Brown
K
Killingly
D
Yanofsky
C
Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli
Proc. Natl Acad. Sci. USA
 , 
1978
, vol. 
75
 (pg. 
4271
-
4275
)
99
Naville
M
Gautheret
D
Premature terminator analysis sheds light on a hidden world of bacterial transcriptional attenuation
Genome Biol.
 , 
2010
, vol. 
11
 pg. 
R97
 
100
Urban
JH
Vogel
J
Two seemingly homologous noncoding RNAs act hierarchically to activate glmS mRNA translation
PLoS Biol.
 , 
2008
, vol. 
6
 pg. 
e64
 
101
Papenfort
K
Pfeiffer
V
Lucchini
S
Sonawane
A
Hinton
JC
Vogel
J
Systematic deletion of Salmonella small RNA genes identifies CyaR, a conserved CRP-dependent riboregulator of OmpX synthesis
Mol. Microbiol.
 , 
2008
, vol. 
68
 (pg. 
890
-
906
)
102
De Las Penas
A
Connolly
L
Gross
CA
SigmaE is an essential sigma factor in Escherichia coli
J. Bacteriol.
 , 
1997
, vol. 
179
 (pg. 
6862
-
6864
)
103
Papenfort
K
Pfeiffer
V
Mika
F
Lucchini
S
Hinton
JC
Vogel
J
SigmaE-dependent small RNAs of Salmonella respond to membrane stress by accelerating global omp mRNA decay
Mol. Microbiol.
 , 
2006
, vol. 
62
 (pg. 
1674
-
1688
)
104
Santiviago
CA
Toro
CS
Hidalgo
AA
Youderian
P
Mora
GC
Global regulation of the Salmonella enterica serovar Typhimurium major porin, OmpD
J. Bacteriol.
 , 
2003
, vol. 
185
 (pg. 
5901
-
5905
)
105
Bossi
L
Figueroa-Bossi
N
A small RNA downregulates LamB maltoporin in Salmonella
Mol. Microbiol.
 , 
2007
, vol. 
65
 (pg. 
799
-
810
)
106
Jones
AM
Goodwill
A
Elliott
T
Limited role for the DsrA and RprA regulatory RNAs in rpoS regulation in Salmonella enterica
J. Bacteriol.
 , 
2006
, vol. 
188
 (pg. 
5077
-
5088
)
107
Coornaert
A
Lu
A
Mandin
P
Springer
M
Gottesman
S
Guillier
M
MicA sRNA links the PhoP regulon to cell envelope stress
Mol. Microbiol.
 , 
2010
, vol. 
76
 (pg. 
467
-
479
)
108
Griffin
JE
Gawronski
JD
Dejesus
MA
Ioerger
TR
Akerley
BJ
Sassetti
CM
High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism
PLoS Pathog.
 , 
2011
, vol. 
7
 pg. 
e1002251
 
109
Gallagher
LA
Shendure
J
Manoil
C
Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq
MBio
 , 
2011
, vol. 
2
 (pg. 
e00315
-
e00310
)
110
Gawronski
JD
Wong
SM
Giannoukos
G
Ward
DV
Akerley
BJ
Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung
Proc. Natl Acad. Sci. USA
 , 
2009
, vol. 
106
 (pg. 
16422
-
16427
)
111
Citron
M
Schuster
H
The c4 repressors of bacteriophages P1 and P7 are antisense RNAs
Cell
 , 
1990
, vol. 
62
 (pg. 
591
-
598
)
112
Ravin
NV
Svarchevsky
AN
Deho
G
The anti-immunity system of phage-plasmid N15: identification of the antirepressor gene and its control by a small processed RNA
Mol. Microbiol.
 , 
1999
, vol. 
34
 (pg. 
980
-
994
)
113
Hobbs
EC
Astarita
JL
Storz
G
Small RNAs and small proteins involved in resistance to cell envelope stress and acid shock in Escherichia coli : analysis of a bar-coded mutant collection
J. Bacteriol.
 , 
2010
, vol. 
192
 (pg. 
59
-
67
)

Author notes

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
Present addresses: Daniel J. Turner, Oxford Nanopore Technologies, Oxford, OX4 4GA, UK.
Minh-Duy Phan, Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD 4072, Australia.
A. Keith Turner, Discuva Ltd., 260 Cambridge Science Park, Milton Road, Cambridge, CB4 0WE, UK.
John Wain, Norwich Medical School, University of East Anglia, NRP Innovation Centre, Norwich Research Park, Colney Lane, Norwich, Norfolk, NR4 7GJ, UK.
Paul P. Gardner, School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Comments

0 Comments