High-throughput comparison of gene fitness among related bacteria

  • Rocio Canals1,

    Affiliated with

    • Xiao-Qin Xia2,

      Affiliated with

      • Catrina Fronick3,

        Affiliated with

        • Sandra W Clifton3,

          Affiliated with

          • Brian MM Ahmer4,

            Affiliated with

            • Helene L Andrews-Polymenis5,

              Affiliated with

              • Steffen Porwollik6 and

                Affiliated with

                • Michael McClelland1, 6Email author

                  Affiliated with

                  BMC Genomics201213:212

                  DOI: 10.1186/1471-2164-13-212

                  Received: 16 November 2011

                  Accepted: 4 April 2012

                  Published: 30 May 2012

                  Abstract

                  Background

                  The contribution of a gene to the fitness of a bacterium can be assayed by whether and to what degree the bacterium tolerates transposon insertions in that gene. We use this fact to compare the fitness of syntenic homologous genes among related Salmonella strains and thereby reveal differences not apparent at the gene sequence level.

                  Results

                  A transposon Tn5 derivative was used to construct mutants in Salmonella Typhimurium ATCC14028 (STM1) and Salmonella Typhi Ty2 (STY1), which were then grown in rich media. The locations of 234,152 and 53,556 integration sites, respectively, were mapped by sequencing. These data were compared to similar data available for a different Ty2 isolate (STY2) and essential genes identified in E. coli K-12 (ECO). Of 277 genes considered essential in ECO, all had syntenic homologs in STM1, STY1, and STY2, and all but nine genes were either devoid of transposon insertions or had very few. For three of these nine genes, part of the annotated gene lacked transposon integrations (yejM, ftsN and murB). At least one of the other six genes, trpS, had a potentially functionally redundant gene encoded elsewhere in Salmonella but not in ECO. An additional 165 genes were almost entirely devoid of transposon integrations in all three Salmonella strains examined, including many genes associated with protein and DNA synthesis. Four of these genes (STM14_1498, STM14_2872, STM14_3360, and STM14_5442) are not found in E. coli. Notable differences in the extent of gene selection were also observed among the three different Salmonella isolates. Mutations in hns, for example, were selected against in STM1 but not in the two STY strains, which have a defect in rpoS rendering hns nonessential.

                  Conclusions

                  Comparisons among transposon integration profiles from different members of a species and among related species, all grown in similar conditions, identify differences in gene contributions to fitness among syntenic homologs. Further differences in fitness profiles among shared genes can be expected in other selective environments, with potential relevance for comparative systems biology.

                  Background

                  When a library of transposon (Tn) integrations is created in a bacterial genome, some insertions are not recovered in the resulting pool of mutants, either because the insertion is in an essential gene or because the gene is required in the media used to grow the bacterium. This fact has been exploited extensively to identify genes under selection when growth conditions are changed [13].

                  Another potential utility of such data, that is explored here, is to compare different strains, serovars, and species to reveal apparent orthologs that have very different levels of fitness in different strains. We perform the first experiments to quantitate this phenomenon in Salmonella.

                  We used high-throughput sequencing to determine the location of tens of thousands of integration sites of a Tn5 derivative in the genome of Salmonella enterica serovar Typhimurium strain ATCC 14028 (STM1) and in Salmonella enterica serovar Typhi Ty2 (STY1) after growth in rich media (Luria Broth). Our datasets were compared to each other and to a series of other published data on the fitness of mutations in Salmonella [46] and Escherichia coli (ECO) [710], including a previously obtained transposon profile in a separate Ty2 isolate, STY2, which differs from STY1 by having mutations in htrA aroC and aroD [3].

                  Differences in the selective pressure on apparent orthologs in the related genomes (STM1, STY1, STY2 and ECO) are of interest because they likely reflect differences in the systems that interact with these otherwise functionally similar genes or their products.

                  Results and discussion

                  Profiling of a library of transposon insertions in Salmonella Typhimurium and Typhi

                  Five independent transposon libraries were constructed in S. Typhimurium ATCC 14028 (STM1) and two in S. Typhi Ty2 (STY1), using the EZ-Tn5 < KAN-2 > Promoter Insertion Kit (Epicentre Biotechnologies) (see methods), and grown in Luria broth (Additional file 1: Table S1). The genomic DNA directly adjacent to each transposon was obtained using a procedure similar to that described in Santiviago et al. [4] and sequenced as described in Additional file 2 and in Additional file 3: Figure S1.

                  We obtained a total of 16,642,379 first-strand Illumina sequencing reads of 100 bases in length. Sequences were subsequently filtered to reveal those reads that contained a complete primer including each unique barcode, followed by two bases of transposon (Tn) beyond the primer. The remainder of the sequence was mapped to the genome to determine the transposon integration site.

                  Each Tn integration site was generally represented by multiple reads, and these reads usually varied in the length of the Salmonella sequence due to the random DNA shearing used in the sequencing protocol. To reduce bias due to preferential PCR of some fragments, duplicate identical shear events were removed. The remaining reads for each transposon integration site were used to determine the number of different “shear events” for that transposon. This filtering resulted in the mapping of 234,152 and 53,556 Tn integration locations, with 2,827,876 and 313,585 unique shear events in STM1 and STY1, respectively. The average density of integrations into the genome was one every 20 bases with an average of about 12 shear events per site in STM1, and one every 90 bases with an average of about 6 shear events per site in STY1.

                  A genome-wide survey of permitted transposon integrations

                  The transposon libraries used in these experiments were constructed and grown in rich media. Those regions of the genome with rare or absent transposon integrations include regions that are essential or under strong selection in rich media. A sizeable subset of these regions should also be essential under all growth conditions. An example of a profile of transposon integrations is shown in Figure 1, which displays a region in the STM1 genome that includes a known essential gene, priA (primosome assembly), and a gene with an essential region, ftsN (involved in cell division). Regions that were essential in Luria broth are identifiable in this plot as having no transposon integrations. Near-essential regions have a lower than average number of integration sites, usually accompanied by a lower number of shear events.
                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-212/MediaObjects/12864_2011_4334_Fig1_HTML.jpg
                  Figure 1

                  Visualization of transposon integrations into an S . Typhimurium 14028 genome region. The number of different sequencing reads originating from transposons (shear events) is plotted, averaged across 500 bases. Red, positive strand; blue, negative strand. The displayed region contains one gene necessary for growth in LB (priA) and one gene where only a segment shows selection (ftsN).

                  Our data also provide information on the orientation of each transposon, which can be informative. For example, in Figure 1, at the beginning of cytR, adjacent to ftsN, the negative strand contains far more transposons; this is the strand in which the heavily expressed antibiotic resistance marker of the transposon is oriented away from ftsN, likely making these integrations less disruptive. Many other examples of general selection and strand-specific selection are seen in Additional file 4: Figure S2, which shows a plot of the frequency of transposon insertions across the entire STM1 genome. A dramatic example of strand-specific selection is seen in the ribosomal RNA operons, for example at position 290,000 in the genome (Additional file 4: Figure S2). In this operon transposons are only permitted in the antisense strand, perhaps because truncated sense strand transcripts produced by the strong antibiotic resistance promoter in the transposon would disrupt ribosome assembly.

                  The transposon frequency analyses for all genes in STM1 (compared with known essentiality information for STY2 and ECO) and STY1 are presented in Additional file 5: Table S2 and Additional file 6: Table S3, respectively. Data for the htrA - aroC - and aroD - mutant STY2 were derived from two selections: a single passage on a solid medium (i) and six passages in Luria broth (ii).

                  Essential genes in E. coli

                  Between E. coli, S. Typhimurium and S. Typhi, over 60% of protein coding genes are syntenic and have over 95% amino acid sequence identity [11]. There are 339 of the approximately 4,000 genes in ECO that have been reported to be essential in at least one of two comprehensive studies, the PEC (Profiling of E. coli Chromosome) database and the Keio collection [710] (sources are summarized in Table 1). Of these potentially essential genes, 277 genes are considered essential in both databases, and all 277 genes have syntenic homologs in STM1. These genes are listed in Additional file 5: Table S2.
                  Table 1

                  Numbers of essential genes under laboratory conditions in relevant E. coli , S. Typhimurium and S. Typhi isolates

                  Species/serovar

                  Strain

                  Essential genes/non- essential

                  Method (type of mutagenesis, medium)

                  Reference

                  E. coli

                  K-12 MG1655

                  302/4477

                  Published literature and MD (medium-scale) and LD (large-scale) deletion mutants (targeted mutagenesis, antibiotic medium 3)

                  Profiling of E. coli chromosome (PEC) database ( http://​shigen.​lab.​nig.​ac.​jp/​ECOli/​pec/​) [9, 10]

                  E. coli

                  K-12 BW25113

                  303/3985

                  Single-gene deletion mutants (targeted mutagenesis, LB)

                  Keio collection [7]

                  E. coli

                  K-12 BW25113

                  299/3864

                  Single-gene deletion mutants (targeted mutagenesis, LB)

                  Update on the Keio collection [8]

                  E. coli

                  K-12 W3110

                  299/4109

                  Published literature

                  PEC database ( http://​shigen.​lab.​nig.​ac.​jp/​ECOli/​pec/​) [9, 10]

                  S. Typhimurium

                  ATCC 14028

                  NA/1,023

                  Single-gene deletion mutants (targeted mutagenesis, LB)

                  [4]

                  S. Typhimurium

                  ATCC 14028

                  257/NA

                  Insertion-duplication mutagenesis (IDM) sequencing (random mutagenesis, LB)

                  [5]

                  S. Typhimurium

                  LT2

                  144 (LB and/or M9/glc)/NA

                  Metabolic reconstruction (in silico approach, M9/glc and LB)

                  [6]

                  S. Typhi

                  Ty2 (STY2)

                  356/4162

                  Random transposon mutagenesis and two types of growtha

                  [3]

                  a Plating on an “aro mix” agar containing L-phe, L-trp, p-aminobenzoic acid and 2,3-dihydroxybenzoic acid (condition 1), six passages of growth in Luria broth (condition 2).

                  We ranked all Salmonella genes based on their density of transposons and the total number of shear events, and set a threshold of the 15th percentile for “highly selected” genes. Exactly 549 genes in STM1, 582 genes in STY1, and 437 genes in STY2 met these criteria. Only six of the 277 genes essential in ECO were not among these highly selected genes in STM1 (Table 2): three narrowly missed the threshold (folK, yejM and trpS) and three had average amounts of transposon insertions (murB ftsN and degS) indicating that mutants were not under selection in LB in this isolate. Two other genes did not meet the “selected” threshold in our STY1 assay (yrfF, gpsA) and one gene, folA, was not found to be selected in the published STY2 data [3].
                  Table 2

                  Essential genes in E. coli that are not as strongly selected in Typhimurium or Typhi*

                  S. Typhimurium 14028 gene symbol

                  Gene name

                  Gene description

                  Best hit in S. Typhimurium LT2

                  Best hit in S. Typhi Ty2a

                  Best hit in ECOa

                  STM1 transposons

                  STM1 reads

                  STY1 transposons

                  STY1 reads

                  STY2 transposonsb

                  STY2 readsb

                  STY2 transposonsc

                  STY2 readsc

                        

                  Percentile rank

                  STM14_0106

                  folA

                  Dihydrofolate reductase

                  STM0087

                  t0090

                  b0048

                  4

                  3

                  8

                  8

                  19

                  19

                  46

                  60

                  STM14_0217

                  folK

                  2-amino-4-hydroxy-6-hydroxymethyldihyropteridine pyrophosphokinase

                  STM0183

                  t0191

                  b0142

                  12

                  22

                  18

                  16

                  25

                  56

                  16

                  21

                  STM14_2754

                  yejM

                  Putative hydrolase

                  STM2228

                  t0626

                  b2188

                  14

                  16

                  30

                  25

                  13

                  19

                  17

                  20

                  STM14_4041 d

                  degS

                  Serine endoprotease

                  STM3349

                  t3265

                  b3235

                  50

                  34

                  14

                  14

                  6

                  7

                  4

                  4

                  STM14_4193

                  trpS

                  Tryptophanyl-tRNA synthetase

                  STM3481

                  t4024

                  b3384

                  18

                  16

                  14

                  14

                  78

                  64

                  12

                  12

                  STM14_4208

                  yrfF

                  Intracellular growth attenuator protein

                  STM3495

                  t4011

                  b3398

                  1

                  4

                  19

                  19

                  70

                  42

                  12

                  27

                  STM14_4460

                  gpsA

                  NAD(P)H-dependent glycerol-3-phosphate dehydrogenase

                  STM3700

                  t3819

                  b3608

                  10

                  10

                  14

                  17

                  45

                  46

                  8

                  11

                  STM14_4921

                  ftsN

                  E cell division protein

                  STM4093

                  t3525

                  b3933

                  42

                  38

                  14

                  15

                  20

                  27

                  15

                  15

                  STM14_4971.J

                  murB

                  UDP-N- acetylenolpyruvoylglucosamine reductase

                  STM4136

                  t3489

                  b3972

                  42

                  48

                  26

                  22

                  8

                  24

                  13

                  21

                  * Details for all genes can be found in Additional file 5: Table S2. Genes were ranked by the number of transposon insertions and number of independent reads per gene. Ranks above the 15th percentile are shown in bold.

                  a Best hits are listed only if gene is syntenic with S. Typhimurium strain 14028 and has at least 95% sequence identity.

                  b Growth on L-agar with “aro mix” (L-phe, L-trp, p-aminobenzoic acid and 2,3-dihydroxybenzoic acid) [3].

                  c Growth after six passages in LB broth [3].

                  d Considered essential in STY2 [3].

                  Visual inspection of Additional file 4: Figure S2 revealed that in three of the genes that are essential in ECO but seemingly not essential in STM1, part of the respective gene was, in fact, devoid of any transposon insertions: yejM, a putative hydrolase; ftsN, which encodes a cell division protein; and murB, a UDP-N-acetylenolpyruvoylglucosamine reductase. Figure 1 shows the ftsN gene as an example. These genes can apparently be disrupted in certain locations without losing their essential function. Thus, because of the high density of transposon integration data, we were able to reveal those cases where only part of the gene is essential.

                  The fourth of the six cases, trpS, encodes a tryptophanyl-tRNA synthetase. It may tolerate transposon insertions in STM1 and STY because of the presence of a distant paralog (trpS2), which does not exist in ECO. TrpS2 may substitute the TrpS function, although it is only 28% identical [12].

                  The fifth gene essential in ECO and not under strong selection in STM1 is degS, a serine endoprotease. This gene was under strong selection in STY2 [3]. That strain (but not our STY1) is an htrA mutant, a paralog of degS [13]. In our STY1 data, transposon insertion into degS was somewhat diminished, but not enough to qualify the gene for the “selected” category, suggesting at least some effect of the lack of a functional HtrA in STY2. However, in STM1 there is no evidence for any degree of transposon underrepresentation in degS, and it seems likely that another paralog, as yet undefined, can perform the proteolytic activity of DegS in this strain, if needed.

                  Finally, folK was somewhat underrepresented in transposon insertion frequency in STM1, but not to a degree that warranted inclusion in the “selected” category.

                  There are three other genes that are essential in ECO and STM1, but seemed not strongly selected in our STY1 survey and/or STY2. The folA gene, involved in folate biosynthesis, was strongly selected in STM1 and STY1 but did not show as much selection in STY2, especially after six passages in LB. Lastly, yrfF and gpsA are two genes essential in ECO that did not meet the “selected” threshold in our STY1 assay. Both these genes were under strong selection in STM1 and in STY2 after six passages in LB, indicating that mutations cannot be maintained for many passages in this medium. The yrfF (igaA) gene encodes an intracellular growth attenuator protein; and gpsA encodes a NAD(P)H-dependent glycerol-3-phosphate dehydrogenase. IgaA has been described as essential in S. Typhimurium unless there are additional mutations in the RcsCDB system, because it acts as a repressor of this system [14, 15]. Expression of igaA is positively regulated by Lon and negatively modulated by Hnr (MviA) through the transcriptional regulator RpoS [16]. S. Typhi Ty2 carries a defect in the rpoS gene [17, 18], which may explain the lesser degree of selection of igaA in STY.

                  Genes that are not essential in E. coli but are under strong selection in Typhimurium and Typhi

                  The list of essential genes in ECO is stringent, generally including the inability to obtain a viable deletion mutant in rich medium. In contrast, the measure available from transposon integrations in STM1 and STY reveals genes that were under strong selection, but not necessarily essential. A group of 159 such genes that are under selection in all Salmonella (i.e. STM1, STY1 and STY2) but not essential in ECO were identified (Additional file 5: Table S2). This list included many of the genes that might be expected to be under selection, such as genes encoding parts of the ribosome and its accessory proteins, as well as some genes encoding replication components. However, there were at least 14 genes that still have an unknown or a poorly understood function (ybaB, ybeD, ybeY, phoL, ycaR, ycdC, yciM, yciS, ygfZ, yhaL, yheM, yheN, wecF, and yigP). Given the conservation of these genes between Salmonella and E. coli, these are particularly interesting targets for future studies to determine their exact function.

                  The approximately 900 genes shared by STM1 and STY that have no synteny in ECO (Salmonella-specific genes) yielded only two strongly and consistently selected genes: STM14_5442 and STM14_2872, which both encode putative cytoplasmic proteins. However, Santiviago et al. reported successful knockout mutations in both of these genes in this same strain, so they are likely not essential [4]. However, the genes may have an effect on growth, because mutants in these genes grow poorly in competitive assays (unpublished data).

                  Genes under greater selection in Typhimurium than in Typhi

                  Genes that were under stronger selection in STM1 than in STY when the transposon libraries were grown in LB are depicted in Table 3A. The most dramatic difference was in hns, with an almost equal effect on the near adjacent gene hnr. Whereas hns is essential in Salmonella unless certain second site mutations are also present [19], no selection against insertion in this gene was found in STY in this study. The most likely explanation for this phenomenon is that S. Typhi Ty2 is known to contain a mutation in the rpoSgene [17, 18]. Mutations in this gene permit second site mutations in hns to be viable [19], and hnr (mviA) is a response regulator which post-transcriptionally modulates RpoS levels [20]. Interestingly, stpA, which encodes a 53% identical paralog of hns, also showed a greater tolerance for transposons in STY than in STM1. Unlike hns, viable hnr mutants can be obtained in S. Typhimurium [21], even though this gene appears to be strongly selected in STM1. Hnr participates in RpoS stability by acting as an adaptor for degradation by the ClpXP protease [22]. Mutants in hnr show reduced growth rate because of an increased RpoS stability, which increases transcription of genes involved in growth arrest and resistance to a variety of stresses [20]. The selection against hnr mutations seen in STM1 might be due to the non-advantageous phenotype of slow cell division in these mutants when they are in competitive growth.
                  Table 3

                  Genes displaying prominent differences in selection between Typhimurium and Typhi*

                  S. Typhimurium 14028 gene symbol

                  Gene name

                  Gene description

                  Best hit in S. Typhimurium strain LT2

                  Best hit in S. Typhi Ty2a

                  Best hit in ECOa

                  STM1 transposons

                  STM1 reads

                  STY1 transposons

                  STY1 reads

                  STY2 transposonsb

                  STY2 readsb

                  STY2 transposonsc

                  STY2 readsc

                        

                  Percentile rank

                  A. Examples of genes with stronger selection in STM1 than in STY1 and STY2

                  STM14_0199.RJ

                  yacC

                   

                  STM0167

                  t0172

                  b0122

                  12

                  13

                  57

                  70

                  53

                  51

                  59

                  51

                  STM14_1028.R

                  potF

                  Putrescine ABC transporter putrescine-binding protein

                  STM0877

                  t2019

                  b0854

                  14

                  15

                  47

                  63

                  35

                  24

                  38

                  32

                  STM14_1092

                   

                  Hypothetical protein

                   

                  t1967

                   

                  13

                  10

                  61

                  59

                  35

                  42

                  75

                  66

                  STM14_1127

                  ycbL

                  Putative metallo-beta- lactamase

                  STM0997

                  t1937

                  b0927

                  13

                  13

                  30

                  40

                  27

                  34

                  23

                  36

                  STM14_1141

                  xisW

                  Excisionase

                  STM1006

                  t1928

                   

                  11

                  9

                  72

                  62

                  53

                  30

                  43

                  24

                  STM14_1152

                  dnaC

                  Putative replication protein

                  STM1015

                  t1917

                   

                  16

                  16

                  27

                  36

                  34

                  31

                  41

                  32

                  STM14_1548

                  yeaM

                  Putative regulatory protein

                  STM1279

                  t1159

                  b1790

                  14

                  17

                  50

                  59

                  36

                  25

                  44

                  38

                  STM14_1599

                  celG

                  Hypothetical protein

                  STM1317

                  t1196

                  b1733

                  15

                  17

                  67

                  75

                  54

                  46

                  50

                  48

                  STM14_1682

                  orf70

                  Hypothetical protein

                  STM1388

                  t1256

                  b1675

                  12

                  11

                  32

                  71

                  34

                  27

                  37

                  29

                  STM14_1877 d

                   

                  Putative coiled-coil protein

                  STM1554

                  t1468

                   

                  15

                  16

                  39

                  37

                  79

                  81

                  78

                  81

                  STM14_1977 d

                   

                  Putative periplasmic binding protein

                  STM1633

                  t1536

                   

                  13

                  16

                  34

                  27

                  86

                  85

                  86

                  83

                  STM14_1978 d

                   

                  Putative ABC transporter permease component

                  STM1634

                  t1537

                   

                  6

                  7

                  17

                  44

                  87

                  91

                  85

                  90

                  STM14_2116

                  hns

                  Global DNA-binding transcriptional dual regulator H-NS

                  STM1751

                  t1662

                  b1237

                  5

                  3

                  32

                  29

                  91

                  89

                  35

                  91

                  STM14_2119

                  hnr

                  Response regulator of RpoS

                  STM1753

                  t1664

                  b1235

                  1

                  1

                  63

                  58

                  69

                  59

                  48

                  31

                  STM14_2120

                  ychK

                  Hypothetical protein

                  STM1754

                  t1665

                  b1234

                  11

                  11

                  64

                  81

                  64

                  61

                  62

                  46

                  STM14_2140

                  chaB

                  Cation transport regulator

                  STM1770

                  t1679

                  b1217

                  12

                  11

                  30

                  29

                  63

                  60

                  73

                  49

                  STM14_2176

                  ldcA

                  L,D-carboxypeptidase A

                  STM1800

                  t1077

                  b1192

                  14

                  15

                  43

                  37

                  28

                  28

                  39

                  39

                  STM14_2186 d

                  gsnB

                  Putative cytoplasmic protein

                  STM1809

                  t1068

                   

                  18

                  14

                  39

                  30

                  43

                  58

                  59

                  70

                  STM14_2278

                   

                  Hypothetical protein

                  STM1873

                  t1004

                  b1839

                  14

                  13

                  37

                  57

                  48

                  28

                  54

                  38

                  STM14_2685

                   

                  Putative 1,2-dioxygenase

                  STM2178

                  t0677

                   

                  15

                  18

                  32

                  33

                  31

                  29

                  24

                  32

                  STM14_2708.RJ

                   

                  Putative DNA-binding protein

                  STM2195

                  t0660

                   

                  17

                  15

                  51

                  44

                  65

                  36

                  62

                  49

                  STM14_2745

                   

                  Bicyclomycin/multidrug efflux system

                   

                  t0634

                   

                  16

                  14

                  65

                  47

                  66

                  43

                  73

                  68

                  STM14_3050

                  purC

                  Phosphoribosylaminoimidazole- succinocarboxamide synthase

                  STM2487

                  t0372

                  b2476

                  11

                  11

                  28

                  32

                  47

                  34

                  34

                  30

                  STM14_4596 d

                   

                  Pseudogene

                  STM3806

                  t0121

                   

                  14

                  12

                  78

                  49

                  53

                  55

                  36

                  51

                  STM14_4883

                  cpxP

                  Periplasmic repressor

                  STM4060

                  t3559

                  b3913

                  16

                  16

                  27

                  46

                  38

                  41

                  43

                  34

                  STM14_5142 d

                   

                  Putative cytoplasmic protein

                  STM4276

                  t4182

                   

                  15

                  13

                  46

                  28

                  16

                  49

                  19

                  47

                  STM14_0109

                  ksgA

                  Dimethyladenosine transferase

                  STM0090

                  t0093

                  b0051

                  37

                  28

                  5

                  5

                  19

                  13

                  5

                  5

                  STM14_2281

                  holE

                  DNA polymerase III subunit theta

                  STM1876

                  t1001

                  b1842

                  63

                  70

                  11

                  11

                  9

                  8

                  25

                  15

                  STM14_2498

                   

                  Hypothetical protein

                  STM2011.1n

                  t0860

                   

                  51

                  43

                  13

                  12

                  8

                  10

                  14

                  17

                  STM14_2665 e

                   

                  Hypothetical protein

                  STM2161

                  t0694

                  b2128

                  37

                  53

                  14

                  13

                  8

                  7

                  14

                  11

                  STM14_3017

                  eutA

                  Reactivating factor for ethanolamine ammonia lyase

                  STM2459

                  t0399

                  b2451

                  26

                  29

                  16

                  16

                  10

                  11

                  16

                  16

                  STM14_3267 e

                   

                  Hypothetical protein

                  STM2666

                  t2621

                  b2598

                  81

                  84

                  17

                  14

                  9

                  7

                  19

                  13

                  STM14_4041 e

                  degS

                  Serine endoprotease

                  STM3349

                  t3265

                  b3235

                  50

                  34

                  14

                  14

                  6

                  7

                  4

                  4

                  STM14_4465

                  yibP

                  Hypothetical protein

                  STM3705

                  t3814

                  b3613

                  63

                  65

                  19

                  18

                  11

                  11

                  10

                  11

                  STM14_4747

                  yifL

                  Putative outer membrane lipoprotein

                  STM3946

                  t3351

                  b4558

                  77

                  65

                  12

                  12

                  22

                  17

                  18

                  16

                  STM14_5345

                  treC

                  Trehalose-6-phosphate hydrolase

                  STM4453

                  t4488

                  b4239

                  50

                  49

                  13

                  13

                  10

                  8

                  5

                  9

                  * Details for all genes can be found in Additional file 5: Table S2. See Table 2 for notes a, b, and c. Ranks above the 20th percentile are shown in bold.

                  d Knockout mutant has been created in STM [4].

                  e Considered essential in STY2 [3].

                  At least 26 additional genes appeared to be under strong selection in STM1 but not in either STY. This class includes, among others, the putrescine ABC transporter potF; an excisionase; a cation transport regulator chaB; the L,D-carboxypeptidase A ldcA; the phosphoribosylaminoimidazole-succinocarboxamide synthase purC; and cpxP, a periplasmic repressor of the envelope stress response pathway. Viable mutants were obtained in this same Typhimurium strain for six of these genes [4] (listed in Table 3). It is not yet known if the remaining 20 genes can be deleted but it is likely that most, if not all, are not essential.

                  Genes under greater selection in Typhi than in Typhimurium

                  Genes that were under greater selection in STY1 and STY2 compared to STM1 are listed in Table 3B. Among the 10 genes under consistent selection in STY2 (both after passage in LB and after growth on aro-mix agar [3]) and our own STY1 data, but not in STM1, were four genes that encode hypothetical proteins and the previously mentioned degS. Other genes in this class include eutA, involved in the ethanolamine utilization pathway, the dimethyladenosine transferase ksgA and treC, a trehalose-6-phosphate hydrolase. The gene holE is an interesting example that is more strongly selected in STY than in STM1. This gene encodes the theta subunit of DNA polymerase III. The STM14_5586 protein encoded on the virulence plasmid in STM1, which is not present in STY, is a paralog that may partially substitute for holE [23].

                  Pseudogenes

                  Integrations in genes that are thought to be pseudogenes in STY and intact in STM1, or vice versa, were inspected. Of approximately 60 putative pseudogenes annotated in the S. Typhimurium 14028 genome, four showed strong selection in STM1 and are annotated as intact in S. Typhi Ty2: STM14_1358, STM14_1498.L, STM14_1778, and STM14_4596. Only one of them, STM14_1358, has an ortholog in ECO (yceQ) and, interestingly, has been reported as essential in this species. In STM14_1358 and STM14_1498.L, levels of selection in STM1 were similar to the levels in both STY. In the other two cases, there was no selection in at least one of the two STY isolates.

                  Of approximately 200 pseudogenes in S. Typhi Ty2, four were strongly selected in at least one of the STY: eda, a keto-hydroxyglutarate-aldolase/keto-deoxy-phosphogluconate aldolase; astA, an arginine succinyltransferase; t2152 (STM14_0843), a putative glycosyltransferase involved in cell wall biogenesis; and t3548 (STM14_4894), a putative cytoplasmic protein. The eda gene is the only case showing a strong selection in both STY1 and STY2.

                  In S. Typhi Ty2, an RNA-seq analysis of the transcriptome was recently published [24] and concluded that the vast majority of pseudogenes had low or undetected transcription. Only nine pseudogenes showed high levels of transcription, none of which correspond to our four strongly selected pseudogenes. A region annotated as a pseudogene and showing strong selection in any environmental condition suggests that a function is encoded in this region, whether it is a partial protein or a regulatory region.

                  Differences between transposon mutant libraries of two strains of Typhi

                  In our study, 53,556 transposon integration sites for STY1 were determined. In a previous work, 370,000 insertion sites were identified in STY2. Over 100 genes showed a difference in fitness between these two strains. Some of these differences may be attributable to the growth conditions used in the two studies. Our STY1 data are from a single LB growth passage whereas the STY2 data were from L-agar supplemented with aromatic compounds as well as from six passages in LB. Furthermore, STY2 is an attenuated strain, CVD908-htrA [3], which differs from STY1 in that it carries additional mutations in aroC aroD, and htrA [25]. Deletions in the aro genes, which encode enzymes involved in the shikimate biosynthesis pathway, render bacteria auxotrophic for the aromatic amino acids p-aminobenzoate (pABA) and 2,3-dihydroxybenzoate [26]. These aro mutations also result in the inability to produce ubiquinone and menaquinone, leading to respiration defects [27], and in defects in some components of the cell envelope [28], unless aromatic precursors are added to the medium. HtrA is a serine protease involved in the degradation of aberrant periplasmic proteins. An htrA mutant presents more susceptibility to oxidative stress than the wild type [29].

                  We found 17 transposons in htrA and one each in aroC and aroD in STY1. The number of transposons in htrA was close to the average random transposon insertion frequency (15.9) whereas aroC and aroD showed selection in both STY1 and STY2. The apparent selection of aroC and aroD in STY2 is explained by the fact that these genes were knocked out in this strain, resulting in a much smaller gene remnant as transposon target area. The htrA mutation may explain at least some differences between the two strains of STY, such as the selection of the periplasmic protein HlpA and the strong selection of DsbA after six passages in LB in STY2, but not in our STY1 study. In E. coli, the skp (hlpA in Salmonella) degP (htrA in STM) double mutant is lethal [30] and the dsbA degP double mutant shows reduced growth [31].

                  STY1 showed selection in some Salmonella Pathogenicity Island 2 (SPI-2) genes [32, 33]. Some of these were also selected in STM1 (ssaI ssaH ssaR ssaT sifB) but not in STY2. Some other SPI genes were interesting from a regulatory point of view. The hilC and rtsA SPI-1 regulators were selected in STY1, but not in STY2 [34]. RtsA is encoded in an operon that also includes the similarly selected rtsB, whose product represses the master regulator of the flagellar regulon, flhDC [35].

                  Flagellar genes

                  Flagellar genes show different patterns of selection comparing growth in LB broth under aeration (STM1, STY1 and STY2) versus on LB agar (STY2). Flagella and motility are highly regulated in S. Typhimurium and involve over 60 genes integrated in a hierarchy of controlled transcription [36]. The flagellar structure consists of three components: the basal body, the hook and the filament. The basal body consists of three rings and a rod which transverses the periplasmic space. These three components are expressed coordinately, first the hook-basal body (HBB) and later the filament, and assembled via a flagellar type III secretion apparatus [37]. The flgM gene encoding the anti-sigma 28 negative regulator of the synthesis of the flagellar filament, was selected in broth media, but not on LB agar. FlgM binds to the sigma 28 factor to prevent transcription of late flagellar genes before the completion of HBB structure [38]. In contrast, flgC flgJ fliI fliK, and fliO were selected only when bacteria were grown on an agar surface. FlgC is one of the structural rod components and FlgJ is the capping protein of the rod which also possesses muramidase activity [39]. FliI is an ATPase that forms part of the flagellar type III export apparatus, although it is not essential [40]. FliK regulates the length of the hook by switching the secretion specificity from rod-hook type substrates to filament-type substrates [41]. FliO is one of the integral membrane proteins of the flagellar secretion system which seems to have a role in stabilizing another protein of this system, FliP [42]. These five proteins are components of the basal body or the flagellar export apparatus, or interact with these structures. Furthermore, fliT, which encodes a protein that acts as a repressor of flagella biosynthesis [43], was selected after six passages in LB in STY2 [3]. The flagellar gene flhE showed selection on agar growth and after six passages in LB in STY2 [3]. Although it is known that the lack of FlhE does not affect flagella biogenesis or swimming motility, these mutants are defective in swarming motility [44]. In agreement with these results, Wang et al. reported that flagellar genes were regulated in a surface-specific manner [45]. Overall, our data indicates selection for genes encoding inhibitors of flagellar biogenesis when bacteria are grown in LB broth, such as flgM and fliT; and selection for genes necessary for flagellar biosynthesis when bacteria are grown on agar.

                  Comparison of fitness data with previous efforts to identify essential genes in Salmonella

                  We compared our data (Additional file 5: Table S2) to previous experiments that had sought to identify essential and non-essential genes in Typhimurium (Table 1). We previously reported 1,023 genes that give viable mutants in rich media in the same strain of Typhimurium as used here [4]. At least 38 of these genes are “selected” in our study (among the 15% with the lowest density of transposon integrations and shear events). The differences may be attributable to measuring essentiality, which is absolute, versus fitness selection, which is relative. In another study, 257 genes were identified as potentially essential genes for in vitro growth in LB using an insertion-duplication mutagenesis (IDM) strategy based on a temperature-sensitive integration plasmid [5]. Of these 257 genes, only 104 were under strong selection in STM1 in our data. It was previously suggested that some of these putative essential genes might be non-essential [46]. It appears that IDM may give a high rate of false positives for essential genes. In another study, a prediction using metabolic reconstruction identified 144 genes that could be essential for growth of S. Typhimurium LT2 in LB, of which 71 were already known to be essential in E. coli [6]. Of the remaining 74 potentially novel essential phenotypes, 19 were under strong selection in STM1. Additionally, 57 genes were predicted to be essential only in minimal media. Six of these genes were under strong selection in rich media in STM1. Given the considerable discrepancy in the observed and predicted genes under selection, fitness data have the great potential to improve systems biology models in the future.

                  An extended motif for transposon integration

                  Tn5 transposition can occur into almost any sequence. Indeed, we were unable to find any bias in integration targeting when we used all the transposon integration data we had available and a variety of motif-finding programs (data not shown), indicating that the vast bulk of integration sites were largely randomly distributed. However, hotspots, where Tn5 integration is preferred, have been reported [47]. Goryshin et al. sequenced 198 integration sites in a plasmid and identified a short degenerate consensus palindromic motif where integrations were more frequent: a-GntYWRanC-t [48]. The dash indicates the site of cleavage of the target that is then duplicated on either side of the transposon integration. To examine whether hotspots for integration in our data might further refine this motif we filtered over 300,000 integration sites for those sites that occurred in more than one of our independent transposon libraries. Then we filtered for integration sites represented by more than the average number of total shear events. This strategy yielded 654 sites that were among the most highly preferred targets for integration. By aligning the sequences surrounding these sites, we were able to refine the originally reported motif and further extend the motif by five bases on either side, including a highly conserved C and G located two and five bases upstream of the nick in the target DNA: cGcgCa-GttYWRaaC-tGcgCg (Figure 2). The opportunity for preferred interactions along the length of this 21-base target motif might stabilize a transposase-DNA pre-cleavage or cleavage complex for co-crystalization or other imaging studies.
                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-212/MediaObjects/12864_2011_4334_Fig2_HTML.jpg
                  Figure 2

                  An extended motif for transposon integration hot spots. Integration sites that occurred in the same location and were overrepresented in more than one transposon library were aligned and a consensus was obtained. Base frequencies over 50% are purple, between 30 and 50% light blue, and below 16%, orange.

                  Conclusions

                  We have identified differences in the ability to tolerate transposon integrations between Salmonella Typhimurium strain 14028 and two strains of S. Typhi Ty2. We also found potential differences in essentiality of homologous genes between Salmonella and E. coli (summarized in Tables  2 and 3).

                  Sometimes, these differences in selection can be explained by the presence of an identifiable paralog, present in one genome but not another, which can take over some or all of the functions of the mutated gene. Examples include trpS and holE. In other cases, a difference in a function encoded elsewhere in the genome differentially impacts the role of orthologs. The best example of this phenomenon in the present study is hns which is essential in STM1 but not in either STY dataset. It is known that rpoS mutants permit mutations in hns in Salmonella in some circumstances [19], and STY1 and STY2 are rpoS mutants [17, 18]. Similarly, hnr also showed a profound selection only in STM1. Although this gene is not essential in Salmonella, the presence of a functional copy of rpoS may also be the reason for this selection, because Hnr acts as a stability moderator for RpoS [22].

                  For some genes, such as yejM, ftsN and murB, we identified ORF segments that are essential whereas other regions of the gene can be freely interrupted by transposon insertions. Likely, these non-essential ORF regions exclude protein domains that are involved in critical functional modules of those genes.

                  Our experiments here involve comparisons among three Salmonella strains grown in rich media. However, the study of fitness profiles in hundreds of different strains in multiple growth conditions has the potential to reveal differences in life strategy not evident from the genome sequences alone and to contribute to understanding natural diversity. Advances in DNA sequencing and the ability to incorporate any number of different barcodes for comparison of multiple samples at the same time mean that comparative analysis of fitness among many different natural strains with different phenotypes has become practical. We speculate that as systems biology models become more refined, the fitness profiles of genomes may become useful for constraining these models.

                  Methods

                  Strains and growth conditions

                  The strains used in this study were Salmonella enterica serovar Typhimurium ATCC 14028 (STM1) and S. Typhi Ty2 strain JSG624 (STY1) provided by Ferric Fang (University of Washington, Seattle, WA). Bacterial cells were grown in LB medium containing 1% Bacto tryptone (Difco), 0.5% Bacto yeast extract (Becton, Dickinson and Company), and 1% NaCl, supplemented with kanamycin at 50 μg/ml, when necessary.

                  Construction of transposon integrations in S. Typhimurium 14028 and S. Typhi Ty2

                  Salmonella cells were made competent by standard methodology. Briefly, cells were grown in LB with shaking at 37°C to logarithmic phase, then washed three times with cold 10% glycerol and concentrated 250 fold in 10% glycerol. Transposome mixtures were prepared mixing 2 μl glycerol, 2 μl EZ-Tn5 < T7/KAN-2 > transposon, and 4 μl EZ-Tn5 transposase. After 3 h of incubation, 1 μl of this mixture was mixed with 50 μl of competent cells and 1 μl TypeOne restriction inhibitor. Transformation was performed at 2.5 kV using 0.2 cm electrode gap cuvettes and a Bio-Rad MicroPulser at EC2 setting. Transformed cells in each cuvette were resuspended in 1 ml of LB and incubated for 1 h at 37°C. After incubation, reaction aliquots were joined, complexity was determined by cell counts of various dilutions on LB agar containing kanamycin, and the remainder was grown overnight at 37°C in LB broth supplemented with kanamycin. Stocks of the different transposon mutant libraries containing 20% glycerol were prepared from the overnight cultures.

                  High-throughput sequencing of transposon insertion sites

                  The entire procedure is illustrated in Additional file 3: Figure S1. In brief, DNA was sheared, poly(A) tailed and PCR amplified using a pair of primers, one located in the transposon and one appended to the poly(A) tail, in a manner similar to that described in Santiviago et al. [4]. Subsequently, Illumina sequencing primers were added by PCR and sequencing performed on a Genome Analyzer GAII.

                  Mapping of transposons to the genome

                  The beginning of each read primer contained a code that defined the particular transposon library used. The codes are listed in Additional file 1: Table S1. The reads were sorted into seven libraries. The sequencing primers were positioned such that the first two bases beyond the primers used for PCR would be the last two bases of the transposon. Reads that included this AG sequence were retained. Most transposons were represented by many shear events. Transposons that were represented by only one or two shear events were distributed throughout the genome, including in known essential regions. Thus, these rare reads were artifactual samples of the whole genome. These sites were eliminated from further consideration.

                  Mapping of genes between genomes

                  Best hits between Typhimurium 14028, Typhimurium LT2, Typhi CT18, Typhi Ty2 and E. coli K-12 were identified by Blast searches of annotated genes against each other in the different genomes. Synteny was determined based on at least one of the two adjacent genes also being a best hit and being adjacent in other genomes. The assigned unique gene numbers differ for all the genome annotations, so all assigned gene numbers and gene symbols in these five genomes are reported in the Additional file 5: Table S2 for the convenience of the reader.

                  Data processing for genome comparisons

                  Because the distribution and number of transposons is not identical between STM1 and STY1, the data were processed to express the ratio of the number of transposon integrations in each gene and intergenic regions versus the number of integrations in that region that would be expected if all the integrations were randomly distributed. The ratio of observed divided by expected numbers were then expressed as a log2. The published STY2 data [3] were recalculated to allow direct comparison.

                  To identify those genes that had large differences in fitness among STM1, STY1 and STY2, all log2 ratios (observed number of insertion events/expected number of insertion events) among all 3,907 genes shared between these genomes were ranked from those with the lowest density of transposons to those with the highest density. Ranks in STM1 and both STYs were subsequently subtracted to identify those genes with the biggest difference in rank order of transposon density.

                  The percentile rank of the number of transposons per base and the number of reads per base (shear events) were calculated for each putative ortholog in each experiment in Salmonella. We arbitrarily defined “selected” genes as those having a sum of the two percentile ranks less than 30% (i.e., averaging less than the 15th percentile).

                  Declarations

                  Acknowledgements

                  This work was supported, in part, by a Beatriu de Pinos fellowship to RC. Other investigators were supported in part by NIH Contract No. HHSN272200900040C and grants AI039557 AI052237, AI073971, AI075093, AI077645 AI083646, USDA grants 2009-03579-30127 and 2011-67017-30127, the Binational Agricultural Research and Development Fund, and CDMRP BCRP W81XWH-08-1-0720.

                  Authors’ Affiliations

                  (1)
                  University of California
                  (2)
                  Institute of Hydrobiology, Chinese Academy of Sciences
                  (3)
                  Washington University
                  (4)
                  Ohio State University
                  (5)
                  Texas A&M University System Health Science Center
                  (6)
                  Vaccine Research Institute of San Diego

                  References

                  1. Badarinarayana V, Estep PW, Shendure J, Edwards J, Tavazoie S, Lam F, Church GM: Selection analyses of insertional mutants using subgenic-resolution arrays. Nat Biotechnol 2001,19(11):1060–1065.PubMedView Article
                  2. Sassetti CM, Boyd DH, Rubin EJ: Comprehensive identification of conditionally essential genes in mycobacteria. Proc Natl Acad Sci U S A 2001,98(22):12712–12717.PubMedView Article
                  3. 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 2009,19(12):2308–2316.PubMedView Article
                  4. 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 2009,5(7):e1000477.PubMedView Article
                  5. Knuth K, Niesalla H, Hueck CJ, Fuchs TM: Large-scale identification of essential Salmonella genes by trapping lethal insertions. Mol Microbiol 2004,51(6):1729–1744.PubMedView Article
                  6. Thiele I, Hyduke DR, Steeb B, Fankam G, Allen DK, Bazzani S, Charusanti P, Chen FC, Fleming RM, Hsiung CA, et al.: A community effort towards a knowledge-base and mathematical model of the human pathogen Salmonella Typhimurium LT2. BMC Syst Biol 2011, 5:8.PubMedView Article
                  7. 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, 2:2006–2008.View Article
                  8. Yamamoto N, Nakahigashi K, Nakamichi T, Yoshino M, Takai Y, Touda Y, Furubayashi A, Kinjyo S, Dose H, Hasegawa M, et al.: Update on the Keio collection of Escherichia coli single-gene deletion mutants. Mol Syst Biol 2009, 5:335.PubMedView Article
                  9. 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 2005,55(1):137–149.PubMedView Article
                  10. Kato J, Hashimoto M: Construction of consecutive deletions of the Escherichia coli chromosome. Mol Syst Biol 2007, 3:132.11.View Article
                  11. 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 2001,413(6858):852–856.PubMedView Article
                  12. Hamilton S, Bongaerts RJ, Mulholland F, Cochrane B, Porter J, Lucchini S, Lappin-Scott HM, Hinton JC: The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms. BMC Genomics 2009, 10:599.PubMedView Article
                  13. Baek KT, Vegge CS, Skorko-Glonek J, Brondsted L: Different contributions of HtrA protease and chaperone activities to Campylobacter jejuni stress tolerance and physiology. Appl Environ Microbiol 2011,77(1):57–66.PubMedView Article
                  14. Cano DA, Dominguez-Bernal G, Tierrez A, Garcia-Del Portillo F, Casadesus J: Regulation of capsule synthesis and cell motility in Salmonella enterica by the essential gene igaA. Genetics 2002,162(4):1513–1523.PubMed
                  15. Costa CS, Pettinari MJ, Mendez BS, Anton DN: Null mutations in the essential gene yrfF (mucM) are not lethal in rcsB , yojN or rcsC strains of Salmonella enterica serovar Typhimurium. FEMS Microbiol Lett 2003,222(1):25–32.PubMedView Article
                  16. Garcia-Calderon CB, Casadesus J, Ramos-Morales F: Regulation of igaA and the Rcs system by the MviA response regulator in Salmonella enterica . J Bacteriol 2009,191(8):2743–2752.PubMedView Article
                  17. Robbe-Saule V, Norel F: The rpoS mutant allele of Salmonella typhi Ty2 is identical to that of the live typhoid vaccine Ty21a. FEMS Microbiol Lett 1999,170(1):141–143.PubMedView Article
                  18. Deng W, Liou SR, Plunkett G, 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 2003,185(7):2330–2337.PubMedView Article
                  19. 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 2006,313(5784):236–238.PubMedView Article
                  20. Bearson SM, Benjamin WH, Swords WE, Foster JW: Acid shock induction of RpoS is mediated by the mouse virulence gene mviA of Salmonella typhimurium . J Bacteriol 1996,178(9):2572–2579.PubMed
                  21. Benjamin WH, Yother J, Hall P, Briles DE: The Salmonella typhimurium locus mviA regulates virulence in Itys but not Ityr mice: functional mviA results in avirulence; mutant (nonfunctional) mviA results in virulence. J Exp Med 1991,174(5):1073–1083.PubMedView Article
                  22. Zhou Y, Gottesman S, Hoskins JR, Maurizi MR, Wickner S: The RssB response regulator directly targets sigma(S) for degradation by ClpXP. Genes Dev 2001,15(5):627–637.PubMedView Article
                  23. Chikova AK, Schaaper RM: The bacteriophage P1 hot gene product can substitute for the Escherichia coli DNA polymerase III theta subunit. J Bacteriol 2005,187(16):5528–5536.PubMedView Article
                  24. Perkins TT, Kingsley RA, Fookes MC, Gardner PP, James KD, Yu L, Assefa SA, He M, Croucher NJ, Pickard DJ, Maskell DJ, Parkhill J, Choudhary J, Thomson NR, Dougan G: A strand-specific RNA-Seq analysis of the transcriptome of the typhoid bacillus Salmonella typhi . PLoS Genet 2009,5(7):e1000569.PubMedView Article
                  25. Tacket CO, Sztein MB, Losonsky GA, Wasserman SS, Nataro JP, Edelman R, Pickard D, Dougan G, Chatfield SN, Levine MM: Safety of live oral Salmonella typhi vaccine strains with deletions in htrA and aroC aroD and immune response in humans. Infect Immun 1997,65(2):452–456.PubMed
                  26. Hoiseth SK, Stocker BA: Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 1981,291(5812):238–239.PubMedView Article
                  27. Hone DM, Harris AM, Chatfield S, Dougan G, Levine MM: Construction of genetically defined double aro mutants of Salmonella typhi . Vaccine 1991,9(11):810–816.PubMedView Article
                  28. Sebkova A, Karasova D, Crhanova M, Budinska E, Rychlik I: aro mutations in Salmonella enterica cause defects in cell wall and outer membrane integrity. J Bacteriol 2008,190(9):3155–3160.PubMedView Article
                  29. Johnson K, Charles I, Dougan G, Pickard D, O’Gaora P, Costa G, Ali T, Miller I, Hormaeche C: The role of a stress-response protein in Salmonella typhimurium virulence. Mol Microbiol 1991,5(2):401–407.PubMedView Article
                  30. Rizzitello AE, Harper JR, Silhavy TJ: Genetic evidence for parallel pathways of chaperone activity in the periplasm of Escherichia coli . J Bacteriol 2001,183(23):6794–6800.PubMedView Article
                  31. Skorko-Glonek J, Sobiecka-Szkatula A, Narkiewicz J, Lipinska B: The proteolytic activity of the HtrA (DegP) protein from Escherichia coli at low temperatures. Microbiology 2008,154(Pt 12):3649–3658.PubMedView Article
                  32. Kuhle V, Hensel M: Cellular microbiology of intracellular Salmonella enterica : functions of the type III secretion system encoded by Salmonella pathogenicity island 2. Cell Mol Life Sci 2004,61(22):2812–2826.PubMedView Article
                  33. Fass E, Groisman EA: Control of Salmonella pathogenicity island-2 gene expression. Curr Opin Microbiol 2009,12(2):199–204.PubMedView Article
                  34. Ellermeier CD, Ellermeier JR, Slauch JM: HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hilA in Salmonella enterica serovar Typhimurium. Mol Microbiol 2005,57(3):691–705.PubMedView Article
                  35. Ellermeier CD, Slauch JM: RtsA and RtsB coordinately regulate expression of the invasion and flagellar genes in Salmonella enterica serovar Typhimurium. J Bacteriol 2003,185(17):5096–5108.PubMedView Article
                  36. Macnab RM: How bacteria assemble. Annu Rev Microbiol 2003, 57:77–100.PubMedView Article
                  37. Macnab RM: Type III flagellar protein export and flagellar assembly. Biochim Biophys Acta 2004,1694(1–3):207–217.PubMedView Article
                  38. Karlinsey JE, Tanaka S, Bettenworth V, Yamaguchi S, Boos W, Aizawa SI, Hughes KT: Completion of the hook-basal body of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription. Mol Microbiol 2000,37(5):1220–1231.PubMedView Article
                  39. Hirano T, Minamino T, Namba K, Macnab RM: Substrate specificity classes and the recognition signal for Salmonella type III flagellar export. J Bacteriol 2003,185(8):2485–2492.PubMedView Article
                  40. Paul K, Erhardt M, Hirano T, Blair DF, Hughes KT: Energy source of flagellar type III secretion. Nature 2008,451(7177):489–492.PubMedView Article
                  41. Minamino T, Ferris HU, Moriya N, Kihara M, Namba K: Two parts of the T3S4 domain of the hook-length control protein FliK are essential for the substrate specificity switching of the flagellar type III export apparatus. J Mol Biol 2006,362(5):1148–1158.PubMedView Article
                  42. Barker CS, Meshcheryakova IV, Kostyukova AS, Samatey FA: FliO regulation of FliP in the formation of the Salmonella enterica flagellum. PLoS Genet 2010,6(9):pii:e1001143.View Article
                  43. Aldridge C, Poonchareon K, Saini S, Ewen T, Soloyva A, Rao CV, Imada K, Minamino T, Aldridge PD: The interaction dynamics of a negative feedback loop regulates flagellar number in Salmonella enterica serovar Typhimurium. Mol Microbiol 2010,78(6):1416–1430.PubMedView Article
                  44. Stafford GP, Hughes C: Salmonella typhimurium flhE , a conserved flagellar regulon gene required for swarming. Microbiology 2007,153(Pt 2):541–547.PubMedView Article
                  45. Wang Q, Frye JG, McClelland M, Harshey RM: Gene expression patterns during swarming in Salmonella typhimurium : genes specific to surface growth and putative new motility and pathogenicity genes. Mol Microbiol 2004,52(1):169–187.PubMedView Article
                  46. Becker D, Selbach M, Rollenhagen C, Ballmaier M, Meyer TF, Mann M, Bumann D: Robust Salmonella metabolism limits possibilities for new antimicrobials. Nature 2006,440(7082):303–307.PubMedView Article
                  47. Lodge JK, Weston-Hafer K, Berg DE: Transposon Tn5 target specificity: preference for insertion at G/C pairs. Genetics 1988,120(3):645–650.PubMed
                  48. Goryshin IY, Miller JA, Kil YV, Lanzov VA, Reznikoff WS: Tn5/IS50 target recognition. Proc Natl Acad Sci U S A 1998,95(18):10716–10721.PubMedView Article

                  Copyright

                  © Canals et al.; licensee BioMed Central Ltd. 2012

                  This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                  Advertisement