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GENETICS AND MOLECULAR BIOLOGY

A HilA-Independent Pathway to Salmonella typhimurium Invasion Gene Transcription

Jennifer L. Rakeman, Heather R. Bonifield, Samuel I. Miller
Jennifer L. Rakeman
Departments of Microbiology and
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Heather R. Bonifield
Departments of Microbiology and
Medicine, University of Washington, Seattle, Washington 98195
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Samuel I. Miller
Departments of Microbiology and
Medicine, University of Washington, Seattle, Washington 98195
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DOI: 10.1128/JB.181.10.3096-3104.1999
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  • Fig. 1.
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    Fig. 1.

    Definition of sirB and sirC DNA. The minimum DNA required for the suppression ofsirA::Tn10d phenotypes as defined by restoration of PrgH::PhoA expression was determined forsirB (A) and sirC (B). +, suppression (PhoA activity restored); −, no suppression (PhoA activity not restored). Activity was determined by a blue colony phenotype on plates containing 5-bromo-4-chloro-3-indolylphosphate and confirmed by quantitative alkaline phosphatase activity measurements of bacteria grown in liquid culture. Abbreviations: A, SacII; I, EcoRI; P,PstI; H, HindIII; N, NruI; S,SalI; V, EcoRV. Plac indicates the relative position of the vector-encoded lac promoter (pWSK29 or pWKS30).

  • Fig. 2.
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    Fig. 2.

    SirC contributes to invasion cooperatively with SirA. Invasion is expressed as a percentage of the initial inoculum, and the numbers above the bars on the graph represent the fold decrease in invasiveness versus that of the wild-type strain, JLR129. The graph depicts results from one experiment performed in triplicate and is representative of several replicate experiments. Error bars represent the standard deviation; the absence of bars indicates that the standard deviation is insignificant. ∗, P = 0.0001. WT, wild type.

  • Fig. 3.
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    Fig. 3.

    sirC expression does not require HilA. The expression of sirC was measured by quantitating the amount of luciferase activity produced by strains containing thesirC::luc transcriptional fusion. RLU, relative light units. Error bars represent the standard deviation; the absence of bars indicates that the standard deviation is insignificant. OD600, optical density at 600 nm.

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    Fig. 4.

    Regulation of hilA expression.hilA expression was measured by quantitating the amount of β-galactosidase activity produced by strains expressing thehilA-iagB::lacZY fusion. (A) Enzyme activity over a growth curve of bacteria grown in L broth with shaking. (B) β-galactosidase activity produced by bacteria grown overnight under microaerophilic conditions in L broth. The cultures used in this assay were the same cultures used in the invasion assay represented in Fig. 2. Error bars represent the standard deviation; the absence of bars indicates that the standard deviation is insignificant. WT, wild type; OD600, optical density at 600 nm.

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    Fig. 5.

    Definition of HilA-independent pathways to invasion gene expression. Squares, expression of the indicated transcriptional fusions in the wild-type background; diamonds, expression in thehilA::kan background; circles, expression in thehilA::kan-plus-plasmid background. (A) Overexpression of sirC from pCJ20. (B) Overexpression ofsirA from pCJ13d. Error bars represent the standard deviation; the absence of bars indicates that the standard deviation is insignificant. OD600, optical density at 600 nm.

  • Fig. 6.
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    Fig. 6.

    Model of HilA-independent and HilA-dependent pathways to invasion gene expression. The HilA-independent pathway is depicted with boldface arrows, and the HilA-dependent pathway is depicted with the smaller arrows. It is possible that SirA interacts directly with theinvF promoter rather than exerting its regulatory effects through other regulators (such as SirC).

Tables

  • Figures
  • Table 1.

    Bacterial strains and plasmids

    Strain or plasmidGenotype or relevant phenotypeSource or reference
    S. typhimurium
     14028sWild typeATCCa
     CS01914028s phoN2 zxx::6251 Tn10d-Cm34
     CS401CS019 Strr; wild-type invasion/TTSSS. I. Miller lab
     CJ010CS019 withsirA::Tn10d-Tc20
     IB040CS019 with prgH::TnphoA, invasion defective3
     DAP3IB040 withsirA::Tn10d20
     CJ022DAP3 with pCJ2020
     JLR020DAP3 with pWKSHRV2.3This work
     JLR002DAP3 with pCJ20aThis work
     JLR010DAP3 with pCJ20bThis work
     JLR016DAP3 with pCJ20cThis work
     JLR018DAP3 with pCJ20dThis work
     CJ023DAP3 with pCJ2220
     HRB002DAP3 with pCJ22aThis work
     HRB003DAP3 with pCJ22bThis work
     HRB004DAP3 with pCJ22cThis work
     HRB005DAP3 with pCJ22dThis work
     HRB090ΔsirB 1.2-kb in-frame deletion in CS401This work
     CS015phoP102::Tn10d-Cm PhoP−35
     CS022pho-24 PhoPc, PhoP-repressed genes constitutively repressed35
     VV341hilA::kan2
     JLR158CS401 with hilA::kanfrom VV341This work
     JLR028sirC::luc in CS401, measures sirC expression in the presence of SirCThis work
     JLR077CS015 withsirC::luc from JLR028This work
     JLR076CS022 with sirC::lucfrom JLR028This work
     JLR027JLR028 withhilA::kan from VV341This work
     JLR040CJ010 with sirC::lucfrom JLR028This work
     HRB094HRB090 withsirC::luc from JLR028This work
     JLR053ΔsirC 748-bp in-frame deletion in CS401This work
     CL87iagB::lacZY measureshilA expression in the presence of HilA, wild-type invasivenessGift of C. Lee
     JLR129CS401 withiagB::lacZY from CL87This work
     JLR130JLR053 with iagB::lacZYfrom CL87This work
     JLR151JLR129 withsirA::Tn10d from CJ010This work
     JLR152JLR130 with sirA::Tn10dfrom CJ010This work
     EE638sspC::Tn5-lacZY1
     EE637invF::Tn5-lacZY1
     EE656prgH::Tn5-lacZY2
     JLR138CS401 withsspC::Tn5-lacZY from EE638This work
     JLR135CS401 withinvF::Tn5-lacZY from EE637This work
     JLR136CS401 withprgH::Tn5-lacZY from EE656This work
     JLR141JLR138 with hilA::kanfrom VV341This work
     JLR149JLR135 withhilA::kan from VV341This work
     JLR140JLR136 with hilA::kanfrom VV341This work
     JLR147JLR141 with pCJ20This work
     JLR150JLR149 with pCJ20This work
     JLR145JLR140 with pCJ20This work
     JLR153JLR141 with pCJ13dThis work
     JLR155JLR149 with pCJ13dThis work
     JLR156JLR140 with pCJ13dThis work
    Plasmids
     pWSK29Ampr, low-copy-number cloning vector9
     pWKS30Ampr, low-copy-number cloning vector9
     pWKSHRV2.3Ampr, 2.3-kbHindIII-EcoRV fragment (contains all oforgA)Gift of C. Lee and V. Bajaj
     pCJ20Ampr, contains 4.4 kb of sirCregion20
     pCJ20a-dAmpr (see Fig. 1)This work
     pCJ22Ampr, contains 4.6 kb of sirB region20
     pCJ22a-dAmpr (see Fig. 1)This work
     pCJ13dAmpr, contains sirA expressed from its own promoter20
     pGPL01Ampr, luciferase suicide vector17
     psirC::lucAmpr, 1-kb HindIII-NruI fragment from pCJ20 in pGPL01This work
     pKAS32Ampr Strs, pGP704-based suicide vector39
     pΔsirCAmpr Strs, used to create deletion of 748 bp of sirCThis work
     pΔsirBAmpr Strs, used to create deletion of 1.2 kb of sirB ORF1 and ORF2This work
    • ↵a ATCC, American Type Culture Collection.

  • Table 2.

    Regulation of sirC expression

    Background (strain)% of maximum luciferase activityaP
    Wild type (JLR028)100
    sirA::Tn10d(JLR040)8.30.018
    ΔsirB(HRB094)26.00.009
    pho-24(JLR076)1.60.001
    • ↵a Optical density at 600 nm = 1.8.

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A HilA-Independent Pathway to Salmonella typhimurium Invasion Gene Transcription
Jennifer L. Rakeman, Heather R. Bonifield, Samuel I. Miller
Journal of Bacteriology May 1999, 181 (10) 3096-3104; DOI: 10.1128/JB.181.10.3096-3104.1999

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A HilA-Independent Pathway to Salmonella typhimurium Invasion Gene Transcription
Jennifer L. Rakeman, Heather R. Bonifield, Samuel I. Miller
Journal of Bacteriology May 1999, 181 (10) 3096-3104; DOI: 10.1128/JB.181.10.3096-3104.1999
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KEYWORDS

Bacterial Proteins
Gene Expression Regulation, Bacterial
Salmonella Typhimurium
Trans-Activators
Transcription, Genetic

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