Article Figures & Data
Figures
- 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.
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.
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.
- 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.
- 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.
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
- Table 1.
Bacterial strains and plasmids
Strain or plasmid Genotype or relevant phenotype Source or reference S. typhimurium 14028s Wild type ATCCa CS019 14028s phoN2 zxx::6251 Tn10d-Cm 34 CS401 CS019 Strr; wild-type invasion/TTSS S. I. Miller lab CJ010 CS019 withsirA::Tn10d-Tc 20 IB040 CS019 with prgH::TnphoA, invasion defective 3 DAP3 IB040 withsirA::Tn10d 20 CJ022 DAP3 with pCJ20 20 JLR020 DAP3 with pWKSHRV2.3 This work JLR002 DAP3 with pCJ20a This work JLR010 DAP3 with pCJ20b This work JLR016 DAP3 with pCJ20c This work JLR018 DAP3 with pCJ20d This work CJ023 DAP3 with pCJ22 20 HRB002 DAP3 with pCJ22a This work HRB003 DAP3 with pCJ22b This work HRB004 DAP3 with pCJ22c This work HRB005 DAP3 with pCJ22d This work HRB090 ΔsirB 1.2-kb in-frame deletion in CS401 This work CS015 phoP102::Tn10d-Cm PhoP− 35 CS022 pho-24 PhoPc, PhoP-repressed genes constitutively repressed 35 VV341 hilA::kan 2 JLR158 CS401 with hilA::kanfrom VV341 This work JLR028 sirC::luc in CS401, measures sirC expression in the presence of SirC This work JLR077 CS015 withsirC::luc from JLR028 This work JLR076 CS022 with sirC::lucfrom JLR028 This work JLR027 JLR028 withhilA::kan from VV341 This work JLR040 CJ010 with sirC::lucfrom JLR028 This work HRB094 HRB090 withsirC::luc from JLR028 This work JLR053 ΔsirC 748-bp in-frame deletion in CS401 This work CL87 iagB::lacZY measureshilA expression in the presence of HilA, wild-type invasiveness Gift of C. Lee JLR129 CS401 withiagB::lacZY from CL87 This work JLR130 JLR053 with iagB::lacZYfrom CL87 This work JLR151 JLR129 withsirA::Tn10d from CJ010 This work JLR152 JLR130 with sirA::Tn10dfrom CJ010 This work EE638 sspC::Tn5-lacZY 1 EE637 invF::Tn5-lacZY 1 EE656 prgH::Tn5-lacZY 2 JLR138 CS401 withsspC::Tn5-lacZY from EE638 This work JLR135 CS401 withinvF::Tn5-lacZY from EE637 This work JLR136 CS401 withprgH::Tn5-lacZY from EE656 This work JLR141 JLR138 with hilA::kanfrom VV341 This work JLR149 JLR135 withhilA::kan from VV341 This work JLR140 JLR136 with hilA::kanfrom VV341 This work JLR147 JLR141 with pCJ20 This work JLR150 JLR149 with pCJ20 This work JLR145 JLR140 with pCJ20 This work JLR153 JLR141 with pCJ13d This work JLR155 JLR149 with pCJ13d This work JLR156 JLR140 with pCJ13d This work Plasmids pWSK29 Ampr, low-copy-number cloning vector 9 pWKS30 Ampr, low-copy-number cloning vector 9 pWKSHRV2.3 Ampr, 2.3-kbHindIII-EcoRV fragment (contains all oforgA) Gift of C. Lee and V. Bajaj pCJ20 Ampr, contains 4.4 kb of sirCregion 20 pCJ20a-d Ampr (see Fig. 1) This work pCJ22 Ampr, contains 4.6 kb of sirB region 20 pCJ22a-d Ampr (see Fig. 1) This work pCJ13d Ampr, contains sirA expressed from its own promoter 20 pGPL01 Ampr, luciferase suicide vector 17 psirC::luc Ampr, 1-kb HindIII-NruI fragment from pCJ20 in pGPL01 This work pKAS32 Ampr Strs, pGP704-based suicide vector 39 pΔsirC Ampr Strs, used to create deletion of 748 bp of sirC This work pΔsirB Ampr Strs, used to create deletion of 1.2 kb of sirB ORF1 and ORF2 This work ↵a ATCC, American Type Culture Collection.
