We constructed a Typhimurium strain that lacks seven regulators, six of which are known to affect the expression of the type III secretion systems of this bacterium. The TTSS1-encoding region of SPI1, which harbors five regulatory genes (hilC, hilD, hilA, invF and sprB) and a co-regulator (sicA), was deleted in a strain that harbors a mutation of the sirA gene (see Materials and Methods). Transduction of an ssrB mutation into this strain resulted in YD038 which lacks seven regulatory genes (hereafter known as the multi-mutant). Chromosomal lacZY transcriptional fusions to ten effector genes were then constructed in YD038. These included fusions to genes that encode effectors secreted by TTSS1 (sopA, sopB and sopE2), TTSS2 (sspH2, sifA, sifB, sseI, and sseG) or both (slrP and sspH1). These ten fusions were also transduced into the wild type strain, 14028, using phage P22HTint.

Low copy number plasmids individually encoding seven regulators of SPI1 or SPI2 (or the vector control) were transformed into each of the ten strains that carry a lacZY fusion in the multi-mutant background. Each plasmid was constructed in such a way as to have the regulatory gene expressed from the vector's lac promoter. Expression and regulatory activity of the resulting constructs was confirmed using complementation tests with known target genes (Figure 2). The only exception was sprB for which the target genes are not known. The resulting 80 strains (and the ten additional strains harboring the same fusions in the wild-type background) were then grown in triplicate to log phase under either SPI1-inducing conditions (LB broth standing at 37°C overnight 42) or SPI2-inducing conditions (MgM broth shaking at 37°C until OD₅₉₅ of 0.2 to 0.8). The β-galactosidase activity of the strains was then determined. The results of SPI1-inducing conditions are shown in Table 1 and the results of SPI2-inducing conditions are shown in Table 2. For all of the discussion below, any effect less than 3-fold is considered to be no effect.

The strains and plasmids used in this study are described in Additional file 1. The bacterial strains were grown in LB (Luria-Bertani) medium at 37°C. For induction in the Magnesium minimal medium (MgM) 46 cells from LB overnight cultures were washed with MgM prior to 1:50 dilution. The following antibiotics were obtained from Sigma and used at the following concentrations when required: kanamycin (kan, 50 μg/ml), ampicillin (amp, 100 μg/ml), chloramphenicol (cam, 20 μg/ml), tetracycline (tet, 20 μg/ml), and hygromycin (hyg, 200 μg/ml). Sucrose was supplemented at 219 mM final concentration to select against sacB. The β-galactosidase chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) was obtained from Molecular Probes (Eugene, OR) and used at a final concentration of 40 μg/ml.

General molecular biology techniques were performed essentially as described 47. Restriction and modification enzymes were purchased from Invitrogen (Carlsbad, CA) or New England Biolabs (Beverly, MA), and used as recommended by the manufacturers. PCR primers were purchased from IDT Inc. (Coralville, IA). Plasmids were extracted using kits from Qiagen (Valencia, CA). P22 transduction was performed as described 48.

The β-galactosidase activities were measured using o-nitrophenyl β-D-galactopyranoside (ONPG, Sigma, St. Louis MO) using the method described by Miller 49 or the Beta-Glo™ assay system (Promega, WI) according to the manufacturer's recommendations. Briefly the assay consisted of mixing an equal volume of a log phase growing culture (OD₅₉₀ 0.2–0.8) and a detection reagent. The latter contains (i) a luciferin-galactoside substrate (6-O-β-galactopyranosyl-luciferin) that is cleaved by the β-galactosidase to form luciferin and galactose, and (ii) a firefly luciferase that catalyzes the luciferin to generate light. After at least 1 hour of incubation at room temperature the light produced is measured in a Turner Designs TD-20/20 luminometer or a Wallac 1420 VICTOR³ plate reader and the results expressed as light per OD₅₉₅ per μl of culture. The results were expressed as the mean ± SD of at least three different experiments.

The invF, sicA, sprB, ssrA, and ssrB genes were replaced with the cat cassette from pKD3 using the λ-red recombination system as described 50.

The TTSS genes of SPI1 (~35 kb), but not the manganese transport genes 51, were deleted using the pRE112 suicide vector 52. This deleted avrA through invH. To do this, PCR was performed using primers BA1093 and BA1094 (see Additional file 2) with 14028 genomic DNA as template. The 1366 bp product contains the 3' end of the invH gene, stm2901, and the 5' end of stm2902. A second PCR was performed using primers BA1095 and BA1096 (see Additional file 2). The 1255 bp product contains the 3' end of sitC, the sitD gene, and the 5' part of the avrA gene. Both products were cloned into pCR2.1 Topo (Invitrogen). The sitD-containing fragment was removed with SacI and cloned into the SacI site of the stm2901-containing clone. This placed the two fragments adjacent to each other and created the Δ(avrA-invH)1 deletion. This 2894 bp fragment was removed with KpnI and PvuII and cloned into the suicide vector pRE112 that had been digested with KpnI and SmaI to yield the plasmid pYD25. This plasmid contains (i) a cat gene that confers a resistance to chloramphenicol, (ii) the R6K replication origin that requires the pir gene, and (iii) the sacB gene of Bacillus subtilis that is toxic in Gram-negative bacteria grown in the presence of high concentrations of sucrose 53. Allelic exchange was performed by mobilizing pYD25 from BW20767 into JVR140 (sirA4::hyg). The chloramphenicol resistant transconjugants harbor the pYD25 plasmid integrated into the chromosome, resulting in the duplication of the region upstream and downstream of TTSS1. Isolated colonies were grown in LB without chloramphenicol selection until the exponential phase and plated onto LB lacking NaCl and containing 219 mM sucrose to select for loss of the integrated plasmid by homologous recombination. The colonies obtained were screened for Δ(avrA-invH)1 using PCR with primers hybridizing upstream and downstream of the TTSS1 region. Finally, the resulting Δ(avrA-invH)1 sirA4::hyg strain was used as a recipient for the P22HTint transduction of an ssrB::cat mutation from MJW129 29 to make the multi-mutant YD038 strain. YD038 lacks the co-regulator sicA, and seven regulators, sirA, ssrB, hilA, hilC, hilD, invF, and sprB.

To make lacZY transcriptional fusions to effector genes, the 3' portion of each gene (including the stop codon but without the transcription terminator) was amplified using PCR and cloned into pCR2.1 TOPO (Invitrogen, Carlsbad, CA). These gene portions were then removed as EcoRI fragments and cloned into the EcoRI site upstream of the promoterless lacZY gene of the pVIK112 pir-dependent suicide vector 54. The resulting plasmids were mobilized from BW20767 into YD038. The correct integration of the plasmids into the chromosome of the resulting kanamycin-resistant transconjugants was verified by PCR. Fusions were transduced using phage P22HTint into other backgrounds as needed. A second fusion to sifA was constructed as described previously 55. First the lambda red recombination system was used to integrate a kanamycin resistance gene flanked by FRT (Flp recombinase target) sites from the pKD4 plasmid 50. The FRT-kan-FRT cassette was placed after the stop codon, but before the transcription terminator, of sifA. Second, the temperature sensitive pCP20 plasmid 56 that encodes Flp recombinase was introduced into the strain. Flp-mediated excision of the kan cassette left a single FRT site that serves as an integration point for the fusion. The third step was to integrate the pCE36 suicide plasmid that has a FRT site directly upstream of the promoterless lacZY 55. After the integration of pCE36, the pCP20 plasmid was cured by growth at 37°C.

Each of the regulatory genes missing in the multi-mutant was amplified using PCR (the primers used are described in Additional file 2), cloned into the pCR2.1 TOPO vector, released by enzymatic digestion and cloned under the control of the lac promoter of pWSK29. The hilD, sirA, and hilA genes were released from the TOPO vector as 1353 bp SacI-XbaI, 936 bp KpnI-XhoI, and 1920 bp EcoRI fragments respectively and cloned into the corresponding sites of pWSK29 to yield the plasmids pYD17, pYD28 and pYD29 respectively. The 971 bp SpeI-XhoI fragment containing the sprB gene was cloned into the XbaI-XhoI sites of pWSK29 to give the pYD15 plasmid. The hilC gene was released as a 1541 bp PvuII-XbaI fragment and ligated to the EcoRV-XbaI sites of pWSK29 to produce the plasmid pYD16. A 852 bp EcoRI fragment and a 622 bp KpnI-XhoI fragment containing the invF and the sicA genes were cloned into the corresponding sites of pWSK29 to yield the plasmids pYD23 and pYD38 respectively. The 5353 bp ApaLI-XhoI fragment from pYD23 was ligated to the 1527 ApaLI-XhoI fragment from pYD38 to give the pYD40 plasmid that harbours the invF and sicA genes as a bi-cistronic operon under the control of the lac promoter. Each construction was verified by the complementation of a corresponding regulatory mutant (Figure 2), except for sprB for which a complementable function has not yet been discovered 18. We sequenced both strands of the P_lac-sprB region of the sprB-encoding plasmid to ensure that the sequence was correct.

The promoter region and the 5'part of the sifA gene was PCR-amplified from 14028 genomic DNA using the primers YD1 and YD2 (see Additional file 2). The product was cloned into the pCR2.1 TOPO vector (Invitrogen) and then released as an EcoRI fragment that was subsequently cloned upstream of the luxCDABE operon of the pSB384 vector 57. The correct orientation of the PsifA promoter in the resulting pYD56 plasmid was verified by PCR.

Liquid cultures were grown in LB or MgM media for six hours. The cultures were brought to similar OD₆₀₀ and 200 μl of cell suspensions were placed in tubes and imaged using the Xenogen IVIS imaging system (Alameda, CA). The images are displayed as pseudocolors with blue representing the lowest and red the highest light intensity. For luminometry the light emitted by 100 μl of culture was measured using a Turner Designs TD-20/20 luminometer.