InvF Is Required for Expression of Genes Encoding Proteins Secreted by the SPI1 Type III Secretion Apparatus inSalmonella typhimurium

Bacterial strains and plasmids.Bacterial strains and plasmids used in this study are described in Table1. Electroporation of plasmids into bacteria was carried out as previously described (36). Plasmids that were manipulated in Escherichia coli were passaged through a restriction-minus (hsd) Salmonella typhimurium LT2 strain (LB5000) (37) prior to electroporation into S. typhimurium SL1344 (19). P22 HT int lysates were harvested and used for transductions as previously described (30).

pDL7-2 (generously provided by Catherine Lee) is a pLAFR2-based clone (11) containing the inv-spa andsicAsipB genes (32). Two subclones were made by digesting pDL7-2 with EcoRI and cloning an ∼8-kb fragment containing invHFGEABC′ and a 6.7-kb fragment containing theinvIJ spaOPQRS sicA sequence into pWKS130 (40), resulting in pHD7 and pHD8, respectively. pHD7 was digested withPstI to subclone a 1.7-kb fragment containinginvF, creating pHD9-1. This fragment includes about 250 bp of sequence upstream of the invF start codon and is transcribed in the same direction as the lacZ promoter in pWKS130. The same PstI fragment was cloned into the medium-copy-number vector pHG329 (39), forming pHD10-1.

To construct pHD14, a 2.5-kb SalI-BamHI fragment containing spaQRS was cloned from pHD8 into pMAK705 (16). A unique BglII site was used to clone a ∼2-kb streptomycin-spectinomycin resistance cassette from pSmUC intospaS, resulting in pHD14.

To construct the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible sigDE clone pHH37, a 3.2-kbEcoRI-BamHI fragment from pHH20 (20) was cloned into pVLT33 (9). Induction of sigDEexpression was done by the addition of IPTG to a final concentration of 100 μM in overnight cultures.

pHD3 (invF-lacZYA) and pHD11 (sicA-lacZYA) were made by PCR amplification (Pfu polymerase; Stratagene) of putative promoter sequences and cloning into pRW50 (29). For pHD3, an invH primer with an EcoRI linker (invH-1 [5′-GGAATTCCGGCGCCATGTTTTTACACAACCGTCAGAAC-3′]) and aninvF primer with a BamHI linker (invF-1 [5′-CGGGATCCCGGCAGCTTTTGCGCGGAACACGTCTGTATAAACC-3′]) (Gibco BRL) were used to amplify ∼460 bp betweeninvF and invH from pDL7-2. The resulting fragment was cloned into the BamHI and EcoRI sites of pRW50. For pHD11, the primers spaS-EcoRI-3 (5′-GGAATTCCGCGGAGAAGGTTGGCGTACCTG-3′) andsicA-BamHI-1 (5′-CGGGATCCCGGCGTGGCGCCTTCACTAACGGCATCC-3′) were used to amplify the intergenic sequence (137 bp) between spaS andsicA along with 192 bp of the 3′ end of spaS and 76 bp of sicA. This amplified product was cloned into pRW50 as described above, and both strands were sequenced with the same primers used for amplification to confirm the sequence.

To construct the sigD chromosomal lacZYAreporter, plasmid pFUSE was used (5). pFUSE contains an R6K origin of replication and cannot replicate in SL1344. A 1-kbXbaI fragment from pHH15 (20) containing 0.4 kb of sequence upstream of sigD and 0.6 kb of sigDcoding sequence was cloned into the unique XbaI site of pFUSE. Ligations were transformed into E. coliS17-1λpir, and clones were screened by restriction digestion with PstI and EcoRI for inserts in the correct orientation relative to lacZYA. One clone was selected and named pHD5. pHD5 was transferred by conjugation into SVM252 (14028s sirA::Tn10dTc) (20, 22), where it integrated into sigD, leaving an intactsigDE⁺ copy in addition to asigD-lacZYA operon fusion. ThesirA::Tn10dTc strain was used as a recipient in order to provide a selectable marker (tetracycline resistance) for S. typhimurium. Correct integration of pHD5 was confirmed in several exconjugants by transduction linkage analysis using SVM167 (sigE::Tn10dTc) (86% linkage). The Φ(sigD-lacZYA) fusion was then transduced into the appropriate strains.

Growth conditions. S. typhimurium and E. coli strains were grown in Luria-Bertani (LB) broth (Difco) at 37°C with aeration on a roller drum or without aeration in standing cultures, as indicated. Antibiotics were used at the following final concentrations: chloramphenicol, 25 μg/ml; kanamycin, 100 μg/ml; streptomycin, 100 μg/ml; spectinomycin, 100 μg/ml; and tetracycline, 15 or 30 μg/ml for single-copy or multicopy tetracycline resistance, respectively. For the detection of β-galactosidase activity, solid medium (LB agar) was supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-Gal) at 40 μg/ml. IPTG was used at a final concentration of 100 μM.

Tissue culture invasion assays.HEp-2 cells were maintained and passaged as recommended by the American Type Culture Collection. For invasion assays, 2 × 10⁵ cells/ml were seeded into Falcon 24-well tissue culture plates (Becton Dickinson, Lincoln Park, N.J.) to obtain about 90% confluent monolayers on the following day. For bacterial cultures, single colonies were inoculated into 2 ml of LB broth and grown for 18 h without shaking at 37°C. Aliquots of 5 μl (10⁷ to 10⁸ CFU) were used per well of tissue culture cells. The invasion assay was performed as previously described (20).

Construction of spaS disruption mutants.To construct the spaS::ΩStr/Sp mutant SMV514, a 2.5-kb SalI-BamHI fragment containingspaQRS was cloned into pMAK705, creating pHD12. ABamHI streptomycin-spectinomycin resistance cassette from pSmUC was cloned into the unique BglII site within the 5′ region of spaS. This plasmid, pHD14, was used to exchange the disrupted spaS allele with the wild-type allele on the chromosome as previously described (16).

To confirm the disruptions on the chromosome, Southern analysis was performed (36). Labeling of a DNA probe and detection of disrupted sequences were done with enhanced chemiluminescence (ECL). Southern blotting detection reagents (Amersham Pharmacia Biotech). To confirm the spaS disruption, chromosomal DNA was digested with BamHI and probed with a spaS PCR-amplified product. As predicted, the spaS probe hybridized to a 7-kb fragment of SL1344 chromosomal DNA and a 9-kb fragment in thespaS-disrupted strains (data not shown). OnespaS::ΩStr/Sp mutant was chosen and called SVM514.

Construction of the invF in-frame deletion mutant.To make an in-frame deletion in invF, pHD10-1 was digested with ClaI and SacII, the 5′ and 3′ single-stranded overhangs were removed with mung bean nuclease (New England Biolabs), and the blunt ends were ligated with T4 DNA ligase (New England Biolabs). Mung bean nuclease can sometimes remove double-stranded DNA in addition to single-stranded DNA; therefore, several clones were sequenced to identify clones with an in-frame deletion in invF. One clone, pHD13, contained an in frame deletion of 465 bp. A 1.25-kb PstI fragment from pHD13 was subcloned into pMAK705, producing pHD15, which was then used to exchange the deletion onto the chromosome as previously described (16).

To screen for the invF deletion on the wild-type chromosome, bacteria with resolved plasmids were pooled in phosphate-buffered saline, subcultured into LB broth, and infected with P22 HTint containing pHH21 (sigD-lacZYA reporter plasmid, medium copy number) (20). If invF were required for the expression of sigD as predicted, the expression of Φ(sigD-lacZYA) would be reduced in an ΔinvF strain. Transductants were grown on MacConkey lactose agar supplemented with ampicillin. Four of several hundred transductants that formed less red or white colonies were purified and subsequently cured of the reporter plasmid by growing bacteria in LB broth for 5 days, subculturing 1:500 on each day. Individual colonies were screened for ampicillin sensitivity on LB agar containing ampicillin. One mutant was chosen and called SVM579. TheinvF mutation in this strain was complemented byinvF in a low-copy-number vector (pHD9-1), suggesting that the phenotype of this mutant (see Results) was due to the deletion ininvF and not another mutation elsewhere on the chromosome. The same technique was used to introduce the invF deletion onto the chromosome of BJ68 (sipC::Tn5lacZY) (34), creating SVM725. The ΔinvF resulted in reducedlacZY expression from thesipC::Tn5lacZY fusion when tested on LB agar plates supplemented with X-Gal.

To confirm the presence of the deletion on the chromosome and the absence of any gross chromosomal rearrangements, Southern analysis was performed (36). Chromosomal DNA was digested withPstI and probed with the 1.7-kb PstI fragment from pHD10-1. The probe hybridized to a 1.7-kb PstI fragment of chromosomal DNA from SL1344 and a 1.2- to 1.3-kb fragment of SVM579 and SVM725 as predicted (data not shown).

To construct the ΔinvF spaS::ΩStr/SpsipC::Tn5lacZY triple mutant, SVM754, the spaS::ΩStr/Sp mutation in SVM514 was transduced into SVM579 (ΔinvF). Nine purified transductants were screened by Southern hybridization to confirm that the invF deletion had not been lost upon transduction due to its spaS-linked location on the chromosome (data not shown). One mutant, SVM733 (ΔinvF spaS::ΩStr/Sp), was used to make a P22 HT int lysate. ThespaS::ΩStr/Sp mutation from SVM733 was transduced into SVM725 (ΔinvF sipC::Tn5lacZY). Tetracycline-, spectinomycin-, and streptomycin-resistant transductants were purified and checked for P22 sensitivity.

Analysis of culture supernatants.Cultures were grown in 5 to 10 ml of LB broth with antibiotics for 18 h without aeration, and equivalent units of optical density at 600 nm were harvested as previously described (20). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (26) on 7.5% gels for silver stain analysis (7) and 5% gels for immunoblot (Western) analysis (ECL Western blotting detection system; Amersham Pharmacia Biotech). It is notable that in these gels (30% acrylamide to 1.6% bisacrylamide), some proteins migrated more slowly than through gels poured with 0.8% acrylamide (data not shown). For immunoblots, proteins were transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore).

Antibodies.To make antibodies to SigD, the first 575 bp ofsigD were cloned into the expression vector pMAL-c1 (New England Biolabs). Expression and affinity purification of the maltose binding protein (MBP)-SigD′ fusion protein expressed from this plasmid were performed as described by the manufacturer. Protein eluted from the amylose column was separated on a 7.5% SDS-polyacrylamide gel, and the fusion protein was excised from the gel. Lyophilized gel slices containing MBP-SigD′ were used to raise rabbit antibodies to SigD (Covance Research Services, Denver, Pa.). The anti-SigD antibodies were preadsorbed with an acetone powder of E. coli DH5α expressing MBP (17) to reduce cross-reactivity of the antibody to proteins other than SigD. The preadsorbed antiserum was used at a 1:1,000 dilution. Anti-rabbit horseradish peroxidase-conjugated secondary antibodies were obtained from Sigma (St. Louis, Mo.).

Enzyme assays.β-Galactosidase assays were performed and values were calculated as previously described (31). Cultures were grown in 5 ml of LB with appropriate antibiotics for 18 h at 37°C in 13- by 100-mm screw-capped tubes. Samples of 1.5 ml were harvested, washed once in 0.88% NaCl, and resuspended in 1 ml of working buffer; 100 μl of each cell suspension was used per assay.

Sequence analysis.Sequencing was performed by using the BigDye Terminator Cycle Sequencing Ready Reaction system (PE Applied Biosystems, Foster City, Calif.). Reactions were analyzed by the Washington University Nucleic Acid Chemistry Laboratory (St. Louis, Mo.). Sequence analyses (homologies, mapping, etc.) were performed using the Wisconsin Sequence Analysis Package (Genetics Computer Group, Inc.).