Supplemental Data Materials and Methods
Immunoblot Detection of Fur-HA
14028s and the furmutant pfur-ha (NC1038) were grown under aerobic conditions in Luria-Bertani (LB) media overnight. Samples were centrifuged at 15,000 x g for 10 min and washed with phosphate buffer and centrifuged again. Concentrated samples were resuspended in phosphate buffer and the optical density at 600 nm (OD600)was determined. Samples were boiled in sample buffer(0.05 mM Tris-HCl pH 6.8, 1% β mercaptoethanol, 2% SDS, 10% glycerol, 0.02% bromophenol blue) for 10 min and subjected to electrophoresis in a 12% acrylamide SDS gel. Following electrophoresis, proteins were transferred to a nitrocellulose membrane in transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol) and blocked overnight in blocking buffer (TBS-T, 5% non-fat dry milk (w/v), 0.05% Tween 20, 25 mM Tris, 150 mM NaCl, pH 7.4). The membrane was briefly washed and primary antibody was added with fresh TBS-T for 3 h. Then, the membrane was washed and secondary antibody was added with fresh TBS-T for 3 h. The membrane was washed and immunoreactivity was visualized by incubating with H2O2 and 3,3 diaminobenzidine. Primary anti-HA (from rabbit) and secondary HRP conjugated antibodies (from goat) were purchased from Abcam Inc (Cambridge, Massachusetts) and used at 1:1,000 and 1:2,000 dilutions, respectively.
Supplemental Data References
1.Altier, C., M. Suyemoto, and S. D. Lawhon. 2000. Regulation of Salmonella enterica serovar typhimurium invasion genes by csrA. Infect Immun 68:6790-7.
2.Bajaj, V., R. L. Lucas, C. Hwang, and C. A. Lee. 1996. Co-ordinate regulation of Salmonella typhimurium invasion genes by environmental and regulatory factors is mediated by control of hilA expression. Mol Microbiol 22:703-14.
3.Bourret, T. J., M. Song, and A. Vazquez-Torres. 2009. Codependent and independent effects of nitric oxide-mediated suppression of PhoPQ and Salmonella pathogenicity island 2 on intracellular Salmonella enterica serovar typhimurium survival. Infect Immun 77:5107-15.
4.Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47-56.
Supplemental Data Figure Legends
Figure S1 – The pfur-ha Plasmid Restores Wild-type Appearance of Siderophore Production in the fur Mutant. (A) Confirmation of fur-tagged hemaglutinin epitope by immunoblotting. Cultures (14028s and NC1038) were grown aerobically in Luria-Bertani media overnight and the optical density at 600 nm (OD600) was determined. Samples were subjected to SDS-PAGE followed by transfer to nitrocellulose membrane followed by immunodetection. Three ODs (~ 1 x 109 cells) were loaded for 14028s and dilutions of the furmutant pfur-ha(NC1038) were used from 3 ODs down to 0.1 OD (~ 4 x 107 cells). An arrow depicts the cross reactivity of antibodies to hemaglutinin. The predicted size of Fur-HA is ~18.2 kDa. (B) Cultures grown in Luria-Bertani media were spread on Tris buffered chrome azurol agar plates (CAS plates,(4) containing 0.3% xylose and incubated at 37°C overnight. The increased production of siderophores was apparent by the yellow halo surrounding growth of the fur mutant (KLM001), which was lacking for 14028s and the furmutant with pfur-ha (NC1038).
Figure S2 – Expression of the PhoP-activated pagP-lacZ is Not Affected by Iron Chelation. Reporter activity of pagP-lacZ(AV0609) was determined throughout anaerobic growth of the culture in LB-MOPS-X media in the presence and absence of 200 μM 2,2’-dipyridyl. Assays were performed as described in the Materials and Methods with data presented in the form of a differential plot (U.ml-1 vs OD600). The slope of this plot determined the specific activity of the reporter. Data shown are from three separate experiments (average ± standard deviation).
Figure S3 – Fur regulation of hilA is lost in the absence of hns. β-Galactosidase activity of hilA-lacZ was determined under anaerobic conditions (A-F). Assays were performed as described in the Materials and Methods with data presented in the form of a differential plot (U.ml-1 vs OD600). The slope of this plot determined the specific activity of the reporter (shown in Fig. 6). Data shown are representative of three separate experiments.
Figure S4 – Deletion of furand Iron Deprivation Reduces Expression of HilA Activated Genes Within SPI-1 Under Anaerobic Conditions. Reporter activity of invF-lacZ(CA701 and NC1112) and sipC-lacZ(RM5385 and NC1116) was determined throughout anaerobic growth of the cultures in LB-MOPS-X media with and without 200 μM 2,2’-dipyridyl. Assays were performed as described in the Materials and Methods with data presented in the form of a differential plot (U.ml-1 vs OD600). The slope of this plot determined the specific activity of the reporter. Data shown were representative of three separate experiments.
Figure S5 – Hfq is Not Required for Anaerobic hilA-lacZ Activation by Fur. Reporter activity of hilA-lacZ was determined throughout anaerobic growth of the 14028s and NC1124 cultures grown in LB-MOPS-X media with and without 200 μM 2,2’-dipyridyl. Assays were performed as described in the Materials and Methods with data presented in the form of a differential plot (U.ml-1 vs OD600). The slope of this plot determined the specific activity of the reporter. Data shown are from three separate experiments (average ± standard deviation).
Table S1 – Bacterial Strains
Strain / Genotype / SourceAV0609 / pagP-lacZ / (3)
CA701 / invF-lacZY / (1)
NC1112 / fur::bla invF-lacZY / This work
RM5385 / sipC-lacZY / (2)
NC1116 / fur::bla sipC-lacZY / This work
NC1016 / hfq::FRT / This work
NC1124 / hfq::FRT hilA-lacZ / This work
NC1038 / fur::bla pfur-ha / This work
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