Article Figures & Data
Figures
- Fig. 1.
Growth curves demonstrating Fe starvation of 4169. (A) Growth curve of 4169 cultured in Chelex-treated CDM (○) and in Chelex-treated CDM–36 μM FeSO4 (•). (B) Growth curve of 4169 cultured in BHI (■) and in BHI–100 μM EDDA (□). Each point represents the mean culture density of three independent cultures. Error bars represent 1 standard deviation of the mean.
- Fig. 2.
Induction of FeRPs by Fe starvation of B. avium. Lanes: 1, molecular mass standards; 2, 4169 in Chelex-treated CDM–36 μM FeSO4; 3, 4169 in Chelex-treated CDM; 4, 4169 in BHI; 5, 4169 in BHI–100 μM EDDA; 6, 838 in BHI; 7, 838 in BHI–100 μM EDDA; 8, 75 in BHI; 9, 75 in BHI–100 μM EDDA. The location of the 84-, 91.5-, 92-, and 95-kDa FeRPs is marked in brackets. The 51-kDa FeRP, which is evident only in lanes 3 and 5, is marked with asterisks. Lanes 2 to 9 each contain 1 μg of total protein. Molecular masses are in kilodaltons. The gel was stained with silver.
- Fig. 3.
SDS-PAGE analysis of the OMPs of B. avium4169 and the transposon mutants Pho-6 and Pho-20. Lanes: 1, molecular mass standards; 2, 4169 in BHI; 3, 4169 in BHI-EDDA; 4, Pho-6 in BHI; 5, Pho-6 in BHI-EDDA; 6, Pho-20 in BHI; 7, Pho-20 in BHI-EDDA. Lanes 2 to 7 each contain 1 μg of total protein. Molecular masses are in kilodaltons. The positions of the 95- and 84-kDa FeRPs that are missing in the outer membrane of Pho-20 are denoted by arrows. The gel was stained with silver.
- Fig. 4.
Growth of 4169 and the transposon mutants Pho-6 and Pho-20. ○, 4169 in Chelex-treated CDM; •, 4169 in Chelex-treated CDM–36 μM FeSO4; □, Pho-6 in Chelex-treated CDM; ■, Pho-6 in Chelex-treated CDM–36 μM FeSO4; ◊, Pho-20 in Chelex-treated CDM; ⧫, Pho-20 in Chelex-treated CDM–36 μM FeSO4. Each point represents the mean culture density obtained from three independent cultures. The error bars represent 1 standard deviation of the mean.
- Fig. 5.
Turkey serum as a source of Fe for 4169 and the transposon mutants Pho-6 and Pho-20. Growth conditions: 1, Chelex-treated CDM; 2, Chelex-treated CDM–36 μM FeSO4; 3, Chelex-treated CDM–1.7% turkey serum; 4, Chelex-treated CDM–1.7% turkey serum–50 μM EDDA; 5, Chelex-treated CDM–1.7% turkey serum–50 μM EDDA–50 μM FeSO4; 6, Chelex-treated CDM–1.7% turkey serum–50 μM EDDA–86 μM FeSO4. Each bar represents the mean stationary-phase culture density obtained from three independent cultures. The error bars represent 1 standard deviation of the mean. An asterisk at the top of a bar indicates that the mean OD600 of the mutant strain is significantly different from the mean OD600 of 4169 for the particular growth condition (P ≤ 0.05). P values are noted for each asterisk.
Tables
- Table 1.
Formula for the CDM for growth ofB. aviuma
Step Ingredient or procedure Amt Base solution KH2PO4 0.5 g Sodium glutamate 10.72 g NaCl 2.5 g KCl 0.2 g MgCl2 · 6H2O 0.1 g Tris 1.525 g α-Ketoglutamic acid 2.0 g Sodium pyruvate 2.0 g CaCl2 20.0 mg Deionized H2O To 909.0 ml Adjust to pH 7.8 with NaOH, autoclave, and store at 4°C CDM supplement Ascorbic acid 0.2 g Glutathione, reduced 1.0 g Deionized H2O To 100.0 ml Store at −20°C l-Cystine supplement Deionized H2O 3.0 ml HCl (11.6 M) 1.0 ml l-Cystine 0.4 g Deionized H2O To 100.0 ml Store at −20°C l-Proline supplement l-Proline 3.0 g Deionized H2O To 200.0 ml Autoclave and store at −20°C l-Phenylalanine supplement l-Phenylalanine 4.0 g Deionized H2O To 400.0 ml Autoclave and store at 4°C Vitamins p-Aminobenzoic acid 10 mg Biotin 1 mg Folic acid 10 mg Pyridoxyl HCl 80 mg Riboflavin 40 mg Thiamine HCl 40 mg Calcium pantothenate 80 mg Nicotinamide 200 mg Deionized H2O 800 ml Add 2.5 N NaOH until solution clears Add deionized H2O to a final volume of 1,000 ml Store at −20°C Prepare final medium Base medium 909 ml l-Cystine stock 10 ml l-Proline stock 16 ml CDM supplement 10 ml l-Phenylalanine stock 40 ml Vitamins 10 ml Sterilize by ultrafiltration (0.2-μm pore size) ↵a CDM may be stored for up to 2 weeks at 4°C. Stock solutions may be stored for up to 3 months.
- Table 2.
Alkaline phosphatase activities of the TnphoA mutants of B. avium
Strain Fe availability (sp act)a Fold inductionb Fe replete Fe limited 4169 0.018 ± 0.001 0.014 ± 0.001 0.8 Pho-6 0.004 ± 0.005 0.129 ± 0.042 32.3 Pho-20 0.033 ± 0.021 0.217 ± 0.045 6.6 ↵a Values are the results from three replicate cultures of each strain. All values are means ± SD. Specific activities (41) are reported as micromoles of NPP hydrolyzed per minute per OD600 unit by the formula
↵b Activity during Fe-limited culture/activity during Fe-replete culture.
