Article Figures & Data
Figures
- Fig. 1.
Detection of the 100-kDa HxuA protein in CS from wild-type and mutant Hib strains. Proteins present in CS from the wild-type Hib strain DL42 (lanes A, C, and E) and from the HibhxuA mutant strain DL42.61 (lanes B, D, and F) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and either stained with Coomassie blue (panel 1) or transferred to nitrocellulose for Western blot analysis with rat antiserum raised against CS from Hib strain DL26 (panel 2) or with the Hib DL42 HxuA-specific monoclonal antibody 4C11 (panel 3). The arrowhead to the left of lane A indicates the position of the HxuA protein. Molecular mass position markers (in kilodaltons) are present on the left side of the figure.
- Fig. 2.
Effect of exogenous Hib HxuA on the ability of wild-type and mutant Hib strains to utilize heme-hemopexin. Heme-starved cells of the wild-type strain DL42 (A) and the hxuA mutant DL42.61 (B) were inoculated into BHI-NAD broth (basal medium) supplemented with the following compounds at the concentrations listed in the text: no supplement (negative control) (circles), heme (Hm) (positive control) (squares), heme-hemopexin (HmHx) (asterisks), Hib DL42 CS (i.e., HxuA) (diamonds), and Hib DL42 CS (i.e., HxuA) and heme-hemopexin (triangles). Growth was monitored by plating samples from each culture on BHIs agar plates at timed intervals. The data points in each graph are the means of results from two separate experiments; error bars are included.
- Fig. 3.
Effect of exogenous NTHI HxuA on the ability of wild-type and mutant Hib strains to utilize heme-hemopexin. Heme-starved cells of the Hib hxuA mutant DL42.61 (A) and the wild-type Hib strain 760705 (B) were inoculated into BHI-NAD broth (basal medium) supplemented with the following compounds: no supplement (negative control) (circles), heme (Hm) (positive control) (squares), heme-hemopexin (HmHx) (asterisks), NTHI N182 CS (i.e., HxuA) (diamonds), and NTHI N182 CS (i.e., HxuA) and heme-hemopexin (triangles). Growth was monitored by plating samples from each culture on BHIs agar plates at timed intervals.
Tables
- Table 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Description or phenotype Reference(s) or source H. influenzae strains DL26 Wild-type Hib strain that expresses the HxuA protein and can utilize heme-hemopexin 16 DL42 Wild-type Hib strain that expresses the HxuA protein and can utilize heme-hemopexin 4, 5 DL42.107d DL42 mutant with a NotI linker inserted into the hxuA structural gene; this strain does not express HxuA and cannot utilize heme-hemopexin 17 DL42.61 DL42.107d with a cat cartridge inserted into the NotI site within the hxuAgene 5 760705 Wild-type Hib strain that does not express the HxuA protein and does not utilize heme-hemopexin 4, 47 N182 Wild-type NTHI strain that expresses the HxuA protein and can utilize heme-hemopexin 4 E. colistrains RR1 Host strain for recombinant plasmids 38 RR1(pBR322) Recombinant strain that does not release HxuA into the CS 38 RR1(pHX1-6) Recombinant strain that releases HxuA into the CS This study Plasmids pBR322 Cloning vector, Ampr Tetr 38 pHX1-6 pBR322 with a 13.6-kb insert of H. influenzae DL42 chromosomal DNA containing the hxuCBAgene cluster 5, 17 - Table 2.
RIP-based detection of the binding of soluble HxuA to heme-hemopexin complexes
Source of soluble CS proteins 125I-heme-hemopexin immuno- precipitated (cpm) by: Antiserum to CS proteinsa Control serumb Hib DL42 206,892 21,892 Hib DL42.107d 15,208 21,880 E. coliRR1(pBR322) 7,906 20,306 E. coliRR1(pHX1-6) 177,312 19,766
