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. 2010 Feb;75(4):900-9.
doi: 10.1111/j.1365-2958.2009.07025.x. Epub 2010 Jan 13.

Genetic analysis of petrobactin transport in Bacillus anthracis

Affiliations

Genetic analysis of petrobactin transport in Bacillus anthracis

Paul E Carlson Jr et al. Mol Microbiol. 2010 Feb.

Abstract

Iron acquisition mechanisms play an important role in the pathogenesis of many infectious microbes. In Bacillus anthracis, the siderophore petrobactin is required for both growth in iron-depleted conditions and for full virulence of the bacterium. Here we demonstrate the roles of two putative petrobactin binding proteins FatB and FpuA (encoded by GBAA5330 and GBAA4766 respectively) in B. anthracis iron acquisition and pathogenesis. Markerless deletion mutants were created using allelic exchange. The Delta fatB strain was capable of wild-type levels of growth in iron-depleted conditions, indicating that FatB does not play an essential role in petrobactin uptake. In contrast, Delta fpuA bacteria exhibited a significant decrease in growth under low-iron conditions when compared with wild-type bacteria. This mutant could not be rescued by the addition of exogenous purified petrobactin. Further examination of this strain demonstrated increased levels of petrobactin accumulation in the culture supernatants, suggesting no defect in siderophore synthesis or export but, instead, an inability of Delta fpuA to import this siderophore. Delta fpuA spores were also significantly attenuated in a murine model of inhalational anthrax. These results provide the first genetic evidence demonstrating the role of FpuA in petrobactin uptake.

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Figures

Figure 1
Figure 1. Construction of B. anthracis mutants
Gene deletion mutants were created by allelic exchange removing either (A) GBAA4766 (fpuA) or (B) GBAA5330 (fatB). For each deletion, the initial 30 and final 30 nucleotides of the gene were fused creating a markerless deletion. Deleted genetic material was replaced by three stop codons, represented by “X” above.
Figure 2
Figure 2. Growth of B. anthracis mutant strains in iron depleted media
(A and B) Wild-type (solid diamonds), ΔfatB (solid squares), ΔfpuA (open triangles), ΔfpuA ΔfatB (open squares), and ΔasbABCDEF (open circles) were grown in either iron depleted media (A) or BHI (B). (C) Wild-type (solid diamonds), ΔfpuA (open triangles), ΔfpuA/pfpuA+ were grown in IDM at 26°C. All cultures were inoculated with vegetative bacilli at an initial OD600 = 0.05 and growth was monitored by measuring change in OD600 over time. Data presented are representative of least three individual experiments.
Figure 3
Figure 3. Addition of supplemental petrobactin does not rescue binding protein mutants
Wild-type (solid diamonds), ΔfpuA (open triangles), and ΔasbABCDEF (open circles) were grown in either (A) iron depleted media or(B) iron depleted media supplemented with 2.5μM petrobactin. Cultures were inoculated with spores and grown in microtiter plates. Data presented are representative of three individual experiments.
Figure 4
Figure 4. Increased petrobactin levels observed in ΔfpuA strains
(A) Extracellular catechol levels were measured over time in culture supernatants using the Arnow assay. Data were normalized to the OD600 of cultures at each timepoint and are presented as percent of wild-type petrobactin levels at three hours. No signal could be detected at timepoints prior to three hours. Error bars represent standard deviation of triplicate samples within one experiment. Data are representative of four individual experiments. (B) Comparison of petrobactin accumulation by HPLC. Supernatants from wild-type and ΔfpuA cultures that were grown for six hours and normalized to equivalent OD600 were observed by HPLC. A peak corresponding to an authentic petrobactin standard is observed in both WT and ΔfpuA traces (arrow).
Figure 5
Figure 5. Attenuation of virulence of the ΔfpuA strain in a murine model of infection
DBA/2J mice were infected by intratracheal infection with WT (filled diamonds) or ΔfpuA (open triangles) spores at 1×105 (A), or 1×106 (B) spores per mouse. Mice were monitored for fourteen days. Survival curves for ΔfpuA were significantly different from wild-type at all doses tested by the log-rank test (p values indicated above).

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