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. 2011;6(6):e20777.
doi: 10.1371/journal.pone.0020777. Epub 2011 Jun 6.

Regulation of petrobactin and bacillibactin biosynthesis in Bacillus anthracis under iron and oxygen variation

Affiliations

Regulation of petrobactin and bacillibactin biosynthesis in Bacillus anthracis under iron and oxygen variation

Jung Yeop Lee et al. PLoS One. 2011.

Abstract

Background: Bacillus anthracis produces two catecholate siderophores, petrobactin and bacillibactin, under iron-limited conditions. Here, we investigate how variable iron and oxygen concentrations influence the biosynthetic output of both siderophores in B. anthracis. In addition, we describe the differential levels of transcription of select genes within the B. anthracis siderophore biosynthetic operons that are responsible for synthesis of petrobactin and bacillibactin, during variable growth conditions.

Methodology/principal findings: Accumulation of bacillibactin in B. anthracis Sterne (34F(2)) and in a mutant lacking the major superoxide dismutase (ΔsodA1) was almost completely repressed by the addition of 20 µM of iron. In contrast, petrobactin synthesis in both strains continued up to 20 µM of iron. Accumulation of petrobactin and bacillibactin showed a slight increase with addition of low levels of paraquat-induced oxidative stress in wild type B. anthracis Sterne. Cultures grown with high aeration resulted in greater accumulation of petrobactin relative to low aeration cultures, and delayed the repressive effect of added iron. Conversely, iron-depleted cultures grown with low aeration resulted in increased levels of bacillibactin. No difference was found in overall superoxide dismutase (SOD) activity or transcriptional levels of the sodA1 and sodA2 genes between iron-depleted and iron-replete conditions at high or low aeration, suggesting that SOD regulation and iron metabolism are separate in B. anthracis. The highest transcription of the gene asbB, part of the petrobactin biosynthetic operon, occurred under iron-limitation with high aeration, but transcription was readily detectable even under iron-replete conditions and in low aeration. The gene dhbC, a member of the bacillibactin biosynthetic operon, was only transcribed under conditions of iron-depletion, regardless of growth aeration.

Conclusion: These data suggest that bacillibactin regulation is highly sensitive to iron-concentration. In contrast, although regulation of petrobactin is less dependent on iron, it is likely subject to additional levels of regulation that may contribute to virulence of B. anthracis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Chemical structures of B. anthracis siderophores petrobactin and bacillibactin.
Figure 2
Figure 2. LCMS traces of extracts from the IDM cultures of B. anthracis showing the presence of petrobactin and bacillibactin with or without addition of iron.
(A) Selected ion monitoring (SIM) chromatogram of petrobactin at the mass range of m/z 719.3 [M+H]+. The peaks at 15.989–16.183 min represent petrobactin. (B) SIM chromatogram of bacillibactin at the mass range of m/z 883.2 [M+H]+. The peaks at 19.235–19.300 min represent bacillibactin.
Figure 3
Figure 3. LCMS chromatogram of petrobactin and bacillibactin in the extracts obtained from the IDM cultures of B. anthracis with or without addition of paraquat.
(A) SIM chromatogram of petrobactin at the mass range of m/z 719.3 [M+H]+. The peaks at 15.989–16.289 min represent petrobactin. (B) SIM chromatogram of bacillibactin at the mass range of m/z 883.2 [M+H]+. The peaks at 19.216–19.300 min represent bacillibactin.
Figure 4
Figure 4. LCMS profiles of petrobactin and bacillibactin from the extracts of the IDM cultures of B. anthracis with or without addition of iron under high and low aeration.
(A) SIM chromatogram of petrobactin at the mass range of m/z 719.3 [M+H]+. The peaks at 15.989–16.271 min represent petrobactin. (B) SIM chromatogram of bacillibactin at the mass range of m/z 883.2 [M+H]+. The peaks at 19.204–19.300 min represent bacillibactin.
Figure 5
Figure 5. Nitroblue tetrazolium salt (NBT) assay of superoxide dismutase (SOD) activity from whole cell lysates of B. anthracis grown in various growth conditions.
(A) Percent inhibition of superoxide production from 5 µg of whole cell lysates of B. anthracis Sterne 34F2, and the mutants ΔsodA1 and ΔsodA2 grown in LB (iron-rich) medium with and without the addition of a high concentration of paraquat (800 µM). (B) Percent inhibition of superoxide production from 5 µg of whole cell lysates of B. anthracis Sterne 34F2 grown in IDM and IDM supplemented with 20 µM Fe(II)SO4•7H2O under high and low aeration. Low O2 indicates cultures grown in a 40% culture to flask volume/volume ratio with slow shaking (125 rpm); High O2 indicates cultures grown in a 10% culture to flask volume/volume ratio with rigorous shaking (300 rpm). Percent inhibition indicates the amount by which the cell lysate inhibits NBT reduction relative to a non-lysate containing control reaction (see Materials and Methods).
Figure 6
Figure 6. End-Point RT-PCR and SYBR-Green qRT-PCR of select genes from the asb and bac operons in iron-depleted, iron-supplemented, and iron-rich conditions.
Ethidium bromide stained agarose gels from endpoint RT-PCR of the second gene in the asb operon (asbB), the second gene in the bac operon (dhbC) and a constitutively expressed elongation factor (fusA) from B. anthracis Sterne 34F2 grown in different media with various iron concentrations. –Fe is Iron-Depleted Medium (IDM); +Fe is IDM with 20 µM Fe(II)SO4 supplementation; LB is Luria Bertani Broth (∼7.6 µM iron concentration). High O2 and low O2 are cultures grown in high or low aeration respectively. LB cultures were grown under high O2 conditions (see Methods). Numbers are approximations of fold differences in transcript abundance as assayed by SYBR-Green qRT-PCR representing the following comparisons: 1 asbB is ∼11-fold more highly expressed in IDM with high aeration than in IDM+Fe with high aeration. 2 asbB is ∼37 fold more highly expressed in IDM with low aeration than in IDM+Fe with low aeration. 3 asbB is ∼21 fold less abundant in LB medium than in IDM with high aeration. Note that there is less than a 2-fold difference in transcript abundance between LB and IDM+Fe. 4 dhbC is greater than 500 fold more highly expressed in IDM with high aeration than in IDM+Fe with high aeration. 5 dhbC is greater than 500 fold more highly expressed in IDM with low aeration than in IDM+Fe with low aeration. 6 dhbC is more than 500 fold less abundant in LB medium than in IDM with high aeration. Note that the abundance of transcript in LB medium as compared to IDM+Fe is approximately the same.

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