Production of the siderophore 2,3-dihydroxybenzoic acid is required for wild-type growth of Brucella abortus in the presence of erythritol under low-iron conditions in vitro

Abstract

Production of the siderophore 2,3-dihyroxybenzoic acid (2,3-DHBA) is required for the wild-type virulence of Brucella abortus in cattle. A possible explanation for this requirement was uncovered when it was determined that a B. abortus dhbC mutant (BHB1) defective in 2,3-DHBA production displays marked growth restriction in comparison to its parent strain, B. abortus 2308, when cultured in the presence of erythritol under low-iron conditions. This phenotype is not displayed when these strains are cultured under low-iron conditions in the presence of other readily utilizable carbon and energy sources. The addition of either exogenous 2,3-DHBA or FeCl(3) relieves this growth defect, suggesting that the inability of the B. abortus dhbC mutant to display wild-type growth in the presence of erythritol under iron-limiting conditions is due to a defect in iron acquisition. Restoring 2,3-DHBA production to the B. abortus dhbC mutant by genetic complementation abolished the erythritol-specific growth defect exhibited by this strain in low-iron medium, verifying the relationship between 2,3-DHBA production and efficient growth in the presence of erythritol under low-iron conditions. The positive correlation between 2,3-DHBA production and growth in the presence of erythritol was further substantiated by the observation that the addition of erythritol to low-iron cultures of B. abortus 2308 stimulated the production of 2,3-DHBA by increasing the transcription of the dhbCEBA operon. Correspondingly, the level of exogenous iron needed to repress dhbCEBA expression in B. abortus 2308 was also greater when this strain was cultured in the presence of erythritol than that required when it was cultured in the presence of any of the other readily utilizable carbon and energy sources tested. The tissues of the bovine reproductive tract are rich in erythritol during the latter stages of pregnancy, and the ability to metabolize erythritol is thought to be important to the virulence of B. abortus in pregnant ruminants. Consequently, the experimental findings presented here offer a plausible explanation for the attenuation of the B. abortus 2,3-DHBA-deficient mutant BHB1 in pregnant ruminants.

FIG. 1.

Growth restriction displayed by the B. abortus dhbC mutant BHB1 during growth in low-iron minimal medium (13) supplemented with 0.5% meso-erythritol.

FIG. 3.

Genetic complementation of the erythritol-induced growth restriction of the B. abortus nonpolar dhbC mutant BHB2 by a plasmid-borne cloned copy of dhbC. Growth was evaluated by determining the OD₆₀₀ of cultures following 36 h of growth in low-iron medium (13) supplemented with 0.1% meso-erythritol. Triplicate samples from each culture were used to calculate standard deviations, and results presented are from a representative experiment.

FIG. 2.

Exogenous FeCl₃ (50 μM) enhances the growth of B. abortus BHB1 (ΔdhbC) in low-iron minimal medium supplemented with 0.5% meso-erythritol. Culture density is presented on a log scale, and results shown are representative of those obtained from multiple experiments.

FIG. 4.

Erythritol-associated stimulation of 2,3-DHBA production by B. abortus 2308 during growth in low-iron minimal medium (13). Samples from an unsupplemented low-iron minimal medium culture (2308) and a low-iron minimal medium culture supplemented with 1.0% erythritol prior to inoculation (2308 Ery) were harvested at the indicated times and analyzed for growth and siderophore production. (A) Growth was recorded as OD₆₀₀ and was plotted on a logarithmic scale. (B) 2,3-DHBA levels were determined by the Arnow assay (2). To account for differences in growth rates between separate cultures, Arnow results are presented as a ratio of Arnow activity over culture density for each sample collected (A₅₁₀/OD₆₀₀). In statistical comparison, normalized Arnow values from erythritol-supplemented cultures were significantly greater than those from non-erythritol-supplemented cultures (P ≤ 0.01; paired t test analysis). p.i., postinoculation.

FIG. 5.

Iron-dependent stimulation of B. abortus 2308 dhbC transcription by erythritol. Transcription of dhbC was measured by monitoring the activity of the dhbC::lacZ reporter in plasmid pP_dhbC::lacZ following 20 h of growth of 2308 in low-iron medium without (−Fe) and with (+Fe) 100 μM FeCl₃ and with or without 0.1% erythritol. The β-galactosidase activity of each culture was determined by using the methods described by Miller (18). The results presented are from a representative experiment, and statistical significance was calculated (P < 0.001) by t test analysis by using replicate samples for each culture condition.

FIG. 6.

Erythritol- and iron-dependent repression of dhbC expression. To determine the amount of exogenous iron needed to repress dhbC transcription, B. abortus 2308 (pP_dhbC::lacZ) was grown in low-iron medium supplemented with increasing amounts of FeCl₃ in either the presence or absence of erythritol. Cells were harvested after 24 h of incubation under these conditions, and the β-galactosidase activity of each culture was determined by using the methods described by Miller (18). In statistical comparison, β-galactosidase activity from erythritol-supplemented cultures was significantly greater than that from non-erythritol-supplemented cultures at each iron concentration examined (P < 0.01; paired t test analysis using replicate samples for each growth condition). Results presented are from a single experimental trial and are representative of the results obtained from multiple experiments.