Metabolism of bismuth subsalicylate and intracellular accumulation of bismuth by Fusarium sp. strain BI

Abstract

Enrichment cultures were conducted using bismuth subsalicylate as the sole source of carbon and activated sludge as the inoculum. A pure culture was obtained and identified as a Fusarium sp. based on spore morphology and partial sequences of 18S rRNA, translation elongation factor 1-alpha, and beta-tubulin genes. The isolate, named Fusarium sp. strain BI, grew to equivalent densities when using salicylate or bismuth subsalicylate as carbon sources. Bismuth nitrate at concentrations of up to 200 muM did not limit growth of this organism on glucose. The concentration of soluble bismuth in suspensions of bismuth subsalicylate decreased during growth of Fusarium sp. strain BI. Transmission electron microscopy and energy-dispersive spectroscopy revealed that the accumulated bismuth was localized in phosphorus-rich granules distributed in the cytoplasm and vacuoles. Long-chain polyphosphates were extracted from fresh biomass grown on bismuth subsalicylate, and inductively coupled plasma optical emission spectrometry showed that these fractions also contained high concentrations of bismuth. Enzyme activity assays of crude extracts of Fusarium sp. strain BI showed that salicylate hydroxylase and catechol 1,2-dioxygenase were induced during growth on salicylate, indicating that this organism degrades salicylate by conversion of salicylate to catechol, followed by ortho cleavage of the aromatic ring. Catechol 2,3-dioxygenase activity was not detected. Fusarium sp. strain BI grew with several other aromatic acids as carbon sources: benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, gentisate, d-mandelate, l-phenylalanine, l-tyrosine, phenylacetate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate, and phenylpropionate.

FIG. 1.

Light micrographs (A and B) (magnification, ×1,300) showing types and morphology of Fusarium sp. strain BI spores. Macroconidia and microconidia (A) and chlamydospores (B) were all present in this isolate. For comparison, light micrographs previously published by Toussoun and Nelson (44) of macroconidia and microconidia (C) (magnification, ×950) and chlamydospores (D) (magnification, ×1,660) from a Fusarium solani strain are shown (reproduced with permission of The Pennsylvania State University Press).

FIG. 2.

Plot of total soluble bismuth versus time in 1.5 mM bismuth subsalicylate suspensions in minimal medium that was uninoculated (□) or inoculated with Fusarium sp. strain BI (Δ). Bismuth concentrations were determined by ICP-OES.

FIG. 3.

TEM (A and B) and EDS (C) analysis of Fusarium sp. strain BI grown in a 1.5 mM bismuth subsalicylate suspension in minimal medium. Intracellular electron-dense granules were visible in cross-sections of hyphal filaments (A) (magnification, ×28,000). A magnified view (B) (magnification, ×45,000) of the boxed area in panel A shows that the electron-dense and electron-transparent regions are of similar size and shape. The spectrum (C) obtained from EDS analysis of the electron-dense region indicated by the arrow in panel B revealed the presence of bismuth, phosphorus, calcium, and traces of magnesium and aluminum in the granule. Uranium appears in the spectrum because the section was stained with uranyl acetate. The carbon and oxygen peaks may represent both the composition of the granule and fixed biological material around the granule, as the beam spot size was slightly larger than the granule. keV, kilo-electron volts.

FIG. 4.

Ratio of bismuth to polyphosphate in extracts of Fusarium sp. strain BI biomass grown on salicylate and bismuth subsalicylate. Biomass was ground and extracted to yield fractions containing polyphosphate of different chain lengths (10). Bismuth concentrations were determined in each fraction by ICP-OES. Polyphosphate concentrations were determined colorimetrically (39) as orthophosphate released after acid hydrolysis of polyphosphate in each fraction (25). Error bars represent the standard deviations of three replicates.

FIG. 5.

Known pathways for salicylate catabolism in fungi and/or bacteria. Intermediates in the proposed primary pathway of bismuth subsalicylate degradation by Fusarium sp. strain BI are shown in boldface.

FIG. 6.

Total dry biomass of Fusarium sp. strain BI grown for 96 h on 0.25 mmol of various substrates as sole carbon sources. Error bars represent the standard deviation of three replicates.