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A bioassay for the detection of benzimidazoles reveals their presence in a range of environmental samples.

Crofts TS, Men Y, Alvarez-Cohen L, Taga ME - Front Microbiol (2014)

Bottom Line: Of the three classes of lower ligands, the benzimidazoles are uniquely found in cobamides, whereas the purine and phenolic bases have additional biological functions.The concentrations of individual benzimidazoles in these samples were measured by liquid chromatography-tandem mass spectrometry.Several benzimidazoles were detected in subpicomolar to subnanomolar concentrations in host-associated and environmental samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California at Berkeley Berkeley, CA, USA.

ABSTRACT
Cobamides are a family of enzyme cofactors that include vitamin B12 (cobalamin) and are produced solely by prokaryotes. Structural variability in the lower axial ligand has been observed in cobamides produced by diverse organisms. Of the three classes of lower ligands, the benzimidazoles are uniquely found in cobamides, whereas the purine and phenolic bases have additional biological functions. Many organisms acquire cobamides by salvaging and remodeling cobamides or their precursors from the environment. These processes require free benzimidazoles for incorporation as lower ligands, though the presence of benzimidazoles in the environment has not been previously investigated. Here, we report a new purification method and bioassay to measure the total free benzimidazole content of samples from microbial communities and laboratory media components. The bioassay relies on the "calcofluor-bright" phenotype of a bluB mutant of the model cobalamin-producing bacterium Sinorhizobium meliloti. The concentrations of individual benzimidazoles in these samples were measured by liquid chromatography-tandem mass spectrometry. Several benzimidazoles were detected in subpicomolar to subnanomolar concentrations in host-associated and environmental samples. In addition, benzimidazoles were found to be common contaminants of laboratory media components. These results suggest that benzimidazoles present in the environment and in laboratory media have the potential to influence microbial metabolic activities.

No MeSH data available.


Related in: MedlinePlus

Optimization of the S. meliloti bluB CF bioassay to quantify benzimidazoles. (A) CF fluorescence of liquid cultures of WT S. meliloti, an S. meliloti bluB mutant (bluB), or S. meliloti bluB grown in the absence (-) or presence of 0.5 μM DMB or 1 μM cobalamin. Error bars represent the SE of three independent experiments. (B) Development of the CFB phenotype as a function of growth. S. meliloti bluB was grown in the presence and absence of 0.5 μM DMB. At the indicated time points, aliquots were removed and incubated with CF for 5 h. The difference in the CF fluorescence of cultures with and without DMB is plotted. (C) Development of the CFB phenotype as a function of incubation time with CF. After growth of S. meliloti bluB with (circles) and without (squares) DMB for 48 h, CF was added and fluorescence was measured at the indicated time points. Triangles represent the difference in fluorescence between the two cultures. (D) CF fluorescence (left axis, circles) and optical density (O.D.600; right axis, triangles) is shown for the S. meliloti bluB mutant incubated in a 96-well plate with the indicated concentrations of DMB for 48 h followed by addition of CF for 5 h. Error bars represent the SE of six independent experiments. Fluorescence is presented in arbitrary units (FU).
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Figure 3: Optimization of the S. meliloti bluB CF bioassay to quantify benzimidazoles. (A) CF fluorescence of liquid cultures of WT S. meliloti, an S. meliloti bluB mutant (bluB), or S. meliloti bluB grown in the absence (-) or presence of 0.5 μM DMB or 1 μM cobalamin. Error bars represent the SE of three independent experiments. (B) Development of the CFB phenotype as a function of growth. S. meliloti bluB was grown in the presence and absence of 0.5 μM DMB. At the indicated time points, aliquots were removed and incubated with CF for 5 h. The difference in the CF fluorescence of cultures with and without DMB is plotted. (C) Development of the CFB phenotype as a function of incubation time with CF. After growth of S. meliloti bluB with (circles) and without (squares) DMB for 48 h, CF was added and fluorescence was measured at the indicated time points. Triangles represent the difference in fluorescence between the two cultures. (D) CF fluorescence (left axis, circles) and optical density (O.D.600; right axis, triangles) is shown for the S. meliloti bluB mutant incubated in a 96-well plate with the indicated concentrations of DMB for 48 h followed by addition of CF for 5 h. Error bars represent the SE of six independent experiments. Fluorescence is presented in arbitrary units (FU).

Mentions: The apparent dose-dependent relationship between the concentration of DMB and the CF phenotype on plates (Figure 2A) suggests that the CF phenotype of the bluB mutant could be used for the quantitative detection of DMB. To investigate whether the CFB response can be adapted for liquid media in a 96-well plate format, WT S. meliloti and a bluB mutant were cultured for 45 h in a 96-well plate, mixed with CF, and the fluorescence was measured in a multiwell plate reader. As on solid media, the bluB mutant showed significantly higher fluorescence than WT S. meliloti, with a 27-fold difference in fluorescence between the two strains (Figure 3A). Culturing the bluB mutant in the presence of DMB or cobalamin resulted in a low level of fluorescence comparable to that of WT S. meliloti, consistent with the phenotype on agar plates (Figure 3A; Campbell et al., 2006). A greater separation of the dim and bright phenotypes was observed as the bacteria were cultured for longer periods prior to the addition of CF (Figure 3B). Further optimization showed that the greatest difference in fluorescence was achieved when the cultures were incubated with CF for 5 h (Figure 3C). Based on these observations, the remaining CF fluorescence measurements were performed by incubating cultures with CF for 5 h following at least 55 h of growth in the 96-well plates.


A bioassay for the detection of benzimidazoles reveals their presence in a range of environmental samples.

Crofts TS, Men Y, Alvarez-Cohen L, Taga ME - Front Microbiol (2014)

Optimization of the S. meliloti bluB CF bioassay to quantify benzimidazoles. (A) CF fluorescence of liquid cultures of WT S. meliloti, an S. meliloti bluB mutant (bluB), or S. meliloti bluB grown in the absence (-) or presence of 0.5 μM DMB or 1 μM cobalamin. Error bars represent the SE of three independent experiments. (B) Development of the CFB phenotype as a function of growth. S. meliloti bluB was grown in the presence and absence of 0.5 μM DMB. At the indicated time points, aliquots were removed and incubated with CF for 5 h. The difference in the CF fluorescence of cultures with and without DMB is plotted. (C) Development of the CFB phenotype as a function of incubation time with CF. After growth of S. meliloti bluB with (circles) and without (squares) DMB for 48 h, CF was added and fluorescence was measured at the indicated time points. Triangles represent the difference in fluorescence between the two cultures. (D) CF fluorescence (left axis, circles) and optical density (O.D.600; right axis, triangles) is shown for the S. meliloti bluB mutant incubated in a 96-well plate with the indicated concentrations of DMB for 48 h followed by addition of CF for 5 h. Error bars represent the SE of six independent experiments. Fluorescence is presented in arbitrary units (FU).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Optimization of the S. meliloti bluB CF bioassay to quantify benzimidazoles. (A) CF fluorescence of liquid cultures of WT S. meliloti, an S. meliloti bluB mutant (bluB), or S. meliloti bluB grown in the absence (-) or presence of 0.5 μM DMB or 1 μM cobalamin. Error bars represent the SE of three independent experiments. (B) Development of the CFB phenotype as a function of growth. S. meliloti bluB was grown in the presence and absence of 0.5 μM DMB. At the indicated time points, aliquots were removed and incubated with CF for 5 h. The difference in the CF fluorescence of cultures with and without DMB is plotted. (C) Development of the CFB phenotype as a function of incubation time with CF. After growth of S. meliloti bluB with (circles) and without (squares) DMB for 48 h, CF was added and fluorescence was measured at the indicated time points. Triangles represent the difference in fluorescence between the two cultures. (D) CF fluorescence (left axis, circles) and optical density (O.D.600; right axis, triangles) is shown for the S. meliloti bluB mutant incubated in a 96-well plate with the indicated concentrations of DMB for 48 h followed by addition of CF for 5 h. Error bars represent the SE of six independent experiments. Fluorescence is presented in arbitrary units (FU).
Mentions: The apparent dose-dependent relationship between the concentration of DMB and the CF phenotype on plates (Figure 2A) suggests that the CF phenotype of the bluB mutant could be used for the quantitative detection of DMB. To investigate whether the CFB response can be adapted for liquid media in a 96-well plate format, WT S. meliloti and a bluB mutant were cultured for 45 h in a 96-well plate, mixed with CF, and the fluorescence was measured in a multiwell plate reader. As on solid media, the bluB mutant showed significantly higher fluorescence than WT S. meliloti, with a 27-fold difference in fluorescence between the two strains (Figure 3A). Culturing the bluB mutant in the presence of DMB or cobalamin resulted in a low level of fluorescence comparable to that of WT S. meliloti, consistent with the phenotype on agar plates (Figure 3A; Campbell et al., 2006). A greater separation of the dim and bright phenotypes was observed as the bacteria were cultured for longer periods prior to the addition of CF (Figure 3B). Further optimization showed that the greatest difference in fluorescence was achieved when the cultures were incubated with CF for 5 h (Figure 3C). Based on these observations, the remaining CF fluorescence measurements were performed by incubating cultures with CF for 5 h following at least 55 h of growth in the 96-well plates.

Bottom Line: Of the three classes of lower ligands, the benzimidazoles are uniquely found in cobamides, whereas the purine and phenolic bases have additional biological functions.The concentrations of individual benzimidazoles in these samples were measured by liquid chromatography-tandem mass spectrometry.Several benzimidazoles were detected in subpicomolar to subnanomolar concentrations in host-associated and environmental samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California at Berkeley Berkeley, CA, USA.

ABSTRACT
Cobamides are a family of enzyme cofactors that include vitamin B12 (cobalamin) and are produced solely by prokaryotes. Structural variability in the lower axial ligand has been observed in cobamides produced by diverse organisms. Of the three classes of lower ligands, the benzimidazoles are uniquely found in cobamides, whereas the purine and phenolic bases have additional biological functions. Many organisms acquire cobamides by salvaging and remodeling cobamides or their precursors from the environment. These processes require free benzimidazoles for incorporation as lower ligands, though the presence of benzimidazoles in the environment has not been previously investigated. Here, we report a new purification method and bioassay to measure the total free benzimidazole content of samples from microbial communities and laboratory media components. The bioassay relies on the "calcofluor-bright" phenotype of a bluB mutant of the model cobalamin-producing bacterium Sinorhizobium meliloti. The concentrations of individual benzimidazoles in these samples were measured by liquid chromatography-tandem mass spectrometry. Several benzimidazoles were detected in subpicomolar to subnanomolar concentrations in host-associated and environmental samples. In addition, benzimidazoles were found to be common contaminants of laboratory media components. These results suggest that benzimidazoles present in the environment and in laboratory media have the potential to influence microbial metabolic activities.

No MeSH data available.


Related in: MedlinePlus