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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

The calcofluor (CF) phenotype of the Sinorhizobium meliloti bluB mutant is influenced by DMB availability and DNA stress. (A) CF fluorescence of S. meliloti bluB on an LB CF plate. Filter disks containing DMB at the indicated concentrations were applied to the plate. (B) CF fluorescence phenotypes of S. meliloti strains (1) bluB mutant expressing Escherichia coli nrdAB on plasmid pMS03 ( Taga and Walker, 2010), (2) Wild type (WT) S. meliloti with empty vector pMS03, (3) bluB mutant with empty vector pMS03, and (4) bluB mutant expressing E. coli metE on plasmid pMS03 ( Taga and Walker, 2010). (C) Strain SmNrdAB+ on an LB CF plate with a filter disk containing hydroxyurea, photographed under white light. (D) Same as (C) but photographed under UV light. (E) Fluorescence of WT S. meliloti on an LB CF plate with a filter disk containing nalidixic acid. Filter disks were applied to the plates at the time of inoculation. (F) Schematic description of the physiological link between the bluB mutation and the CF fluorescence phenotype. The dashed line indicates that the mechanism is unknown.
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Figure 2: The calcofluor (CF) phenotype of the Sinorhizobium meliloti bluB mutant is influenced by DMB availability and DNA stress. (A) CF fluorescence of S. meliloti bluB on an LB CF plate. Filter disks containing DMB at the indicated concentrations were applied to the plate. (B) CF fluorescence phenotypes of S. meliloti strains (1) bluB mutant expressing Escherichia coli nrdAB on plasmid pMS03 ( Taga and Walker, 2010), (2) Wild type (WT) S. meliloti with empty vector pMS03, (3) bluB mutant with empty vector pMS03, and (4) bluB mutant expressing E. coli metE on plasmid pMS03 ( Taga and Walker, 2010). (C) Strain SmNrdAB+ on an LB CF plate with a filter disk containing hydroxyurea, photographed under white light. (D) Same as (C) but photographed under UV light. (E) Fluorescence of WT S. meliloti on an LB CF plate with a filter disk containing nalidixic acid. Filter disks were applied to the plates at the time of inoculation. (F) Schematic description of the physiological link between the bluB mutation and the CF fluorescence phenotype. The dashed line indicates that the mechanism is unknown.

Mentions: We previously found that the CFB phenotype of the S. meliloti bluB mutant can be rescued by the addition of DMB, which is used to produce cobalamin (Campbell et al., 2006; Taga et al., 2007). Bioassays, such as the bacterial assay that enabled the first purification of cobalamin (Rickes et al., 1948), can provide inexpensive, high-throughout, and highly sensitive means to detect molecules of interest (Kelleher and Broin, 1991). To investigate the possibility that this phenotype could be used to detect DMB, we first used a filter disk assay to examine the effect of DMB concentration on the CFB phenotype of a bluB mutant. This showed that the size and intensity of the CF-dim zone surrounding the DMB-soaked disks were positively correlated with the DMB concentration, suggesting that the CF phenotype could be used for quantification of DMB (Figure 2A).


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)

The calcofluor (CF) phenotype of the Sinorhizobium meliloti bluB mutant is influenced by DMB availability and DNA stress. (A) CF fluorescence of S. meliloti bluB on an LB CF plate. Filter disks containing DMB at the indicated concentrations were applied to the plate. (B) CF fluorescence phenotypes of S. meliloti strains (1) bluB mutant expressing Escherichia coli nrdAB on plasmid pMS03 ( Taga and Walker, 2010), (2) Wild type (WT) S. meliloti with empty vector pMS03, (3) bluB mutant with empty vector pMS03, and (4) bluB mutant expressing E. coli metE on plasmid pMS03 ( Taga and Walker, 2010). (C) Strain SmNrdAB+ on an LB CF plate with a filter disk containing hydroxyurea, photographed under white light. (D) Same as (C) but photographed under UV light. (E) Fluorescence of WT S. meliloti on an LB CF plate with a filter disk containing nalidixic acid. Filter disks were applied to the plates at the time of inoculation. (F) Schematic description of the physiological link between the bluB mutation and the CF fluorescence phenotype. The dashed line indicates that the mechanism is unknown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230183&req=5

Figure 2: The calcofluor (CF) phenotype of the Sinorhizobium meliloti bluB mutant is influenced by DMB availability and DNA stress. (A) CF fluorescence of S. meliloti bluB on an LB CF plate. Filter disks containing DMB at the indicated concentrations were applied to the plate. (B) CF fluorescence phenotypes of S. meliloti strains (1) bluB mutant expressing Escherichia coli nrdAB on plasmid pMS03 ( Taga and Walker, 2010), (2) Wild type (WT) S. meliloti with empty vector pMS03, (3) bluB mutant with empty vector pMS03, and (4) bluB mutant expressing E. coli metE on plasmid pMS03 ( Taga and Walker, 2010). (C) Strain SmNrdAB+ on an LB CF plate with a filter disk containing hydroxyurea, photographed under white light. (D) Same as (C) but photographed under UV light. (E) Fluorescence of WT S. meliloti on an LB CF plate with a filter disk containing nalidixic acid. Filter disks were applied to the plates at the time of inoculation. (F) Schematic description of the physiological link between the bluB mutation and the CF fluorescence phenotype. The dashed line indicates that the mechanism is unknown.
Mentions: We previously found that the CFB phenotype of the S. meliloti bluB mutant can be rescued by the addition of DMB, which is used to produce cobalamin (Campbell et al., 2006; Taga et al., 2007). Bioassays, such as the bacterial assay that enabled the first purification of cobalamin (Rickes et al., 1948), can provide inexpensive, high-throughout, and highly sensitive means to detect molecules of interest (Kelleher and Broin, 1991). To investigate the possibility that this phenotype could be used to detect DMB, we first used a filter disk assay to examine the effect of DMB concentration on the CFB phenotype of a bluB mutant. This showed that the size and intensity of the CF-dim zone surrounding the DMB-soaked disks were positively correlated with the DMB concentration, suggesting that the CF phenotype could be used for quantification of DMB (Figure 2A).

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