Quantitative detection of vitamin B12 in algae by bioassay and ICP-MS/MS

Sunnyjoy Dupuis, Stefan Schmollinger, Sabeeha S. Merchant

Published: 2022-04-21 DOI: 10.17504/protocols.io.14egn7726v5d/v1

Disclaimer

E. coli strains were generously provided by Michi Taga.

Abstract

This protocol describes two methods for determining the amount of vitamin B₁₂ present in the spent medium and cell lysate of algae cultures. The first method is a bioassay, adapted from Mok, Hallberg, & Taga (2022), which estimates the B₁₂ concentration in solution from the growth of a B₁₂-requiring Escherichia coli mutant. The second method uses the direct detection of cobalt via Inductively Coupled Plasma Mass Spectrometry (ICP-MS/MS) as a proxy for vitamin B₁₂. We describe the preparation of spent medium and cell extract fractions from the chlorophyte alga Chlamydomonas reinhardtii for each method, preparation of standard cyanocobalamin solutions, and the correlation between cobalt and cyanocobalamin in algal cells.

We thank Michi Taga and Alison Smith for their guidance in optimizing the bioassay for C. reinhardtii .

Citation

Kenny C. Mok, Zachary F. Hallberg, Michiko E. Taga 2022 Purification and detection of vitamin B12 analogs Methods in Enzymology https://doi.org/10.1016/bs.mie.2021.11.023

Citation

Kirmiz N, Galindo K, Cross KL, Luna E, Rhoades N, Podar M, Flores GE 2020 Comparative Genomics Guides Elucidation of Vitamin B12 Biosynthesis in Novel Human-Associated Akkermansia Strains. Applied and environmental microbiology https://doi.org/pii:e02117-19.10.1128/AEM.02117-19

Steps

E. coli B12 Bioassay

Note

This protocol makes use of the Escherichia coli ΔmetE and ΔmetE ΔmetH mutant strains. MetE is the cobalamin-independent homocysteine transmethylase, and MetH is a cobalamin-dependent methionine synthase. The E. coli ΔmetE strain requires either methionine or vitamin B₁₂supplementation for growth. The ΔmetE ΔmetH strain can only grow when provided methionine. Samples of interest are provided to both ΔmetE and ΔmetE ΔmetH , and the growth of each is compared to that supported by either vitamin B₁₂ standards or methionine standards, respectively. Thus B₁₂ concentration in a solution can be measured and can be distinguished from methionine.

Prepare cell culture fractions:

Note

The following procedure is effective for the preparation of spent medium and cell lysate from both the chlorophyte Chlamydomonas reinhardtii (both cell wall containing and cell wall reduced strains) as well as the α-proteobacteria Mesorhizobium loti and Sinorhizobium meliloti when grown in minimal media.

2.1.

Collect 2mL culture into a 2 ml screw-cap tube.

Note

We use USA Scientific 2.0ml Self-Standing Grad Microcentrifuge Tubes, Catalogue Number 1420-9700.Snap-cap tubes can be used if desired, but are prone to popping open during boiling (Step 2.5). If snap-cap tubes are used, secure lids with a special tube rack or a weight while boiling.

2.2.

Centrifuge at >8000rcf. Quickly transfer 950µL of the spent medium supernatant to each of two 1.5 ml screw-cap tubes without disturbing the pellet.

Note

Work quickly to prevent cells from dispersing from the pellet and contaminating the supernatant fraction. This is especially important for motile strains.

2.3.

To wash the pellet, resuspend in 1mL 0.85% NaCl, then centrifuge again at >8000rcf and discard the supernatant.

2.4.

Resuspend the pellet in 1.9mL 0.85% NaCl.

2.5.

Boil both cell suspension and spent medium fractions at 100°C for 0h 10m 0s to extract B₁₂ from the cells.

2.6.

Centrifuge all samples at >8000rcf,4°C.

Note

This centrifugation step should be performed cold to decrease the temperature of the samples before further handling.

2.7.

Transfer the supernatant into convenient portions (2 or more) for downstream testing.

Note

This is to avoid repeated freeze-thaw of samples.

2.8.

Flash freeze all samples in liquid nitrogen to store at -80°C.

Note

Ensure tubes are sealed tightly to avoid sample loss during freezing or thawing.

Prepare cyanocobalamin (CNCbl) and methionine standards:

3.1.

Prepare a series of CNCbl standards up to 10X the target concentration in purified water (Millipore) by serial dilution. Aliquot at least 250µL of each standard into screw-cap tubes.

Note

We have found serial dilutions to the following concentrations produces a useful range of 10X CNCbl standards: 900, 600, 400, 267, 178, 119, 79, 53, 35, 23 ng/L.Standards can also be prepared in minimal media that were used for test samples if desired. However, this should not impact results, since B₁₂ will be the limiting nutrient for E. coli growth during the bioassay.

3.2.

Prepare a series of 1mL methionine standards to 10X the target concentration in milliQ H₂O.

Note

We have found serial dilutions of the following concentrations produces a useful range of 10X methionine standards: 300, 150, 75, 38, 19 mg/L

3.3.

Boil standards at 100°C for 0h 10m 0s.

Note

This ensures that any degradation of B₁₂ or methionine that may occur in the samples also occurs in the standards. However, we have shown that boiling for 10 min does not significantly decrease the amount of CNCbl detected via this bioassay:

Boiling does not impact B12 detected by bioassay.

3.4.

Centrifuge the standards at >8000rcf,4°C.

3.5.

Flash freeze the standards in liquid nitrogen to store at -80°C.

Conduct the bioassay:

4.1.

Inoculate starter cultures from single colonies of Δ metE and Δ metE Δ metH mutant strains from LB agar plates into2mL of M9 + 0.2% glucose minimal medium with 1 mg/mL methionine. Grow at 37°C 250rpm for 24h 0m 0s to saturation.

Note

We use Falcon 14 mL Polystyrene Round-Bottom Tubes, Catalogue Number 352051 for all E. coli cultures.

Note

M9 recipe can be found here: M9 recipe can be found here: http://cshprotocols.cshlp.org/content/2010/8/pdb.rec12295.short

4.2.

Generate pre-cultures by transferring 1% (vol/vol) of the saturated starter cultures into fresh 2mL M9 + 0.2% glucose minimal medium with 1 mg/mL methionine and grow at 37°C 250rpm for 24h 0m 0s to saturation.

Note

Each bioassay culture will require ~20 uL of 2X pre-culture (Step 4.7), so plan the number of 2 mL pre-cultures you will need to provide sufficient inoculum for the bioassay.

4.3.

On the day of the assay, prepare bioassay cultures: add 1mL of 2X M9 glucose, the desired amount of your sample or standard, and sterile Millipore water up to 2mL final volume.

The following control and test cultures should be included:

Sample to be inoculated with Δ metE
Sample to be inoculated with Δ metE Δ metH
M9 medium alone without B₁₂ or methionine supplementation for each E. coli strain (important to test efficacy of E. coli innocula washing)
B₁₂ and methionine standard series for each E. coli strain (important to include with every bioassay you perform, as maximum OD₆₀₀ of strains can vary slightly from assay to assay)
Controls for the highest concentrations of B₁₂ and methionine in the standard series, and for one unknown sample, that will not be inoculated with E. coli (important to test for contamination or failure to kill study species during sample preparation)
Note
Complete this step prior to harvesting E. coli pre-cultures.

4.4.

Next, harvest 2 mL of each E. coli pre-culture in snap-cap tubes and pellet by centrifuging at 10000rcf. Discard the supernatant.

4.5.

Resuspend in 1mL sterile 0.85% NaCl, and repeat this wash twice more.

4.6.

Resuspend washed pellet in 1mL 0.85% NaCl then measure OD₆₀₀.

4.7.

Use washed cells to inoculate the bioassay tubes at a starting OD₆₀₀ of 0.01.

Note

The volume of washed inocula needed per should be 10-30 uL.

4.8.

Incubate cells at 37°C 250rpm for 24h 0m 0s, then read final OD₆₀₀.

4.9.

Establish a standard curve that correlates the provided B₁₂ and methionine concentrations in the standards to the OD_{600 of each E. coli strain} E. coli strain. Determine the amount of B₁₂ and methionine in your unknown samples by comparing the OD₆₀₀ reached by each strain to the standard curves.

Citation

Example standard curves for cyanocobalamin and methionine.

Sample preparation for ICP-MS Detection of B12-Derived Cobalt

Collect and wash samples:

Note

This protocol is effective for preparing spent media and cells of Chlamydomonas reinhardtii for ICP-MS/MS elemental analysis. The recommended cell densities are useful to ensure effective cell digestion and proper signal-to-noise for detecting elements of interest.It is important to work in a clean environment free from dust, which could contaminate samples. All reagents should be prepared with trace-metal grade chemicals, water, and vessels. Avoid unnecessary exposure of samples, tubes, and pipettes to air.If clean tubes and pipettes are not available, they can be prepared using the following steps:Soak pipettes and tubes in detergent overnight.Rinse thoroughly with de-ionized water.Submerge in 10 % nitric acid (normal grade) and soak for a week at 50°C.Rinse thoroughly with Millipore grade water. Avoid unnecessary exposure of opened tubes to air.

5.1.

Collect 1x10⁸ cells in a 50ml falcon tube.

5.2.

Centrifuge samples 3500rpm. Quickly transfer the supernatant to a clean tube for spent medium analysis.

Note

Work quickly to prevent cells from dispersing from the pellet and contaminating the supernatant fraction. This is especially important for motile strains.

5.3.

Resuspend the pellet in 5-10mLof 1mM EDTA pH 8.0 (Washing Buffer 1), then fill up to 50mL.

Note

Resuspend cell pellets by swirling or rotating the tube in a large radius with your arms, rather than pipetting or vortexing. This ensures cells will not be sheared during the wash steps.

5.4.

Repeat wash step: centrifuge samples 3500rpm. Quickly discard the supernatant, then resuspend pellet in 5-10mLof Washing Buffer 1, and fill up to 50mL.

5.5.

Wash cells again: centrifuge samples 3500rpm. Quickly discard the supernatant, then resuspend pellet in 1mLof Washing Buffer 1, then transfer to a 15 ml Falcon tube. To ensure all cells have been collected, add 5mL more Washing Buffer 1 to the 50 ml Falcon tube and transfer to the same 15 ml Falcon tube.

5.6.

Centrifuge samples 3500rpm. Quickly discard the supernatant, then resuspend the pellet in 2-5mL of Millipore water, then fill up to 10mL of Millipore water.

5.7.

Centrifuge samples 3500rpm. Quickly discard the supernatant. Then centrifuge samples 3500rpm, and remove the remaining supernatant completely using a filtered pipette tip.

5.8.

Store the dry pellet and the spent medium at -20°C until further processing.

Digest cell pellet samples:

6.1.

Thaw pellet at 65Room temperature, then centrifuge again 3500rpm to compact the pellet.

6.2.

Carefully overlay the pellet with 286µL trace metal grade nitric acid (we use Fischer Chemical Trace Metal Grade Nitric Acid, Catalogue Number A509-P212).

Note

Take care to leave the compacted pellet undisturbed, as any cell material dissolving from the pellet will not be digested properly.

6.3.

Digest samples for 24h 0m 0s at 65Room temperature, then incubate for 2h 0m 0s at 65°C.

6.4.

Add 9.5mL Millipore water for 2 % HNO3 final concentration. Vortex the sample thoroughly.

Samples are now ready for ICP-MS.

Prepare spent media samples: transfer 2mL spent medium to a 15ml Falcon tube, and add 5mLof 2.8% HNO3 for 2% final concentration. Spent media samples are now ready for ICP-MS.

(A) Chemical structure of cyanocobalamin showing coordinated cobalt atom. (B) Correlation between intracellular cobalt detected by ICP-MS, normalized per sulfur as a proxy for biomass, and intracellular B12 detected using the E. coli bioassay in C. reinhardtii.Note that ICP-MS detection of vitamin B12 is only suitable for eukaryotic cells that are not supplied with any other source of cobalt in their growth medium.

45.

Prepare (CNCbl) and standards:

Quantitative detection of vitamin B12 in algae by bioassay and ICP-MS/MS

Disclaimer

Abstract

Steps

E. coli B12 Bioassay

Sample preparation for ICP-MS Detection of B12-Derived Cobalt

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