Materials and Reagents

Sinorhizobium meliloti (S. meliloti 1021, Galibert et al., 2001)
Note: The protocol can be also applied to other bacteria.
Bio-Rad Protein Assay Kit I (Sigma-Aldrich, catalog number: 500-0001 )
50 mM Tris-HCl buffer (pH 7.5) (5 ml per sample)
1x BugBuster reagent (Novagen, catalog number: 70921 )
0.5 M EDTA (pH 8.0) (2.5 µl per sample)
GTP (Guanosine 5’-triphosphate sodium salt hydrate) (Sigma-Aldrich, catalog number: G8877 )
HPLC Standard: 6,7-Dimethylpterin (Schircks Laboratories, catalog number: 11.503 )
Biotin
YMB medium (Somerville and Kahn, 1983) (see Recipes) (1 plate per sample)
MMNH₄ medium (Somerville and Kahn, 1983) (see Recipes) (13 ml per sample)
Lysis buffer (see Recipes) (1 ml per sample)
Desalting buffer (see Recipes) (3 ml per sample)
RibA assay buffer (see Recipes) (2.5 µl per sample)
Derivatization reagent (see Recipes) (50 µl per sample)
HPLC mobile phase (see Recipes)
Note: Except as otherwise noted, all other chemicals were obtained from Sigma-Aldrich.

Equipment

14 ml Falcon tubes (BD Biosciences, catalog number: 352059 )
Eppendorf 1.7 ml tubes (Thermo Fisher Scientific, catalog number: 14-222-168 )
0.22 µm syringe filter (Microsolv Technology, catalog number: 58022-N04-C )
2 ml Zeba Spin Desalting Columns (Thermo Fisher Scientific, catalog number: 87768 )
Mini-centrifuge
Centrifuge
Shaker
HPLC: Waters Alliance 2695 HPLC system linked to a 2475 fluorescence detector
XTerra MS C18 column (4.6 x 100 mm, 5 µm) (Waters, part number: 186000486 )

Procedure

Cell extract preparation
1. Grow S. meliloti on YMB plate for 48 h at 30 °C. The optimal temperature for S. meliloti growth is between 28 °C and 30 °C.
2. Inoculate S. meliloti from the stock YMB plate to an OD₆₀₀ of ~0.1 in 3 ml MMNH₄ medium.
3. Grow cells for 48 h at 30 °C, 250 rpm in 14 ml Falcon or glass tubes.
4. Dilute the cells 20-fold into 10 ml fresh MMNH₄ medium and grow overnight at 30 °C, 250 rpm in 50 ml glass tubes or flasks.
5. Harvest cells from 10 ml culture by centrifugation at 3,600 x g for 15-20 min at 4 °C in 14 ml Falcon tubes.
6. Discard the supernatant and wash the cells with 5 ml 50 mM Tris buffer at 4 °C.
7. Resuspend the pellet in 1 ml of lysis buffer at 4 °C.
8. Incubate at room temperature for 15 min, without shaking.
9. Centrifuge the lysate at maximum speed for 15 min at 4 °C.
10. Measure protein concentration in the cell lysates using Bio-Rad Protein Assay Kit.
Enzymatic assay
This assay protocol was adapted from a method for assaying GTP cyclohydrolase II activity in the purified enzyme (Bacher et al., 1997) and modified for using with bacterial extracts.
1. Desalt the cell lysate using 2 ml Zeba Spin Desalting Columns following the manufacturer’s instructions. The column was equilibrated with 3 ml desalting buffer.
2. Add 25 µl of the desalted whole cell lysate form the previous step in a total volume of 50 µl reaction mixture containing 10 µl 5 x RibA assay buffer, 5 µl 10 mM GTP (prepare GTP in ultrapure water).
3. Incubate the assay at 37 °C for 30 min.
4. Terminate the assay by adding 2.5 µl EDTA (0.5 M, pH 8.0).
5. Derivatize the product by adding 50 µl of the derivatization reagent, followed by incubation at 70 °C for 20 min.
6. Clear the samples by centrifugation at 3,000 x g for 10 min at 4 °C.
7. Filter the supernatant using a 0.22 µm syringe filter.
8. Analyze the derivatization products using HPLC with fluorescence detection.
HPLC purification and signal detection
1. Separate the fluorescent products on a XTerra MS C18 column (4.6 x 100 mm, 5 µm) using a Waters Alliance 2695 HPLC system linked to a 2475 fluorescence detector. Excitation and emission wavelengths are 330 nm and 435 nm, respectively.
Data analysis
1. Quantify the products by comparison to standards. A standard curve was obtained by running sequential dilution of 6,7-dimethylpterin (0.25 nM, 2.5 nM, 25 nM, 0.25 µm, 2.5 µM and 25 µM) on HPLC.
2. Normalize the enzyme activity against protein concentration and assay incubation time in nmol/min/mg protein.
3. Perform three replicates on each assay. Data is the average ± S.E. of three replicates.
  Note: The activities of GTP cyclohydrolase II vary in different organisms. The activity of purified recombinant enzymes have been reported from about 0.1- 182 nmol/min/mg protein, the activity in bacterial extracts can be lower (Herz et al., 2000; Kaiser et al., 2002; Yurgel et al., 2014).

Representative data

Figure 1. HPLC chromatograms of 2.5 µM 6,7-dimethylpterin standard (A), product of GTP cyclohydrolase II assay from bacterial extracts (B) and co-elution of product from GTP cyclohydrolase II assay with 6,7-dimethylpterin standard (C). HPLC mobile phase: 10% methanol and 90% phosphoric acid (v/v). Excitation and emission wavelengths were 330 nm and 435 nm respectively.

Recipes

YMB media for Rhizobium

1 L
Concentration

Yeast Extract
1 g

Mannitol
10 g
54.9 mM

Agar
15 g

Autoclave, cool to 55 °C, then add

YMB Salt I
10 ml

YMB Salt II
10 ml

YMB Salt I
1 L
Concentration

K₂HPO₄
50 g
287.06 mM

NaCl
10 g
171.15 mM

d-H₂O
960 ml

YMB Salt II
1 L
Concentration

MgSO₄^.7H₂O
20 g
81.11 mM

d-H₂O
1 L

MMNH₄ (minimal mannitol ammonia media for Rhizobium)

	per 1 L	Concentration
Mannitol	10.0 g	54.9 mM
NH₄Cl	0.5 g	9.34 mM
Agar (for plates preparation)	15.0 g
d-H₂O	970 ml

Autoclave, cool to 55 °C, then add:

Biotin (0.2 mg/ml in 50% EtOH)	1.0 ml
Thiamine (2 mg/ml), filter sterilized	1.0 ml
Min Man Salts I	10.0 ml
Min Man Salts II	10.0 ml

Min Man Salts I

	per 1 L	Concentration
K₂HPO₄	100 g	574.12 mM
KH₂PO₄	100 g	734.8 mM
Na₂SO₄	25 g	174.8 mM
d-H₂O	1 L

Min Man Salt II

	per 1 L	Concentration
FeCl₃^.6H₂O	1.0 g	3.7 mM
Concentrated HCl	adjust pH to ~7.0 (~1drop)
CaCl₂^.2H₂O	10.0 g	68 mM
MgCl₂^.6H₂O	25.0 g	123 mM
d-H₂O	1 L
Autoclave

Lysis buffer
50 mM Tris-HCl (pH 7.5)
10 mM MgCl₂
1 mM Tris (hydroxypropyl) phosphine
1x BugBuster reagent
Desalting buffer
50 mM Tris-HCl (pH 7.5)
1 mM Tris (hydroxypropyl) phosphine
10% glycerol
RibA assay buffer
100 mM Tris-HCl (pH 8.5) buffer
5 mM MgCl₂
5 mM dithiothreitol (DTT)
Derivatization reagent
1% (v/v) diacetyl
15% (w/v) trichloroacetic acid
HPLC mobile phase
10% (v/v) methanol
90% phosphoric acid (27 mM phosphoric acid)

Acknowledgments

This protocol was adapted from Yurgel et al., 2014. This work was supported by the Agricultural Research Center (WNP-00773) at Washington State University and grant DE-FG0396ER20225 Vol. 27, No. 5, 2014 / 445 from the Energy Biosciences Program at the United States Department of Energy, and by grant NSF-MCB 1052492 to S. Rajamani. This activity was funded, in part, with an Emerging Research Issues Internal Competitive grant from the Agricultural Research Center at Washington State University, College of Agricultural, Human, and Natural Resource Sciences and Biologically-Intensive Agriculture and Organic Farming (BIOAg) Internal Competitive grant from The Center for Sustaining Agriculture and Natural Resources (CSANR) at Washington State University to S. Yurgel. We thank M. Kahn for discussions and the Washington State University Laboratory for Biotechnology and Bioanalysis for sequencing support.

References

Bacher, A., Richter, G., Ritz, H., Eberhardt, S., Fischer, M. and Krieger, C. (1997). Biosynthesis of riboflavin: GTP cyclohydrolase II, deaminase, and reductase. Methods Enzymol 280: 382-389.
Galibert, F., Finan, T. M., Long, S. R., Puhler, A., Abola, P., Ampe, F., Barloy-Hubler, F., Barnett, M. J., Becker, A., Boistard, P., Bothe, G., Boutry, M., Bowser, L., Buhrmester, J., Cadieu, E., Capela, D., Chain, P., Cowie, A., Davis, R. W., Dreano, S., Federspiel, N. A., Fisher, R. F., Gloux, S., Godrie, T., Goffeau, A., Golding, B., Gouzy, J., Gurjal, M., Hernandez-Lucas, I., Hong, A., Huizar, L., Hyman, R. W., Jones, T., Kahn, D., Kahn, M. L., Kalman, S., Keating, D. H., Kiss, E., Komp, C., Lelaure, V., Masuy, D., Palm, C., Peck, M. C., Pohl, T. M., Portetelle, D., Purnelle, B., Ramsperger, U., Surzycki, R., Thebault, P., Vandenbol, M., Vorholter, F. J., Weidner, S., Wells, D. H., Wong, K., Yeh, K. C. and Batut, J. (2001). The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293(5530): 668-672.
Herz, S., Eberhardt, S. and Bacher, A. (2000). Biosynthesis of riboflavin in plants. The ribA gene of Arabidopsis thaliana specifies a bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase. Phytochemistry 53(7): 723-731.
Kaiser, J., Schramek, N., Eberhardt, S., Puttmer, S., Schuster, M. and Bacher, A. (2002). Biosynthesis of vitamin B2. Eur J Biochem 269(21): 5264-5270.
Somerville, J. E. and Kahn, M. L. (1983). Cloning of the glutamine synthetase I gene from Rhizobium meliloti. J Bacteriol 156(1): 168-176.
Yurgel, S. N., Rice, J., Domreis, E., Lynch, J., Sa, N., Qamar, Z., Rajamani, S., Gao, M., Roje, S. and Bauer, W. D. (2014). Sinorhizobium meliloti flavin secretion and bacteria-host interaction: role of the bifunctional RibBA protein. Mol Plant Microbe Interact 27(5): 437-445.