D glucose

For this purpose, needles were obtained in batches from about 20 L total of bacteria growing in M9-type minimal media (Studier, 2005 (link)) supplemented with 2 g ml⁻¹¹⁵N labelled ammonium chloride and U–¹³C₆ labelled d-glucose (Cambridge Isotope Laboratories/CK Gas). Overnight cultures of 5 ml M9 minimal Media with labelled glucose and NH₄Cl, were inoculated with ΔmxiH (pACT3mxiH; (Shen et al., 2010 (link))), with kanamycin and chloramphenicol. The cultures were incubated at 37 °C with shaking at 180 rpm. After 16 hrs, the optical density at 600 nm (OD₆₀₀) was measured. If the OD₆₀₀ was >1.00, all of the 5 ml culture was used to inoculate 1 L of M9 minimal media, labelled as above, in a 5 L sterile conical flask, with 200 μM IPTG and incubated at 37 °C with shaking at 180 rpm. After 16 hrs, the OD₆₀₀ was measured again, if OD₆₀₀ was >1.10, growth was halted and needles purified as previously described (Cordes et al., 2005 (link), Fujii et al., 2012 (link)). The final pellet was resuspended in 0.01% of the initial culture volume in sterile 20 mM Tris–HCl pH 7.4, 100 mM NaCl, 10% w/v d-(+)-Trehalose (Sigma) and 0.02% w/v sodium azide, generally attaining 3–7 mg ml⁻¹ in protein concentration, and then flash frozen in liquid nitrogen and stored at −80 °C until use. From 20 L of bacterial culture about 7 mg of labelled needles were obtained.

B. subtilis wild-type or Δthi cells were cultured in the minimal medium containing [U-13C6, 99%] labeled D-glucose (1%) (Cambridge Isotope Laboratories, Inc.) in 200 ml with 0.4g shaking at 30°C for 2 d. Simultaneously, A. nidulans were cultured in minimal medium containing normal D-glucose in 200 ml for 2 d. The B. subtilis cells were collected by centrifuge, whereas the A. nidulans cells were collected using Mira cloth and washed. Then they were co-cultured in minimal medium containing D-glucose 200 ml for 2 d. The fungal cells were sieved through Mira cloth and washed thoroughly with milliQ water to remove any bacterial cells attached to the surface. Extraction of labeled thiamine from the cell extract followed the same protocol as of extraction of thiamine described above.

The ¹⁵N, ¹³C-labeled hMEX3B KH1, KH2, and KH1/2 proteins used for NMR experiments were expressed by growing the bacteria in LeMaster and Richards medium (2.5 g/L D-Glucose [Cat# CLM-1396-PK, Cambridge Isotope Laboratories, Inc.], 0.5 g/L ¹⁵NH₄Cl [Cat# NLM-467-PK, Cambridge Isotope Laboratories, Inc.], 24 g/L KH₂PO₄, 5 g/L NaOH, 100 μM CaCl₂, and 2.2 mM MgSO₄) and purified by the same procedure as described above. Backbone resonances of the KH1 and KH2 domains were assigned using 0.5 mM ¹⁵N,¹³C-labeled protein in NMR buffer (50 mM sodium phosphate, pH 6.5, 100 mM NaCl, 5 mM TCEP, 10% D₂O [Cat# DLM-7005, Cambridge Isotope Laboratories, Inc.]) using CBCANH, CBCA(CO)NH, HNCA, and HN(CO)CA on a Bruker DMX 600 MHz at 298 K. All NMR data were processed using NMR Pipe^{40 (link)}, and the spectra were assigned using Sparky^{41 (link)}. The chemical shift assignments for KH1 and KH2 have been deposited in BioMagResBank (http://www.bmrb.wisc.edu) under accession numbers 52147 and 52148, respectively.