Genetic basis of thiaminase I activity in a vertebrate, zebrafish Danio rerio

Sequence analysis

Protein sequence searches were conducted in the GenBank nr database with BLASTP42 using default parameters, including automatically adjusting parameters for short input sequences (Table S1). Conserved domain searches were run against the GenBank Conserved Domain Database (CDD)43. Sequence alignments were conducted in CLC Main Workbench 20.0.4 (Qiagen) with the fast alignment algorithm, gap open cost = 10, and gap extension cost = 1. Biochemical properties of the fish putative thiaminase I protein sequences were predicted with the Create Sequence Statistics function in CLC Main Workbench 20.0.4 (Qiagen, Hilden, Germany). The molecular weights were calculated from the sum of the amino acids in the sequence, and the isoelectric points (pIs) were calculated from the pKa values ​​for the individual amino acids in the sequence.

Bacteria culture

Pure cultures of P.thiaminolyticus strain 818822 were cultured at 37 °C in Terrific Broth (MO BIO Laboratories, Carlsbad, CA) in either a shaking incubator or in a beveled flask with a stir bar and were harvested after 48–80 h of culture. Upon harvest, cultures were processed immediately or frozen whole in 50 mL Falcon tubes at − 80 °C. Fresh or thawed cultures were spun at 14,000×Gand culture supernatant was concentrated using Amicon-ultra 10 kDa molecular weight cut-off (MWCO) filters (EMD Millipore, Billerica, MA).

The zebrafish and alewife candidate thiaminase I genes were cloned and overexpressed in E. coli to determine whether they produced functional thiaminases. The recombinant thiaminase I gene from P.thiaminolyticus was overexpressed in E. coli as a positive control. Candidate and control genes were synthesized (Integrated DNA Technologies, Inc., Coralville, Iowa) and placed into the pET52b vector (EMD Millipore). Insert sequences are provided in Supplementary Figs. S10-S13. The empty pET52b vector was used as a negative control. The plasmid was transformed into E. coli (Rosetta 2(DE3)pLysS Singles Competent Cells, EMD Millipore) according to the manufacturer’s instructions, and expression of candidate genes was induced by the addition of IPTG. Cells were lysed in 1X BugBuster (Millipore) according to the manufacturer’s instructions in the presence of benzonase nuclease, and soluble and insoluble fractions were separated by centrifugation.

tissue collections

Adult common carp were captured from Lake Erie using short-set gill nets. Adult alewife and quagga mussels (Dreissena bugensis) were collected from Sturgeon Bay, Lake Michigan using bottom trawls. Fish collections were completed during July 2007. Sex of sampled fish was not identified. Upon collection, unanesthetized animals were immediately euthanized by flash freezing between slabs of dry ice and stored at − 80 °C. Fish were harvested by the Great Lakes Science Center, US Geological Survey (USGS). Laboratory use of frozen animal tissues and wild type and recombinant bacteria was in accordance with institutional guidelines and biosafety procedures at Oregon State University and USGS. Animal care and use procedures were approved by the Great Lakes Science Center, USGS. All USGS sampling and handling of fish during research are carried out in accordance with guidelines for the care and use of fishes by the American Fisheries Society44. All methods are reported in accordance with applicable ARRIVE guidelines ( Zebrafish from OSU’s zebrafish facility were anesthetized and euthanized by overdose with waterborne 200 ppm ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma-Aldrich, St. Louis, MO) following protocols approved by the OSU Animal Institutional Care and Use Committee and were frozen at − 80 °C after euthanization. Gills, liver, spleen, and the intestinal tract were dissected, and gill tissue was homogenized separately from liver, spleen, and gut, which were homogenized together and designated “viscera.” Homogenization and protein preparation procedures were the same as that for alewife. Zebrafish from Columbia Environmental Research Center (CERC), USGS cultures were anesthetized and euthanized by overdose with 200 ppm ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma-Aldrich, St. Louis, MO) in water following protocols approved by CERC Institutional Animal Care and Use Committee (IACUC). Whole fish (0.2–0.6 g) were homogenized in 10 mL cold phosphate buffer, pH 6.5. Whole common carp and alewife were thawed until they could just be dissected. Preliminary extraction trials on alewife stomach and intestines, spleen, and gills revealed similar results and revealed that gills and spleen tissue produced the cleanest protein preparations. Therefore, subsequent extractions for common carp and alewife used gill tissue. Samples were pooled from 3 to 5 individual fish, haphazardly chosen from the sampled fish without exclusions. Quagga mussels were thawed just sufficiently to be husked from their shell and were used whole. Researchers were aware of the species and tissue designation of each sample throughout the experiments. Animal tissues were placed in ice-cold (4 °C) beakers containing cold extraction buffer (16 mM K3HPO484mM KH2PO4, 100 mM NaCl, pH 6.5 with 1 mM DTT, 2 mM EDTA, 3 mM Pepstatin, 1X Protease inhibitor cocktail (Sigma), and 1 mM AEBSF). All extractions were carried out at 4 °C in pre-chilled glassware. Samples were mechanically homogenized using a rotor-stator tissue grinder. Samples were gently stirred for several hours to overnight at 4 °C, centrifuged at 14,000×G to remove debris, and strained through cheesecloth to remove any insoluble lipids. Extracts were then subjected to 30–75% ammonium sulfate precipitation. Pellets from the precipitation were resuspended in buffer (83 mM KH2PO417mM K2HPO4and 100 mM NaCl), centrifuged to remove any remaining debris, and stored in 30% glycerol at − 20 °C.

protein electrophoresis

Native PAGE was run using either pre-cast TGX gels (BioRad, Hercules, California) of varying percentage (7.5% to 12% or 8–16% gradient gels) or on hand-cast gels (TGX FastCast, BioRad) made according to the manufacturer’s instructions.

Blue-native PAGE was used to estimate the mass of thiaminases in their native conformation. Blue native PAGE45 gels were run using the NativePage Novex Bis–Tris system (Life Technologies) or hand-cast equivalents46. Light blue cathode buffer was used to facilitate visualization of the activity stain.

Standard denaturing SDS-PAGE was used to estimate the molecular mass of thiaminases after denaturation. Denaturing SDS-PAGE was run using one of three relatively equivalent methods: pre-cast TGX gels (BioRad) according to the manufacturer’s instructions, hand-cast Tris-HCl gels using standard Laemmli chemistry47 with an operating pH of approximately 9.5, or hand-cast Bis–Tris gels (MOPS buffer) with an operating pH of approximately 7. For all denaturing and non-denaturing SDS-PAGE applications, standard Laemmli sample buffer was used, and samples were heated to 75 °C for 15 min to facilitate denaturation followed by brief centrifugation to eliminate any precipitated debris.

Non-denaturing PAGE was used as an alternative to denaturing PAGE for the common carp thiaminase that could not be renatured (ie, activity could not be recovered) following a denaturing SDS-PAGE. Non-denaturing PAGE was conducted using any of the three aforementioned gel chemistries with SDS-containing running buffers including reductant (DTT), but samples were not heated prior to application to the gel. Samples for non-denaturing PAGE were allowed to incubate in sample buffer at room temperature for 30 min prior to gel loading. This preserves the charge-shift induced by SDS but does not result in protein denaturation, facilitating in-gel analysis of thiaminase I activity after separation.

To visualize proteins following electrophoresis, gels were stained with Coomassie stain (CBR-250 at 1 g/L in methanol/acetic acid/water (4:5:1) and destained with methanol/acetic acid/water (1.7:1:11.5 Mini-gels were run on BioRad’s mini-protean gel rigs, Midi-gels (16 cm length) were run on Hoefer’s SE660, and large-format gels (32 cm length) were run on a BioRad’s Protean Slab Cell. Blue-native PAGE was always run at 4 °C.

Two-dimensional electrophoresis (2DE) proteins separated in the first dimension based on pI and in the second dimension based on mass (either native or denatured). 2DE was performed by combining in-gel IEF with either denaturing SDS-PAGE, non-denaturing SDS-PAGE, or native PAGE. IPG strips were incubated in TRIS-buffered equilibration solution48 either with 6 M urea, SDS, and iodacetamide (denaturing) or without urea, SDS, and iodacetamide (non-denaturing) for 20 min. Low melting point agarose was used to solidify IGP strips in place. Agarose was cooled to just above the gelling temperature, as hot agarose inactivated thiaminase I activity.

Isoelectric focusing

Isoelectric focusing (IEF) was conducted both in-gel and in-liquid. In-gel IEF was conducted in immobilized pH gradient (IPG) strips using a Multifor II (GE Healthcare Life Sciences). Prior to rehydration, all protein preparations were desalted in low-salt (~ 5 to 10 mM) sodium or potassium phosphate buffer (pH 6.5) using 10 kDA MWCO filters. All samples were applied using sample volumes and protein concentrations recommended by the manufacturer. For standard denaturing in-gel IEF, rehydration solution consisted of 8 M urea, 2% CHAPS, 2% IPG buffer of the appropriate pH range, 1% bromophenol blue, and 18 mM DTT. The IEF was conducted at a maximum of 2 mA total current and 5 W total power, with an EPS3500 XL power supply in gradient mode. Voltage gradients were based on standard protocols recommended by the manufacturer. In-gel IEF was also performed under native conditions to allow thiaminase I activity staining of IPG strips. Protocols were essentially the same as those for denaturing conditions, with the following exceptions: (1) urea was eliminated and the CHAPS concentration was reduced to 0.5% in the rehydration solution; (2) rehydration was conducted at 14°C; and (3) the water in the cooling tray was cooled to 4 °C.

In-liquid IEF was conducted using a Rotofor (BioRad) according to the manufacturer’s instructions. Non-denaturing in-liquid IEF was also conducted using a focusing solution including no urea, 2% pH 3-10 biolyte, 0.5% CHAPS, 20% glycerol, and 5 mM DTT. The addition of glycerol helped retain activity but also increased focusing times. The Rotofor was run at a constant 15 W with a maximum current of 20 mA and voltage set for a maximum of 2000 V. Samples containing 8 M urea were cooled to 14 °C while focusing to avoid urea precipitation, samples lacking urea were cooled to 4 °C during focusing. Protein extracts in salt solutions greater than 10 mM were destroyed directly in focusing solution using a 10 kDA MWCO filter. Focusing runs were allowed to proceed until the voltage stabilized and fractions were harvested with the needle array and vacuum pump. Ampholytes were removed by addition of NaCl to 1 M and then samples were desalted into phosphate buffer using a 10kD MWCO filter.

Thiaminase I activity measurements

For quantitative measurements of thiaminase I activity, we conducted a radiometric assay at CERC as previously described49. Zebrafish homogenates were diluted 1:8, 1:16, or 1:32 in cold phosphate buffer, pH 6.5. Two replicates per dilution were assayed. Activity was calculated from the greatest dilution that gave activity within the linear range of the assay and was reported as pmol thiamine consumed per g tissue (wet weight) per minute (pmol/g/min).

Thiaminase I activity staining

After electrophoresis, gels were stained for thiaminase I activity using a previously described diazo-coupling reaction19.50. Briefly, gels were washed 3 times in water, twice in 25 mM sodium phosphate buffer with 1 mM DTT, and once in 25 mM sodium phosphate buffer without DTT. Gels were then incubated in 0.89 mM thiamine-HCl and co-substrate (1.45 mM pyridoxine, 24 mM nicotinic acid, or 20 mM pyridine) in 25 mM sodium phosphate buffer for 10 min. Gels were briefly rinsed in water and placed in a lidded container and incubated at 37 °C for 30 min to allow thiamine degradation by any thiaminases in the gel. The diazo stain19.50 was then applied to detect remaining thiamine in the gel for five minutes with gentle agitation. Stained gels were rinsed with water and photographed, and further stained with Coomassie to visualize proteins.

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