Media and chemicals used
The media used in this study were: minimal medium (MM) = six mineral salts and 10.0 g/L sucrose , MSB = six mineral salts containing 0.5% Bacto Peptone , MSC = MSB containing reduced quantity (0.3%) Bacto Peptone, MSY = six mineral salts + 0.5% yeast extract, MS-DON = six mineral salts + 200 μg/mL DON, SE = sterile water-soluble soil extract , PDB = potato dextrose broth, LB = Luria Bertani, NB = nutrient broth and 1/10 NB = one-tenth strength NB. The pH of the media was adjusted as needed before autoclaving. Solid media were prepared by adding 1.5% agar. The media ingredients and prepared media were purchased from Sigma-Aldrich (Oakville, ON, Canada), Fisher Scientific (Fair Lawn, New Jersey, USA), and/or Difco (Sparks, Maryland, USA). All trichothecene mycotoxins were obtained from Biomin (Romer Lab, Union, MO, USA).
Enrichment and functional stability of trichothecene de-epoxidizing microorganisms
A soil microbial culture having low level of DON de-epoxidation activity originating from southern Ontario, Canada was used for enrichment . An aliquot (20 μL) of the culture was transferred to a 1.5 mL eppendorf tube containing 0.5 mL MSB (pH 7.0). The culture was spiked with 200 μg/mL pure DON and incubated with continuous shaking at 220 revolutions per minute (rpm) at room temperature. After 7–10 days of incubation, an aliquot (10 μL) of the culture was transferred to fresh MSB containing 200 μg/mL DON. In negative controls no DX100 was added. This procedure was repeated twelve times and the microbial consortium was designated DX100. Before every transfer to fresh MSB the de-epoxidation activity of the culture was examined by using a standard method, liquid chromatography-ultraviolet-mass spectrometry (LC-UV-MS) . Briefly, equal volumes of the bacterial culture and methanol were mixed and the samples were incubated for 30 min at room temperature to lyse the bacterial cells. Insoluble cell materials and media components in the mixture were eliminated by passing it through a 0.45 μm polyvinylidene difluoride membrane filter (Whatman®, Florham Park, New Jersey, USA). The percentage of DON to dE-DON (de-epoxy deoxynivalenol), also known as DOM-1, transformation in the filtrate was determined by LC-UV-MS technique .
To determine the stability of the microbial de-epoxidation activity an active culture was subcultured for one hundred generations in MSB containing 50 μg/mL DON. The final subculture was tested for its de-epoxidation activity using LC-UV-MS technique.
16S rRNA metagenomic library preparation and sequencing
Genomic DNA was extracted from the highly enriched bacterial culture using Gentra Puregene Yeast/Bacteria Kit (Qiagen Inc., Mississauga, Ontario, Canada). Amplicon libraries of the V3-4 region of the 16S rRNA gene were prepared following the procedure of Illumina 16S Metagenomic Sequencing Library Preparation Guide (Rev. B). Briefly, a ~ 550 bp fragment of the 16S rRNA V3-4 region was amplified using the primers Bakt 341F (5-cctacgggnggcwgcag) and Bakt 805R (5-gactachvgggtatctaatcc) . The Bakt 341F and Bakt 805R primers contained 5-tcgtcggcagcgtcagatgtgtataagagacag and 5-gtctcgtgggctcggagatgtgtataagagacag 5’ Illumina overhang adapter sequences, respectively. Each 25 μl volume reaction mixture contained 12.5 ng of template DNA, 200 nM of each primer and 1x KAPA HiFi HotStart ReadyMix (VWR, Mississauga, ON, Canada). PCR was performed following the conditions: 95 °C (3 min), 25 cycles of 95 °C (30 s), 55 °C (30 s) and 72 °C (30 s), followed by 72 °C (5 min). The PCR amplicons were purified using Ampure XP beads (Beckman Coulter, Mississauga, ON, Canada). Sequencing adapters containing 8 bp indices were incorporated into the 3’ and 5’ ends of the purified amplicons by PCR using the Nextera XT Index kit (Illumina, San Diego, CA, USA). The 50 μl PCR reaction contained 5 μl PCR amplicon, 5 μl each indexing primer, and 25 μl 2x KAPA HiFi HotStart ReadyMix. PCR was performed under the following conditions: 95 °C (3 min), 8 cycles of 95 °C (30 s), 55 °C (30 s) and 72 °C (30 s), followed by 72 °C (5 min). The PCR amplicons were purified with Ampure XP bead and quantified using the Quant-iT PicoGreen double-stranded DNA assay (Invitrogen/Life Technologies Inc., Burlington, ON, Canada). After pooling equimolar amounts of amplicons, 5% equimolar of PhiX DNA (Illumina) was added, and sequencing was performed on an Illumina MiSeq instrument, using the Illumina MiSeq 600-cycle v3 kit. Taxonomic composition of the sequences was determined with Illumina MiSeq Reporter v.2.5.1 using the metagenomics workflow.
Transformation of A- and B type trichothecene mycotoxins
DX100 was examined for its ability to degrade five type-A trichothecene mycotoxins (diacetoxyscirpenol, HT-2 toxin, neosolaniol, T-2 toxin and T2-triol) and five type-B trichothecene mycotoxins (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, fusarenonX, nivalenol and verrucarol). One hundred and fifty micrograms of each mycotoxin were added to 1.5 mL NB in 5 mL microcentrifuge tubes (Sigma Aldrich), resulting in a 100 μg/mL mycotoxin solutions. Each tube was inoculated with 100 μL of an overnight DX100 culture, and the cultures in NB + mycotoxin were incubated at 28 °C with continuous shaking at 180 rpm, while the negative controls lacked DX100. The percentage of trichothecene transformation to different compounds (including de-epoxy and de-acetylated forms) by the culture was analyzed . Five replicate cultures were evaluated for each trichothecene mycotoxin.
Effects of DON concentration, substrate, temperature, pH, and aeration on microbial de-epoxidation activity
To determine the effects of DON concentration on the growth and de-epoxidation activity of DX100, tubes containing 3 mL of NB or MSB spiked with low (20 μg/mL), high (200 μg/mL) or very high (500 μg/mL) levels of DON were inoculated with DX100. The cultures were grown for 16 h with continuous shaking at 220 rpm and then the cell density was adjusted to 0.25 OD600nm (107 cells/mL). While incubating the culture at 28 °C with shaking at 180 rpm, 1.5 ml was removed every 12 h and the OD600nm was measured. The same volume of microbial culture (bacteria and broth) was analyzed for de-epoxidation activity by LC-UV-MS technique . Three replicate cultures for each mycotoxin were tested.
The effect of oxygen was evaluated by inoculating 5 mL of NB or MSB in 15 mL falcon tubes with an overnight culture of DX100. A moderate amount of DON (50 μg/mL) was added to the cultures and they were incubated at 28 °C with shaking at 180 rpm. Bacterial growth (OD600nm) and de-epoxidation activity were determined every 6 h . The assay was repeated with MSB medium under anaerobic conditions . Three replicate cultures were considered for each experiment.
To examine the effects of substrate on microbial de-epoxidation activity DX100 was grown at 28 °C for 72 h in NB, 1/10 NB, MM, MS-DON, MSB, MSC, MSY, SE, PDB or LB containing 50 μg/mL DON. The media pH values for this assay were maintained at pH 7.0 ± 0.2. To evaluate the effect of temperature, MSB containing 50 μg/mL DON was inoculated with DX100 and incubated at 10, 15, 20, 25, 30, 35, 40, 45 or 50 °C. The effect of pH on growth and de-epoxidation of DX100 was studied with MSB + 50 μg/mL DON adjusted to pH 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0, and the cultures were grown at 28 °C for 72 h. Three replicate cultures were tested for each experiment.
Effects of bacterial energy inhibitor, and localization of microbial de-epoxidation enzyme
In order to determine if the trichothecene de-epoxidation is bacterial and enzymatic, overnight cultures of DX100 grown in MSB containing 50 μg/mL DON were treated with 0.01% and 0.001% (w/v) of the bacterial energy inhibitor, sodium azide (NaN3) (Sigma-Aldrich), which blocks gram-negative bacterial electron transport . Controls were prepared without SA, and three replicate cultures were maintained for each treatment. The de-epoxidation activity of the DX100 culture was analyzed after incubation at 28 °C for 72 h with shaking at 180 rpm .
In a separate assay, bacterial cell lysates were prepared using B-PER II Bacterial Protein Extraction Reagent following the manufacturer’s instruction (PI 78260, Thermo Scientific, Rockford, IL, USA). DX100 was cultured for 48 h in 1.5 mL MSB under the conditions described above. After collecting the cell pellet by centrifuging one-third (0.5 mL) of DX100 culture at 5000 × g for 10 min 2 mL B-PER II reagent was added to it. The cells were disrupted by pipetting the pellet suspension up and down several times. Immediately 20 μL Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL, USA) was added to the cell lysate. To prepare culture filtrate the DX100 culture (0.5 mL) was passed through a 0.22 μm EMD filter MillexTM Sterile Syringe Filters: DuraporeTM PVFD Membrane (Fisher Scientific). The remaining 0.5 mL of the DX100 culture was used as positive control. Cell lysate, culture filtrate and control were spiked with 50 μg/mL DON and incubated at 28 °C. The de-epoxidation activity of the cell lysate, culture filtrate and control were analyzed after a 72 h incubation . Three replicate cultures were tested.
Data were organized using Microsoft Excel Program 2007, and the statistical analyses were carried out using SAS procedures of General Linear Model for Windows version 9.1 (SAS Institute, Cary, NC, USA, 2002–03). Average values of the replications were expressed as means ± standard errors. The mean differences among treatments were considered significant at p < 0.05 according to Tukey’s-HSD test.