Denaturing Gradient Gel Electrophoresis

DNA extractions from intestinal luminal contents were prepared as described previously [14 (link)]. In brief, DNA extracts and plasmids were quantified by using Quant-iT PicoGreen reagent (Invitrogen, UK) and all adjusted to 1 ng DNA/µl.
The abundance of specific intestinal bacterial groups was measured by qPCR with group-specific 16S rRNA gene primers (Tib MolBiol, Germany) as decribed previously [15 (link),16 (link)]. As reference for quantification standard curves with tenfold serial dilutions of plasmids (ranging from 2×10⁸ to 2×10² copies) were generated for each run. The real-time PCR primers were first used to amplify cloned 16S rDNA of reference strains (see Table 1). The number of 16S rRNA gene copies / ng DNA of each sample was determined. Frequencies of the given bacterial groups were calculated proportionally to the eubacterial (V3) amplicon.
Genetic fingerprints were generated by PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) as described previously [14 (link)].

DGGE ITS2 diversity typing was performed following the protocol detailed in LaJeunesse (2002) . Briefly, the Symbiodinium ITS2 region was amplified with the primer pair ITSintfor2 and ITS2CLAMP using PCR conditions described in LaJeunesse et al. (2003) with the following modifications: the annealing temperature was maintained at 52 °C for 27 cycles after 20 cycles of touchdown amplification. PCR products were mixed with 10 μL Ficoll-based loading buffer and concentrated by a speed vacuum before loading on an 8% polyacrylamide gel using a Cipher DGGE kit (CBS Scientific Company, Del Mar, CA). Gels were run at 150 V for 15 h, stained for 30 min with 1× SYBR Green (Invitrogen, Carlsbad, CA) and visualized on a Dark Reader Transilluminator (Clare Chemical Research, Dolores, CO). Prominent band(s) were excised from the DGGE gel with a sterile scalpel. Each band was transferred into an Eppendorf tube that contained 500 μL DNase-free water and incubated at 4 °C for 24 h. Two microlitres of this were used for reamplification as described in LaJeunesse (2002) and purified with Illustra ExoStar (SelectScience, Bath, UK) enzyme mix following the manufacturer's instructions. Successful amplification was verified by running products on a 1% agarose gel stained with 1× SYBR Safe (Invitrogen). Samples were sent for bidirectional Sanger sequencing at the KAUST BioScience Core Laboratory (Thuwal, Saudi Arabia). Sequences were processed in CodonCode Aligner (CodonCode Corporation, Centerville, MA). After quality trimming, forward and reverse sequences were assembled into contigs. For phylogenetic assignment of ITS2 sequences, we built a custom BLAST database (File S1, Supporting information) of ITS2 types collected from 409 ITS2 sequences taken from GeoSymbio (Franklin et al. 2012 (link)) (denoted as GS), 7 ITS2 sequences from Scott Santos’ database (www.auburn.edu/∼santosr/sequencedatasets.htm) (denoted as ST) and 17 DGGE ITS2 sequences from Todd LaJeunesse's SD2-GED database (https://131.204.120.103/srsantos/symbiodinium/sd2_ged/database/views.php) denoted as LJ. ITS2 sequences were assigned to the ITS2 types that represented highest identity in the BLASTN hits.

PCR-DGGE analyses were performed to investigate lactobacilli populations; for each sampling location, 17 (out of 20) DNA extracted from individual guts were processed. The PCR and subsequent denaturing gradient gel electrophoresis (DGGE) analysis, using the Dcode Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA, USA), were performed as described by Alberoni et al. (2018) (link). Denaturing gradient was established at 35–65%. Fingerprinting analyses were carried out using the Bionumerics v 7.1 (Applied Maths, St. Martens-Latem, Belgium) and the UPGMA algorithm based on the Pearson correlation coefficient with an optimization of 1% was applied. Microbial diversity was analyzed with the following parameters: Shannon–Wiener index (H), Simpson index (S), and band evenness (EH), calculated according to Hill et al. (2003) (link). Moreover, principal components analysis (PCA) was carried out by using Bionumerics. Relevant bands were excised from the gels and processed to achieve purified amplicons to be sequenced (Gaggìa et al., 2015 ). Sequencing was carried out by Eurofins Genomics (Ebersberg, Germany) and obtained sequences were assigned to bacterial species using megablast algorithm.²

For community analysis (amplicon metabarcoding and Denaturing Gradient Gel Electrophoresis, DGGE), total DNA from both the original consortia and the consortia obtained after specific time points following serial transfers in N-FIX medium was analyzed using an FastDNA™ SPIN Kit for Soil (MP Biomedicals) per the standard protocol. For metagenomic analysis, a consortium sample obtained after 10 transfers via the N-FIX medium was used for DNA extraction. To improve cell recovery, 1 mL of 10% (volume/mass of approximately 0.03 M) sodium dodecyl sulfate solution was added to each flask containing culture medium, with a final concentration of approximately 0.25% (0.0008 M). The contents of three glass bottles (120 mL) containing the full-grown consortia were filtered through sterile 0.22-µm Millipore membranes; the membranes were then directly subjected to DNA extraction, and instead of the single lysis step in the FastPrep™ system (Bio 101, Inc., La Jolla, CA, USA), we performed the lysis step again. We used the FastPrep™ system at 6.0 rpm for 40 s. Nanodrop™ and Qubit™ (Thermo Fisher Scientific) were used to assess the quality (260/230 and 260/280 ratios) and amount of the extracted DNA, respectively. Electrophoresis was performed using 1.0% agarose gel for 40 min at 90 V, after which the gels were stained with SYBR^™ Safe (Thermo Fisher Scientific) and observed under an ultraviolet (UV) transilluminator to assess DNA integrity.