Abi prism bigdye terminator v3.1 reagent kit

Real-time PCR data were validated in selected samples (one to three samples for each genotype of the detected variants) by Sanger sequencing of the PCR products. The nucleotide sequence of PCR products was determined using the ABI PRISM BigDye Terminator v3.1 reagent kit (Thermo Fisher Scientific, Waltham, MA, USA) followed by the analysis of the reaction products on DNA sequencer Applied Biosystem 3500 DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s protocol.

The DNA of the strains was extracted from bacterial biomass using Diatom™ DNA Prep 100 (Isogen Laboratory, Moscow, Russia) according to the manufacturer’s recommendations. The purified DNA samples were used as the PCR templates. Pure cultures of bacteria were identified by 16S rRNA gene analysis using the Bacteria-universal primer set 27F–1492R [31 ]. DNA sequencing was performed according to the Sanger method on an ABI 3730 DNA Analyzer automatic sequencer using the ABI PRISM^® BigDye™ Terminator v. 3.1 reagent kit (Applied Biosystems, Waltham, MA, USA).
Initial taxonomic assignment of strains HO-A22^T and SHC 2-14 was carried out using EzBioCloud [32 (link)]. The phylogenetic position of the isolated strains was determined using 16S rRNA gene sequences of type strains of the family Rhizobiaceae. The 16S rRNA gene sequences were aligned by MUSCLE [33 (link)], and the maximum-likelihood tree was performed with a GTR+F+I+G4 model recommended by ModelFinder [34 (link)] in IQ-TREE [35 (link)]. Branch supports were obtained with 10,000 ultrafast bootstraps [36 (link)]. Maximum parsimony and neighbor-joining trees were reconstructed with MPBoot [37 (link)] and MEGA11 [38 (link)], respectively.

The E. anophelis strain ML-44 was isolated via Endo agar medium (HiMedia) in raw milk monitoring studies. An unpasteurized milk sample came from a farm in the Nizhny Novgorod region of Russia. The sample was serially diluted and aseptically plated on Endo agar. After 24 h, a single colony with a smooth glittering surface and white-colored was detected. For further research, a colony was streaked onto tryptic soy agar (HiMedia).
Strain identification was performed through 16s rRNA gene sequencing as follows. A single colony was suspended in 100 µL of sterile deionized water and lysed by incubation at 95 °C for 10 min. PCR reaction was carried out with the universal bacterial primers specific for 16s rRNA gene: 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R: 5′-TACGGYTACCTTGTTACGACTT-3′ [46 (link)]. PCR product was extracted and purified from the agarose gel to sequence using the ABI PRISM BigDye Terminator v. 3.1 reagent kit (Applied Biosystems) according to the manufacturer’s protocols, followed by an analysis of the reaction products and an automatic sequencer Applied Biosystems 3730 DNA Analyzer. The 16S rRNA nucleotide sequences were assembled using the Unipro UGENE (v39.0) software [47 (link)]. The obtained consensus was compared to other bacterial 16s rRNA genes with the EzTaxon server database [48 (link)].

DNA was isolated from the biomass of the Sphingomonas sp. AR_OL41 strain using a set of DiatomTM DNA Prep 100 reagents (IsoGen Inc., Moscow, Russia) according to the manufacturer’s recommendations. The purified DNA preparation was used as a matrix for PCR. Pure cultures were identified by 16S rRNA gene sequence analysis using the bacteria-universal primer set 27F–1492R [47 ]. DNA sequencing was performed according to the Sanger method on an ABI 3730 DNA analyzer automatic sequencer using the ABI PRISM^® BigDye™ Terminator v. 3.1 reagent kit (Applied Biosystems, Waltham, MA, USA). Taxonomic affiliation of the strains was determined using EzBioCloud [48 (link)].
Genomic DNA from the AR_OL41 strain was used for genome sequencing using the Illumina HiSeq 2500 platform (Illumina, Inc., San Diego, CA, USA). The library for Illumina sequencing was prepared using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). Sequencing on the Illumina MiSeq generated 825,013 paired-end reads (2 × 300 nt, ~497 Mbp in total). Overlapping paired-end reads were merged using FLASH v.1.2.11 [49 (link)], and low-quality bases were trimmed using Sickle v.1.33 (https://github.com/najoshi/sickle; accessed on 28 January 2023) [50 ]. The reads were assembled de novo using SPAdes v. 3.15.4 software [51 (link)]. Default parameters were used for all software.

Sequencing of a nearly full-length 16S rRNA gene was implemented for identification. Bacterial DNA was isolated via thermal lysis of cell suspension at 95°C for 20 min (a single colony from an agar plate was used). We performed a polymerase chain reaction with the following universal primers: 27F 5’-AGAGTTTGATCMTGGCTCAG-3′ and 1492R 5’-TACGGYTACCTTGTTACGACTT-3′ (Weisburg et al., 1991 (link)). PCR products of ~1,500 bp were detected in 1% agarose gel. Amplicons were extracted from the gel and purified using a DNA extraction kit (Dia-m, Moscow, Russia). Further DNA sequencing was performed using the ABI PRISM BigDye Terminator v. 3.1 reagent kit (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s protocols, followed by an analysis of the reaction products on an automatic sequencer Applied Biosystems 3,730 DNA Analyzer. Obtained forward and reverse sequences were assembled into consensus using UGENE (v41.0) software (Okonechnikov et al., 2012 (link)). The EzTaxon server database was utilized to compare the resulting consensus to the sequences of other bacterial 16S rRNA genes (Yoon et al., 2017 (link)). To further clarify the strain identity, the average nucleotide identity (ANI) value, as well as DDH in silico, were calculated.