Clean up purification columns

Total genomic DNA was extracted while using a Genomic Mini AX Bacteria Spin Kit (A&A Biotechnology, Gdynia, Poland and then stored at −20 °C. All of the PCR amplifications were carried out with PCR mix RAPID (A&A Biotechnology, Gdynia, Poland), according to the manufacturer’s recommendations. The primer sequences and PCR conditions used are listed in Table 3. The amplified PCR products were purified with Clean-Up purification columns (A&A Biotechnology, Gdynia, Poland) and then sequenced in Genomed S.A. (Warsaw, Poland). Preliminarily, the 16S rRNA gene sequences were compared with the EzBioCloude database. All of the obtained sequences were analyzed while using BLAST available on the NCBI website. Multiple sequence alignment matrices of the individual gene sequences were created using ClustalW included in the MEGA 6.06 software [56 (link)]. The sequence identity values were calculated while using BioEdit 7.0.5 software.
The 16S rRNA gene sequence similarity threshold value of 98.7% between the isolate and species type strain was used as an indicator that an isolate can be a member of a given species [34 ]. The identification of the isolates at the species level was considered to be final when the searching results that were based on 16S rRNA gene sequences were concordant with those based on the rpoB, rpoD, or recA gene sequences.

The genomic DNA was extracted using a bacterial genomic DNA extraction kit (GeneMATRIX Tissue & Bacterial DNA Purification Kit, EURx). The 16S rRNA gene was amplified and sequenced with the bacterial universal primers fD1 and rD1 described by Weisburg et al.^{40 (link)}. PCR amplification reactions were carried out with ReadyMix™ Taq PCR Reaction Mix (Sigma) according to the manufacturer’s recommendations. The amplified products were purified with Clean-Up purification columns (A&A Biotechnology) and sequenced with BigDye Terminator Cycle sequencing kit using the 3500 Genetic Analyzer according to the manufacturer’s procedures (Life Technologies) as described elsewehere^{41 (link)}. The 1337 bp long 16S rRNA gene sequence fragments of MMK2^T and MMK3 were deposited in GenBank under the accession numbers OQ799602 and OQ799603. The sequence similarity searches were performed by using the BLAST algorithm. Phylogenetic tree based on 16S rRNA gene sequences were constructed using the software MEGA X^{42 (link)} and the maximum likelihood algorithm with Tamura 3-parameter model^{43 (link)}. Bootstrap values were derived from 1000 replications.