DNA Gyrase

We adapted the protocol by Wu et al. for phylogenomic reconstructions (Wu and Eisen, 2008 (link)). In a first step, the individual clusters of CSCG-encoded proteins were aligned using MUSCLE (Edgar, 2004 (link)), and HMMs were built for each cluster using hmmbuild from the HMMER package (Eddy, 2011 (link)). Then, the models were used as queries to search against other genomes and the resulting alignments were trimmed adapting scripts from AMPHORA (Wu and Eisen, 2008 (link)). In a next step the trimmed alignments were concatenated with one another into a master alignment, which was further refined using Gblocks (Talavera and Castresana, 2007 (link)) to remove the less conserved columns. Finally, the refined master alignment was used as the input for PhyML (Guindon et al., 2010 (link)) for phylogenetic reconstruction.
The CSCG tree of Epsilonproteobacteria (Figure 3A) included six additional draft or complete genomes that were published after our initial steps of data collection. These included Sulfurospirillum barnesii SES-3, Uncultured Sulfuricurvum sp. RIFRC-1, Arcobacter butzleri ED-1 (Toh et al., 2011 (link)), Arcobacter sp. L (Toh et al., 2011 (link)), Sulfurovum sp. AR (Park et al., 2012 (link)), as well as the single-cell genomes of Thiovulum sp. ES (Marshall et al., 2012 (link)). To accommodate the incompleteness of draft genomes, we selected a subset of the CSCG-encoded proteins that occurred once in every draft genomes, and used only these as markers for tree construction. As a result, 194 of the CSCG-encoded proteins were used in the above procedure to construct the local phylogeny for Epsilonproteobacteria.
The global bacterial phylogeny was constructed with 37 globally conserved single copy markers (Figure 4). In addition to the 31 applied in the AMPHORA package (Wu and Eisen, 2008 (link)), we identified six additional phylogenetic markers using the HMM of core proteins: DNA gyrase subunit B (gyrB), Tryptophanyl-tRNA synthetase (TrpRS), SSU ribosomal protein S12p (S23e), LSU ribosomal protein L17p, SSU ribosomal protein S4p (S9e), and SSU ribosomal protein S15p (S13e). Among these new marker genes, GyrB (Kasai et al., 2000 (link); Holmes et al., 2004 (link); Peeters and Willems, 2011 (link)) and TrpRS (Rajendran et al., 2008 (link)) have been used in previous studies to determine the phylogeny of selected taxonomic groups, and the rest are ribosomal proteins.
The global bacterial tree in Figure 4 was rooted using mid-point rooting. The 16S and CSCG trees in Figure 3 were rooted based on the relative positions of different epsilonproteobacterial species at the global bacterial tree and using all other bacteria as an outgroup. As indicated with a black arrow in Figure 4, the root of Epsilonproteobacteria is located between Nautiliales and the other examined lineages.

The molecular docking analysis was performed as described in Reference [70 (link)]. Protein structures of four antioxidant proteins (namely, Lipoxygenase (PDB: 1N8Q), CYP2C9 (PDB: 1OG5), NADPH Oxidase (PDB: 2CDU), and Bovine Serum Albumin (PDB: 4JK4)) and three antimicrobial proteins (DNA gyrase topoisomerase II from E. coli (PDB: 1KZN), Enoyl-Acyl Carrier Protein Reductase from S. aureus (PDB: 3GNS), and Glucosamine-6-Phosphate (PDB: 2VF5)) were retrieved from RCSB Protein Data Bank (PDB) in a crystallographic 3D structure and adopted as docking targets, using Autodock Tools (version 1.5.6). The protein structures were stripped of H₂O molecules, metal atoms, co-crystalized ligands, and other non-covalently bound substances. Following the addition of Kollman charges, polar hydrogens and the merge of nonpolar hydrogens, the target file was saved as an appropriate pdbqt format. Ligands identified in D. ambrosioides essential oils were constructed as follows: sdf (3D conformer) file was downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) (accessed on 5 March 2022) and then converted to a pdb file by using PyMol. The ligand final pdbqt file was obtained by using Autodock Tools (version 1.5.6). Rigid molecular docking was executed with Autodock Vina’s embedded scoring function [71 (link)]. The grid box representing the docking search space was resized to best match the active binding site. Table 6 shows the grid box coordinates. The docked ligand complexes’ data were given as ΔG binding energy values (kcal/mol). Discovery Studio 4.1 (Dassault Systems Biovia, San Diego, CA, USA) was used to examine protein–ligand binding interactions and in the construction of 2D schemes of molecular interactions.

The transcription of urease (ureACD) and accessory gene regulator (agr) effector (RNAIII) genes in UTI-ST1 and UTI-ST5 was detected by Quantitative reverse transcription PCR (RT-qPCR). Complementary DNA (cDNA) was synthesized from total RNA using the PrimeScript™ reverse transcriptase kit (Takara), according to the manufacturer’s instructions. Thereafter, the cDNA samples were amplified using the FastStart Universal SYBR Green Master kit (Roche). The reactions were performed on a 7500 Sequence Detector (Applied Biosystems). Purified chromosomal DNA (0.005–50 ng/mL) was used to construct a standard curve. The reactions were performed in triplicate, and DNA gyrase subunit B (gyrB) was used as an internal reference.