Additional file 1 of Garvicin Q: characterization of biosynthesis and mode of action
Creators: name: Desiderato, Christian K.
name: Hasenauer, Katharina M.
name: Reich, Sebastian J.
name: Goldbeck, Oliver
name: Holivololona, Lalaina
name: Ovchinnikov, Kirill V.
name: Reiter, Alexander
name: Oldiges, Marco
name: Diep, Dzung B.
name: Eikmanns, Bernhard J.
name: Riedel, Christian U.
Publisher: name: figshare
Year: 2022
Subjects: subject: Microbiology
subject: FOS: Biological sciences

description: Additional file 1: Table S1. Primer and codon optimized gene sequences used in present study. Table S3. Whole genome sequence alignment of Lactococcus sp. B1726 to other Lactococcus species. JSpeciesWS was used for performing the alignment. A tetra correlation search for Isolate B1726 was done to find similar genomes. Identified genomes were compared by ANIm (average nucleotide index based on MUMer). The percentage of aligned nucleotides is also indicated as well as the source, safety level and potential bacteriocins. Table S4. Predicted virulence factors and proteins important for survival in fish encoded on the genome of L. petauri B1726. The annotated genome of L. petauri B1726 was searched for virulence factors or proteins importat for survival in the host described for pathogenic strains of L. garvieae. Table S7. Impact of different growth conditions on garvicin Q production by L. petauri B1726. L. petauri B1726 was cultivated in 5 ml medium in glass tubes overnight. The antimicrobial activity was determined by growth inhibition assay with L. innocua LMG2785 as sensor strain Table S8. Ammonium sulphate (AS) precipitation of garvicin Q. Cell-free culture supernatant of an overnight L. petauri B1726 culture was subjected to AS precipitation applying different AS concentrations. The precipitate was dissolved in demin H2O (dissolved precipitate). The dissolved precipitate was clarified by centrifugation to remove insoluble components and the clarification supernatant was used in a growth inhibition assay (clarification). The percentage of recovered activity after clarification compared to the dissolved precipitate was then calculated (recovered activity). The antimicrobial activity was determined by growth inhibition assay with L. innocua LMG2785 as sensor strain. Although the recovered activity was highest for 20–30 % of AS, the highest overall activity was determined in the dissolved precipitate using 70 % AS. These results indicate that &gt;50 % of AS reduces solubility of garvicin Q. Figure S2. Concentration of Lactococcus sp. B1726 peptide by cation exchange (CIEX) and reverse-phase chromatography (RPC). CIEX of ammonium sulphate precipitation of cell-free supernatants of Lactococcus sp. B1726 A and RPC of the active CIEX elution fraction B. The black arrow indicates the active fraction. Antimicrobial activity was tested by spot on lawn assay with L. innocua LMG2785 as sensor strain. Figure S5. α-hemolysis by L. petauri B1726. L. petauri B1726 and L. lactis IL1403 were streaked on tryptone soya agar supplemented with 5 % sheep blood (Thermo ScientificTM). The agar plates were incubated at 37 °C. Incubation of L. petauri B1726 for ~4 days led to a clear discoloration of the blood agar indicating α-hemolysis. Figure S6. Garvicin Q operon of L. petauri B1726. Nucleotide sequence of garQ (red), garI (blue), garC (orange) and garD (green) are given. Predicted promoter sequences (− 10 and − 35 boxes) are displayed in italic and bold letters. Rho-independent terminator sequences are underlined. A putative ribosome binding site and start codon within garD is highlighted by capital and bold letters. FGENESB and BPROM were used for prediction of promoter and transcription units. ARNold was used for finding terminators. Figure S9. QTOF LC-MS analysis of natural garvicin Q in pooled RPC elution fractions harbouring antimicrobial activity. Graphs are derived from the spectrum shown in Fig. 4D zoomed in on the peaks with a mass to charge ratio (m/z) of 1069.1500 (left), 891.1142 (middle) and 763.9628 (right), which correspond to the 5-fold ([M+5H]5+; m/z spacing of 0.2), 6-fold ([M+6H]6+; m/z spacing of 0.17), and 7-fold ([M+7H]7+; m/z spacing of 0.14) positively charged ion of garvicin Q. Figure S10. Predicted interaction of garvicin Q with the IIC and IID subunit of the L. monocytogenes EGDe group I PTSMan. Garvicin Q (GarQ) is indicated in blue. The PTSMan subunits IIC and IID are indicated in pink and green. The surface of garvicin Q and PTSMan residues in close vicinity to garvicin Q (within 5 angstrom) is transparent. A and C Amino acid residues that were previously described to be involved in the interaction of garvicin Q and PTSMan by Tymoszewska et al. [5] are indicated in red. B and D Amino acid residues that were previously described to be involved in the interaction of pediocin and PTSMan by Zhu et al. 2022 [6] are indicated in cyan. AlphaFold was used for structure prediction and ChimeraX for visualization. Figure S11. interaction of pediocin PA-1 with the IIC and IID subunit of the L. monocytogenes EGDe group I PTSMan (PDB: 7VLY). Pediocin PA-1 (Ped) is indicated in blue. The PTSMan subunits IIC and IID are indicated in pink and green. In B, amino acid residues that were previously described to be involved in the interaction of pediocin and PTSMan by Zhu et al. [6] are indicated in cyan. ChimeraX was used for visualization. Figure S12. Characterisation of L. lactis IL1403/pNZ-pHin2Lm. A Fluorescence imaging of the recombinant strain L. lactis IL1403 pNZ-pHin2Lm and the wild type strain. The recombinant strain showed fluorescence compared to the wild type strain. This indicates synthesis of pHluorin2 protein. Imaging was performed in an iBrightTM FL 1000 imaging system with filter settings for green fluorescence. B Excitation spectrum of L. lactis IL1403 pNZ-pHin2Lm. Addition of the pore-forming bacteriocin nisin (green) and garvicin Q (blue) is shifting the ratio of the excitation maxima in comparison to the LMB buffer negative control (red). Figure S13. Establishment of recombinant garvicin Q production by C. glutamicum. A Schematic map of expression vector pPBEx2-garQICDCgl. Upstream of each gene the sequence of the ribosomal binding site (RBS) of the elongation factor TU was inserted. B Influence of induction of gene expression during pre-cultivation of C. glutamicum CR099/pPBEx2-garQICDCgl. Garvicin Q activity was determined by pHluorin2 assay using L. lactis IL1403/pNZ-pHin2Lm as a sensor. Samples were taken 4 h after inoculation of the main culture. Addition of 0.05 mM IPTG during pre-cultivation led to a more pronounced decrease in the fluorescence ratio indicating pore formation/garvicin Q activity. Trypsin treatment completely abolished antimicrobial activity indicated that the antimicrobial compound is a protein/peptide. C High-cell density cultivation of C. glutamicum CR099/pPBEx2-garQICDCgl for garvicin Q production. D Activity in supernatants of C. glutamicum CR099/pPBEx2-garQICDCgl or the control strain harbouring the empty plasmid pPBEx2 at 4 and 24 h of cultivation in a high-cell density approach. Assay were performed with L. lactis IL1403/pNZ-pHin2Lm as indicator. Assay buffer (LMB) served as negative control and HIC purified garvicin Q (GarQ) was used as a positive control. Values in B and D are ratios of fluorescence intensity at 520 nm after excitation at 400 and 480 nm (ratio RFU 400/480) and are mean of n = 2–3 independent experiments (supernatants of independent cultivations). Figure S14. QTOF LC-MS analysis of recombinant garvicin Q in pooled RPC elution fractions harbouring antimicrobial activity. The graph is derived from the spectrum shown in Fig. 7D zoomed in on the peak with a mass to charge ratio (m/z) of 1069.2010, which corresponds to the 5-fold positively charged ion of garvicin Q ([M+5H]5+; m/z spacing of 0.2).