Additional file 1 of Phylogeny-guided genome mining of roseocin family lantibiotics to generate improved variants of roseocin
Creators: name: Chaudhary, Sandeep
name: Kishen, Shweta
name: Singh, Mangal
name: Jassal, Sunanda
name: Pathania, Reeva
name: Bisht, Kalpana
name: Sareen, Dipti
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. (A) Pie chart of the obtained hits, with most of the hits being from actinobacteria and cyanobacteria phylum. (B) Phylum-dependent clade formation of 100 RosM homologs in Bayesian analysis. Figure S2. Complete BGCs of selected 42 LanMs based on BAGEL 4 and antiSMASH 5.0 prediction. Annotation is based on CDD analysis. Figure S3. Multiple sequence alignment of roseocin family lanthipeptide precursor sequences to determine the conserved motif. Figure S4. Bayesian analysis of two LanMs of the same biosynthetic gene cluster from roseocin and lacticin 3147 families. Figure S5. (A) Distantly related BGCs of roseocin family from Walker et al. 2020 study, with multiple numbers of precursors in gene clusters. (B) Rosα homologs of these BGC are unusually short but contain the conserved motif S/TxxxxTxGCC. Figure S6. (A) Pairwise sequence alignment and percent identity of 42 LanM sequences (B) and the comparative cumulative frequency of identity percentage among leader, core, and LanM protein sequences of 42 BGCs. Figure S7. Sequence logo from multiple sequence alignment (A) Rosα and (B) Rosβ homologs from type 2 subclade showed highly conserved positions and possible sites of evolutionary variation. Figure S8. ESI-MS data showed that variants of Rosα were post-translationally modified by RosM, in E. coli BL21(DE3). Figure S9. Evaluation of antimicrobial activity in synergism with Rosβ after leader removal with proteinase K. Figure S10. Multiple sequence alignment of Rosα homologs to predict the ring topology in (A) Rosα and (B) Rosβ. Figure S11. Schematic representation of the primer design for site-directed mutagenesis. The arrowheads represent the site of mutation in the primer-primer overlapping region. Figure S12. A tanglegram between the 16S rRNA and the lanM gene tree indicates that intra-phyla horizontal gene transfer (HGT) is a major source of lanthipeptide diversity. Figure S13. Maximum likelihood phylogenetic tree of 16S rRNA from bacterial species of (A) actinobacteria and (B) cyanobacteria, in correlation of their BGCs organization. Figure S14. (A, B) Tanglegram between the phylogenetic tree of LanM and their associated lanthipeptide leader/core in Dendroscope. 4. Table S1. 42 selected hits from RosM search in the NCBI database. Table S2. A comparison of the roseocin and lacticin 3147 families of two LanM-two precursor genes. Table S3. Accession number of ProcM family LanMs from Cubillos-Ruiz et al. 2017. Table S4. Calculated (by Expasy) and observed (by ESI-MS) average mass of full length (with leader region) post-translationally modified Rosα-wild type and its variants. Selected positions for mutant generation are bold and italicized. Table S5. Calculated (by Expasy) and observed (by MALDI-TOF MS) monoisotopic mass of Rosα variants after leader cleavage using endoproteinase GluC (leaving an overhang of four amino acid residues) reduced with TCEP only &amp; TCEP reduced peptides alkylated with IAA. Table S6. b and y ions obtained in MS-MS fragmentation of Rosα variants. Table S7. The list of primers used for the SDM-PCR in the generation of variants (F-forward and R-reverse primer).