Additional file 2 of Diversity of enzymes for exopolysaccharide synthesis in the fructophilic honeybee symbiont Apilactobacillus kunkeei

Additional file 2: Figure S1. Sanger sequencing chromatograms of the PCR products of primer pairs targeting GH genes A) A1401_12760-70 (GS2 and BrS segments), B) H3B111M_12570-80 (BrS), C) FHON2_13560-70 (GS2), D) H3B209X_13360-70 (GS2), E) G0102_12710-20 (GS1). Putative stop codons are highlighted in grey, and differences with the original sequence are highlighted in red. R = ruler, - = negative control. Figure S2. Phylogenetic relationships of GH70 and GH32 domains. Phylogenetic trees were inferred from the amino acid alignments of the A) 907 core proteins, B) 97 GH70 domains plus four outgroups and C) 25 GH32 domains in 38 representative strains of A. kunkeei. Strain names and locus tags are colored by phylogroup designations according to the core protein phylogeny by Dyrhage et al. [18]. The catalytic activities of protein domains with experimentally confirmed functions are indicated in the yellow boxes. Proteins annotated as GTFB in Limosilactobacillus reuteri and Limosilactobacillus fermentum were used as outgroups in the GH70 domain phylogeny. The trees were inferred with maximum likelihood methods. Numbers on nodes show bootstrap support values ≥50. Figure S3. Possible domain organizations of the GH70 and GH32 family proteins. Organization of domains and sequence repeats in a subset of A) GH70 and B) GH32 enzymes with and without glucan-binding repeats in the 38 reference A. kunkeei strains. All domains and repeats are based on InterProScan predictions, except for signal peptides, which are based on SignalP6 (slow mode) predictions; glucan-binding domains, which are based on AlphaFold predictions, and serine-rich repeats, which are based on protein sequence. GS = glucansucrases, BrS = branching sucrases, NGB = non-glucan binding GH70, PTL = proteins with the N-terminal segment of a GH70 domain, S1-3 = GH32 subtypes, of which S3 lacks glucan-binding domains. Figure S4. Pairwise BLASTN comparisons of a 40 kb segment of the A. kunkeei genome with the genes coding for GH70 and GH32 enzymes. The order of strains is based on the core protein phylogeny shown in Figure S2A. Strain names are colored by phylogroup designations. Forward BLASTN matches are colored in grey and reverse matches, in red. GH70 and GH32 are colored depending on the subtype, and transposons are colored in black. Figure S5. Pairwise BLASTN comparisons of a 40 kb segment of the A. kunkeei genome with the genes coding for the NGB enzymes. The order of strains is based on the core protein phylogeny shown in Figure S2A. Strain names are colored by phylogroup designations. Forward BLASTN matches are colored in grey and reverse matches, in red. NGB is colored in dark blue-green, phage genes in purple, ABC transporters in green, CRISPR genes in brown and transposons in black. Figure S6. Phylogenetic relationships of GH70 domains in bacteria. Phylogenetic trees were inferred with the maximum likelihood method from the amino acid alignments of GH70 domains in members of the Lactobacillaceae, Streptococcaceae and Enterococcaceae. The clades containing GSs, BrSs and NGBs in the reference A. kunkeei strains and A. apinorum are schematically indicated by circles colored in orange. Species names and gene IDs are colored by family designations. The catalytic activities of protein domains with experimentally confirmed functions are indicated by AS (alternansucrase), DS (dextransucrase), MS (mutansucrase), RS (reuteransucrase) or BrS labels, all marked in yellow. Proteins annotated as GTFB in Limosilactobacillus reuteri and Limosilactobacillus fermentum were used as outgroups. The subtypes within which the domains cluster (GS, BrS, NGB or GTFB) are indicated by dotted rectangles. Numbers on nodes show bootstrap support values ≥50. Figure S7. Comparison of GH70 domain tree topologies. Comparisons of A, B) tree topologies and C, D) quartet-based internode certainty scores for GH70 and core proteins. Phylogenetic trees and were inferred based on the amino acid alignments of the A) GS1 and B) NGB domains in the two subsets of A. kunkeei strains that contain genes for these proteins. The tree topologies were compared to the topologies obtained from phylogenetic inferences based on 907 core proteins in the two subsets of A. kunkeei strains, respectively. The trees were inferred with the maximum likelihood method. Bootstrap support values of 100% and ≥ 95% are shown as black and grey dots, respectively. Quartet-based internode certainty score comparisons of the core protein phylogenies and the C) GS1 and D) NGB domain phylogenies. The branch values display the lowest quartet internode certainty (LQ-IC), the quadripartition internode certainty (QP-IC) and the extended quadripartition internode certainty (EQP-IC) scores, where values close to 1 indicate support of the internal branch, values close to 0 indicate high levels of incongruence and values close to -1 contest it. Branches with LQ-IC scores close to 1 are highlighted. The GH70 and core protein input phylogenies were unrooted in the QuartetScore analysis, and the resulting trees were midpoint rooted for visualization purposes. Strain names and locus tags are colored by phylogroup designations. Figure S8. Recombination breakpoint plot, showing the genes in the genomic region for GH70 and GH32 family proteins. The lines at the top of the plot represent predicted breakpoints. The dashed red line represents the join point between the two alignments. Gaps are shown as grey rectangles. The continuous black line represents the number of predicted breakpoints per 200 nt windows along the alignment. The two shaded areas behind the black line represent the confidence intervals (CIs), where the strongly-shaded area represents the 95% CI and the faintly-shaded area represents the 99% CI. Green triangles represent parts of the alignment where the number of predicted breakpoints is above the 95% CI, if less saturated, or the 99% CI, if more saturated. The coding sequences along the alignment are shown at the bottom. Figure S9. ClonalFrameML plot showing recombination along the A. kunkeei chromosome for A) phylogroup A and B) phylogroups B-C. Dark blue = predicted recombination. Light blue = no change. White, yellow and orange represent the degree of homoplasy, with white being the lowest and orange, the highest.