Additional file 1 of Calcium regulates the mycophagous ability of Burkholderia gladioli strain NGJ1 in a type III secretion system-dependent manner
Creators: name: Yadav, Sunil Kumar
name: Das, Joyati
name: Kumar, Rahul
name: Jha, Gopaljee
Publisher: name: figshare
Year: 2020
Subjects: subject: Microbiology
subject: FOS: Biological sciences

description: Additional file 1: Fig. S1. Effects of different divalent cations on bacterial mycophagy. Confrontation of NGJ1 with R. solani on PDA plates either containing 10 mM concentration of different divalent cationic salts (CaCl2, MgCl2 and FeCl2) or 4 mM concentration of their respective chelators (EGTA, EDTA and Dipyridyl). The mycophagous ability was enhanced on EGTA containing plates while inhibited on CaCl2 containing plates. However, other divalent cationic salts (MgCl2 and FeCl2) as well as their chelators (EDTA and Dipyridyl) did not alter mycophagous ability. Notably, the presence of different divalent cationic salts and their respective chelators did not alter the growth of R. solani as well as NGJ1. Similar results were obtained in at least three independent biological experiments and only representative photographs are shown. Fig. S2. Mycophagous behaviour of NGJ1 on R. solani in CaCl2 supplemented semi-synthetic minimal media. (A) NGJ1 shows mycophagy on CDA (without supplementation) plates and is able to forage over the fungal mycelium. While supplementation of 5 mM, 10 mM and 20 mM concentration of CaCl2 onto CDA plates gradually reduced the mycophagy. (B) Bacterial abundance in CDB broth with or without CaCl2 supplementation. NGJ1 showed limited growth in CDB broth while the growth was enhanced in presence of R. solani mycelia. However, supplementation of different concentration of CaCl2 suppressed the bacterial growth in CDB containing R. solani mycelia. Similar results were obtained in at least three independent biological experiments and only representative images are shown. Graphs show mean values± standard deviation. Fig. S3. Low calcium condition regulates mycophagy in a functional T3SS dependent manner. Confrontation of R. solani with NGJ1 or NGJ12, a T3SS deficient mutant strain on PDA plates with or without EGTA/CaCl2 supplementation at 3 dpi. Presence of EGTA or CaCl2 didn’t alter the mycophagy defect of NGJ12. Similar results were obtained in at least three independent biological experiments and only representative images are shown. Fig. S4. Expression profile of T3SS apparatus encoding genes of NGJ1. RT- PCR analysis reflected expression of T3SS apparatus encoding genes to be upregulated in presence of R. solani at 48 h. However presence of 10 mM and 20 mM CaCl2 reduced their expression. The differential expression of these genes was estimated during NGJ1 growth in presence of R. solani with respect to that observed in absence of R. solani using 16S rRNA gene as endogenous control. The experiment was independently repeated three times with minimum three technical replicates. Asterisks * and ** indicate statistical significant difference between indicated groups at P &lt; 0.05 and P &lt; 0.001, respectively (estimated using one-way ANOVA). Graphs show mean values ± standard deviation. Fig. S5. HrcC (an important constituent of T3SS) protein sequence based phylogenetic relationship of NGJ1 with other bacteria. The phylogenetic tree was constructed using maximum likelihood method. The bootstrap values are indicated at branch node. The information about calcium mediated regulation of the T3SS of different bacteria has been mentioned and the related reference has been cited. Fig. S6. Conservation of T3SS signal sequence in Bg_9562 orthologs. Multiple sequence alignment of orthologs of Bg_9562 protein in different bacteria revealed that potential T3SS signal (9 amino acids sequence at N-terminus) is conserved in different B. gladioli strains. Predicted T3SS signal sequence is highlighted in blue-square. Rsol: Ralstonia solanacearum; Bpse: B. pseudomallei; Bsta: B. stagnalis; Bcen: B. cenocepacia; Bglu: B. glumae; Bubo: B. ubonensis; Bgla: B. gladioli; Pbacid: Paraburkholderia acidipaludis; Pbterri; Paraburkholderia terricola. Fig. S7. Complementation with T3SS signal deleted variant of Bg_9562 failed to restore mycophagy in the Bg_9562 mutant bacterium. (A) Strategy adopted to clone T3SS signal sequence deleted (pGD5) as well as full length Bg_9562 (pGD3). (B) Effect of Bg_9562 mutant (NGJ101) and mutant strains complemented with pGD3 (NGJ102) and pGD5 (NGJ104) on the germination and growth of R. solani sclerotia. At both high (109cells/ml) and low (105 cells/ml) concentration, the NGJ101 as well as NGJ104 were defective in mycophagous ability. While the wild type (NGJ1) as well as NGJ102 were proficient in mycophagy. Fig. S8. Expression profile of potential T3SS effector encoding genes of NGJ1 during mycophagous interaction with R. solani. RT-PCR analysis revealed the T3SS effector encoding genes of NGJ1 to be induced during 48 h of mycophagous interaction in PDB. The differential gene expression was estimated during mycophagous interaction of NGJ1 with R. solani with respect to that observed during NGJ1 growth in absence of R. solani using 16S rRNA gene as endogenous control. The experiment was independently repeated three times with minimum three technical replicates. Asterisks * indicate significantly different at P &lt; 0.001 (estimated using one-way ANOVA). Graphs show mean values± standard deviation. Fig. S9. Phylogenetic analysis of glucanase like proteins in Burkholderia sp. and various other bacteria. The amino acid sequence of proteins of different bacterial species has been obtained from NCBI and used for construction of phylogenetic tree using maximum likelihood method. The red box depicts the endo-β-1, 3-glucanase of NGJ1. The bootstrap values are indicated at branch node. Table S1. Mycophagous behaviour of NGJ1 on R. solani in presence of different cationic salts and their chelators. Table S2. Sclerotial formation on B. gladioli strain NGJ1 and R. solani confrontation plates upon various cationic salts supplementation. Table S3. Germination rate of secondary sclerotia of R. solani isolated from NGJ1 confrontation plates. Table S4. Calcium concentration measurement through ICP-MS. Table S5. Bonafide T3SS effector proteins of NGJ1. Table S6. Effect of the endo-beta- 1, 3- glucanase mutant (NGJ105) and complement (NGJ106) strains of NGJ1 on secondary sclerotia production by R. solani.Table S7. Bacterial strains and plasmids used in this study. Table S8. Primers used in this study.