Supplementary Material for

Synergism Between Different Germinant Receptors in the Germination of Bacillus subtilis Spores

Xuan Yi1, Jintao Liu2, James R. Faeder3 and Peter Setlow1*

1Department of Molecular, Microbial and Structural Biology; University of Connecticut Health Center; Farmington, CT 06030-3305

2Department of Physics and Astronomy and 3Department of Computational and Systems Biology; University of Pittsburgh; Pittsburgh, PA 15260

*Corresponding Author

Address: Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030-3305

Phone: 860-679-2607

Fax: 860-679-3408

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Fig. S1 A) Amount of DPA release from wild-type B. subtilis spores germinating with L-valine and/or AGFK and B) degree of synergy when both germinants were used. Spores of B. subtilis strain PS533 (wild-type) were germinated with various concentrations of L-valine and/or L-asparagine, with GFK each at 10 mM in germinations with AGFK, and amount of DPA (in arbitrary units) released at 30 min after addition of germinants are shown (similar results were obtained for other time points). The symbols used in A) are:  - L-valine germination;  - AGFK germination;  - sum of the rates with L-valine and AGFK germination alone; and  - experimental curve with AGFK plus L-valine germination.Degree of synergy was defined as the ratio between experimentally measured rates with AGFK plus L-valine germination and the sum of the corresponding rates with L-valine and AGFK germination alone. Data were from the same experiment shown in Fig. 1.

Fig. S2A) Amount of DPA release from gerB*B. subtilis spores germinating with L-valine and/or L-asparagine and B) degree of synergy when both germinants were used. Spores of B. subtilis FB10 (has the GerB* GR) were germinated with various concentrations of L-valine and/or L-asparagine,and amount of DPA (in arbitrary units) released at 30 min after addition of germinants are shown (similar results were obtained for other time points). The symbols used in A) are:  - L-valine germination;  - L-asparagine germination;  - sum of the rates with L-valine and L-asparagine germination alone; and  - experimental curve with L-valine plus L-asparagine germination.Degree of synergy was defined as the ratio between experimentally measured rates with L-valine plus L-asparagine germination and the sum of the corresponding rates with L-valine and L-asparagine germination alone. Data were from the same experiment shown in Fig. 2.

Fig. S3A,B)Amount of DPA release from B. subtilis spores carrying the GerB* GR with or without GerD and germinating with multiple germinants and C) degree of synergy when mixtures of germinants were used. Spores of B. subtilis strains: A) FB10 (has the GerB* GR); and B) PS3929 (has the GerB* GR, and lacks GerD) were germinated with various concentrations of L-valine and/or L-asparagine,and amount of DPA (in arbitrary units) released at 30 min after addition of germinants are shown (similar results were obtained for other time points). The symbols used in A,B) are:  - L-valine germination;  - germination with L-asparagine alone;  - experimental curve for germination with L-valine plus L-asparagine; and  - sum of the rates with L-valine and L-asparagine germination alone.The symbols used in C) are:  - FB10 spores;  - PS3929 spores. Degree of synergy was defined as the ratio between experimentally measured rates with L-valine plus L-asparagine germination and the sum of the corresponding rates with L-valine and L-asparagine germination alone. Data were from the same experiment shown in Fig. 4.

Fig. S4A,B)Amount of DPA release from B. subtilis spores carrying the GerB* GR with or without the overexpressed GerA GR and germinating with multiple germinants and C) degree of synergy when mixtures of germinants were used. Spores of B. subtilis strains: A) FB10 (has the GerB* GR); and B) PS3501 (has the GerB* GR, and the GerA GR is overexpressed) were germinated with various concentrations of L-valine and/or L-asparagine,and amount of DPA (in arbitrary units) released at 30 min after addition of germinants are shown (similar results were obtained for other time points). The symbols used in A,B) are:  - L-valine germination;  - germination with L-asparagine alone;  - experimental curve for germination with L-valine plus L-asparagine; and  - sum of the rates with L-valine and L-asparagine germination alone. The symbols used in C) are:  - FB10 spores;  - PS3501 sproes. Degree of synergy was defined as the ratio between experimentally measured rates with L-valine plus L-asparagine germination and the sum of the corresponding rates with L-valine and L-asparagine germination alone. Data were from the same experiment shown in Fig. 5.