Pyrazines from bacteria and ants: convergent chemistry within an ecological niche

Ants use pheromones to coordinate their communal activity. Volatile pyrazines, for instance, mediate food resource gathering and alarm behaviors in different ant species. Here we report that leaf-cutter ant-associated bacteria produce a family of pyrazines that includes members previously identified as ant trail and alarm pheromones. We found that L-threonine induces the bacterial production of the trail pheromone pyrazines, which are common for the host leaf-cutter ants. Isotope feeding experiments revealed that L-threonine along with sodium acetate were the biosynthetic precursors of these natural products and a biosynthetic pathway was proposed.


Figure S13
. GC-MS spectra of 3-ethyl-2,5-dimethylpyrazine (2), molecular ion with m/z 136, from five dissected poison glands in A, five gasters of Atta sexdens rubropilosa in B, and Sigma-Aldrich standard of 1 in C.  Figure S14. GC-MS analysis of VOCs produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose and in A, and cultivated on the same medium added with 1.5% of L-threonine in B, 2.0% of L-threonine in C, 1.5% of L-valine in D, 1.5% of L-serine in E and 1.5% of L-alanine in F. 2,5-dimethylpyrazine (1) peaks are highlighted with blue rectangle and 3-ethyl-2,5-dimethylpyrazine with red rectangle (2).

Figure S15
. GC-MS spectra of 2,5-dimethylpyrazine (1), retention time 7.5 min and molecular ion with m/z 108, produced by S. marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose and 1.5% of L-threonine in B and same medium with 2.0% of L-threonine in C. Compound 1 was not detected on cultures of S. marcescens 3B2 on M9 agar medium supplemented with 0.2% of glucose in A and M9 agar medium supplemented with 0.2% of glucose and 1.5% of L-valine in D, 1.5% of L-serine in E and 1.5% of L-alanine in F. Figure S16. GC-MS spectra of 3-ethyl-2,5-dimethylpyrazine (2), retention time 15.4 min and molecular ion with m/z 136, produced by S. marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose and 1.5% of L-threonine in B and same medium with 2.0% of L-threonine in C. Compound 2 was not detected on cultures of S. marcescens 3B2 on M9 agar medium supplemented with 0.2% of glucose in A and M9 agar medium supplemented with 0.2% of glucose and 1.5% of L-valine in D, 1.5% of L-serine in E and 1.5% of L-alanine in F. Figure S18. GC-MS analysis of VOCs produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose in A, and cultivated on the same medium added with 0.5% of L-threonine in B, 1.0% of L-threonine in C, 1.5% of L-threonine in D and 2.0% of L-threonine in E. 2,5-dimethylpyrazine (1) peaks are highlighted with blue rectangle and 3-ethyl-2,5-dimethylpyrazine (2) with red rectangle. Figure S19. GC-MS spectra of 2,5-dimethylpyrazine (1), retention time 7.5 min and molecular ion with m/z 108, produced by S. marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose and 1.0% of L-threonine in C, 1.5% of L-threonine in D and 2.0% of Lthreonine in E. Compound 1 was not detected when the bacterium was cultivated on M9 agar medium supplemented with 0.2% of glucose in A and M9 agar medium supplemented with 0.2% of glucose and 0.5% of L-threonine in B. Figure S20. GC-MS spectra of 3-ethyl-2,5-dimethylpyrazine (2), retention time 15.4 min and molecular ion with m/z 136, produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose and 1.0% of L-threonine in C, 1.5% of L-threonine in D and 2.0% of L-threonine in E. Compound 2 was not detected when the bacterium was cultivated on M9 agar medium supplemented with 0.2% of glucose in A and M9 agar medium supplemented with 0.2% of glucose and 0.5% of L-threonine in B.  Figure S21. GC-MS analysis of VOCs produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine and 0.5% of L-alanine in A and the control medium in B. 2,5-dimethylpyrazine (1) peak is highlighted with blue rectangle and 3-ethyl-2,5-dimethylpyrazine (2) with red rectangle. Figure S22. GC-MS spectra of 2,5-dimethylpyrazine (1), retention time 7.5 min and molecular ion with m/z 108, produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine and 0.5% of L-alanine, in A. GC-MS spectra of VOCs from the culture media in 7.5 min showing the absence of 1, in B.  Figure S24. Extracted ion chromatograms from UHPLC-ESI-HRMS analyses of 2,5dimethylpyrazine (1) in A and 3-ethyl-2,5-dimethylpyrazine (2) in B produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of Lthreonine and 0.5% of L-alanine.    Figure S31. GC-MS analysis of VOCs produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine, 0.5% of L-alanine and 0.15% of non-labelled sodium acetate in A and cultivated on the same medium added with 0.15% of U-13 C-sodium acetate in B. 2,5-dimethylpyrazine (1) peaks are highlighted with blue rectangle and 3-ethyl-2,5-dimethylpyrazine (2) with red rectangle. Figure S32. GC-MS spectra of 2,5-dimethylpyrazine (1), retention time 7.5 min, produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine, 0.5% of L-alanine and 0.15% of non-labelled sodium acetate (molecular ion with m/z 108) in A and cultivated on the same medium added with 0.15% of U-13 C-sodium acetate (molecular ion with m/z 108) in B.  Figure S34. High resolution mass spectra of 2,5-dimethylpyrazine (1) produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of Lthreonine, 0.5% of L-alanine and 0.15% of non-labelled sodium acetate in A and cultivated on the same medium added with 0.15% of U-13 C-sodium acetate in B.
[M+H] + C6H9N2 with m/z 109.076. Figure S35. High resolution mass spectra of 3-ethyl-2,5-dimethylpyrazine (2) produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine, 0.5% of L-alanine and 0.15% of non-labelled sodium acetate in A and cultivated on the same medium added with 0.15% of U-13 C-sodium acetate in B.  Figure S36. GC-MS analysis of VOCs produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine and 0.5% of non-labelled L-alanine in A and cultivated on the same medium added with 0.5% of 15 N-L-alanine in B. 2,5dimethylpyrazine (1) peaks are highlighted with blue rectangle and 3-ethyl-2,5-dimethylpyrazine (2) with red rectangle. Figure S37. GC-MS spectra of 2,5-dimethylpyrazine (1), retention time 7.5 min, produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine and 0.5% of non-labelled L-alanine (molecular ion with m/z 108) in A and cultivated on the same medium added with 0.5% of 15 N-L-alanine (molecular ion with m/z 108) in B.  Figure S39. High resolution mass spectra of 2,5-dimethylpyrazine produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of Lthreonine and 0.5% of non-labelled L-alanine in A and cultivated on the same medium added with 0.5% of 15 N-L-alanine in B.
A B A B Figure S41. GC-MS analysis of VOCs produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine and 0.5% of non-labelled L-alanine in A and cultivated on the same medium added with 0.5% of 3-13 C-L-alanine in B. 2,5dimethylpyrazine (1) peaks are highlighted with blue rectangle and 3-ethyl-2,5-dimethylpyrazine (2) with red rectangle. Figure S42. GC-MS spectra of 2,5-dimethylpyrazine (1), retention time 7.5 min, produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine and 0.5% of non-labelled L-alanine (molecular ion with m/z 108) in A and cultivated on the same medium added with 0.5% of 3-13 C-L-alanine (molecular ion with m/z 108) in B.  Figure S44. High resolution mass spectra of 2,5-dimethylpyrazine (1) produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of Lthreonine and 0.5% of non-labelled L-alanine in A and cultivated on the same medium added with 0.5% of 3-13 C-L-alanine in B.
[M+H] + C6H9N2 with m/z 109.076 and [M+H] + C5 13 CH9N2 with m/z 110.079. Figure S45. High resolution mass spectra of 3-ethyl-2,5-dimethylpyrazine (2) produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine, 0.5% of non-labelled L-alanine in A and cultivated on the same medium added with 0.5% of 3-13 C-L-alanine in B.  Figure S46. Mass spectra of 2,5-dimethylpyrazine (1) Figure S48. GC-MS spectra of pyrazine 4 produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine, 0.5% of non-labelled L-alanine in A, cultivated on the same medium added with 0.5% of 15 N-L-alanine in B, added with 0.5% of 3-13 C-L-alanine in C, and added with 1.5% of L-[U-13 C, 15 N]-threonine in D. Figure S49. GC-MS spectra of pyrazine 5 produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine, 0.5% of non-labelled L-alanine in A, cultivated on the same medium added with 0.5% of 15 N-L-alanine in B, added with 0.5% of 3-13 C-L-alanine in C, and added with 1.5% of L-[U-13 C, 15 N]-threonine in D.  Figure S50. GC-MS spectra of pyrazine 6 produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine, 0.5% of non-labelled L-alanine in A, cultivated on the same medium added with 0.5% of 15 N-L-alanine in B, added with 0.5% of 3-13 C-L-alanine in C, and added with 1.5% of L-[U-13 C, 15 N]-threonine in D. Figure S51. GC-MS spectra of pyrazine 3 produced by Serratia marcescens 3B2 cultivated on M9 agar medium supplemented with 0.2% of glucose, 1.5% of L-threonine, 0.5% of non-labelled L-alanine in A, cultivated on the same medium added with 0.5% of 15 N-L-alanine in B, added with 0.5% of 3-13 C-L-alanine in C, and added with 1.5% of L-[U-13 C, 15 N]-threonine in D.