Fig. 1.

Performing BONCAT-FACS on soil samples. a Details of the incubation conditions of the 76 cm and 30 cm soil samples. Samples were incubated in triplicate and three time points were sorted. b The gate drawing was done in two steps, first the cells were separated from the background particles based on their DNA dye staining SYTO59 fluorescence (Ex: 640 nm/Em: [655–685 nm]), as pictured by the blue gate on the top panel. The SYTO+ cells were further analyzed for their BONCAT fluorescence with the FAM Picolyl dye (Ex: 488 nm/Em: 530 nm). The middle panel shows an example of a control sample that was water incubated and clicked (water –HPG control), the BONCAT gate (in green) was set such that less than 0.5 % of events would fall in that gate (false positive). The bottom panel is an example of how the BONCAT+ and BONCAT – gates where set in a HPG incubated sample. Note that the green gate is the same than in the control sample. c Total extracted cell counts over time showing ~20 million cells per gram at 30 cm and ~5 million cells per gram at 76 cm. d Temporal dynamics of BONCAT+ (express as a percent of the extractable cells) labeling for the 30 cm and the 76 cm sample. Error bars represent standard deviation (n = 3)