Mak189

Citrate synthase, succinyl-CoA synthetase, and α-ketoglutarate dehydrogenase enzyme activities were measured using the assay kits according to the manufacturer’s protocols (Sigma-Aldrich; MAK193, MAK217, and MAK189). Briefly, each parental strain was cultured in Trypticase soy broth medium for 48 h. The bacterial cell pellets were collected and lysed using the specific assay buffer provided in each kit. The amount of total protein in each strain was measured using the bicinchoninic acid (BCA) protein assay (Pierce/Thermo Scientific) and used as a reference to compare enzyme activities between samples. One-way ANOVA with Tukey’s test for multiple comparisons was used to examine differences in enzyme activities between ground control and space-surviving strains.
The ATP production in each strain was measured by using CellTiter-Glo luminescent cell viability assay according to the manufacturer’s protocol (Promega; G7571). Briefly, each strain was cultured and lysed as described previously above. Lysates with 10 μg total protein per sample were used to compare each sample-specific amount of ATP. One-way ANOVA with Tukey’s test for multiple comparisons was used to analyze the statistical significance of ATP production between ground controls and space-surviving strains.