Extended Data Fig. 3: Impacts of biotinylation and bacterial cell density on the detection of interactions via BASEHIT.
From: A host–microbiota interactome reveals extensive transkingdom connectivity
a, Four bacterial strains with differing interaction profiles were grown and labeled with a titration of biotin ranging from 50 nM to 500 µM and then screened by BASEHIT. The enrichments of each protein hit are shown across all conditions, along with the enrichments of two predicted inert proteins — the coronaviral spike protein 229E-S1, and the arylsulfatase ARSA, which serve as internal negative controls. The biotin concentration used for labelling in our large-scale screen (5 µM) is highlighted in teal. Across all tested interactions, 5 µM biotin exhibited enrichments within two-fold of the “optimal” condition, and no appreciable enrichment of inert proteins was observed under any conditions. Data represent the mean ± s.d. from n = 3 independent experiments. b, Five strains were screened via BASEHIT at bacterial amounts ranging from 50 µL of 0.25 OD/mL to 10 OD/mL per well. The enrichments of hits identified in the BASEHIT screen, as well as the predicted inert proteins 229E-S1 and ARSA. The density used in our large-scale BASEHIT screen, 5 OD/mL, is highlighted in each graph. Across all tested interactions, an input of 50 µL of 5 OD/mL provided enrichment within two-fold of the “optimal” condition, and no appreciable enrichment of inert proteins was observed under any conditions. The density of bacterial particles was determined via volumetric counts for 97 strains used in our large-scale BASEHIT screen (all strains were at ~5 OD/mL). The five strains selected approximated the lower and upper bounds of particle density (~1 × 107 to ~3 × 108 particles/mL).
