Fig. 4: Competitive fitness assays in wild-type B. theta uncovered novel functions in carbon utilization.

a Consistency of strain fitness values between two replicates across 93 pairs of experiments with 6 pooled libraries. Gray points represent fitness scores <5. Dashed lines separate fitness scores of ≥5. Barcodes with fitness scores ≥5 whose regions overlap with other high-scoring barcodes are highlighted in green. b d-Glucosamine and GlcNAc share a metabolic pathway in Bacteroidales. ROK kinase and hexokinase hits from our screen are displayed based on biochemical evidence. c A hexokinase conferred a growth advantage on d-glucosamine when overexpressed in B. theta. d In vitro end-point enzyme assays coupled to NADH depletion were performed for hexokinase to reveal a substrate preference for d-glucosamine (GlcN), glucose, and mannose. A representative experiment with replicates of three is shown. Data is shown for mean values ± standard deviation. e B. theta expressing RafT is more fit growing on raffinose and FOS as the sole carbon source. f An AraC regulator provides a fitness gain on heparin. g A genomic region containing an ssDNA-binding protein (HMPREF1071_RS19060) is beneficial on l-fucose. For all growth curves, individual strains were cloned to identify the causative region and constructs with “+ upstream” indicate the inclusion of the upstream 200 bp. The VB minimal medium was used for each condition except for heparin where a different defined minimal medium (MM)⁴⁴ was used (Supplementary Data 4). See Supplementary Data 7 for the genomic boundaries of library fragments verified by growth assays of individual strains.