Extended Data Fig. 2: Generation of mutant strains, growth, and gene profiles in B. theta and P. merdae strains.

a, PCR verification of counter-selected deletion and complemented clones of the BT0416 gene in B. theta VPI-5482 Δtdk (WT). Lane 1: B. theta Δtdk (WT), lane 2: B. theta ΔtdkΔsult_sult⁺ (complemented strain), lane 3: B. theta ΔtdkΔsult (KO), and lane 4: NEB 1 kb DNA ladder. PCR primers, BT0416_F/ BT0416_R. Data presented represent individual PCR reactions. b, Growth profiles of B. theta cultured in BHI + media displayed no difference between B. theta WT, KO, and Δsult_sult⁺. Culture turbidity was measured as optical density at 600 nm (OD₆₀₀). Values are mean ± SEM, n = 3 biological replicates. c, Growth profiles of B. theta VPI-5482 Δtdk and VPI-5482 Δsult show no difference when incubated with either Ch (30 µM) or Ch-S (30 µM) in BHI + media. Culture turbidity was measured as optical density at 600 nm (OD₆₀₀). Values are mean ± SEM, n = 3 biological replicates. d, PCR of counter-selected colonies for the deletion of BT0412 in B. theta VPI-5482 Δtdk. Lane L: NEB 1kb DNA ladder, Lanes 1-4,5: negative cultures, Lane 4: B. theta ΔBT0412 (KO). Lane 6: Wild type culture amplification. PCR primers: BT0412_UF2, BT0412_DR2. Data presented represent individual PCR reactions. e, Growth curves plotting OD₆₀₀ of B. theta WT, ΔBT0412, and ΔBT0413-5 cultures in BHI + media with 30 µM Ch, data shown are mean ± SEM, n = 3 biological replicates. f-h, OD₆₀₀ measures of WT, ΔBT0412, and ΔBT0413-5 cultures at i) 4h, j), 8h, and k) 24h, showing significant defects in the growth of both knockout strains. Data are shown as mean ± SEM with one-way ANOVA multiple comparisons to WT with Dunnett’s correction, n = 3 biological replicates. I, Representative extracted ion chromatogram (EIC) UPLC-MS traces (left) and quantified production (right) show that B. theta and B. theta ΔBT0412 produces Ch-S when incubated with Ch (100 µM) over 72 hours while B. theta ΔBT0413-5 does not produce Ch-S under the same conditions. Data are presented as mean ± SEM, n = 3 biological replicates. j, PCR of counter-selected colonies for the deletion of BT0413-5 in B. theta VPI-5482 Δtdk. Lane L: NEB 1kb DNA ladder, Lanes 1: Wild type culture amplification, Lanes 2-12,14,16: negative cultures, Lanes 13,15: B. theta ΔBT0413-5 (KO). PCR primers: BT0413-5_UF2, BT0413-5_DR2. Data presented represent individual PCR reactions. k, B. theta ΔBT0413-5 culture lysate produced Ch-S when chemically complemented with PAPS (100 µM). No Ch-S production was detected in the absence of PAPS. Data are shown as mean ± SEM, n = 3 replicates. l, Endpoint overlapping PCR of genes in BtSULT cluster with gDNA (lanes 2, 4, 6, 8, 10, and 12) and paired cDNA (lanes 3, 5, 7, 9, 11, and 13) derived from extracted mRNA. Representative samples (n = 3) run on 0.8% agarose gel with NEB 1kb ladder (L). m, As measured by RT-qPCR, the expression of BT0416 in B. thetaiotaomicron was not affected by the addition of cholesterol at 1, 6, and 8 hours post Ch addition (n = 3 biological replicates per group, data are normalized to 16S rRNA and shown as mean ± SEM with Welch’s t-test with 2-tailed p-value). n-o, Cultures of B. theta in BHI + media with Ch (30 µM) were harvested at 48 and 168 hours and Ch-S levels were assessed in whole culture, media supernatant, and washed pellet. The data are reported as percentage of Ch-S in pellet and supernatant compared to paired whole cell culture. n = 3 biological replicates, mean ± SEM with Welch’s t-test with two-tailed correction. p, PCR verification of counter-selected clones for deletion of the PARMER_01922 gene in P. merdae ATCC 43184 (WT). Lane 1: P. merdae (WT), lane 2: P. merdae Δsult (KO), and lane 4: NEB 1 kb DNA ladder. PCR primers, PARMER01922_F/ PARMER01922_R. q, Growth profiles of P. merdae cultured in BHI + media displayed no difference between P. merdae WT and Δsult. Culture turbidity was measured as optical density at 600 nm (OD₆₀₀). Values are mean ± SEM, n = 3 biological replicates. Data presented represent individual PCR reactions.

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