Figure 3. Competition assays reveal functional specificity for predicted redundant transporters in vitro.

(A) A B. thetaiotaomicron strain encoding only BtuB1 is dramatically outcompeted in competition with the wildtype parent in conditions in which B₁₂ transport is required for growth, while a strain encoding only BtuB2 shows no competitive defect in these conditions and BtuB3 provides an intermediate phenotype. The slight competitive defect observed in methionine-replete conditions is complemented by expression of the deleted genes in trans. Wildtype, mutant and complemented strains were inoculated at an initial ratio of 1:8:1, passaged by daily 1:1,000 dilution for five days, and strain abundances determined by qPCR. Normalized mean ratios and standard deviations of mutant and complemented strains to the wildtype from triplicate cultures are shown. Asterisks indicate significant differences (t-test, * p < 0.05 of Log10 transformed data). (B) Direct competition of B. thetaiotaomicron strains encoding only BtuB1 or BtuB3 reproduces functional preferences observed in wildtype-mutant competition assays in media with methionine or B₁₂. Corrinoid structure also determines the relative fitness of these strains. Strains were inoculated at an initial ratio of 1:1, passaged, and quantified as above and mean percentages and standard deviations of each strain across triplicate cultures are shown. (C) Molecular structure of vitamin B₁₂ (cyanocobalamin) and the lower ligands of other corrinoids purified and examined in this study; abbreviations describe the corresponding cobamides (Cba) (Allen and Stabler, 2008; Renz, 1999). Dashed lines outline the structural precursor cobinamide (Cbi) and the lower ligand, which varies between corrinoids. See also Figure S3.