(A) We grew E. coli in pairwise conditions on CCA with either H. alvei, G. candidum or P. camemberti. Asterisks indicate significant differences in growth of E. coli as compared to growth alone at day 3 (Dunnett’s test, adjusted p-value≤5%) (B) Using the E. coli RB-TnSeq library, we identified genes with negative fitness in each pairwise condition at three timepoints (days 1, 2, 3). Each volcano plot shows fitness values of all 3298 genes at all timepoints (three points per gene). We identified 145 genes with a negative fitness in the presence of H. alvei in at least one timepoint, 131 genes in pairwise culture with G. candidum and 142 genes in pairwise culture with P. camemberti. Altogether, they constitute 153 genes with negative fitness in pairwise conditions. (C) Comparing these genes (dark blue) to the 160 genes with a negative fitness during E. coli growth alone (black), we obtained a total of 235 unique genes and identified 78 genes that have a negative fitness both during growth alone and all pairwise conditions (conserved negative fitness), 75 genes that have a negative fitness in at least one pairwise condition but not alone (pairwise-induced negative fitness) and 82 genes with a negative fitness in growth alone but not in at least one pairwise condition (pairwise-alleviated negative fitness). (D) We selected a gene to illustrate conserved negative fitness (corA, 37 insertion mutants), pairwise-induced negative fitness (lpoB, 31 insertion mutants), and to illustrate pairwise-alleviated negative fitness (argH, 50 insertion mutants). For each gene, we display the number of sequencing reads for associated insertion mutants in the T0 sample, in growth alone day 3 and growth with G. candidum day 3. These sequencing reads are the raw data accounting for mutant abundance and used for fitness calculation (f represents each gene's fitness value). While reads are not rarefied in the fitness calculation pipeline, we used rarefied reads for the purpose of the figure. Asterisks indicate genes with significant fitness values (consistent decrease in the number of reads per insertion mutant in the condition compared to T0). (E) We mapped the genes associated with conserved, pairwise-induced, and pairwise-alleviated negative fitness to the KEGG BRITE database. 41/75, 45/82 and 33/77 genes had hits.