Identification of essential genes

LB-I, LB-II, and LB-III were analyzed to identify the essential genes in S. Typhimurium 14028 in LB medium. We used two different tools for Tn-seq essential gene analysis. First, TRANSIT [46] analysis of essentiality on gaps in entire genome was conducted using tn5gaps algorithm. Only the insertions inside the 5% of N-terminal and 10% of C-terminal ends of all open reading frames (ORF) were included and even insertions with only one reads were considered for the analysis. The gene was considered essential if its p value ≤0.05. Secondly, Tn-Seq Explorer [48] was used for essential gene analysis by applying a 550 window size. The insertions in the 5% of N-terminal and 10% of C-terminal ends of all ORFs were removed, and insertions with only one reads were included in the analysis. The gene was considered essential if its Essentiality Index (EI) was ≤2. Then, the essentiality analysis results by both methods were combined. Finally, we demanded that for a gene to be considered essential for growth on LB medium (both LB agar and LB broth) the following three criteria should be met: (i) the gene is essential in LB-III by both tools, Tn-Seq Explorer and TRANSIT, and (ii) the gene is essential in at least 5 out of the 6 analyses that were performed for the LB-I, LB-II, and LB-III by the two analysis tools (Additional file 2: Figure S4). We made exceptions for the 17 genes for which the requirements for essential genes in at least 5 out of the 6 analyses were reduced to 4 out of 6. These exceptions were based on the observations that these 17 genes were previously identified as essential genes both in S. Typhimurium SL326 [13] and E. coli K12 [10, 11] and slightly missed the threshold for calling essential genes in this study. The list of all essential genes we identified according to our stringent conditions is shown in Additional file 1: Table S3. The same strategy was used to identify essential genes under iron-restricted conditions (Dip250-I, Dip250-II, and Dip400).