Additional file 1 of Metabolic stress constrains microbial L-cysteine production in Escherichia coli by accelerating transposition through mobile genetic elements
Creators: name: Heieck, Kevin
name: Arnold, Nathanael David
name: Brück, Thomas Bartholomäus
Publisher: name: figshare
Year: 2024
Subjects: subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics

description: Additional file 1: Table S1. Oligo sequences used for assembly of plasmids pCYS_i and pCYS_m. Table S2. Average number of generations after each passage of the simulated fermentation of W3110 and MDS42 with integrated pCYS, pCYS_i and pCYS_m. Each strain was cultivated in biological triplicates. Table S3. Average growth rates of samples taken after each passage of the simulated long-term cultivation of W3110 and MDS42 with integrated pCYS, pCYS_i and pCYS_m. Each strain was cultivated in biological triplicates. Growth rates were calculated according to the formula found in the manuscripts’ methods section of “Measurement of population growth rates”. Table S4. Mapping statistics overview. For each sample, the following statistics are provided: Reads mapped: the total number of reads mapped to the reference genome. Unique: number of uniquely mapped reads, i.e. read can only be mapped to one reference locus. Reference covered: reference bases covered by at least one read. Mean read coverage: average read coverage of the reference sequence. HGP: high generation population, LGP: low generation population. Table S5. Expression profiling statistics. For each sample, the following statistics are provided: Effective library size: The total number of reads mapped to reference features. Normalized library size: The total number of reads mapped to reference features normalized by the associated normalization factor, which can be derived by dividing the normalized library size by the effective library size. No feature: The number of reads mapping to the reference sequence that could not be assigned to any annotated feature, i.e. mapping positions and feature positions do not overlap. Filtered: The number of reads that were filtered due to insufficient mapping quality or ambiguous mapping location. These reads were ignored for read counting. Table S6. Table showing accession numbers, gene names, features, logarithmic fold changes (logFC) and p-values of all differentially expressed genes (DEGs) of W3310_pCYS with p-values &lt;0.05. LogFC and logCPM were calculated by dividing values of the later generation population (LGP) by values of the early generation population (EGP). *: Genes were excluded because they did not fall within the FC range of the metabolic cluster. Table S7. Table showing accession numbers, gene names, features, logarithmic fold changes (logFC) and p-values of all differentially expressed genes (DEGs) of MDS42_pCYS with p-values &lt;0.05. LogFC and logCPM were calculated by dividing values of the later generation population (LGP) by values of the early generation population (EGP). Table S8. Table showing accession numbers, gene names, features, logarithmic fold changes (logFC) and p-values of all differentially expressed genes (DEGs) of W3110_pCYS_i with p-values &lt;0.05. LogFC and logCPM were calculated by dividing values of the later generation population (LGP) by values of the early generation population (EGP). Table S9. Table showing accession numbers, gene names, features, logarithmic fold changes (logFC) and p-values of all differentially expressed genes (DEGs) of MDS42_pCYS_i with p-values &lt;0.05. LogFC and logCPM were calculated by dividing values of the later generation population (LGP) by values of the early generation population (EGP). Table S10. Table showing accession numbers, gene names, features, logarithmic fold changes (logFC) and p-values of all differentially expressed genes (DEGs) of W3110_pCYS_m with p-values &lt;0.05. LogFC and logCPM were calculated by dividing values of the later generation population (LGP) by values of the early generation population (EGP). Table S11. Table showing accession numbers, gene names, features, logarithmic fold changes (logFC) and p-values of all differentially expressed genes (DEGs) of MDS42_pCYS_m with p-values &lt;0.05. LogFC and logCPM were calculated by dividing values of the later generation population (LGP) by values of the early generation population (EGP). Table S12. Values of calculated plasmid DNA contents extracted from early and late generation populations (EGP, LGP). Plasmid DNA was extracted from 10 ml cultures and eluted in 50 µl each. Cell dry weights were extrapolated with the factor of 0.33 g/L/OD600= 1.0 for E. coli K-12 MG1655 cells according to Sauer et al. (2). Table S13. Single-nucleotide polymorphism (SNP) variant table. The SNP calling was done using VarSCan2 (3). Allele frequency cut-off used for variant calling was 1%. For each sample, the following variant summary is provided: Strain, plasmid, population. Additionally, POS: Position at which the variant was observed, BB/ORF: Location affected by the mutation (BB: backbone, ORF: open reading frame), REF: Reference base, ALT: Alternative base, Allele Freq: Variant allele frequency in percentage, Alt Depth: Depth of variant-supporting bases, total depth: Depth of variant-supporting bases and reference-supporting bases. Mutations which showed allele-frequencies &gt;95% were assumed to be originally present in the plasmids. Figure S1. Plots of total L-cysteine yields in mg/OD600 = 1.0. Cultivation and subsequent L-cysteine yields determination was carried out in biological triplicates of W3110 (A) and MDS42 (B) with the three different plasmids pCYS, pCYS_i and pCYS_m. Figure S2. Per base coverage depths (x) of sequenced pCYS extracted from early and late generation populations (EGPs and LGPs) of E. coli W3110 and MDS42. Sequencing was conducted with Illumina Novaseq paired end 2x150bp. Figure S3. Per base coverage depths (x) of sequenced pCYS_i extracted from early and late generation populations (EGPs and LGPs) of E. coli W3110 and MDS42. Sequencing was conducted with Illumina Novaseq paired end 2x150bp. Figure S4. Per base coverage depths (x) of sequenced pCYS_m extracted from early and late generation populations (EGPs and LGPs) of E. coli W3110 and MDS42. Sequencing was conducted with Illumina Novaseq paired end 2x150bp. Note 1. Spectrophotometric protocol for L-Cysteine determination adapted from Gaitonde (1).