DOI: 10.5281/zenodo.6952399
title: E. coli-ΦX174 genotype to phenotype map reveals flexibility and diversity in LPS structures

description: Here are the raw sequencing data of all <em>E. coli</em> C and ΦX174 strains obtained during this study. A short description of each file's content can be found below. <em>Bacteria</em> "E.coliC_res.zip": Whole genome sequencing results of <em>E. coli</em> C WT and ΦX174-resistant strains (generated by fluctuation experiments). All<em> E. coli</em> C samples were prepared for whole-genome sequencing from one millilitre of stationary-phase cultures. Genomic DNA was extracted using the Wizard® Genomic DNA purification Kit (Promega, Germany). Bacterial samples were tested for quality, pooled, and sequenced by the Max-Planck Institute for Evolutionary Biology (Plön, Germany) using an Illumina Nextera DNA Flex Library Prep Kit to produce 150 bp paired-end reads. "E.coliC_res_excluded.zip": Whole genome sequencing results of the four ΦX174-resistant <em>E. coli</em> C strains (generated by fluctuation experiments) that have been excluded from the analyses<em>: E. coli </em>C R1, R3, R15, and R19. We found that both<em> E. coli</em> C R3 and R15 were not isogenic. While whole-genome sequencing from their respective glycerol stocks showed only a single mutation in <em>galE</em>, whole-genome sequencing carried on colony re-streaks showed that additional mutations were systematically associated with the single mutation in <em>galE</em> (<strong>see file "E.coliC_res_excluded_10_clones</strong>). <em>E. coli</em> C R1 displayed an unstable resistant phenotype. Finally, <em>E. coli</em> C R19 was partially resistant to ΦX174 WT. It carries a single mutation in the <em>yajC</em> gene, which encodes for a periplasmic protein. No apparent link to the LPS biosynthesis or assembly has been discovered yet, but <em>yajC</em> might play a role in phage DNA injection into the bacterium’s cytoplasmic membrane. "E.coliC_res_excluded_10_clones.zip": Whole genome sequencing results of 10 randomly chosen isolates of<em> E. coli </em>C R1, R3, and R15. "EcoliC.gb": <em>E. coli</em> C WT strain used as reference. <em>Bacteriophages</em> “PhiX174_PCR_399r_400f.zip”: ΦX174 samples were prepared for whole genome re-sequencing from 1 mL of phage lysate. Genomic DNA was extracted using the QIAprep Spin Miniprep Kit (QIAGEN), then amplified by performing 20 cycles of PCR using Q5® High-Fidelity 2X Master Mix (NEB). The sets of primers used for the amplification of ΦX174 whole genome are: PhiX174_399_r: CTTGACTCATGATTTCTTACC PhiX174_400_f: TTACTGAACAATCCGTACGTTTC DNA samples were tested for quality, pooled, and sequenced by the Max-Planck Institute for Evolutionary Biology (Plön, Germany). Sequencing was performed using an Illumina MiSeq DNA Flex Library Prep Kit to produce 150 bp paired-end reads. “PhiX174_PCR_2361r_2362f.zip”: ΦX174 samples were prepared for whole genome re-sequencing from 1 mL of phage lysate. Genomic DNA was extracted using the QIAprep Spin Miniprep Kit (QIAGEN), then amplified by performing 20 cycles of PCR using Q5® High-Fidelity 2X Master Mix (NEB). The sets of primers used for the amplification of ΦX174 whole genome are: PhiX174_2361_r: TCGCTTGGTCAACCCCTCAG PhiX174_2362_f: AGCGCGGTAGGTTTTCTGCT DNA samples were tested for quality, pooled, and sequenced by the Max-Planck Institute for Evolutionary Biology (Plön, Germany). Sequencing was performed using an Illumina MiSeq DNA Flex Library Prep Kit to produce 150 bp paired-end reads. “PhiX174_excluded.zip”: Since we removed R19 from the final analysis, we also removed its corresponding evolved phage obtained during the first evolution experiment (ΦX174 R19 T1). We also removed the phage infecting R5 (ΦX174 R5 T2) because it was not isogenic (confirmed by Sanger Sequencing). “PhiX174_Sequencing_Sanger.zip”: Both <em>F</em> and <em>H</em> genes were amplified by performing 35 cycles of PCR using Phusion® High-Fidelity PCR Master Mix with HF Buffer. The sequencing primers are listed in the<strong> Information_Sanger_Samples_ID.xlsx</strong> file. “PhiX174_ref.gb”: PhiX174 WT strain used as reference. We also include the raw data and pictures from our spotting tests and plaque assays used to complete the final matrix of infection. Spotting_tests_reanalyzed.xlsx”: Raw data from the pictures (see <strong>Photos_matrices</strong><strong>.zip</strong>) used to generate the Hierarchical agglomerative clustering analysis of the host ranges of evolved phage and Infection matrix of evolved ΦX174 phages on the 31 resistant E. coli C strains. The <strong>Photos_matrices</strong><strong>.zip</strong> folder contains “SSA_matrix_Bact_Lawn”: pictures of all evolution experiments obtained from the spotting test method where we spotted phages on bacterial lawns. “SSA_matrix_Phi_Lawn”: pictures of all evolution experiments obtained from the spotting test method where we spotted bacteria on phage lawns. “Mismatches”: We performed plaque assays when combinations of phages and bacteria showed discrepancies between the two spotting test methods “Plaque_assays_R12_R14”: pictures of plaque assays where we tested the sensitivity of <em>E. coli</em> C R12 and R14 toward a subset of evolved phages “Plaque_assays_of_PhiX174R22T1c1/R28T1c1_vs_WT”: pictures of plaque assays where we tested the sensitivity of <em>E. coli</em> C WT toward phages infecting R22 and R28. “displayImage.R”. Script made to look for the desired combination of phage and bacterium. Finally, we include the raw OD measurments: “All_EcoliC_growth_raw_data.xlsx”: raw OD measurements of each <em>E. coli</em> C resistant strains used in this study.