Mutation accumulation and whole genome sequencing of bacteriophage lambda

[Lien vers la fiche source] Mutation Rate and Spectrum of lambda determined with Mutation Accumulation and whole genome sequencing. Mutation Accumulation: From the WT_L1 phage stock of lambda-cI857, a mutation accumulation experiment was conducted on 30 parallel lines, for 150 MA cycles. For the first MA cycle, the MG1655 was used to amplify lambda and generate independent lysis plaques. In each cycle, lambda phages from a liquid stock were streaked on the surface of LB agar plates (15 mL of LB Agar per 60 mm plate). After 5-10 minutes, 50 µL of overnight bacterial culture mixed with 1 mL of 0.22% top agarose supplemented with 0.2% maltose and 10 mM MgSO4 was poured onto the plates, which were incubated for 16 hours at 37°C. Then, for all remaining cycles, one single lysis plaque from the previous cycle was streaked, using a toothpick, onto a fresh 60 mm LBA plate with either 50 µg/mL kanamycin or 25 µg/mL chloramphenicol, on which top agarose mixed with bacteria was poured, as described above. Two bacterial strains were used alternatively to amplify lambda, using antibiotic resistance to counter select bacteria that might have acquired phage resistance. For kanamycin resistance, the MG1655 stfR::kanR (JL01) bacterium was used, for chloramphenicol resistance, the MG1655 tfaQ::cat (MD19) bacterium was used. One cycle of MA was initiated each morning, with an 8h incubation time at 37°C, another one was initiated in the afternoon, with a 16 hours incubation time at 37°C for 16h. At the end of each week and at the end of the 150th cycle, a single plaque per line was isolated using a wide-bore 1 ml pipette tip and resuspended in 300 µL of LB. Chloroform was added (1:10 dilution) and centrifuged for 5 min at 8000g (to kill all bacteria). 100µL of the supernatant was sampled without disturbing the pellet and kept at 4°C. Phage DNA Purification and DNA Sequencing: For all lines after 150 MA cycles and also the ancestral lambda phage stock, phage amplification was performed in WT cells, by mixing 10% of phages with 1 mL of cells at an OD600 = 0.8. The infection was carried over for 3 hours in 10 mL of LB. Phages were centrifuged for 20 min at 4500g, filtered through a 0.2 µm pore size filter and treated with Turbo DNase and RNase I. Then the phages were precipitated overnight at 4°C with polyethylene glycol 10% and NaCl 0.5 M, then centrifuged and treated with proteinase K. Finally, lambda DNA was purified using phenol:chloroform:isoamylalcohol (25:24:1) and kept at -20°C. Nanodrop and Qubit measurements were performed to access DNA purity and concentration. The integrity of the DNA was checked using 1% agarose gel electrophoresis. Libraries were prepared by Eurofins for INVIEW resequencing, using a NovaSeq™ 6000 sequencer (Illumina®, US). Read quality was controlled using fastQC v0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were aligned to the reference genome NC_001416.1 from NCBI and variants were called using Breseq v0.38.1 (http://barricklab.org/breseq). The number of true mutations was determined after exclusion of 193 nucleotide variants that were identical to the sequence of the defective prophages Dlp12, Qin, and Rac. Independently, the mutation spectrum was determined for true mutations, and the minimal recombination frequency was estimated.