Mycoplasmoides pneumoniae
Protocols: Wild-type M. pneumoniae strain M129 (WT) was grown in modified Hayflick medium at 37°C under 5% CO2 in tissue culture flasks. To generate M. pneumoniae mutant libraries, 2μg of mini-transposon plasmid DNA (pMTnCat_BDTer) was electroporated as previously described. The resulting transformants were selected during 5 days in 5ml of culture medium supplemented with 20 μg/ml of chloramphenicol, and then harvested in 1ml of fresh medium. This cell stock was referred to as passage 0 (P0). To assess mutant fitness, transformants were serially cultured through 10 consecutive passages as follows. Five ml of Hayflick medium supplemented with 20 μg/ml of chloramphenicol were inoculated with 25μl of P0. After 4 days of culture (approximately 10 cell divisions), transformants were scraped off from the flask in the culture medium, and 1 ml of cell culture (T1) was used for genomic DNA isolation using the MasterPureTMDNA Purification Kit (Epicentre, Cat. No. MCD85201). In parallel, 25μl of P1 were inoculated to obtain the next passage, repeating this procedure until passage 10 (T10). To account for sampling batch effect, the cell passaging process and sample collection was performed in duplicate. 1 ml of cell culture used for genomic DNA isolation using the MasterPureTMDNA Purification Kit (Epicentre, Cat. No. MCD85201) Between 10 and 500 ng genomic DNA were fragmented to 200-300 bp using a Covaris S2 instrument. End repair and adaptor ligation was performed using the NEBNext Ultra DNA Library Prep kit for Illumina according to manufacturer's instructions with the exception that the adaptor used contained only the read 1 adaptor sequence and not the standard Illumina Y-shaped adaptor containing read 1 and read 2 adaptor sequences. The adaptor ligated dnA was amplified with NEBNext Q5 Hot Start HiFi PCR Master Mix in a 50 µL reaction with primers R1 PA primer and Tn select PA primer (0.2 µM final concentration) using the following PCR program: 98 °C, 30 seconds; 8 cycles of 98 °C, 10 seconds and 65 °C 25 seconds; followed by a final extension of 5 minutes at 65 °C. An estimation of PCR cycles required for library amplification was performed by qPCR NEBNext Q5 Hot Start HiFi PCR Master Mix in a 50 µL reaction adding SYBR Green I (10.000x in DMSO Sigma Aldrich) to the PCR reaction at 0.1x final concentration. PCR was performed in a Roche Light Cycler LC480 for 30 cycles using the same conditions as for the first PCR reaction. 1 µL of the first PCR was used as template and Universal PCR Primer (NEB) and R2 TN select nested primer mix were used at 0.2 µM final concentration. The remaining 49 µL of the first PCR were purified using 1.8x volumes of AMPure XP beads (Beckman Coulter) according to the manufacturer's protocol. The purified product was eluted in 48 µL of buffer EB (Qiagen). 15 µL of the purified first PCR product were then used for a second PCR with the number of cycles estimated from the previous qPCR (cycle number close to plateau minus 3 cycles due to the increased amount of template). PCR conditions were the same as in the qPCR using NEBNext Q5 Hot Start HiFi PCR Master Mix in a 50 µL reaction and Universal PCR Primer (NEB) and R2 TN select nested primer mix were used at 0.2 µM final concentration but omitting the SYBR Green I. The second PCR was purified using 1x volume of AMPure XP beads and eluted in 20 µL of buffer EB. To complete adaptor sequences and add sample barcodes, a third PCR was performed with NEBNext Q5 Hot Start HiFi PCR Master Mix in a 50 µL reaction using 19 µL of the purified second PCR as templates. Primers used were Universal PCR Primer and a suitable NEBNext Multiplex primer for Illumina at 0.6 uM final concentration (Table S1). The PCR program used was: 98 °C, 30 seconds; 4 cycles of 98 °C, 10 seconds and 65 °C 75 seconds; followed by a final extension of 5 minutes at 65 °C. After the third PCR libraries were purified using 1x volume AMPure XP beads and eluted in 20 µL buffer EB.
strain
Mycoplasma pneumoniae M129 (lab strain)
isolate
not applicable
replicate
1
organism
Mycoplasmoides pneumoniae
organism
Mycoplasmoides pneumoniae
growth condition
10 passage (100 divisions)
collection date
not collected
scientific_name
Mycoplasmoides pneumoniae
common name
Filterable agent of primary atypical pneumonia
genotype
Random mutagenesis with pMTnCat_BDter
geographic location (country and/or sea)
not collected