Blunting enzyme mix

After fixation and permeabilization as described above for immunofluorescence, cells were treated for DI-PLA. Coverslips were washed twice for 5 min in 1× CutSmart buffer (NEB) and once in 1× blunting buffer (NEB). Afterwards, blunting was performed at room temperature for 60 min in a final volume of 50 μl for each coverslip [38.5 μl H₂O, 5 μl 10× blunting buffer (NEB), 5 μl dNTP 1 mM (NEB), 0.5 μl BSA (molecular biology grade, 20 mg/ml; NEB) and 1 μl blunting enzyme mix (NEB)]. Coverslips were then washed twice in 1× CutSmart buffer and twice in 1× T4 Ligase buffer (NEB). Then, in situ ligation was performed overnight at 16°C in a sealed humid chamber, in 100 μl final volume per coverslip using: 2 μl T4 Ligase (NEB), 5 μl 10 μM biotinylated linker (5′-TACTACCTCGAGAGTTACGCTAGGGATAACAGGGTAATATAGTTT[biotin–dT]TTTCTATATTACCCTGTTATCCCTAGCGTAACTCTCGAGGTAGTA-3′), 10 μl 10× T4 Ligase buffer (NEB), 1 μl dATP solution 100 mM (NEB), 1 μl BSA (molecular biology grade, 20 mg/ml; NEB) and 81 μl H₂O. Coverslips were washed twice in PBS and processed as described for PLA using a primary antibody against biotin partnered with a primary antibody directed against the protein under investigation (Table S1; Galbiati et al., 2017 (link)).

TnSeq was conducted as described before^{39 (link),40 (link)}; Briefly, cultures of strains MA1 and MA73 were mated with donor strain E.coli MFD λ pir, which contains the pSC189 suicide plasmid encoding the mariner transposon. Approximately 200,000 colonies/replicate were recovered from plates containing Kan50 to select for transposon insertions and 1mM IPTG to allow the expression of ShyA^L109K. Libraries were then resuspended in 30 mL of LB broth, and 1/5 of the culture was used for genomic DNA (gDNA) extraction and the rest was frozen in 30% glycerol at −80°C. Samples were prepared for sequencing as follows. The extracted gDNA was sheared by sonication (9 seconds, 30% amplitude), followed by blunting (Blunting Enzyme Mix, NEB), A-tailing, ligation of specific adaptors and PCR amplification of the transposon-DNA junctions using transposon- and adaptor- specific primers. Libraries were sequenced using Illumina MiSeq as described previously ^{41 (link)}. To determine gene essentiality, data analysis was performed using the Matlab-based pipeline ARTIST^{40 (link)}. Genetic regions predicted to be conditionally essential/enriched were inspected using the genome browser Artemis^{42 (link)} and insertion plots were generated using the tidyverse package in R.

TnSeq was conducted as described before [42 (link),43 (link)]; Briefly, cultures of strains MA1 and MA73 were mated with donor strain E.coli MFD λ pir, which contains the pSC189 suicide plasmid encoding the mariner transposon. Approximately 200,000 colonies/replicate were recovered from plates containing Kan50 to select for transposon insertions and 1mM IPTG to allow the expression of ShyA^L109K. Libraries were then resuspended in 30 mL of LB broth, and 1/5 of the culture was used for genomic DNA (gDNA) extraction and the rest was frozen in 30% glycerol at -80°C. Samples were prepared for sequencing as follows. The extracted gDNA was sheared by sonication (9 seconds, 30% amplitude), followed by blunting (Blunting Enzyme Mix, NEB), A-tailing, ligation of specific adaptors and PCR amplification of the transposon-DNA junctions using transposon- and adaptor- specific primers. Libraries were sequenced using Illumina MiSeq as described previously [44 (link)]. To determine gene essentiality, data analysis was performed using the Matlab-based pipeline ARTIST [43 (link)]. Genetic regions predicted to be conditionally essential/enriched were inspected using the genome browser Artemis [45 (link)] and insertion plots were generated using the tidyverse package in R.