Fig. 1: Flowchart of the construction of the NeMeSys 2.0 complete collection of mutants in N. meningitidis 8013.
As for similar efforts in other bacteria10,12,13, we first selected protein-coding genes to be targeted by systematic mutagenesis, excluding 85 genes (4.1%, highlighted in black in the first pie chart) because they encode transposases of repeated insertion sequences, or correspond to short remnants of truncated genes or cassettes (Supplementary Data 1). We then followed a two-step mutagenesis approach explained in the text and in Supplementary Fig. 1. In brief, we first selected a subset of sequence-verified Tn mutants from a previously constructed arrayed library16,17. Mutations were re-transformed in strain 8013 and PCR-verified. We thus selected 801 Tn mutants with a disrupting transposon in the corresponding target genes (highlighted in black in the second and third pie charts). Next, we systematically mutagenised the remaining 1174 target genes using a validated no-cloning mutagenesis method relying on sPCR20. For each successful transformation, two colonies were isolated and PCR-verified. To minimise false-positive identification of essential genes, each transformation that yielded no transformants was repeated at least three times. In total, we could construct an additional 783 mutants (highlighted in grey in the third pie chart), generating an ordered library of defined mutants in 1584 meningococcal genes (Supplementary Data 3). This effort also identified 391 candidate essential genes, which could not be disrupted, encoding proteins required for N. meningitidis growth on rich medium (Supplementary Data 4).
