Construction of a complete set of Neisseria meningitidis defined mutants – the NeMeSys 2.0 collection – and its use for the phenotypic profiling of the genome of an important human pathogen

One of the great challenges in biology is to determine the function of millions of genes of unknown function. Even in model bacterial species, there is a sizeable proportion of such genes, which has fundamental and practical consequences. Here, we constructed a complete collection of defined mutants in protein-coding genes – named NeMeSys 2.0 – in the human pathogen Neisseria meningitidis , consisting of individual mutants in 1,584 non-essential genes. This effort identified 391 essential genes – broadly conserved in other bacteria – leading to a full overview of the essential meningococcal genome, associated with just four underlying basic biological functions: 1) expression of genome information, 2) preservation of genome information, 3) cell membrane structure/function, and 4) cytosolic metabolism. Subsequently, we illustrated the utility of the NeMeSys 2.0 collection for determining gene function by identifying 1) a novel and conserved family of histidinol-phosphatase, 2) 20 genes, including three new ones, involved in the biology of type IV pili, a widespread virulence factor, and 3) several conditionally essential genes found in regions of genome plasticity encoding antitoxins and/or immunity proteins, which become dispensable when the gene encoding the cognate toxin is deleted. These findings have implications beyond the meningococcus. The NeMeSys 2.0 collection is an invaluable resource paving the way for a global phenotypic landscape in a major human bacterial pathogen. ### Competing Interest Statement The authors have declared no competing interest.