As in the S. aureus project, a mini-transposon derived from the eukaryotic Himar1 mariner transposon was used to generate an ordered library of 4548 mutants in N. meningitidis , a human pathogen which is one of the leading causes of fatal sepsis and meningitis worldwide. However, transposition was performed in vitro on N. meningitidis chromosomal DNA, which was subsequently reintroduced into the bacterium by natural transformation where it integrated in the chromosome via allelic exchange. Transposon insertion sites were amplified by ligation-mediat-ed PCR from the chromosomal DNAs of each mutant, which were prepared and stored in 96-well plates, and directly sequenced. As in the previous case, mutagen-esis was performed in a strain 8013 whose genome sequence is not known but is currently being finished (our unpublished data), which temporarily hinders the exploitation of the sequencing results. Nevertheless, using the available N. menin-gitidis genome sequences it was possible to tentatively map 3221 transposon inser tions out of the 3881 that could be sequenced, and to determine that insertions occurred in at least 940 different ORFs, or approximately 45% of the 2100 expected N. meningitidis coding sequences (our unpublished data). Since PCR-amplified DNA is a suitable target for in vitro mariner transposition as well , the missing ORFs are currently being systematically amplified by PCR and subjected to targeted mutagenesis to generate the missing mutants. This hybrid mutagenesis strategy, unlike the previous examples, is expected to result in the creation of a comprehensive collection of mutants.
Another difference to the previous examples is that this toolbox has been optimized for functional studies by using mini-transposons that were engineered to contain unique identifying DNA barcodes, as in signature-tagged mutagenesis or in the Saccharomyces Genome Deletion Project, which allows up to 48 mutants to be screened simultaneously . The 4548 mutants have already been assayed in three experimental conditions [29, 31, 32], which assigned phenotypic traits to dozens of meningococcal genes, many of which were previously undescribed. For example, the simultaneous assay of pools of mutants for fitness in human serum identified 18 genes required for resistance to complement-mediated lysis including almost all the genes expected from earlier studies . Although this key virulence attribute of N. meningitidis was well characterized, four previously unde-scribed candidates were identified. However, the identification of the genes required for type IV pilus (Tfp) formation in N. meningitidis, which play a critical role in its pathogenic lifestyle by facilitating bacterial attachment to human cells, is probably the best illustration of the use of this resource for the identification of all the genes involved in a defined biological process . Mutants affected as to piliation were identified by their impaired ability to form aggregates, another phe-notype mediated by these organelles. Fifteen genes, of which only seven were previously characterized in Neisseria species, were found to be essential for Tfp biogenesis, a number similar to the number of genes essential for Tfp biogenesis in other well-studied bacteria. Importantly, this study also pinpointed another advantage of genome-wide collections of mutants, and that is the possibility of adding to the results generated by direct phenotypic screens by complementary reverse genetics analysis of all the other genes of interest that were not detected. The mutants in the corresponding genes, which may have attracted interest on the basis of particular sequence homologies or data available in the literature, can be readily retrieved in the library and analyzed immediately in the same phenotypic screens. For example, a gene that was homologous to a known Tfp biogenesis gene (pilZ) from P. aeruginosa  was, surprisingly, found to be dispensable for fiber biogenesis in N. meningitidis. This negative result is interesting since it suggested that there were some subtle differences between otherwise extremely conserved Tfp biogenesis machineries in two different piliated bacterial species.
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