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. 2003 Mar;13(3):391-8.
doi: 10.1101/gr.664303.

Large-scale analysis of the meningococcus genome by gene disruption: resistance to complement-mediated lysis

Marie-Claude Geoffroy  1 , Stéphanie Floquet, Arnaud Métais, Xavier Nassif, Vladimir Pelicic
Affiliations

Large-scale analysis of the meningococcus genome by gene disruption: resistance to complement-mediated lysis

Marie-Claude Geoffroy et al. Genome Res. 2003 Mar.

Abstract

The biologic role of a majority of the Neisseria meningitidis 2100 predicted coding regions is still to be assigned or experimentally confirmed. Determining the phenotypic effect of gene disruption being a fundamental approach to understanding gene function, we used high-density signature-tagged transposon mutagenesis, followed by a large-scale sequencing of the transposon insertion sites, to construct a genome-wide collection of mutants. The sequencing results for the first half of the 4548 mutants composing the library suggested that we have mutations in 80%-90% of N. meningitidis nonessential genes. This was confirmed by a whole-genome identification of the genes required for resistance to complement-mediated lysis, a key to meningococcal virulence. We show that all the genes we identified, including four previously uncharacterized, were important for the synthesis of the polysialic acid capsule or the lipooligosaccharide (LOS), suggesting that these are likely to be the only meningococcal attributes necessary for serum resistance. Our work provides a valuable and lasting resource that may lead to a global map of gene function in N. meningitidis.

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Figures

Figure 1.
Figure 1.
Mapping of 826 sequenced transposon insertions on the genome sequence of N. meningitidis Z2491.
Figure 2.
Figure 2.
Survival of N. meningitidis 8013 (triangles) and an isogenic unencapsulated mutant (squares) in native (A) and heat-inactivated serum (B). Error bars indicate standard errors of the means (n = 4).
Figure 3.
Figure 3.
Functional analysis of LOS mutants. (A) Representation of N. meningitidis 8013 LOS. (B) Silver-stained SDS-PAGE of LOS preparations with wild-type (WT) included as a control.
Figure 4.
Figure 4.
Functional analysis of capsule mutants. The amount of surface-bound capsular polysaccharide relative to the wild-type was determined by whole-cell ELISA quantitation. Error bars indicate standard errors of the means (n = 3).
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