NeMeSys: a biological resource for narrowing the gap between sequence and function in the human pathogen Neisseria meningitidis
- Christophe Rusniok†1, 5,
- David Vallenet†2,
- Stéphanie Floquet3, 6,
- Helen Ewles4,
- Coralie Mouzé-Soulama3, 7,
- Daniel Brown4,
- Aurélie Lajus2,
- Carmen Buchrieser1, 5,
- Claudine Médigue2,
- Philippe Glaser1 and
- Vladimir Pelicic3, 4Email author
© Rusniok et al.; licensee BioMed Central Ltd. 2009
Received: 18 August 2009
Accepted: 9 October 2009
Published: 9 October 2009
Genome sequences, now available for most pathogens, hold promise for the rational design of new therapies. However, biological resources for genome-scale identification of gene function (notably genes involved in pathogenesis) and/or genes essential for cell viability, which are necessary to achieve this goal, are often sorely lacking. This holds true for Neisseria meningitidis, one of the most feared human bacterial pathogens that causes meningitis and septicemia.
By determining and manually annotating the complete genome sequence of a serogroup C clinical isolate of N. meningitidis (strain 8013) and assembling a library of defined mutants in up to 60% of its non-essential genes, we have created NeMeSys, a biological resource for Neisseria meningitidis systematic functional analysis. To further enhance the versatility of this toolbox, we have manually (re)annotated eight publicly available Neisseria genome sequences and stored all these data in a publicly accessible online database. The potential of NeMeSys for narrowing the gap between sequence and function is illustrated in several ways, notably by performing a functional genomics analysis of the biogenesis of type IV pili, one of the most widespread virulence factors in bacteria, and by identifying through comparative genomics a complete biochemical pathway (for sulfur metabolism) that may potentially be important for nasopharyngeal colonization.
By improving our capacity to understand gene function in an important human pathogen, NeMeSys is expected to contribute to the ongoing efforts aimed at understanding a prokaryotic cell comprehensively and eventually to the design of new therapies.
By revealing complete repertoires of genes, genome sequences provide the key to a better and eventually global understanding of the biology of living organisms. It is widely accepted that this will have important consequences on human health and economics by leading to the rational design of novel therapies against pathogens infecting humans, livestock or crops . For example, identifying genes essential for cell viability or pathogenesis would uncover targets for new antibiotics or drugs that selectively interfer with virulence mechanisms of pathogenic species, respectively. The major obstacle to this is the fact that hundreds of predicted coding sequences (CDSs) in every genome remain uncharacterized. Unraveling gene function on such a large scale requires suitable biological resources, which are lacking in most species.
As shown in Saccharomyces cerevisiae, the model organism for genomics, the most valuable toolbox for determining gene function on a genome scale is likely to be a comprehensive archived collection of mutants . In bacteria, archived collections of mutants containing mutations in most or all non-essential genes have been constructed by systematic targeted mutagenesis in model species (Escherichia coli and Bacillus subtilis) and the genetically tractable soil species Acinetobacter baylyi [3–5]. Incidentally, this defined the genes necessary to support cellular life (the minimal genome) as those not amenable to mutagenesis. For a few other bacterial species (Corynebacterium glutamicum, Francisella novicida, Mycoplasma genitalium, Pseudomonas aeruginosa and Staphylococcus aureus) transposon mutagenesis followed by sequencing of the transposon insertion sites has been used to generate large (but incomplete) archived libraries of mutants [6–11]. However, multiple factors often hinder the effectiveness of these toolboxes in contributing to large-scale unraveling of gene function and/or the design of novel therapies, including: slow growth and complex nutritional requirements (M. genitalium); the fact that many of these species do not cause disease in humans (C. glutamicum, F. novicida); the use of strains for which no accurate genome annotation is available; and the frequent lack of publicly accessible online databases for analysis and distribution of the mutants.
Neisseria meningitidis (the meningococcus) possesses several features that make it a good candidate among human pathogens for the creation of such a biological resource. The meningococcus, which colonizes the nasopharyngeal mucosa of more than 10% of mankind (usually asymptomatically), grows on simple media with a rapid doubling time and has a relatively compact genome of approximately 2.2 Mbp [12–15]. Furthermore, it is naturally competent throughout its growth cycle and is therefore a workhorse for genetics. Yet, it is a feared human pathogen because, upon entry in the bloodstream, it causes meningitis and/or septicemia, which can be fatal within hours . Each year there are approximately 1.2 million cases of meningococcal infections worldwide, mostly in infants, children and adolescents, leading to an estimated 135,000 deaths .
Here we have exploited these meningococcal features to design NeMeSys, a toolbox for N. meningitidis systematic functional analysis. We opted for strain 8013 (serogroup C), which was isolated at the Institut Pasteur in 1989 from the blood of a 57-year-old male. This strain belongs to the ST-18 clonal complex, often associated with disease in countries from Central and Eastern Europe. It was chosen primarily because it is well-characterized (extensively used to study adhesion to human cells and type IV pilus (Tfp) biology) and has been previously used to produce an archived library of approximately 4,500 transposon mutants . We created NeMeSys by sequencing the genome of strain 8013, the annotation of which has been performed manually using MicroScope, a powerful platform for microbial genome annotation , and sequencing/mapping the transposon insertion sites in 83% of the above mutants, which showed that 924 genes were hit. Taking advantage of N. meningitidis natural competence for transformation, we designed a targeted in vitro transposon mutagenesis approach useful for completing the library in the future and validated it by constructing 26 mutants. The current library contains mutants in 947 genes of strain 8013. All these datasets were stored in a publicly accessible thematic database (NeisseriaScope) within MicroScope . Furthermore, to maximize the potential of NeMeSys for functional analysis and foster its use in the Neisseria community where multiple strains are used, we have manually (re)annotated the following publicly available genome sequences: four N. meningitidis clinical isolates from the different clonal complexes MC58 (ST-32, serogroup B), Z2491 (ST-4, serogroup A), FAM18 (ST-11, serogroup C) and 053442 (ST-4821, serogroup C) [12–15]; one unencapsulated N. meningitidis carrier isolate (strain α14) ; one isolate of the commensal N. lactamica (ST-640), which shares the same ecological niche as N. meningitidis; and two clinical isolates of the closely related human pathogen N. gonorrhoeae (strains FA 1090 and NCCP11945), which colonizes a totally different niche (the urogenital tract) . As above, these genomes have been stored in NeisseriaScope and are publicly accessible. Finally, we present evidence obtained through functional and comparative genomics illustrating how NeMeSys can be used to narrow the gap between sequence and function in the meningococcus.
Results and discussion
First component of NeMeSys: the genome sequence of strain 8013
General features of N. meningitidis based on six (re)annotated genome sequences
N. meningitidis strain
G+C content (%)
Coding density (%)
To achieve an annotation as accurate as possible, we annotated 8013's genome manually by taking advantage of all the functionalities of the MicroScope platform . This previously described annotation pipeline has three main components: numerous embedded software tools and bioinformatics methods for annotation; a web graphical interface (MaGe) for data visualization and exploration; and the large Prokaryotic Genome DataBase (PkDGB) for data storage, which contains more than 400 microbial genomes. We devoted particular care to identifying and duly labeling gene remnants and silent cassettes because these do not encode functional proteins and are, therefore, not targets for mutagenesis. We identified 69 truncated genes (either in 5' or 3'), which we labeled with the prefix 'truncated'. For example, the truncated rpoN encodes an inactive RNA polymerase sigma-54 factor with no DNA-binding domain . In addition, there are also three types of putative transcriptionally silent cassettes (25 in total), which we named tpsS, mafS and pilS. These cassettes have an important role in nature, generating antigenic variation upon recombination within the tpsA and mafB multi-gene families, which encode surface-exposed proteins (but this is yet to be demonstrated) or pilE, which encodes the main subunit of Tfp [25, 26]. Altogether, 8013's genome contains the information necessary to encode 1,967 proteins. Fifty-five of these proteins are encoded by out of phase genes that we labeled with the suffix 'pseudogene', most of which (94.5%) are inactivated by a single frameshift and are thus present as two consecutive CDSs. Since these pseudogenes result from the slipping of the DNA polymerase through iterative motifs , they are usually switched on again during successive rounds of replication (a process known as phase variation) and are, therefore, bona fide targets for mutagenesis. As is usual in MicroScope , 8013's genome annotation has been stored within PkDGB in a thematic database named NeisseriaScope. To facilitate access to this thematic database, we have designed a simple webpage  with direct links to some of the most salient features in MicroScope. Once in MicroScope, the user then has access to a much larger array of exploratory tools .
Second component of NeMeSys: a growing collection of defined mutants in strain 8013
General features of the collection of defined mutants in strain 8013
Transposon insertion sites sequenced
Insertion sites mapped
Insertions in genes
Insertions between genes
Number of genes mutated (total)
Statistical distribution of transposon insertions within genes
Gene size (bp)
Number of genes
% genes hit
Number of hits
% genes missed
% missed genes in DEG
Although the number of essential genes in bacteria vary in different species , a likely estimate of 350 genes being essential for growth in N. meningitidis suggests that the library contains mutants with insertions in 57.1% of the remaining 1,617 genes that might be amenable to mutagenesis. Although an increase in saturation could be achieved by assembling a much larger library of mutants, this would come at a high cost - that is, a substantial increase in both mutant redundancy and insertions in intergenic regions. We therefore took advantage of 8013's natural competence and strong tendency towards homology-directed recombination to design an alternative targeted mutagenesis strategy, robust enough to be used to complete the library (Table 2). We modified our original mutagenesis method in which genes are amplified, cloned, submitted to in vitro transposition and directly transformed in N. meningitidis  because although it could be used in strain 8013 (we generated mutants in six genes involved in Tfp biology), its efficiency was too variable for high-throughput use. The rationale of the new method was to positively select mutagenized target plasmids in E. coli before transforming them into the meningococcus. We therefore subcloned the mini-transposon into a plasmid with a R6K origin of replication that requires the product of the pir gene for stable maintenance . This allows positive selection of target plasmids with an inserted transposon in target genes after transformation of the in vitro transposition reactions in an E. coli strain lacking pir (see Materials and methods). As initially shown with comP and NMV_0901 (genes with suspected roles in Tfp biology; see below), plasmids suitable for N. meningitidis mutagenesis could be readily selected. This method was further validated by constructing 18 mutants in missed genes encoding two-component systems and helix-turn-helix-type transcriptional regulators. Interestingly, although we obtained plasmids suitable for mutagenesis, we could not disrupt fur, which encodes a ferric uptake helix-turn-helix-type regulator, and NMV_1818, which encodes the transcriptional regulator of a two-component system (Table 2). At this stage, we have at our disposal a library of mutants in 947 genes of strain 8013 (approximately 60% of the genes that might be amenable to mutagenesis; Table 2), including almost all those involved in Tfp biology and transcriptional regulation, and a robust mutagenesis method for completing it in the future.
Functional genomics: NeMeSys facilitates identification of gene function and genes essential for viability
Essential genes are defined as those not amenable to mutagenesis. During targeted mutagenesis, the absence of transformants with plasmids generated by the above method is strong evidence that the corresponding genes are essential since transformation of strain 8013 with plasmids is usually very efficient (up to 1,000 transformants per microgram of DNA). For example, although we obtained plasmids suitable for mutagenesis, we could not obtain mutants in fur and NMV_1818, which suggests that these genes are essential, at least in strain 8013. Furthermore, genes without transposon insertions that are almost certainly essential could readily be highlighted by a statistic analysis. For example, we found that non-repeated genomic regions devoid of transposons that are significantly larger than the average distance between insertions (700 bp) predominantly contain genes listed in the Database of Essential Genes (DEG) . DEG, which lists bacterial genes essential for viability in different species, has therefore been integrated into MicroScope to facilitate this analysis. This is best illustrated by the largest such region (Figure 2), which starts at 130,211, is 36.6 kbp long, and contains 47 genes but not a single tranposon insertion. At least 44 of these genes are almost certainly essential according to DEG, such as the 32 genes that encode protein components of the ribosome. Similarly, this holds true for most of the large genes that were missed (Table 3). Of the four genes longer than 3 kbp that were missed, three are almost certainly essential (rpoC, rpoB and dnaE) as they are involved in basic RNA and DNA metabolism. Of the 36 genes between 2 and 3 kbp long that were missed, approximately 80% are almost certainly essential, such as those encoding 7 tRNA-synthetases or proteins involved in DNA metabolism (dnaZ/X, ligA, gyrA, gyrB, nrdA, parC, pnp, priA, rne, topA and uvrD). Interestingly, not all genes listed in DEG are essential in the meningococcus, as we found insertions in ftsE and ftsX (involved in cell division), which are essential in E. coli, or fba (fructose-bisphosphate aldolase), which is essential in P. aeruginosa. This points to interesting differences between N. meningitidis and these species.
Third component of NeMeSys: eight additional (re)annotated Neisseria genomes
Summary of the (re)annotation effort of eight Neisseria genomes
Manually edited CDSs
CDSs deleted from previous annotation
Comparative genomics: NeMeSys facilitates whole-genome comparisons
Genes shared by six N. meningitidis strains and N. lactamica that are absent in two N. gonorrhoeae strains, some of which may play a role in nasopharyngeal colonization
Conserved hypothetical protein
Putative glycosyl transferase (pseudogene)
Adenosine phosphosulfate reductase (APS reductase)
Sulfate adenylyltransferase small subunit
Sulfate adenylyltransferase large subunit
Conserved hypothetical integral membrane protein
Hypothetical membrane-associated protein
We have designed a biological resource for large-scale functional studies in N. meningitidis that, as illustrated here, has the potential to rapidly improve our global understanding of this human pathogen by promoting and facilitating functional and comparative genomics studies. NeMeSys is viewed as an evolving resource that will be improved, for example, through completion of the collection of mutants (either through gene-by-gene or systematic targeted mutagenesis of the missed genes), further improvement of the accuracy of the annotation by taking into account any new experimental evidence, improvement of the website design and content, and addition of new Neisseria genomes as they become available. There is no doubt that NeMeSys would requite these efforts (thereby justifying its name, which was inspired by an ancient Greek goddess seen as the spirit of divine retribution) by further improving our capacity to understand gene function in N. meningitidis. Ideally, such studies could contribute to the ongoing efforts aimed at comprehensively understanding a prokaryotic cell and help in the design of new therapies.
Materials and methods
Bacterial strains and growth conditions
The sequenced strain (also known as clone 12 or 2C43) is a naturally occurring pilin antigenic variant of the original clinical isolate N. meningitidis 8013, which expresses a pilin mediating better adherence to human cells . Meningococci were grown at 37°C in a moist atmosphere containing 5% CO2 on GCB agar plates containing Kellog's supplements and, when required, 100 μg/ml kanamycin. E. coli TOP10 (Invitrogen, Paisley, Renfrewshire, UK), DH5α or DH5α λpir were grown at 37°C in liquid or solid Luria-Bertani medium (Difco, Oxford, Oxfordshire, UK), which contained 100 μg/ml ampicillin, 100 μg/ml spectinomycin and/or 50 μg/ml kanamycin, when appropriate.
The complete genome sequence of strain 8013 [EMBL:FM999788] was determined by a whole genome shotgun using a library of small inserts in pcDNA 2.1 (Invitrogen). We obtained and assembled 32,338 sequences using dye-terminator chemistry, which gave an approximately nine-fold coverage of the genome. End sequencing of large inserts in a pBeloBAC11 library aided in assembly verification and scaffolding of contigs.
Strain 8013's genome was annotated using the previously described MicroScope annotation pipeline , which has embedded software for syntactic analysis and more than 20 well-known bioinformatics methods (InterProScan, COGnitor, PRIAM, tmHMM, SignalP, and so on). In brief, potential CDSs were first predicted by the AMIGene software  using three specific gene models identified by codon usage analysis, tRNA were identified using tRNAscan-SE , rRNA using RNAmmer  and other RNA by scanning the Rfam database . CDSs were assigned a unique NMV_ identifier and were submitted to automatic functional annotation in MicroScope . Functional annotation, syntactic homogeneity and start codon position of each CDS present in the genome were then refined manually during three rounds of inspection of the results obtained using the above bioinformatics methods. This led to four major classes: CDSs encoding proteins of known function (high homology to proteins of defined function), for which the SwissProt annotation was most often used; CDSs encoding proteins of putative function (conserved protein motif/structural features or limited homology to proteins of defined function), which were labeled with the prefix 'putative'; and CDSs encoding proteins of unknown function defined either as 'conserved hypothetical protein' (significant homology to proteins of unkown function outside of Neisseria species) or 'hypothetical protein' (no significant homology outside of Neisseria species). However, adjectives were added when localization of the corresponding proteins could be predicted through tmHMM  or SignalP  (for example, 'hypothetical periplasmic protein' or 'conserved hypothetical integral membrane protein') or protein motifs not allowing functional predictions were identified through InterProScan  (for example, 'conserved hypothetical TPR-containing protein'). Importantly, during the manual curation of CDSs encoding proteins of unknown function, the dubious ones (typically those with less than 50% coding probability, shorter than 150 bp, overlapping with highly probable CDSs or RNA on the opposite strand, and so on) were deleted. During this process, self-explanatory comments mostly based on InterProScan entries and links to relevant literature in PubMed (139 in total) were entered manually in the database.
To define truncated genes, for which only partial homologies could be detected, or out of phase genes, for which homology was complete but involved at least two consecutive CDSs, we used BLASTP and coding probability results. The corresponding open reading frames were trimmed to their biologically significant portions (both on 5' and 3') and labeled with the prefix 'truncated' or the suffix 'pseudogene', respectively. During this process, putative frameshifts or sequencing errors in 42 CDSs were amplified and resequenced.
All Neisseria genomes available in GenBank (MC58, Z2491, FAM18, 053442, α14, FA 1090 and NCCP11945) or at the Sanger Institute (N. lactamica) were (re)annotated in MicroScope using the same approach as above. AMIGene was used to predict the CDSs, labeling the new ones with a distinct identifier (for example, NEIMA instead of NMA in Z2491), which were submitted to automatic functional annotation in MicroScope. The functional annotation in N. meningitidis strain 8013 was then automatically transferred to all clear orthologs, stringently defined as genes endoding proteins showing at least 90% BLASTP identity over at least 80% of their length. All the remaining CDSs were then annotated manually using the same procedure as for strain 8013, starting with Z2491 and transferring this new annotation to the remaining genomes using the same cutoff. This was then done iteratively in the order MC58, FAM18, 053442, N. lactamica, FA 1090, NCCP11945 and α14. Importantly, previously predicted CDSs that were not recognized as such by AMIGene were deleted during the process.
All the genomic analyses were performed within MicroScope using embedded software. Whole-genome comparisons of gene content (using the mentioned cutoffs) were done using the PhyloProfile Synteny functionality , which combines BLASTP, BBH and/or synteny results. Graphical representation of whole-genome syntenies were generated using LinePlot functionality . Graphical circular representation of the strain 8013 genome with transposon insertions was generated using the CGView software . Characterization of the sulfate reduction pathway in Neisseria strains colonizing the nasopharynx was done using metabolic pathway predictions built with the Pathway Tools software . GIs of putative horizontally transferred genes were identified in each N. meningitidis clinical isolate using the Genomic Island functionality tool . This tool combines detection of synteny break points in the query genome in comparison with closely related genomes, searches for mobility genes, tRNA and direct repeats (if any) at the borders of the synteny break points and finally searches for compositional bias in the query genome.
Genome-wide collection of defined mutants
The construction of an archived library of undefined transposon mutants in strain 8013 and the design/validation of a method for large-scale characterization of transposon insertion sites based on ligation-mediated PCR have been described . Each mutant is assigned a unique x/y identifier, where x indicates the half microtitre plate and y the position of the mutant. Genomic DNA for each mutant, prepared using the Wizard Genomic DNA Purification kit (Promega, Southampton, Hampshire, UK), was used to try to amplify sequences flanking the inserted transposons mainly by ligation-mediated PCR (other techniques have been tested as well). Amplified fragments were sequenced with outward-reading primers ISL or ISR internal to the transposon . Sequences were trimmed to eliminate regions of poor quality or corresponding to the transposon and subsequently mapped on 8013's genome using BLASTN.
Additional mutants were engineered by in vitro transposon mutagenesis on PCR products cloned into pCRII-TOPO or pCR8/GW/TOPO vectors (both from Invitrogen). Initially, mutants in six genes involved in Tfp biology (pilM, pilN, pilO, pilT, pilU and pilZ), four of which have been described previously , were constructed by directly transforming transposition reactions into strain 8013. We used as a donor the pSM1 vector in which the transposon is cloned within a plasmid with a ColE1 origin of replication . However, the efficiency was low, with only zero to two mutants per transposition reaction. Subsequently, we modified this method for high-throughput use by subcloning the mini-transposon into plasmid pGP704, which has a R6K origin of replication. The mini-transposon, extracted from pSM1 on a XbaI-EcoRI fragment, was cloned into XbaI-EcoRI-cut plasmid pGP704 . The resulting plasmid pYU29 can replicate only in the presence of Pir, which is found in E. coli strains such as DH5α λpir. Therefore, upon transformation of an aliquot of the in vitro transposition reaction in DH5α and selection on plates containing kanamycin (cassette in the mini-transposon) and spectinomycin (cassette in the target vector), target plasmids with an inserted mini-transposon can be positively selected. As seen initially with the comP and NMV_0901 genes, hundreds of Spr, Kmr transformants could easily be obtained while no transformants were obtained when no transposase was added in the transposition reaction (data not shown). Restriction analysis of recombinant plasmids confirmed that they contained an inserted transposon (data not shown). Transformants containing plasmids suitable for N. meningitidis mutagenesis - that is, with an insertion approximately in the middle of the target gene - were readily identified by colony-PCR by using a mix of ISL and ISR, and the forward primer used to amplify the target gene. Plasmids were then extracted, used to sequence the site of transposon insertion with ISL or ISR, and transformed in N. meningitidis. This method was validated by constructing mutants in 20 genes (NMV_0125, NMV_0126, NMV_0323, NMV_0419, NMV_0433, mtrR, NMV_0658, NMV_0757, NMV_0773, NMV_0774, NMV_0901, hexR, iscR, NMV_1093, NMV_1134, NMV_1850, NMV_2068, NMV_2160, comP and NMV_2258).
Tfps were detected by immunofluorescence microscopy using the 20D9 monoclonal antibody, which is specific for the pilin in strain 8013 as described elsewhere . This was done using a Nikon Eclipse E600 microscope and digital images were recorded with a Nikon DXM1200 digital camera mounted onto the microscope.
As usual in MicroScope , all the datasets generated during this study have been stored within PkDGB in a thematic sub-database named NeisseriaScope, which is publicly accessible through MaGe. The MaGe web interface can be used to visualize genomes (simultaneously with synteny maps in other microbial genomes, one of its main features), perform queries (by BLAST or keyword searches) and download all datasets in a variety of formats (including EMBL and GenBank). However, to facilitate access to the genome (re)annotations and distribution of mutants to the scientific community, we have designed a straightforward webpage  providing direct links to some of the most salient features in MicroScope. If needed, once in NeisseriaScope, the user has unlimited access to the whole array of exploratory tools within the MicroScope platform. Eventually, upon completion, the library of mutants will be made entirely and freely available. In the meantime, up to ten mutants can be requested simultaneously.
Additional data files
The following additional data are available with the online version of this paper: a figure showing global pairwise genome syntenies between strain 8013 and each sequenced N. meningitidis strain (Additional data file 1); a table listing genes in strain 8013 that have been disrupted in the collection of mutants (Additional data file 2); a table listing genes shared by all N. meningitidis clinical isolates (Additional data file 3); a table listing the genomic islands in each N. meningitidis clinical isolate likely to have been acquired by horizontal transfer (Additional data file 4).
adenosine 5'-phosphosulfate: BBH: bi-directional best BLASTP hit
Database of Essential Genes
Prokaryotic Genome DataBase
type IV pilus.
This work was funded by INSERM and grants from Institut Pasteur/CHU Necker-Enfants Malades, MRT and ANR. We are grateful to Elisabeth Couvé (Institut Pasteur) for help with the construction of DNA libraries, Zoé Rouy (CEA-Institut de Génomique-Génoscope) for help with database managing and Olivera Francetic (Institut Pasteur) for the kind gift of pGP704. We thank Xavier Nassif (INSERM) for support. We thank Stephen Bentley (Wellcome Trust Sanger Institute) for permission to use the unpublished N. lactamica genome sequence. We thank Chiara Recchi (Imperial College London), Jean-Marc Reyrat (INSERM) and Christoph Tang (Imperial College London) for critical reading of the manuscript.
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