Identification of pathogen-specific genes through microarray analysis of pathogenic and commensal Neisseria species

Richard A. Stabler1,{dagger}, Gemma L. Marsden1,{ddagger}, Adam A. Witney1, Yanwen Li2, Stephen D. Bentley3, Christoph M. Tang2 and Jason Hinds1

1 Bacterial Microarray Group, St George's Hospital Medical School, London SW7 0RE, UK
2 Centre for Molecular Microbiology and Infection, Department of Infectious Diseases, Imperial College London, London SW7 2AZ, UK
3 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge CB10 1SA, UK

Correspondence
Richard A. Stabler
Richard.Stabler{at}lshtm.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The release of the complete genome sequences of Neisseria meningitidis MC58 and Z2491 along with access to the sequences of N. meningitidis FAM18 and Neisseria gonorrhoeae FA1090 allowed the construction of a pan-Neisseria microarray, with every gene in all four genomes represented. The microarray was used to analyse a selection of strains including all N. meningitidis serogroups and commensal Neisseria species. For each strain, genes were defined as present, divergent or absent using GACK analysis software. Comparison of the strains identified genes that were conserved within N. meningitidis serogroup B strains but absent from all commensal strains tested, consisting of mainly virulence-associated genes and transmissible elements. The microarray was able to distinguish between pilin genes, pilC orthologues and serogroup-specific capsule biosynthetic genes, and to identify dam and drg genotypes. Previously described N. meningitidis genes involved in iron response, adherence to epithelial cells, and pathogenicity were compared to the microarray analysis. The microarray data correlated with other genetic typing methods and were able to predict genotypes for uncharacterized strains and thus offer the potential for a rapid typing method. The subset of pathogen-specific genes identified represents potential drug or vaccine targets that would not eliminate commensal neisseriae and the associated naturally acquired immunity.


Abbreviations: FUN, function unknown; MLEE, multilocus enzyme electrophoresis; MLST, multilocus sequence typing

Supplementary microarray data are available with the online version of this paper.

{dagger}Present address: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.

{ddagger}Present address: Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Neisseria meningitidis is an important cause of septicaemia and meningitis in childhood. Although the bacterium can cause fulminant bacteraemic disease, N. meningitidis is commonly found as a harmless commensal in the upper airway, where it shares this niche with other bacteria, including non-pathogenic Neisseria species. Indeed up to 40 % of the adult population harbour N. meningitidis in the nasopharynx.

The epidemiology of N. meningitidis has been widely studied. The bacterium can be characterized on the antigenic properties of the polysaccharide capsule it expresses. Strains which express the A, B, C, or W135 serogroup capsules are most associated with human disease. Multilocus enzyme electrophoresis (MLEE) and more recently multilocus sequence typing (MLST) have been used to differentiate isolates on a phenotypic and genetic basis. The results have shown that there are several lineages of related strains that circulate in human populations and there is considerable diversity between lineages. An important feature of the bacterium, which has a considerable influence on its evolution, is its ability to take up and integrate exogenous DNA. The natural competence of N. meningitidis for DNA uptake has led to a mosaic gene structure, in which regions of individual open reading frames have been acquired from other bacteria. The impact of horizontal transfer is demonstrated by the mosaic structure of genes encoding housekeeping functions (Feil et al., 1996; Zhou & Spratt, 1992; Zhou et al., 1997) and structures that are targets of host immune responses (Feavers, 2000). N. meningitidis has also acquired resistance to clinically important antibiotics such as penicillin through the transfer of alleles from Neisseria flavescens (Spratt et al., 1989). Thus, differences in gene composition and content in pathogenic neisseriae may be important in determining the pathogenic potential in strains.

A growing number of pathogenic bacteria have been analysed by comparative genomics using DNA microarrays (Behr et al., 1999; Dorrell et al., 2001; Marokhazi et al., 2003; Salama et al., 2000). In this way the information provided by genome sequencing projects can be expanded to provide an indication of variation in gene content between strains. At present four strains of Neisseria have been sequenced, with a further two more in progress. The sequences currently available cover the commonly used laboratory strains of N. meningitidis and N. gonorrhoeae, namely N. meningitidis MC58 serogroup B (Tettelin et al., 2000), N. meningitidis Z2491 serogroup A (Parkhill et al., 2000), N. meningitidis FAM18 serogroup C (The Wellcome Trust Sanger Institute, unpublished data) and N. gonorrhoeae FA1090 (ATCC 700825) (University of Oklahoma, unpublished data). Comparative analysis of the genome sequences reveals a propensity for large- to small-scale genome rearrangements.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains.
The strains (Table 1) used consisted of 38 Neisseria strains, including 12 commensal species and 18 N. meningitidis serogroup B strains (Barrett & Sneath, 1994; Pizza et al., 2000; Rokbi et al., 2000; Virji et al., 1993). MLST clonal complex data were taken from the Neisseria MLST database (http://pubmlst.org/neisseria/) unless stated. The three hypervirulent clusters ET-5, ET-37 (N. meningitidis serogroup C) and IV-1 (N. meningitidis serogroup A) as well as serogroups W-135, X, Y and Z were represented by at least one strain.


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Table 1. Strains analysed by pan-Neisseria microarray

 
Microarray design.
The pan-Neisseria multi-strain/species microarray was designed using the approach described by Hinds et al. (2002a) to cover the genome sequences of three strains of N. meningitidis (covering serogroups A, B and C) and one strain of N. gonorrhoeae. The first step in this process was the generation of a gene pool representative of all the genes in each of the four genomes. The sequence and annotation for N. meningitidis MC58 (NMB) was used as the base strain of the microarray and therefore all genes from this strain were included in the gene pool (Table 2). The NMB genes were then used as a reference to identify unique or divergent genes present in the other strains using a cumulative BLAST filtering process. For every gene in each additional strain, the BLASTN bit score for a gene against itself was determined and compared to the BLASTN bit scores against other genes already present in the gene pool; if the hit versus self bit score was twice that of any hit versus gene pool bit score then the gene was deemed strain/species-specific and added to the gene pool. This iterative approach was used to sequentially add to the gene pool any strain/species-specific genes identified in N. meningitidis Z2491 (NMA), N. meningitidis FAM18 (NMC) and N. gonorrhoeae FA1090 (NG) respectively (Table 2).


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Table 2. Composition of gene pool

 
Ten pairs of gene-specific primers were designed to each sequence in the resulting gene pool using Primer3 (Rozen & Skaletsky, 2000). Primer design parameters used were: length of 20–25 bp, matched Tm of 70±5 °C, and amplicon size range from 100 to 800 bp with an optimum size of 500 bp. From the number of potential PCR products designed for each gene, a single PCR product was selected for inclusion on the array. This selection was based on BLASTN analysis of the PCR products against genes. Firstly, the PCR products were compared to each input genome to ensure that the selected PCR product matched all BLAST-determined orthologues, thus ensuring that each gene in every strain was represented on the array. Secondly, the PCR products were compared to each gene in the gene pool to make certain that the selected PCR product had the least similarity to any other paralogues in the gene pool.

Amplification of microarray reporter elements.
PCR primers were synthesized by MWG Biotech and supplied in 96-well format to enable high-throughput amplification of PCR products using a liquid handling and PCR amplification robot (RoboAmp 9600, MWG Biotech). PCRs were performed with 10 ng DNA template, 5 U HotStar Taq DNA polymerase (Qiagen), 0·5 µM primers, 1·5 mM MgCl2 and 200 µM dNTPs. Thermocycling was performed using denaturation of 95 °C for 15 min, 40 cycles of 95 °C for 1 min, 52 °C for 1 min and 72 °C for 1 min followed by a final extension of 72 °C for 5 min.

Subsequent rounds of PCR amplification with modified conditions were performed until a single product of predicted size was obtained for all genes that were not amplified successfully under standard conditions. Additional validation was undertaken by sequencing 5 % of the amplified genes. PCR products were concentrated by 2-propanol/glycogen precipitation and resuspended in 50 % DMSO print buffer. Microarrays were constructed by robotic spotting of the PCR products in duplicate on UltraGaps amino-silane coated glass slides (Corning) using a MicroGrid II (BioRobotics) (Hinds et al., 2002b). The microarrays were post-print processed according to the slide manufacturer's instructions using hydration and UV irradiation, and stored in a dark, dust-free environment.

Microarray hybridization.
Hybridizations were performed by modifying a previously described protocol (Dorrell et al., 2001; Hinchliffe et al., 2003). Briefly, 2–3 µg test genomic DNA was labelled with Cy3-dCTP and 2 µg Cy5-dCTP-labelled N. meningitidis MC58 genomic DNA was used as a common reference for all hybridizations. Microarray slides were prehybridized in 3·5x SSC, 0·1 % SDS, 10 mg BSA ml–1 at 65 °C for 20 min before washing in distilled water for 1 min and a subsequent 1 min wash in 2-propanol. Test strain labelled DNA was mixed with reference strain labelled DNA, purified using a MiniElute kit (Qiagen), denatured at 95 °C and mixed to achieve a final 23 µl hybridization solution of 4x SSC, 0·3 % SDS. Using a 22x22 mm LifterSlips (Eyrie Scientific) a microarray was hybridized overnight sealed in a humidified hybridization chamber (Telechem International) and immersed in a water bath at 65 °C for 16–20 h. Slides were washed once in 400 ml 1x SSC, 0·06 % SDS at 65 °C for 2 min and twice in 400 ml 0·06x SSC for 2 min. The microarrays were scanned using a 428 Array Scanner (Affymetrix) and intensity fluorescence data acquired using ImaGene 5.5 (BioDiscovery).

Data analysis.
Data were initially processed and normalized using GeneSpring (Silicon Genetics). For each spot the median pixel intensity for the local background of each spot was subtracted from the median pixel intensity of each spot. Background subtracted pixel intensities for the test strain channel were divided by those for the reference strain channel. The resulting log ratios were normalized by applying the LOWESS intensity-dependent normalization using 50 % of the data. If the value for the reference channel was less than 10 then a value of 10 was used instead. This normalization scheme counteracted any dye effects at low intensity and enabled inter-array comparisons by equalizing the median ratio on each array.

Designation of genes in each strain as present, divergent and absent (highly divergent) was determined using GACK software without the need for arbitrarily defined cut-offs (Fig. 1) (Kim et al., 2002). GACK analysis used LOWESS normalized log ratio data from GeneSpring that was not flagged as absent by ImaGene and all genes were given equal weighting. GACK used the normal distribution to calculate an EPP (estimated probability of presence) value for each gene; in this way the overall genomic sequence deviation (data spread) was taken into account. A gene was designated present (assigned ‘1’) if it had a calculated EPP of 100 %, as divergent (assigned ‘0’) if it had an EPP between 0 % and 100 %, and as deleted (assigned ‘–1’) if it had an EPP of 0 %. An EPP of 0 % indicates a zero per cent chance of being falsely assigned as a divergent gene and an EPP of 100 % indicates a minimum assurance that a gene was present (Kim et al., 2002). Divergence, although primarily indicating sequence divergence of a particular gene, can also indicate a change in copy number of members within a related gene family (e.g. IS elements and transposons). GACK software was also used to model presence of non-N. meningitidis MC58 (additional) genes in the test strain by performing GACK analysis on the inverse of the log ratio data. Strain-specific present genes were designated present (‘1’) in both analyses (Fig. 1).



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Fig. 1. (a) Histogram of the GACK output for N. meningitidis serotype B strain 1000. Red line, data; green line, estimated present genes; blue line, EPP (estimated present percentage); dashed lines, EPP cut-off values [orange, 0 % EPP; black, 100 % EPP]; ABC, GACK output; XYZ, GACK output from mirrored data; A, deleted (–1) genes; B, divergent genes (0); C, present genes; Z, additional (–1) genes; Y, divergent genes (0); X, present genes; P, true present gene set; B+Y, divergent gene set. (b) Scatter plot showing GACK output. Red, present genes (P); black, marginal genes (B+Y); green, absent genes (A); blue, extra genes (Z).

 
The GACK output for all genes was used for phylogeny inference, calculated using a Bayesian phylogenetic algorithm (MrBayes v3.0B4 http://morphbank.ebc.uu.se/mrbayes/info.php) using 1 million iterations with a heat of 0·5. Due to the binary requirement of MrBayes, divergent data and additional non-N. meningitidis MC58 genes were attributed to the present gene category. Phylogeny inference was therefore based on a conservative estimation of gene loss and non-MC58 gene acquisition. The dendrogram was viewed by TREEVIEW (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) (Fig. 2).



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Fig. 2. Bayesian cluster analysis of GACK data from all Neisseria strains. All N. meningitidis strains are serogroup B except F6124, 2059001 and Z2491 (serogroup A), 90/18311 (serogroup C), A22 (serogroup W135), E26 (serogroup X), 860800 (serogroup Y) and E32 (serogroup Z). Commensal strains consist of: L, N. lactamica; P, N. polysaccharea; F, N. cinerea; K, N. flavescens; H, N. perflava.

 

   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Phylogenetic relationships based on microarray analysis
The pan-Neisseria microarray was designed on the genomic sequences from three N. meningitidis strains and one N. gonorrhoeae strain. To investigate the conservation of genes in Neisseria spp. we analysed eleven isolates (including seven species) of non-pathogenic neisseriae. DNA from organisms with substantial gene sequence divergence compared with the PCR products on the microarray will fail to hybridize, producing a weak signal or no signal for a large proportion of genes. As expected, the N. meningitidis and N. gonorrhoeae strains tested produced high-quality hybridization data. Of the commensal species, Neisseria cinerea, N. mucosa, N. flavescens, N. lactamica and N. polysaccharea strains also produced good signal intensities across the whole microarray, while N. cuniculi and N. elongata strains produced high-intensity signal for only a limited number of genes, indicating that a large number of genes were too divergent or absent to make further statistical analysis reliable.

The phylogeny dendrogram based on GACK scores for every gene represented on the microarray (Fig. 2) showed that N. meningitidis strains clustered closely together, with commensal Neisseria species at greatest distance and N. gonorrhoeae occupying an intermediate position between the two groups. Within the N. meningitidis group, N. meningitidis serogroup A strains formed a distinct cluster, with the MLST clonal complex ST-4/subgroup IV strains (2059001 and Z2491) being marginally more closely related to each other than F6124 (MLST clonal complex ST-5/subgroup III). Strains of MLST clonal complex ST-41/44 complex/lineage 3 strains (BZ147, BZ198, NGH36 and NGH15) clustered with each other. Both strains 528 and 1000 are from the ST-18 clonal complex and occur close to the root of the dendrogram together with NGE28.

BZ83 and H44/76 are from the same ST-32 complex/ET-5 clonal complex as N. meningitidis MC58 and were remarkably conserved in gene content and sequence. N. meningitidis C311 clusters closely with BZ83 and H44/76 but is of unassigned clonal cluster. GACK analysis scored 36 N. meningitidis MC58 genes absent in BZ83 whereas H44/76 and C311 had 52 and 57 genes absent respectively. Microarray data of the ‘absent’ genes showed that the majority of genes [12 (33 %), 23 (44 %), 37 (65 %) respectively] clustered in a region between NMB1123 and NMB1197 and had a ratio of approximately 0·5 (twofold decrease in signal intensity). This region contains a number of phage-related genes and was part of a larger section of the genome that showed high variability between strains. Therefore, this area probably represents a cryptic phage; the ratio data are consistent with this region being duplicated in MC58. BLAST analysis of the region confirms that the NMB1123–1159 region has been duplicated (at NMB1161–1197) but this region was only present as a single copy in the other sequenced isolates [N. meningitidis Z2491 (NMA1332–1369), FAM18 (NMC1063–1098) and N. gonorrhoeae FA1090 (NG0801–0775)]. The ratio data suggest that this duplication has been relatively recent, as the closely related species appear to have only a single copy, which may be a result of serial laboratory passage. NMB1123 (and hence NMB1161), encoding a hypothetical protein, shows regions of >=98 % identity with at least seven other N. meningitidis MC58 genes; these may act as IS-like elements and allow genetic rearrangement.

Gene composition of pathogenic neisseriae
Analysis of the microarray data identified 1499 present and divergent genes in all N. meningitidis serogroup B strains (core genes), of which 1160 genes were present in all N. meningitidis and N. gonorrhoeae isolates examined. The remaining 339 genes were found in at least one of the non-serogroup B N. meningitidis or N. gonorrhoeae strains tested, but not all, and therefore no gene was specific to serogroup B.

Analysis of the commensal strains was restricted to N. cinerea, N. mucosa, N. flavescens, N. lactamica and N. polysaccharea (seven strains) due to the sequence divergence of N. cuniculi and N. elongata (including N. elongata var. glycolytica). The commensal present and divergent gene set consisted of 1225 genes (core gene set), 1024 of which were also present in the N. meningitidis serogroup B core gene set; 862 genes were common to all strains tested.

Within the commensal group, 105 genes were absent by GACK analysis in all strains, 55 of which were present in the N. meningitidis serogroup B core gene set (Table 3). All 55 genes were also confirmed to be absent from the N. lactamica ST-640 sequence available at the Sanger Centre by BLAST analysis, which at the time of analysis had a theoretical genome coverage of 99·99 %. The N. meningitidis serogroup B shared gene list has an over-representation of ‘cell processes' and ‘other’ functional categories compared to the N. meningitidis MC58 genome. The 55 N. meningitidis serogroup B shared genes contain 10 transposon-associated genes, 3 phage-related and 23 encoding conserved/hypothetical proteins of unknown function (FUN). Eight genes show homology to the non-functional pilS gene cluster. Additionally a gene encoding a putative TonB-dependent receptor (NMB0293{equiv}NMA2193, NMC1887) was present. TonB has a function in transport of iron from receptors on the outer membrane to the bacterial cytoplasm. A number of the hypothetical proteins possess integral membrane motifs and membrane transport motifs; in addition to these, the N. meningitidis serogroup B conserved genes include ones encoding a putative periplasmic type I secretion system protein, and a putative cytolysin secretion ABC transporter. Furthermore a capsule polysaccharide modification protein gene lipB was present. dca has evidence of phase variability and was previously shown to be absent from N. lactamica (Snyder et al., 2001); it may have a role in competence.


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Table 3. N. meningitidis serogroup B core gene set absent from all commensal Neisseria species

 
opcB (NMA2183{equiv}NMB0304, NMC1877) encodes an outer-membrane protein that mediates adhesion and invasion (Zhu et al., 1999), although this appears to be a pseudogene in the three N. meningitidis sequenced genomes. opcB probably resulted from a duplication event (Zhu et al., 1999) due to the presence of opcA (NMB1053{equiv}NMA1251); however opcA was absent from N. meningitidis FAM18. Both opcA and opcB are present in all serogroup B strains with the exception of opcA in N. meningitidis 528, B16B6, BZ232, NGE28 and NGH38. N. gonorrhoeae lacks both but contains a unique putative opc orthologue (NG1596). All three orthologues were absent from all commensal strains. Seiler et al. (1996) reported that an opc probe did not hybridize with ET-37 complex strains; however the FAM18 genome sequence contains opcB. Microarray data for 90/18311 indicated that opcA and opcNg were absent but also indicated the presence of an opcB orthologue. BLAST analysis of the opc fragment used by Seiler et al. (1996) (accession no. M80195) was homologous with opcA but was not similar to opcB.

Analysis of the complete pilin locus (NMA0264-0272{equiv}NMB0018–0026) (Fig. 3) demonstrated that this locus is absent from all the commensal strains (except a possible copy of pilS7 which was classified as divergent by GACK). Of the N. meningitidis strains, N. meningitidis F6124 (serogroup A), N. meningitidis B16B6, NGE28 and 2996 (serogroup B) and N. meningitidis 90/18311 (serogroup C) show sequence divergence of the locus but to a lesser extent than the commensals (mean locus ratio of 0·5 compared to 0·2 respectively). GACK analysis of B16/B6 scored all pilS genes as divergent except pilS2, pilS5 and pilS6, which were absent; however these were slightly over the divergent/absent cut-off and the result could be due to a reduced copy number as well as sequence divergence. GACK analysis indicated that pilE was absent from NGE28 but divergent in N. lactamica L18. The ratio of pilE from NGE28 shows that the gene was close to the divergent cut-off and its apparent absence may be a result of sequence divergence, whereas pilE from L18 was just inside the divergent cut-off but has a lower microarray ratio than NGE28 due to the overall sequence divergence of the N. lactamica and may not be present.



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Fig. 3. N. meningitidis pilS loci. (a) pilE and pilS regional organization. All pilS reading frames are partial gene sequences. (b) pilE and pilS microarray ratio data. Each column represents one CDS; each row represents hybridization data from each Neisseria species tested. Blue, deleted/highly divergent; yellow, present. All N. meningitidis are serotype B unless indicated in square brackets. Absent, all except pilS7 scored as absent by GACK; Div, divergent (majority of genes scored as divergent by GACK); Prt, present (majority of genes scored as present by GACK).

 
The N. gonorrhoeae FA1090 pilin pilE and pilS regions have been previously described (Hamrick et al., 2001). The authors identified two pilE genes and five regions consisting of 19 silent pilS genes. Although the DNA sequence is essentially the same as the N. gonorrhoeae FA1090 genome sequence the open reading frames have been annotated differently, resulting in a slightly different number of pilin genes. The two pilE genes (pilEc1 and pilEc2) correspond to NG1959 and NG1960 respectively. The pilS1 locus (pilS1c1 to c5) equates to NG1968–1971, the pilS2 locus (pilS2c1 to c6) to NG1962–1966, the pilS3 locus (pilS3c1 to c3) to NG1937–1938, the pilS6 locus (pilS6c1 to c3) to NG1939–1941 and the pilS7 locus (pilS7c1) to NG1420. The N. gonorrhoeae pilin loci had low-level homology to the NMA-Z1491 pilin locus but were represented by specific products on the microarray. N. gonorrhoeae pilin appears to be absent from all commensal strains but present in all the N. meningitidis strains due to similar low-level cross-hybridization with the NMA-Z1491 pilin locus; indeed as expected the loci were scored as extra genes by GACK only for N. gonorrhoeae NMNG. The only exception was NG1937 (pilS3c1), which had a good similarity to NMA0268 (pilS4), which was scored as present in all strains including the commensals but divergent for N. gonorrhoeae.

Phase-variable PilC is required for pilus assembly and pilus-mediated adherence, and most strains carry two pilC loci (pilC1 and pilC2) which are homologous but not identical; however, only PilC1 mediates adherence of N. meningitidis (Rytkonen et al., 2004). Analyses of pilC1 and pilC2 using microarray data and/or GACK scores (Table 4) are confounded by cross-hybridization between genes and between species due to the slight sequence divergence. pilC1 was represented on the microarray by three reporters, NMA0609, NMB1847 ({equiv}NMC0371) and NG0046, and pilC2 by NMB0049 ({equiv}NMA0293), NMC0033 and NG1812. pilC1 and pilC2 were absent from all commensal strains tested except that a marginally positive GACK score was detected for pilC1NMA with both N. lactamica strains and N. mucosa, and pilC1NMB/C with N. lactamica L18 (Table 4). pilC is probably absent given the lack of the remainder of the pilin biogenesis locus; additionally, BLAST analysis against the N. lactamica ST-640 sequence failed to identify a pilC2 homologue. Of the pilC1 reporters, pilC1NMB/C and pilC1NG gave weak homologies with N. lactamica ST-640; however, the pilC1NMA reporter showed a region of identity up to 522/633 bp (82 %), indicating a potential orthologue and explaining the divergent GACK prediction.


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Table 4. Presence of pilC orthologues within Neisseria strains

P, present; P+, present (marginal towards extra); P–, present (marginal towards absent; may indicate sequence divergence or cross-hybridization); A, absent; X, extra (the microarray reporter for NMB1847{equiv}NMC0371 and NMB0049{equiv}NMA0293).

 
Analysis of the N. meningitidis Z2491 genome for capsule-related genes identified two putative operons: NMA0199-0203 consists of sacABCD and galE, and NMA0198-0195 represents ctrABCD genes. The 3' end of ctrA is highly conserved among the meningococci and is used as a PCR-based diagnostic test for the presence of Neisseria; however the 5' end varies and can be used to distinguish between serogroups 29E, X and Z (Bennett et al., 2004). N. meningitidis MC58 also possesses ctrABCD orthologues (NMB0071–0074) and galE (NMB0064), although there is sufficient sequence divergence between orthologues to design differentiating reporters for ctrB (NMA0197 and NMB0072) and ctrC (NMA0196 and NMB0073). sacABCD are responsible for a serogroup A specific capsule polysaccharide (Parkhill et al., 2000) and are absent from N. meningitidis MC58 and N. meningitidis FAM18. N. meningitidis MC58 contains a polysialic acid capsule biosynthesis operon adjacent to the ctrABCD orthologues, siaDCB and synX (NMB0067–0070), which are absent from N. meningitidis Z2491. siaD (NMB0067) in MC58 has been truncated by the insertion of an erm cassette and was not represented on the microarray. N. meningitidis FAM18 (NMC0052–72) possesses the same genes and arrangement as N. meningitidis MC58; however siaD (NMC0051) has not been truncated and the operon has a final gene in the operon (NMC0050) which encodes a hypothetical protein. The NMC0051 siaD reporter only matched NMB0067 along the first 321 bp with 68 % identity and could not be used to detect NMB0067. It is therefore possible to distinguish between the three operons on the microarray through analysis of their unique genes. The three N. meningitidis serogroup A strains all have positive scores for the Z2491 operon, and N. meningitidis serogroup C strain has the full siaD and NMC0050. The majority of N. meningitidis serogroup B strains have the complete MC58 operon except 528 and 297-0. Strain 528 does not possess any of these capsule biosynthesis genes and strain 297-0 contains ctrACDNMB/C shared genes and two of the NMA-specific genes (sacB and sacC). N. gonorrhoeae does not express a capsule and N. gonorrhoeae NMNG microarray results indicated a lack of these capsule genes. The commensal strains and the serogroup X strain (E26) all lack all capsule biosynthesis operons entirely as expected. However, serogroup X strains have been shown to have three unique capsule biosynthesis genes (xcbABC) between ctrA and galE where sacABCD were present in N. meningitidis Z2491 (Tzeng et al., 2003). These unique genes were not present on the microarray, explaining the apparent lack of capsule genes in N. meningitidis E26.

DNA adenine methyltransferase (Dam) is involved in the bacterial mismatch repair system; the loss or absence of the gene impairs this repair system, which may increase the occurrence of frameshifts in homopolymeric tracts or simple base repeats (Bucci et al., 1999). This is an important bacterial mechanism for alteration of gene expression, allowing the on–off switching of loci that can confer heritable niche-specific adaptation. N. meningitidis FAM18 possess a functional dam gene (NMC0327); however in N. meningitidis MC58 and N. meningitidis Z2491 only a non-functional dam gene remnant remains – the rest has been replaced by the gene drg (dam-replacing gene), which encodes a restriction enzyme. This genetic exchange has been linked with increased virulence and conserved within particular ST types (Jolley et al., 2004). The pan-Neisseria microarray had a reporter for dam (NMC0327) and drg (NMB1896) from which it was possible to establish whether a strain was dam+ or drg+ (Table 5). Martin et al. (2004) listed a number of dam+ and dam strains which correlated with the microarray data (Table 5), and Jolley et al. (2004) identified ST lineages that were drg+, which was also consistent with the microarray data (Table 5).


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Table 5. Pan-Neisseria microarray results for the presence of dam or drg genes

NMC0327 reporter indicated the presence (Pos) or absence (Neg) of dam (GACK score of ‘extra’ or ‘core’ respectively); NMB1896 ({equiv}NMA0560, NG0007) reporter indicated the presence (Pos) or absence (Neg) of drg (GACK score of ‘core’ or ‘deleted’ respectively).

 
Hypotheticals
The FUN (function unknown) gene set [defined as putative or hypothetical genes that encode a protein with a given location (e.g. periplasmic) and/or type (e.g. lipoprotein) in the annotation of N. meningitidis Z2491 and N. meningitidis MC58] consists of 1014 genes, 447 of which occurred in the N. meningitidis serogroup B core gene set, indicating that they are conserved within the serogroup. Twenty-nine FUN genes are absent from all commensal strains, 14 of which are conserved in all N. meningitidis serogroup B strains (Fig. 4) and 3 are absent in only one N. meningitidis serogroup B strain.



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Fig. 4. Conservation of FUN genes absent from all commensal strains. a{dagger}, NMA1987 (NMB1731a); b{dagger}, NMA1219 (NMB1000a); c{dagger}, NMA1218 (NMB1000a); d, NMA0695 (NMB0514); e{dagger}, NMA0510 (NMB1942a); f{dagger}, NMA0184 (NMB0084); g{dagger}, NMA0029 (NMB0229); h, NMB2014; i, NMB2013; j{dagger}, NMB1782; k, NMB1775; l, NMB1772; m{dagger}, NMB1268; n, NMB1195; o, NMB0899; p, NMB0883; q, NMB0864; r, NMB0656; s{dagger}, NMB0654; t, NMB0511; u, NMB0499; v{dagger}, NMB0473; w, NMB0248; x{dagger}, NMB0240; y{dagger}, NMB0239; z{dagger}, NMB0230; {alpha}{dagger}, NMB0226; {beta}, NMB0175; {Delta}, NMB0104. All N. meningitidis are serotype B unless indicated in square brackets. Blue, deleted/highly divergent FUN gene; yellow/red, conserved FUN gene; NMA, N. meningitidis Z2491 designed and derived products that represent the N. meningitidis serotype B (MC58) orthologue gene, which may have been absent from the original annotation; {dagger}, conserved FUN gene in all N. meningitidis serotype B strains (core gene set).

 
Grifantini et al. (2003) identified a subset of 231 N. meningitidis MC58 genes by microarray that showed differential regulation during growth in response to iron-depleted or -replete conditions, of which 77 were of unknown function. Thirty-nine of these are conserved within the N. meningitidis serogroup B core gene set but only three were absent from all commensal strains (see supplementary Fig. S1 with the online version of this paper). Grifantini et al. (2002) also identified 344 genes that were differentially regulated on adherence to epithelial cells over a 3 h period. Of these, 96 were FUN genes and 36 were conserved in the N. meningitidis serogroup B core gene set but none of these were absent in all commensal strains. Two FUN genes in the epithelial infection gene list were not found in any of the commensal strains but these were not conserved throughout the N. meningitidis serogroup B strains (supplementary Fig. S2).

Sun et al. (2000) identified through signature-tagged mutagenesis 16 N. meningitidis MC58 hypothetical genes essential for bacteraemic disease; an additional 10 hypothetical genes have since been identified as being required for colonization of the nasopharynx (unpublished results). Twenty-one of the genes were present in the N. meningitidis serogroup B core gene set but none of these were absent from all commensal strains (Fig. 5). NMB0103 (putative bacteriocin resistance protein) was not found in the commensal strains but was present in all N. meningitidis strains except NGE28. Thirteen genes were present in all strains tested and may be part of general stress-response pathways. NMB1596 was present only in ST-32 complex/ET-5 complex cluster strains, and NMB0065 was absent from all serogroup A strains but also serogroup B strains 528 and 297-0.



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Fig. 5. Conservation of genes essential for bacteraemic disease or colonization. a, NMB1971; b, NMB1954*; c, NMB1829; d, NMB1812; e, NMB1726*; f, NMB1694*; g, NMB1681; h, NMB1638; i, NMB1596; j, NMB1564*; k, NMB1523; l, NMB1012; m, NMB0840; n, NMB0734*; o, NMB0673*; p, NMB0572; q, NMB0541; r, NMB0349*; s, NMB0329*; t, NMB0317*; u, NMB0313*; v, NMB0287*; w, NMB0188*; x, NMB0183*; y, NMB0103; z, NMB0065. All N. meningitidis are serotype B unless indicated in square brackets. Blue, deleted/highly divergent gene; yellow/red, conserved; grey, no data; *, conserved in all strains.

 
Tettelin et al. (2000) identified 96 putative pathogenicity genes in N. meningitidis MC58 through homology matches with known virulence genes. Out of the 96 putative genes, 49 were found in the N. meningitidis serogroup B core gene set but only 7 were absent from the commensal strains (supplementary Fig. S3). A further 22 putative pathogenicity genes were absent from all commensal strains but were not conserved in the N. meningitidis serogroup B strains. Out of the 11 colonization genes identified by Tettelin et al. (2000), nine are in the N. meningitidis serogroup B core gene set. Of the remaining two colonization genes, NMB0547 occurs in all N. meningitidis serogroup B strains except 528 and occurs in the N. lactamica, N. cinerea and N. flavescens strains tested. NMB1994 was absent from the majority of strains tested but was present in N. meningitidis BZ83 and C311, which appear closely related to N. meningitidis MC58 but not H44/76.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
GACK results/core gene sets
Analysing the microarray data defined genes that are conserved across the range of serogroup B strains tested, which includes representatives of each major clonal lineage. However, none of these genes were specific to serogroup B isolates, each gene being present in at least one other N. meningitidis serogroup or N. gonorrhoeae. We were able to identify genes that are absent from all the commensals (and from the N. lactamica sequencing project) but present in all N. meningitidis serogroup B species tested. These genes are potentially important drug or vaccine targets against pathogenic neisseriae. Given the role of commensal Neisseria spp. in the acquisition of natural immunity against the meningococcus, strategies that do not lead to the eradication of non-pathogenic species may be preferable.

Barratt & Sneath (1994) used 155 phenotypic tests to characterize four major groups within the Neisseriaceae. The majority (33/38) of strains tested for our work fall within the area A, which includes N. meningitidis, N. gonorrhoeae, N. polysaccharea, N. lactamica and N. cinerea. Area B species included N. mucosa while area C was represented by N. elongata (including var. glycolytica) and N. flavescens. Area D, which is described as the ‘false Neisseria’, was represented by N. cuniculi [synonym Moraxella (Branhamella) cuniculi]. Our microarray analysis of N. cuniculi and N. elongata (including var. glycolytica) is entirely consistent with this classification of strains. The DNA from strains of areas C and D produced poor hybridization signals to a large number of microarray reporters. Interestingly, for one isolate, there is a discrepancy between the phenotypic analysis and our microarray results. N. flavescens has been classified as an area C strain but microarray analysis showed no apparent significant sequence divergence from area A strains.

Smith et al. (1999) analysed Neisseria species relatedness using MLST of three housekeeping genes (recA, argF and rho) and 16S rRNA, and placed them in five related groups. Group 1 (meningitidis group) consists of the species closely related to and including N. meningitidis: N. gonorrhoeae, N. lactamica, N. polysaccharea and N. perflava M7. Group 2 (flavescens group) was represented by N. flavescens K3, group 3 (cinerea group) by N. cinerea F12, and group 5 (elongata group) by N. elongata I1 and N. elongata var. glycolytica J3 and J15. The strains used for our microarray analysis included four of these five groups. Strains from groups 2 and 3 did not differ sufficiently from group 1 to affect the microarray hybridizations. This finding was supported by 16S rRNA sequence typing, which places groups 2 and 3 in the same sub-branch as N. meningitidis group 1 strains (Smith et al., 1999). Dendrograms based on phenotypic traits of argF and recA place N. cinerea as a distant relative of N. meningitidis serogroup, whereas rho and 16S RNA identify the species as a close relative (Smith et al., 1999); our data are consistent with N. cinerea being closely related. Our results confirm that the group 5 species are highly divergent from pathogenic neisseriae.

Our microarray phylogeny was concordant with those obtained by MLST analysis, as strains of the same clonal complex still clustered together. The MLST dendrogram indicates clustering together of the hypervirulent MLST type strains, which is also reproduced with the microarray phylogeny. However, in the MLST phylogeny SWZ107 appears as a close relative to the ST-32/ET-5 clonal complex (e.g. MC58) whereas the nearest relatives of NG3/88 and NG6/88 belong to ET-37 complex (e.g. serogroup C); on the microarray phylogeny SWZ107 also clustered with the ST-32/ET-5 clonal complex but had greatest similarity with NG3/88 and NG6/88.

Interestingly we also detected a major split within the strains tested. One group contains the majority (12/18) of serogroup B strains plus E32 (serogroup Z) (‘B’ arm), with the second group containing, contains all the non-serogroup B strains including the commensals (‘A’ arm), as well as three serogroup B strains.

On the microarray phylogeny the ET-37 complex strain (9018311) occurred on a different major branch (‘A’ arm) of the dendrogram to the ST-32/ET-5 clonal complex (‘B’ arm). A further MLST cluster consists of BZ198, NGH15, NGH36, BZ147, NGH38, BZ232 and 297-0, which is replicated within the microarray phylogeny, down to sub-branch levels, except 297-0, which showed divergence from this group.

Congruence of MLST clonal complexes with the array data was surprising given the high degree of lateral gene transfer and genetic rearrangements that occur within Neisseria species (Feavers, 2000). This shows that MLST conservation is a good estimation of the conservation within the whole genome but not necessarily of gene arrangement.

Pathogen-specific genes
Bioinformatic analysis of the complete genome sequence identified 55 potential virulence genes in N. meningitidis. These genes were universally present in the N. meningitidis serogroup B strains analysed, and absent from commensal strains and the available N. lactamica genome sequence. The majority of known genes have some role on the bacterial cell surface, including pilin genes, secretion system proteins, capsule modification, and an adhesion and invasion protein. The presence of 13 transposon and phage genes may indicate a route of acquisition. The pilin locus cluster consists of a number of pseudogenes that have the potential to recombine with pilE to form functional pilin subunits and may have a role in altering the surface antigens to assist in avoiding the host cell immune system. These genes appear to have some sequence divergence between strains, which is likely to have evolved through host immune pressure; this is borne out by the fact that N. gonorrhoeae has a distinct pilin locus from N. meningitidis. Although pili are an important virulence factor for pathogenic neisseriae the presence in commensals is less established and our data suggest that they lack the pilE pilS loci; however, other pili-related genes appear to be conserved. In all commensal strains pilD (NMB0332), pilF (NMB0329), pilT (NMB0052) and pilT2 (NMB0768) were scored as present and pilQ (NMB1812) was scored either present or divergent by GACK. Although pilE (NMB0018) was found to be absent from all commensals, an orthologue found in N. meningitidis FAM18 (NMC0210) was found to be present in both N. lactamica strains and was confirmed to be present in N. lactamica ST-640 by BLAST analysis. Expressed pilin can be categorized into two structurally distinct classes: class I (e.g. N. gonorrhoeae MS11, N. meningitidis MC58) and class II (e.g. N. meningitidis FAM18). The monoclonal antibody SM1, used to distinguish these two classess, binds to a highly conserved region within the 5' end of class I PilE but this epitope was lacking from the more structurally divergent class II PilE (Aho et al., 2000; Kahler et al., 2001). The SM1 minimum epitope has been determined as the linear peptide EYYLN (Virji et al., 1989). Analysis of the translated genome sequences indicates that this was present in the class I pilin strains N. meningitidis NM58 and N. gonorrhoeae NMNG but absent in the class II strain N. meningitidis FAM18. In N. meningitidis MC58 the SM1 epitope occurred in pilE and four of the silent pilS genes; this was also replicated in N. meningitidis Z2491. The pilE reporters on the pan-Neisseria cDNA microarray cannot distinguish between these slight sequence changes, especially as the gene has a number of highly variable regions, but identification could be achieved using specifically designed oligonucleotides on an oligo-microarray.

The acquisition of a polysaccharide capsule has been suggested as a vital step in the evolution of non-pathogenic neisseriae into pathogenic neisseriae. N. meningitidis MC58 and N. meningitidis FAM18 have identical capsule genes with the exception of siaD. The N. meningitidis Z2491 capsule region contained a number of different serogroup A specific capsule biosynthesis genes. This enables the microarray to distinguish between the three N. meningitidis serogroup capsules. This is currently achieved by examining the capsule biosynthesis locus, where the presence of different alleles of the transferase genes (siaD) can differentiate between serogroup B and C strains (Borrow et al., 1997) and the presence of sacB for serogroup A. None of the capsule loci were present in the commensal species but they were also absent from N. gonorrhoeae NMNG and N. meningitidis E26 (serogroup X). This was consistent with N. gonorrhoeae not possessing a capsule and the unique serogroup X capsule genes not being present on the microarray. The microarray was able to distinguish serogroups A, B and C using what is known about the capsule locus, but with the addition of reporters from the variable region of ctrA and serotype X specific capsule genes it may also be possible to distinguish the other serogroups. Additionally analysing a number of strains from the rarer serogroups may identify other genes that may discriminate between the serogroups.

The pan-Neisseria microarray has been used to analyse the genomic content of a number of documented pathogenic Neisseria strains plus comparison with a number of commensal strains. The microarray was able to identify highly divergent strains through lack of hybridization across the majority of reporters. Reduction of the stringent hybridization condition could allow for greater identification of divergent genes but the data would be less reliable due to increased cross-hybridization. Previous phylogenetic studies using MLST and MLEE were comparable with the microarray data, highlighting both the usefulness of these typing methods and the accuracy of the microarray data. The co-clustering was surprising given the natural competence of Neisseria and evidence of gene acquisition through G+C content variation. This may indicate that virulent strains rarely encounter enhancing exogenous DNA and use the propensity for genetic rearrangement for phase variation and evasion of host immunity. The microarray data also concurred with other virulence determinants, such as dam/drg genotype, characterizing which opc orthologue was present, selectively identifying which pilin subunits and pilC orthologue were present and identifying capsule type in one test. As further virulence determinants are identified the dataset can be used retrospectively, so long as the gene existed in the four design species, to assess all strains tested, and the microarray can be expanded to include additional genes. The microarray can also be used to assess the gene content of a clinical specimen; however, with the need for rapid diagnosis due to the rapid onset of the disease it would be unlikely a microarray would have any benefit over the developing diagnostic TaqMan real-time PCR. Microarrays have been used to identify unique characteristics of an outbreak strain and follow the epidemiology (Rajakumar et al., 2004).

The microarray data were used to identify a subset of genes that appear to be present in all Neisseria species; this may be an overestimate, as only the closely related species produced usable microarray data under the stringent conditions used. Potentially more importantly, the identification of pathogen-specific genes has potential for targeted drug and vaccine design without reducing the beneficial commensal neisseriae. The majority of these genes with known function are related to the cell surface and pathogenicity, which would be expected; from this we could speculate that the FUN genes within this group must also have important roles in pathogenicity and could provide further insights into Neisseria pathogenesis.


   ACKNOWLEDGEMENTS
 
We acknowledge The Wellcome Trust for funding the multi-collaborative microbial pathogen microarray facility under its Functional Genomics Resources Initiative.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aho, E. L., Keating, A. M. & McGillivray, S. M. (2000). A comparative analysis of pilin genes from pathogenic and nonpathogenic Neisseria species. Microb Pathog 28, 81–88.[CrossRef][Medline]

Barrett, S. J. & Sneath, P. H. (1994). A numerical phenotypic taxonomic study of the genus Neisseria. Microbiology 140, 2867–2891.[Medline]

Behr, M. A., Wilson, M. A., Gill, W. P., Salamon, H., Schoolnik, G. K., Rane, S. & Small, P. M. (1999). Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284, 1520–1523.[Abstract/Free Full Text]

Bennett, D. E., Mulhall, R. M. & Cafferkey, M. T. (2004). PCR-based assay for detection of Neisseria meningitidis capsular serogroups 29E, X, and Z. J Clin Microbiol 42, 1764–1765.[Abstract/Free Full Text]

Borrow, R., Claus, H., Guiver, M., Smart, L., Jones, D. M., Kaczmarski, E. B., Frosch, M. & Fox, A. J. (1997). Non-culture diagnosis and serogroup determination of meningococcal B and C infection by a sialyltransferase (siaD) PCR ELISA. Epidemiol Infect 118, 111–117.[CrossRef][Medline]

Bucci, C., Lavitola, A., Salvatore, P., Del Giudice, L., Massardo, D. R., Bruni, C. B. & Alifano, P. (1999). Hypermutation in pathogenic bacteria: frequent phase variation in meningococci is a phenotypic trait of a specialized mutator biotype. Mol Cell 3, 435–445.[CrossRef][Medline]

Dorrell, N., Mangan, J. A., Laing, K. G. & 9 other authors (2001). Whole genome comparison of Campylobacter jejuni human isolates using a low-cost microarray reveals extensive genetic diversity. Genome Res 11, 1706–1715.[Abstract/Free Full Text]

Feavers, I. M. (2000). ABC of meningococcal diversity. Nature 404, 451–452.[CrossRef][Medline]

Feil, E., Zhou, J., Maynard Smith, J. & Spratt, B. G. (1996). A comparison of the nucleotide sequences of the adk and recA genes of pathogenic and commensal Neisseria species: evidence for extensive interspecies recombination within adk. J Mol Evol 43, 631–640.[Medline]

Grifantini, R., Bartolini, E., Muzzi, A. & 14 other authors (2002). Previously unrecognized vaccine candidates against group B meningococcus identified by DNA microarrays. Nat Biotechnol 20, 914–921.[CrossRef][Medline]

Grifantini, R., Sebastian, S., Frigimelica, E., Draghi, M., Bartolini, E., Muzzi, A., Rappuoli, R., Grandi, G. & Genco, C. A. (2003). Identification of iron-activated and -repressed Fur-dependent genes by transcriptome analysis of Neisseria meningitidis group B. Proc Natl Acad Sci U S A 100, 9542–9547.[Abstract/Free Full Text]

Hamrick, T. S., Dempsey, J. A., Cohen, M. S. & Cannon, J. G. (2001). Antigenic variation of gonococcal pilin expression in vivo: analysis of the strain FA1090 pilin repertoire and identification of the pilS gene copies recombining with pilE during experimental human infection. Microbiology 147, 839–849.[Medline]

Hinchliffe, S. J., Isherwood, K. E., Stabler, R. A. & 7 other authors (2003). Application of DNA microarrays to study the evolutionary genomics of Yersinia pestis and Yersinia pseudotuberculosis. Genome Res 13, 2018–2029.[Abstract/Free Full Text]

Hinds, J., Witney, A. A. & Vass, J. K. (2002a). Microarray design for bacterial genomes. Methods Microbiol 33, 67–82.

Hinds, J., Laing, K. G., Mangan, J. A. & Butcher, P. D. (2002b). Glass slide microarrays for bacterial genomes. Methods Microbiol 33, 83–99.

Jolley, K. A., Sun, L., Moxon, E. R. & Maiden, M. C. (2004). Dam inactivation in Neisseria meningitidis: prevalence among diverse hyperinvasive lineages. BMC Microbiol 4, 34. doi:10.1186/1471-2180-4-34[CrossRef][Medline]

Kahler, C. M., Martin, L. E., Tzeng, Y. L., Miller, Y. K., Sharkey, K., Stephens, D. S. & Davies, J. K. (2001). Polymorphisms in pilin glycosylation locus of Neisseria meningitidis expressing class II pili. Infect Immun 69, 3597–3604.[Abstract/Free Full Text]

Kim, C. C., Joyce, E. A., Chan, K. & Falkow, S. (2002). Improved analytical methods for microarray-based genome-composition analysis. Genome Biol 3, research0065. doi:10.1186/gb-2002-3-11-research0065

Marokhazi, J., Waterfield, N., LeGoff, G., Feil, E., Stabler, R., Hinds, J., Fodor, A. & ffrench-Constant, R. H. (2003). Using a DNA microarray to investigate the distribution of insect virulence factors in strains of Photorhabdus bacteria. J Bacteriol 185, 4648–4656.[Abstract/Free Full Text]

Martin, P., Sun, L., Hood, D. W. & Moxon, E. R. (2004). Involvement of genes of genome maintenance in the regulation of phase variation frequencies in Neisseria meningitidis. Microbiology 150, 3001–3012.[CrossRef][Medline]

Parkhill, J., Achtman, M., James, K. D. & 25 other authors (2000). Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 404, 502–506.[CrossRef][Medline]

Pizza, M., Scarlato, V., Masignani, V. & 43 other authors (2000). Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287, 1816–1820.[Abstract/Free Full Text]

Rajakumar, K., Shafi, J., Smith, R. J. & 8 other authors (2004). Use of genome level-informed PCR as a new investigational approach for analysis of outbreak-associated Mycobacterium tuberculosis isolates. J Clin Microbiol 42, 1890–1896.[Abstract/Free Full Text]

Rokbi, B., Renauld-Mongenie, G., Mignon, M., Danve, B., Poncet, D., Chabanel, C., Caugant, D. A. & Quentin-Millet, M. J. (2000). Allelic diversity of the two transferrin binding protein B gene isotypes among a collection of Neisseria meningitidis strains representative of serogroup B disease: implication for the composition of a recombinant TbpB-based vaccine. Infect Immun 68, 4938–4947.[Abstract/Free Full Text]

Rozen, S. & Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132, 365–386.[Medline]

Rytkonen, A., Albiger, B., Hansson-Palo, P., Kallstrom, H., Olcen, P., Fredlund, H. & Jonsson, A. B. (2004). Neisseria meningitidis undergoes PilC phase variation and PilE sequence variation during invasive disease. J Infect Dis 189, 402–409.[CrossRef][Medline]

Salama, N., Guillemin, K., McDaniel, T. K., Sherlock, G., Tompkins, L. & Falkow, S. (2000). A whole-genome microarray reveals genetic diversity among Helicobacter pylori strains. Proc Natl Acad Sci U S A 97, 14668–14673.[Abstract/Free Full Text]

Seiler, A., Reinhardt, R., Sarkari, J., Caugant, D. A. & Achtman, M. (1996). Allelic polymorphism and site-specific recombination in the opc locus of Neisseria meningitidis. Mol Microbiol 19, 841–856.[CrossRef][Medline]

Smith, N. H., Holmes, E. C., Donovan, G. M., Carpenter, G. A. & Spratt, B. G. (1999). Networks and groups within the genus Neisseria: analysis of argF, recA, rho, and 16S rRNA sequences from human Neisseria species. Mol Biol Evol 16, 773–783.[Abstract]

Snyder, L. A., Saunders, N. J. & Shafer, W. M. (2001). A putatively phase variable gene (dca) required for natural competence in Neisseria gonorrhoeae but not Neisseria meningitidis is located within the division cell wall (dcw) gene cluster. J Bacteriol 183, 1233–1241.[Abstract/Free Full Text]

Spratt, B. G., Zhang, Q. Y., Jones, D. M., Hutchison, A., Brannigan, J. A. & Dowson, C. G. (1989). Recruitment of a penicillin-binding protein gene from Neisseria flavescens during the emergence of penicillin resistance in Neisseria meningitidis. Proc Natl Acad Sci U S A 86, 8988–8992.[Abstract/Free Full Text]

Sun, Y. H., Bakshi, S., Chalmers, R. & Tang, C. M. (2000). Functional genomics of Neisseria meningitidis pathogenesis. Nat Med 6, 1269–1273.[CrossRef][Medline]

Tettelin, H., Saunders, N. J., Heidelberg, J. & 39 other authors (2000). Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287, 1809–1815.[Abstract/Free Full Text]

Tzeng, Y. L., Noble, C. & Stephens, D. S. (2003). Genetic basis for biosynthesis of the (alpha 1->4)-linked N-acetyl-D-glucosamine 1-phosphate capsule of Neisseria meningitidis serogroup X. Infect Immun 71, 6712–6720.[Abstract/Free Full Text]

Virji, M., Heckels, J. E., Potts, W. J., Hart, C. A. & Saunders, J. R. (1989). Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of their encoding sequences in the genomes of Neisseria spp. J Gen Microbiol 135, 3239–3251.[Medline]

Virji, M., Saunders, J. R., Sims, G., Makepeace, K., Maskell, D. & Ferguson, D. J. (1993). Pilus-facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin. Mol Microbiol 10, 1013–1028.[Medline]

Zhou, J. & Spratt, B. G. (1992). Sequence diversity within the argF, fbp and recA genes of natural isolates of Neisseria meningitidis: interspecies recombination within the argF gene. Mol Microbiol 6, 2135–2146.[Medline]

Zhou, J., Bowler, L. D. & Spratt, B. G. (1997). Interspecies recombination, and phylogenetic distortions, within the glutamine synthetase and shikimate dehydrogenase genes of Neisseria meningitidis and commensal Neisseria species. Mol Microbiol 23, 799–812.[CrossRef][Medline]

Zhu, P., Morelli, G. & Achtman, M. (1999). The opcA and (psi)opcB regions in Neisseria: genes, pseudogenes, deletions, insertion elements and DNA islands. Mol Microbiol 33, 635–650.[CrossRef][Medline]

Received 7 April 2005; revised 6 June 2005; accepted 8 June 2005.



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