Genome Sequencing Reveals Widespread Virulence Gene Exchange among HumanNeisseria Species
Pradeep Reddy Marri
Mary Paniscus
Nathan J Weyand
María A Rendón
Christine M Calton
Diana R Hernández
Dustin L Higashi
Erica Sodergren
George M Weinstock
Steven D Rounsley
Magdalene So
* E-mail:pradeepm@email.arizona.edu
Conceived and designed the experiments: PRM NJW SDR MS. Performed the experiments: PRM MP NJW MAR DRH DLH ES GMW. Analyzed the data: PRM MP NJW MAR CMC DRH DLH ES GMW SDR MS. Contributed reagents/materials/analysis tools: GMW SDR MS. Wrote the paper: PRM NJW CMC MS.
Contributed equally.
Roles
Received 2010 May 24; Accepted 2010 Jun 1; Collection date 2010.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Abstract
Commensal bacteria comprise a large part of the microbial world, playing important roles in human development, health and disease. However, little is known about the genomic content of commensals or how related they are to their pathogenic counterparts. The genusNeisseria, containing both commensal and pathogenic species, provides an excellent opportunity to study these issues. We undertook a comprehensive sequencing and analysis of human commensal and pathogenicNeisseria genomes. Commensals have an extensive repertoire of virulence alleles, a large fraction of which has been exchanged amongNeisseria species. Commensals also have the genetic capacity to donate DNA to, and take up DNA from, otherNeisseria. Our findings strongly suggest that commensalNeisseria serve as reservoirs of virulence alleles, and that they engage extensively in genetic exchange.
Introduction
The genusNeisseria is a large group of β-Proteobacteria that are obligate symbionts of humans and animals. At least eight species of commensalNeisseria colonize human mucosal surfaces[1],[2]. These sites are also infected by two pathogenicNeisseria:Neisseria meningitidis, which causes epidemics of meningitis and septicemia, andNeisseria gonorrhoeae, a sexually transmitted bacterium. Because of their importance to global public health, research has focused mainly on the two pathogens, leading to the identification of many virulence factors that are important for infection in humans. Recent reports indicate that commensalNeisseria also possess virulence genes[3],[4],[5]. However, these studies focused either on a limited number of virulence genes or on only one commensal genome, that ofNeisseria lactamica. The total virulence gene content of theNeisseria genus is unknown.
The rich diversity ofNeisseria species at human mucosal surfaces raises the possibility of genetic exchange among these bacteria[6],[7]. Indeed, interspecies exchange of antibiotic resistance genes can occurin vitro[8]. These considerations, combined with the limited glimpse of the virulence gene content of commensals, led us to undertake a comprehensive analysis of commensalNeisseria genomes and to determine the extent of interspecies genetic exchange.
Results
We generated high quality draft genome sequences of eight species of human commensalNeisseria, and compared them to 11 publishedN. meningitidis,N. gonorrhoeae andN. lactamica genomes (seeTable S1 for all strains used and their corresponding accession numbers). Commensal genomes were sequenced using a combination of the Roche 454 and Illumina platforms. Commensal genomes range between 1.8 and 2.8 Mb and contain 2,000 to 2,842 predicted genes. In contrast, pathogen genomes are ∼2.2 Mb and encode ∼2,000 predicted genes (Table S2). Genes for common metabolic processes, DNA replication, recombination, transcription and translation are conserved in all commensals (Data Set S1 andText S1).
The coreNeisseria genome – the set of genes present in allNeisseria species – consists of 896 genes, mostly with housekeeping function assignments. To determine the phylogenetic relationship ofNeisseria species, we concatenated the DNA sequences from a subset of 636Neisseria core genes that are shared withChromobacterium violaceum, the outgroup used to root the tree. TheN. meningitidis andN. gonorrhoeae genomes form a distinct monophyletic clade that is derived from the commensal genomes (Figure 1A). The topology of this tree is consistent with that generated by concatenating all 896Neisseria core genes (Figure 1B), and with previously derived single gene phylogenies[2].
Figure 1. Phylogenetic relationship of humanNeisseria species.
(A) Rooted maximum likelihoodNeisseria species tree (GTR + I + γ) based on concatenating the DNA sequences of a subset of 636 coreNeisseria genes that are shared with the outgroupC. violaceum. A dagger denotes a bootstrap value of 100. The threeN. meningitidis carrier strains are denoted by asterisks. (B) Maximum likelihoodNeisseria species tree (GTR + I + γ) based on concatenating the DNA sequences of all 896 coreNeisseria genes.
Repetitive elements are important features of pathogenicNeisseria genomes, playing key roles in genetic transformation, gene expression and genome rearrangements[9],[10]. The 10 bp DNA Uptake Sequence (DUS)GCCGTCTGAA is essential for genetic transformation ofN. meningitidis andN. gonorrhoeae. As in pathogenicNeisseria, the DUS is the most prominent repetitive element in commensals, occurring over 2,000 times in most species (Figure 2A). Intriguingly,Neisseria sicca andNeisseria mucosa, which form a distinct clade on theNeisseria species tree (Figure 1), have far fewer copies of the canonical DUS. Instead, they have >3,400 copies of a variant DUS (DUS1) with a one base mismatch: GtCGTCTGAA. This DUS1 functions in DNA uptake/transformation inN. meningitidis andN. gonorrhoeae, albeit less efficiently than the canonical sequence[11],[12]. Thus, all commensals appear to have functional DNA uptake sequences.
Figure 2. Prevalence of repetitive elements in humanNeisseria genomes.
(A) Numbers of repeat sequences (RS), insertion sequences (IS), Correia elements, and DNA Uptake Sequences (DUS) are given for eachNeisseria species, with complete copies in parentheses. DUS refers to the canonical DNA uptake sequence GCCGTCTGAA. DUS1 refers to the DUS variant GtCGTCTGAA. Note the high copy number of DUS1 inN. sicca andN. mucosa, highlighted in bold. (B) Absence of repeat elements in thepgd/lpxC locus region in commensals. Nel:N. elongata; Nsi:N. sicca; Nmu:N. mucosa; Nsu: N. subflava; Nfl:N. flavescens; Nci:N. cinerea; Npo: N. polysaccharea; Nla:N. lactamica 23970; Nme:N. meningitidis MC58. Black bars represent dRS3 elements; blue bars represent Correia elements. Orthologous genes are the same color; orange represents a γ-glutamyltranspeptidase present in four commensal genomes; grey represents hypothetical proteins unique to each genome. (C) Absence of large repeat arrays in themurI-tbpA/B-potD-1 locus in commensals. Ngo:N. gonorrhoeae FA1090. Forward slashes (//) represent a break in chromosome contiguity. Orthologous genes are the same color; light blue denotes a hypothetical protein. All other features are the same as in (B).
Correia and dRS3 elements[13] are the second most abundant repeats in commensalNeisseria. These elements function in gene regulation and sequence variation in pathogenicNeisseria[14],[15]. Commensals have fewer Correia and dRS3 elements than the pathogens. The only exception isN. sicca, which has more Correia elements than either of the pathogens (Figure 2A, 2B and 2C;Text S1). Moreover, commensal dRS3 elements are not arranged into large Intergenic Mosaic Element arrays, unlike their counterparts in pathogenicNeisseria (Figure 2B and 2C). This suggests that expansion of dRS3 elements occurred more recently in the pathogens.
In pathogenicNeisseria, repetitive elements facilitate two important immune evasion mechanisms: phase and antigenic variation. ManyN. meningitidis andN. gonorrhoeae genes for outermembrane proteins undergo high frequency ON/OFF (phase) switching that results from intragenic recombination of repeat sequences in or near the coding sequence. Over 70% of the genes (52/72) known or hypothesized to be phase variable in pathogenicNeisseria[14],[16],[17] are present in commensals (Table S3). However, many of these (26/52) do not have tandem repeats, indicating that recombination-based phase switching of surface proteins occurs less frequently in commensals.
Studies of a few handpicked genes and whole genome microarrays centered onN. lactamica detected virulence genes in commensals[3],[4],[5]. To examine virulence gene content in the newly sequenced commensal genomes, we determined the distribution of 177 genes that have been reported to play a role inNeisseria virulence (Table S4)[4],[18],[19]. Seventy-five percent of these genes (133/177) are present in one or more commensals (Figure 3A), and 40% (70/177) are in all of them (Data Set S2). A large subset ofNeisseria virulence genes is therefore present in the commensals.
Figure 3. Virulence gene exchange among humanNeisseria species.
(A) Number of genes present in differentNeisseria species, out of the 177 genes known or hypothesized to be important for virulence (seemethods for details). (B) Heatmap of AU testp-values obtained by comparing individual virulence gene tree topologies. The map is a 69×69 matrix of 69 virulence genes that are present in allNeisseria species (y-axis) and their corresponding topologies (x-axis). Blue indicates ap-value of 1 (trees are similar); red indicates ap-value of 0 (trees are significantly different).
Among the virulence genes found in all commensal species are those encoding the Type IV pilus (Tfp), a surface structure that in pathogenicNeisseria promotes twitching motility, DNA uptake/genetic transformation, attachment, and host cell signaling[20]. Commensals have a complete set of Tfp biogenesis genes (Figure S1), including:pilE, encoding the Tfp structural subunit;pilD, encoding a pilin peptidase;pilF, encoding a pilin assembly ATPase; andpilT, encoding an ATPase required for Tfp retraction and DNA uptake. Commensals also have one or more pilin modification genes. The four commensals closest to the pathogen clade,N. lactamica,Neisseria polysaccharea,Neisseria cinerea, andNeisseria flavescens, have apilC ortholog. However, the function of commensalpilC cannot be deduced due to sequence differences from pathogenicNeisseria pilC1 andpilC2. Commensals therefore have the genetic capacity to produce Tfp. The functions of commensal Tfp, if expressed, remain to be elucidated.
Tfp systems of commensal and pathogenicNeisseria differ in one major respect. In pathogenicNeisseria, pilin antigenic variation results from recombination ofpilE with a silent variant pilin pseudogene, orpilS[21]. PathogenicNeisseria have as many as 19 copies ofpilS, while commensals have only 2–5 copies (Table S5). Moreover, in commensals the region upstream ofpilE lacks the guanine-repeat element that is essential forpilE/pilS recombination[22]. Pilin antigenic variation is therefore unlikely to occur in commensals, at least via the guanine-repeat element mechanism. As pseudogenes lacking a function are rapidly lost from the population[23], the presence ofpilS in commensal genomes is curious. The phylogeny ofNeisseria (Figure 1) indicates that commensals are basal to the two pathogenic species, which form a single, derived monophyletic clade. The most parsimonious explanation for these observations is thatpilE/pilS antigenic variation is a novel function that arose in the pathogens from a system originally evolved for another, yet unidentified purpose.
The Opa family of outermembrane proteins promoteN. gonorrhoeae andN. meningitidis attachment, invasion, immune cell signaling and inflammation. Pathogen genomes harbor multiple variantopas;N. meningitidis has 3–4opas, whileN. gonorrhoeae has approximately 11[24]. Most commensals lackopa genes and thus do not interact with host cells via this virulence factor. However,N. polysaccharea,N. flavescens andN. lactamica have 1, 2 and 3 variantopas, respectively (Table S5). All of these contain variable numbers of the CTCTT pentameric repeat that, in pathogenicNeisseria, undergoes slip-strand misrepair resulting in Opa phase and antigenic variation[25],[26]. Commensal Opas are therefore likely to undergo phase switching, but limited antigenic variation.
Iron scavenging from the host is an important virulence attribute of bacterial pathogens[27]. CommensalNeisseria have a diverse arsenal of iron acquisition genes (Figure 4).N. lactamica andN. cinerea have genes for acquiring iron from human transferrin and lactoferrin (tbpA/tbpB andlbpA/lbpB, respectively)[28]. These loci are important forN. gonorrhoeae infection and fitness[28]. Commensals also havehmbR andhpuAB, which are required for acquiring hemoglobin iron[28]. InN. meningitidis, HmbR promotes replication in the blood[29]. All commensals have thetonB/exbB/exbD iron transport locus, andfur, the iron-responsive regulatory element. Notably, commensals most distal to the pathogens have iron uptake genes that are missing from pathogenicNeisseria. These include genes for transport of hemin, Fe+3 and Fe+2, and for siderophore receptors and transport (Figure 4). Because Fe+2 is often complexed with protein in foodstuff, the Fe+2 uptake system, if expressed, may allow oral commensals to absorb iron from the host diet[28]. Thus,Neisseria species possess a diverse array of iron uptake genes. This diversity may help differentNeisseria species to colonize the same niche without being affected by antibodies directed against the iron acquisition components of other species[30].
Figure 4. Iron utilization genes in humanNeisseria species.
Species and strain names are listed in the phylogenetic tree.N. meningitidis carrier strains are marked with asterisks. Each box represents an individual gene. Genes of the same color belong to the same iron utilization system. Genes connected by a horizontal line are contiguous in the chromosome. Loci confirmed by sequencing to be pseudogenes have a forward slash (/).N. meningitidis andN. gonorrhoeae gene names appear below each column. Genes are listed as locus tags if their iron utilization functions were deduced from other bacteria. General functions appear below the gene names.
Several genes are found uniquely in commensals. Only four genes - encoding hypothetical proteins - are present in all commensals, while several are present in one or more commensals. Intriguingly, some commensal-specific genes encode proteins that function as hemolysins, adhesins or invasins in other bacterial pathogens (Data Sets S3 andS4). Others encode components of the Type IV and Type VI Secretion Systems and lipooligosaccharide modification enzymes (Text S1). Commensal-specific genes are generally smaller, have a lower %G+C content thanNeisseria core genes, and have homologs in other bacterial genera, indicating that they were acquired recently via horizontal gene transfer.
We also identified several pathogen-specific genes (Data Set S5). Only 16 genes are present in all sequencedN. meningitidis genomes, and 81 in all sequencedN. gonorrhoeae genomes. Genes for biosynthesis of the polysaccharide capsule, which confers resistance to complement killing and phagocytosis, are unique toN. meningitidis (Figure S2). ThectrABCD andlipAB loci, encoding polysaccharide transporters, are found in some commensals (Text S1). However, thesiaABC locus, which is required for capsule production, appears only inN. meningitidis. ThetdfF locus which is essential for intracellular iron acquisition and thus forNeisseria intracellular replication, is found only in pathogen genomes (Figure 4)[31].
Some genes were found in all sequencedN. meningitidis and/orN. gonorrhoeae genomes, but only rarely in commensal species. For example,iga, encoding an IgA protease that cleaves human IgA and lysosomal membrane protein Lamp1[20], is present only in pathogenicNeisseria genomes with one exception: an intactiga2 allele is found in bothN. lactamica genomes. The 57 kb Gonococcal Genetic Island (GGI)[32] is largely absent from commensals. However, genes with varying degrees of nucleotide sequence identity to GGI encodeddsbC, topB, andparA are present in all commensal genomes (Text S1). InN. gonorrhoeae,dsbC andparA play roles in genetic transformation by secreting DNA[32]. Nf, an M13-like filamentous phage, is the only feature that distinguishes hyperinvasiveN. meningitidis isolates from noninvasive ones[33],[34]. Nf genes are detected inN. flavescens andN. lactamica.N. flavescens has only a subset of Nf genes, whileN. lactamica contains all the major M13-like life cycle genes.
Finally, it should be noted that NMB1646, encoding a putative hemolysin, was proposed to be specific to pathogenicNeisseria. Microarray studies found NMB1646 inN. meningitidis andN. gonorrhoeae, but absent fromN. lactamica[4]. In our study, NMB1646 is also missing fromN. lactamica. However, it was found in all other commensal genomes. Thus, NMB1646 is present in both commensal and pathogenicNeisseria.
Our study shows that commensals have a large number of virulence genes, as well as genes for DNA secretion and uptake, and an abundance of DUS. Additionally, previous work has demonstrated gene exchange amongNeisseria species[8],[35],[36],[37]. Combined, these results suggest that genetic exchange amongNeisseria may be more widespread than previously thought. We therefore determined the extent of genetic exchange amongNeisseria species, using a phylogenetic method to analyze 69 virulence genes shared by all 19Neisseria genomes (Data Set S2). This method relies on the phylogenetic congruence of individual gene trees: a gene tree that does not match the species tree indicates the gene in question has been transferred laterally. We generated individual gene trees for each of the 69 virulence genes using PhyML[38] and looked for the rejection of a set of these topologies by the 69 genes using an Approximate Unbiased (AU) test[39]. Thep-value heatmap (Figure 3B) indicates that most gene trees showed divergent histories (depicted as groups of topologies with dissimilarp-value patterns) that are most likely due to horizontal gene transfer. A small group of topologies (topologies 58–69) were rejected by all the genes. Consistent with these findings, 45% (31/69) of the virulence genes rejected the concatenated tree, compared to 34% of the core genes (average of three replicates of 69 randomly selected core genes). These results suggest that there is widespread genetic exchange amongNeisseria species, with virulence genes being exchanged at a slightly higher frequency than core genes.
The AU test relies on the rejection of a tree by a gene, rather than on its acceptance, and less divergent or saturated alignments may result in false predictions[40]. Moreover, high rates of recombination betweenN. meningitidis strains can also result in conflicting phylogenies. Therefore, we also looked for evidence of horizontal gene transfer using the program RDPv3.18[41] to detect homologous intragenic recombination in the 69 virulence genes mentioned above. Evidence of recombination was detected in 53 of the 69 genes, and over half of these events (31/53) involved at least one commensal genome (Table S6). The intragenic recombination and AU tests provide strong evidence of genetic exchange among commensal and pathogenicNeisseria, suggesting that commensals act as reservoirs of new virulence alleles.
Discussion
In this comprehensive analysis of human commensalNeisseria genomes, we have identified the gene content of commensalNeisseria species, generated an extensive phylogenetic tree of this genus, and determined the distribution of virulence genes in its members. CommensalNeisseria have an extensive repertoire of virulence alleles from pathogenicNeisseria as well as other bacterial genera. Moreover, they have the genetic machinery for exchanging DNA with each other, and nearly half ofNeisseria virulence genes have undergone intra- and interspecies recombination. High frequency horizontal gene transfer can increase pathogen fitness, accelerate host adaptation, and affect bacterial virulence. The prevalence of numerousNeisseria species in the same niches in the human body provides an opportunity for DNA exchange. The repertoire of virulence genes inNeisseria populations is therefore likely to be dynamic.
The large virulence gene set in commensalNeisseria raises an intriguing question: why are these bacteria generally not pathogenic? Although there are no ready answers to this question, we can offer possible explanations. First, a productive infection is determined by multiple factors, including the immediate environment of the infection site and host immune status. Commensals may not be able to express the entire constellation of virulence genes necessary for initiating infection under these circumstances. In this context, it is interesting to note thatN. lactamica, which occasionally causes infections, has the largest set of virulence alleles of all the commensals. Secondly, there may be additional factors governing commensalism that have yet to be defined.N. meningitidis can cause asymptomatic infections in humans[42]. Yet, there is very little difference between the genomes of carrier and invasive strains[18]. This suggests that the ON/OFF phase switching of genes may be an important pathogenesis determinant. Indeed, many commensal orthologs of phase-variable genes lack the repeat elements that participate in phase variation. Third, a virulence allele in a commensal may differ from its pathogenic counterpart in its ability to cause disease. For example,N. gonorrhoeae expressing theN. lactamica porB allele is less able to invade cells[43]. Finally, our results add to the growing body of evidence that suggests the definition of virulence factor needs to be reassessed. Virulence factors have been defined as components whose absence attenuates bacterial virulence but not viability[44]. For example, Tfp is considered a virulence factor because it promotes attachment[45]. However, recent studies demonstrate that Tfp activates pro-survival pathways in epithelial cells[46],[47], a situation that benefits both host and microbe. The presence of Tfp biogenesis genes in commensals supports this idea. Thus, some virulence factors may be more appropriately termed “host adaptation” factors, as they allow bacteria to interact with and adapt to the host environment, but do not specifically promote virulence. The distinction between a true virulence factor versus a host adaptation factor will be clarified as the genomes of more commensal bacteria are sequenced and characterized.
Accession Numbers
Genome sequences generated in this study were deposited in GenBank. SeeTable S1 for the GenBank accession numbers for all genome sequences discussed in this paper. Updated versions of the eight commensalNeisseria genome sequences generated in this study are available athttp://www.u.arizona.edu/~pradeepm/data.html.
Materials and Methods
Bacterial strains
The following strains were obtained from the American Type Culture Collection (ATCC):N. lactamica (ATCC 23970),N. polysaccharea (ATCC 43768),N. cinerea (ATCC 14685),N. sicca (ATCC 29256),N. mucosa (ATCC 25996), andNeisseria elongata subsp.glycolytica (ATCC 29315).Neisseria subflava (NJ9703) was provided by J.B. Kaplan[48].N. flavescens (NRL30031 or H210) was a gift from E.L. Aho[49],[50]. All commensalNeisseria strains were grown on chocolate agar with 3.6% GC agar base (BD), 1% Isovitalex (BD), and 1% hemoglobin (Oxoid) for 18–24 hours in 5% CO2 at 37°C.
DNA isolation
Chromosomal DNA was isolated as described previously[51]. Briefly, cells from half of a 10 cm diameter petri dish were harvested with a sterile Dacron swab into 0.5 ml of 50 mM Tris-Cl, 20 mM EDTA, 50 mM NaCl (pH 8). SDS (1% final concentration) and RNAse A (Qiagen, 1 mg/ml) were added, and cells were lysed for five minutes. Two extractions with phenol-chloroform-isoamyl alcohol (25∶24∶1) and one with chloroform were used to remove proteins. DNA was precipitated by adding two volumes of isopropanol followed by suspension in TE (10 mM Tris, 1 mM EDTA, pH 8). Ammonium acetate was added to 2.5 M followed by precipitation with two volumes of ethanol. Following 70% ethanol washes and drying, DNA was suspended in water or 10 mM Tris, 0.1 mM EDTA (pH 8).
Genome sequencing and assembly
454 sequencing was performed using the Titanium platform (Roche) according to the manufacturer's instructions. Paired-end sequencing was completed using the Illumina Genome Analyzer II following protocols specified by the manufacturer. Draft genomes were assembled using the Newbler (Roche) and Velvet[52] assemblers.
Genome Annotation
Annotation was performed using a combination of RAST[53] and the Washington University Genome Center's gene prediction pipeline. Select loci were manually curated using previously publishedN. meningitidis andN. gonorrhoeae genome sequences as references.
Orthologs and core genome
Orthologous proteins were identified by BLASTP using reference proteins from previously sequencedN. meningitidis andN. gonorrhoeae genomes where possible. Predicted proteins were designated orthologs of a reference protein if the ratio of reference self-hit bitscore to predicted protein bitscore was greater than 0.4. The orthology of proteins unique to draft genomes was determined in the same manner, except randomly chosen predicted proteins were used as reference proteins. The orthology was confirmed by genome context whenever required. The 896 genes present in all 19Neisseria genome sequences used in this study were designated as the coreNeisseria genome.
Generation ofNeisseria species tree
The subset of 636 coreNeisseria genes also present inC. violaceum was used to find phylogenetic relationships amongNeisseria species. The concatenated DNA sequences of these 636 genes were aligned using MAFFT[54] and the resulting alignment edited using Gblocks[55] to remove any gaps. A maximum likelihood tree (using the GTR + I + γ model in PAUP) was generated using the concatenated orthologous sequences fromC. violaceum as the outgroup. Support for each branch was obtained by performing the bootstrap analysis with 100 replicates. Using the same method, an unrooted tree was also obtained with the concatenated sequences from all 896 coreNeisseria genes.
Identification of repeat elements
Repetitive elements of commensalNeisseria were identified using the ‘fuzznuc’ application of EMBOSS[56]. DNA Uptake Sequences (DUS) were identified in each genome by looking for the patternGCCGTCTGAA. Non-canonical DUS were found by allowing one mismatch to the canonical DUS pattern. The dRS3 elements were detected in each genome using the pattern ATTCCCNNNNNNNNGGGAAT[13],[14]. Each identified site was then manually checked to see if it had a lone dRS3 repeat or a complete RS element. Relaxing the search to allow one or two mismatches did not detect any additional RS elements. Correia repeats (CR) were identified by searching for the following patterns[15],[57], with three mismatches allowed per pattern:
TATAG[CT]GGATTAACAAAAATCAGGAC,TATAG[CT]GGATTAAATTTAAACCGGTAC,TATAG[CT]GGATTAACAAAAACCGGTAC,TATAG[CT]GGATTAAATTTAAATCAGGAC.
Each identified repeat was then manually checked to see if it had a lone CR or a complete Correia element.
Identification of virulence genes
A list of 177 virulence genes was created by searching published literature and available databases forNeisseria genes that are known or hypothesized to have virulence functions[4],[18],[19]. The protein sequences of these 177 genes were used as queries in a TBLASTN search against all commensal genome sequences with an E-value cutoff set at 1e−5. A gene was identified as present if it had >50% identity to the query protein and an alignment length of ≥75% of the query length.
Identification of phase variable genes
Searches of the published literature identified 72Neisseria genes known or hypothesized to be phase variable[14],[16],[17]. The protein sequences of these 72 genes were used as queries in a TBLASTN search against all commensal genome sequences with an E-value cutoff set at 1e−5. A gene was identified as present if it had >50% identity to the query protein and an alignment length of ≥75% of the query length. To confirm the presence of the phase variable (PV) repeat, the corresponding gene from the commensal genome sequence was extracted and aligned to the appropriate reference gene sequence. The alignments were manually examined for the presence of the PV repeat.
Gene tree-to-species tree congruence
A phylogenetic method was used to detect the extent of genetic exchange amongNeisseria species by examining 69 of the 70 virulence genes that are shared across all 19Neisseria genome sequences. ThepilE gene was not analyzed, as multiple copies ofpilE in several of the genomes made identifying orthologs difficult. Phylogenies for each of the 69 genes were derived in PhyML[38] using a GTR + I + γ model of evolution with BIONJ starting tree. The site likelihood for each tree was computed using baseml (PAML package)[58]. The AU test[39] was then applied using Consel[59].
Detection of intragenic recombination
To provide a secondary method for measuring genetic exchange, the extent of intragenic recombination was also examined. Alignments for each of the 69 virulence genes were generated and recombination breakpoints identified using the program RDPv3.18[41].
Confirmation of pseudogenes
Genes of interest were amplified from purified genomic DNA by PCR. All PCR reactions were performed in a 50 µl reaction volume using 50 ng of purified DNA as template, 1X Phusion HF master mix (Finnzymes) and 0.1 mM of the gene-specific primers (Table S7). Thermocycler conditions were as follows: 1 cycle at 98°C for 30 sec; 35 cycles at 98°C for 10 sec, 60°C for 30 seconds and 72°C for 15 sec; 1 cycle at 72°C for 10 min. The resulting PCR products were purified using a column-based method (Qiagen). Purified products were then sequenced using appropriate forward and reverse primers (Table S7) by the Sanger method at the University of Arizona Genetics Core (Tucson, AZ).
Supporting Information
Supplemental text and references.
(0.11 MB DOC)
HumanNeisseria type IV pilus (Tfp) biogenesis and pilin modification genes. Species and strain names are listed in the phylogenetic tree on the left.N. meningitidis carrier strains are denoted by asterisks. Each box represents an individual gene. Genes connected by a horizontal line are contiguous on the chromosome. A forward slash (/) indicates genes that have been confirmed as pseudogenes by sequencing. A backwards slash (\) represents hypothetical pseudogenes whose status has not been confirmed by experimentation. Genes in grey are not involved in Tfp biosynthesis or pilin modification. Tfp genes that have not yet been named are listed by their locus tag designations. The designations of commensalpilC orthologs are based on genome context and sequence homology topilC1 orpilC2 of pathogenicNeisseria.
(0.89 MB TIF)
Capsular polysaccharide genes of humanNeisseria species. TheNeisseria capsular locus is made up of five regions: A, B, C, D, and E. Some sequences also contain a duplicate of the D region, denoted as D'. A composite of the multi-species capsular locus is depicted at the bottom of the figure and includes gene names based on published nomenclature. Each box represents an individual gene, except for numbered boxes, which represent genes of the number listed. Genes of the same color are part of the same pathway; grey boxes denote genes whose functions are unknown or unrelated to the capsule. Genes connected by a horizontal line are contiguous on the chromosome. A backwards slash (\) represents hypothetical pseudogenes whose status has not been confirmed by experimentation. Note that inN. subflava, the C and E regions as well as gene NLA0370 are in an inverted orientation compared to the other sequences. Also, whetherN. elongata,N. sicca, andN. mucosa contain a D or D' region cannot be ascertained from the available data.
(0.74 MB TIF)
Genome sequences used in this study.
(0.07 MB PDF)
General characteristics ofNeisseria genomes.
(0.07 MB PDF)
Presence or absence of phase variable genes in commensalNeisseria. Targets were identified by searching the literature for genes known or hypothesized to be phase variable in pathogenicNeisseria.
(0.17 MB PDF)
Genes known or hypothesized to be involved inNeisseria virulence. The 177 targets were identified by searching the literature for genes previously identified as having a role inNeisseria virulence. Genes in bold type are present in all 19Neisseria genomes used in the study.
(0.13 MB PDF)
Copy number ofpilE,pilS andopa genes inNeisseria species. See supplemental references (Text S1) for additional information onN. meningitidis MC58[3],[20] andN. gonorrhoeae FA1090[20],[21].
(0.08 MB PDF)
Recombination of virulence genes amongNeisseria species. The RDPv3.18 program was used to test for recombination in 69 virulence genes that are shared by allNeisseria species.
(0.13 MB PDF)
Primers used for PCR amplification and sequencing of potential pseudogenes.
(0.07 MB PDF)
Common metabolic pathways present in commensalNeisseria. Genes from theN. meningitidis MC58 genome are included as a reference.
(0.18 MB XLS)
Virulence genes present in all 19Neisseria genomes. Note that theN. subflava andN. cinerea genome sequences both contain two copies ofpilE; the second copy is listed directly below the first (see rows 74–75).
(0.06 MB XLS)
Neisseria strain-specific genes. The name on each tab indicates the species included for the given table.
(0.26 MB XLS)
CommensalNeisseria-specific genes. Genes present in two or more commensal genomes but absent from allN. meningitidis andN. gonorrhoeae genomes. See supplemental reference[19] (Text S1) for abbreviations of the COG categories.
(0.22 MB XLS)
PathogenicNeisseria-specific genes. Tab “N. meningitides” includes the sevenN. meningitidis strains analyzed; tab “N. gonorrhoeae” includes the threeN. gonorrhoeae genomes studied. See supplemental reference[19] (Text S1) for abbreviations of the COG categories.
(0.04 MB XLS)
Acknowledgments
We gratefully acknowledge M. Wororbey, C.N. Cornelissen, L.A. Snyder, J.P. McCutcheon, M.W. van Passel, and N. Biais for their helpful comments. We thank J.B. Kaplan and E.L. Aho for providing strains, and J. Parkhill for providing access to theN. lactamica 020-06 genome sequence. We also thank J.S. Knapp, S. Hoffmann and J.G. Cannon for assistance in tracing the provenance ofN. flavescens NRL30031.
Footnotes
Competing Interests:The authors have declared that no competing interests exist.
Funding:This work was supported in part by National Institutes of Health (NIH) grant R01 AI068033-01 and by funds from the BIO5 Institute and College of Medicine, University of Arizona, awarded to MS. Additional support was provided by NIH grant U54HG004968 to GMW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
References
- 1.Janda WM, Knapp JS. Neisseria and Moraxella catarrhalis. In: Murray PR, editor. Manual of clinical microbiology. Washington, D.C.: ASM Press; 2003. pp. 585–608. 8th ed. [Google Scholar]
- 2.Tonjum T. Genus Neisseria. In: Garrity GM, editor. Bergey's manual of systematic bacteriology. New York: Springer-Verlag; 2005. pp. 777–798. 2nd ed. [Google Scholar]
- 3.Bennett JS, Thompson EA, Kriz P, Jolley KA, Maiden MC. A common gene pool for the Neisseria FetA antigen. Int J Med Microbiol. 2009;299:133–139. doi: 10.1016/j.ijmm.2008.06.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Snyder LA, Saunders NJ. The majority of genes in the pathogenic Neisseria species are present in non-pathogenic Neisseria lactamica, including those designated as ‘virulence genes’. BMC Genomics. 2006;7:128. doi: 10.1186/1471-2164-7-128. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Stabler RA, Marsden GL, Witney AA, Li Y, Bentley SD, et al. Identification of pathogen-specific genes through microarray analysis of pathogenic and commensal Neisseria species. Microbiology. 2005;151:2907–2922. doi: 10.1099/mic.0.28099-0. [DOI] [PubMed] [Google Scholar]
- 6.Knapp JS. Historical perspectives and identification of Neisseria and related species. Clin Microbiol Rev. 1988;1:415–431. doi: 10.1128/cmr.1.4.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Saez Nieto JA, Marcos C, Vindel A. Multicolonization of human nasopharynx due to Neisseria spp. Int Microbiol. 1998;1:59–63. [PubMed] [Google Scholar]
- 8.Catlin BW. Interspecific transformation of Neisseria by culture slime containing deoxyribonucleate. Science. 1960;131:608–610. doi: 10.1126/science.131.3400.608-a. [DOI] [PubMed] [Google Scholar]
- 9.Liu SV, Saunders NJ, Jeffries A, Rest RF. Genome analysis and strain comparison of correia repeats and correia repeat-enclosed elements in pathogenic Neisseria. J Bacteriol. 2002;184:6163–6173. doi: 10.1128/JB.184.22.6163-6173.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Snyder LA, McGowan S, Rogers M, Duro E, O'Farrell E, et al. The repertoire of minimal mobile elements in the Neisseria species and evidence that these are involved in horizontal gene transfer in other bacteria. Mol Biol Evol. 2007;24:2802–2815. doi: 10.1093/molbev/msm215. [DOI] [PubMed] [Google Scholar]
- 11.Goodman SD, Scocca JJ. Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae. Proc Natl Acad Sci U S A. 1988;85:6982–6986. doi: 10.1073/pnas.85.18.6982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Qvarnstrom Y, Swedberg G. Variations in gene organization and DNA uptake signal sequence in the folP region between commensal and pathogenic Neisseria species. BMC Microbiol. 2006;6:11. doi: 10.1186/1471-2180-6-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Parkhill J, Achtman M, James KD, Bentley SD, Churcher C, et al. Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature. 2000;404:502–506. doi: 10.1038/35006655. [DOI] [PubMed] [Google Scholar]
- 14.Bentley SD, Vernikos GS, Snyder LA, Churcher C, Arrowsmith C, et al. Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18. PLoS Genet. 2007;3:e23. doi: 10.1371/journal.pgen.0030023. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Snyder LA, Cole JA, Pallen MJ. Comparative analysis of two Neisseria gonorrhoeae genome sequences reveals evidence of mobilization of Correia Repeat Enclosed Elements and their role in regulation. BMC Genomics. 2009;10:70. doi: 10.1186/1471-2164-10-70. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Jordan PW, Snyder LA, Saunders NJ. Strain-specific differences in Neisseria gonorrhoeae associated with the phase variable gene repertoire. BMC Microbiol. 2005;5:21. doi: 10.1186/1471-2180-5-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Snyder LA, Saunders NJ, Shafer WM. 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. 2001;183:1233–1241. doi: 10.1128/JB.183.4.1233-1241.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Schoen C, Blom J, Claus H, Schramm-Gluck A, Brandt P, et al. Whole-genome comparison of disease and carriage strains provides insights into virulence evolution in Neisseria meningitidis. Proc Natl Acad Sci U S A. 2008;105:3473–3478. doi: 10.1073/pnas.0800151105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Tettelin H, Saunders NJ, Heidelberg J, Jeffries AC, Nelson KE, et al. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science. 2000;287:1809–1815. doi: 10.1126/science.287.5459.1809. [DOI] [PubMed] [Google Scholar]
- 20.Merz AJ, So M. Interactions of pathogenic neisseriae with epithelial cell membranes. Annu Rev Cell Dev Biol. 2000;16:423–457. doi: 10.1146/annurev.cellbio.16.1.423. [DOI] [PubMed] [Google Scholar]
- 21.Hill SA, Davies JK. Pilin gene variation in Neisseria gonorrhoeae: reassessing the old paradigms. FEMS Microbiol Rev. 2009;33:521–530. doi: 10.1111/j.1574-6976.2009.00171.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Cahoon LA, Seifert HS. An alternative DNA structure is necessary for pilin antigenic variation in Neisseria gonorrhoeae. Science. 2009;325:764–767. doi: 10.1126/science.1175653. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.van Passel MW, Marri PR, Ochman H. The emergence and fate of horizontally acquired genes in Escherichia coli. PLoS Comput Biol. 2008;4:e1000059. doi: 10.1371/journal.pcbi.1000059. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Dehio C, Gray-Owen SD, Meyer TF. The role of neisserial Opa proteins in interactions with host cells. Trends Microbiol. 1998;6:489–495. doi: 10.1016/s0966-842x(98)01365-1. [DOI] [PubMed] [Google Scholar]
- 25.Muralidharan K, Stern A, Meyer TF. The control mechanism of opacity protein expression in the pathogenic Neisseriae. Antonie Van Leeuwenhoek. 1987;53:435–440. doi: 10.1007/BF00415499. [DOI] [PubMed] [Google Scholar]
- 26.Murphy GL, Connell TD, Barritt DS, Koomey M, Cannon JG. Phase variation of gonococcal protein II: regulation of gene expression by slipped-strand mispairing of a repetitive DNA sequence. Cell. 1989;56:539–547. doi: 10.1016/0092-8674(89)90577-1. [DOI] [PubMed] [Google Scholar]
- 27.Payne SM. Iron acquisition in microbial pathogenesis. Trends Microbiol. 1993;1:66–69. doi: 10.1016/0966-842x(93)90036-q. [DOI] [PubMed] [Google Scholar]
- 28.Cornelissen CN. Iron transport system in pathogenic bacteria: Neisseria. In: Cronin A, editor. Iron transport in bacteria. Washington, D.C.: ASM Press; 2004. [Google Scholar]
- 29.Stojiljkovic I, Hwa V, de Saint Martin L, O'Gaora P, Nassif X, et al. The Neisseria meningitidis haemoglobin receptor: its role in iron utilization and virulence. Mol Microbiol. 1995;15:531–541. doi: 10.1111/j.1365-2958.1995.tb02266.x. [DOI] [PubMed] [Google Scholar]
- 30.Snyder LA, Davies JK, Ryan CS, Saunders NJ. Comparative overview of the genomic and genetic differences between the pathogenic Neisseria strains and species. Plasmid. 2005;54:191–218. doi: 10.1016/j.plasmid.2005.04.005. [DOI] [PubMed] [Google Scholar]
- 31.Hagen TA, Cornelissen CN. Neisseria gonorrhoeae requires expression of TonB and the putative transporter TdfF to replicate within cervical epithelial cells. Mol Microbiol. 2006;62:1144–1157. doi: 10.1111/j.1365-2958.2006.05429.x. [DOI] [PubMed] [Google Scholar]
- 32.Hamilton HL, Dominguez NM, Schwartz KJ, Hackett KT, Dillard JP. Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system. Mol Microbiol. 2005;55:1704–1721. doi: 10.1111/j.1365-2958.2005.04521.x. [DOI] [PubMed] [Google Scholar]
- 33.Bille E, Zahar JR, Perrin A, Morelle S, Kriz P, et al. A chromosomally integrated bacteriophage in invasive meningococci. J Exp Med. 2005;201:1905–1913. doi: 10.1084/jem.20050112. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Kawai M, Uchiyama I, Kobayashi I. Genome comparison in silico in Neisseria suggests integration of filamentous bacteriophages by their own transposase. DNA Res. 2005;12:389–401. doi: 10.1093/dnares/dsi021. [DOI] [PubMed] [Google Scholar]
- 35.Bowler LD, Zhang QY, Riou JY, Spratt BG. Interspecies recombination between the penA genes of Neisseria meningitidis and commensal Neisseria species during the emergence of penicillin resistance in N. meningitidis: natural events and laboratory simulation. J Bacteriol. 1994;176:333–337. doi: 10.1128/jb.176.2.333-337.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Linz B, Schenker M, Zhu P, Achtman M. Frequent interspecific genetic exchange between commensal Neisseriae and Neisseria meningitidis. Mol Microbiol. 2000;36:1049–1058. doi: 10.1046/j.1365-2958.2000.01932.x. [DOI] [PubMed] [Google Scholar]
- 37.Maiden MC. Horizontal genetic exchange, evolution, and spread of antibiotic resistance in bacteria. Clin Infect Dis. 1998;27(Suppl 1):S12–20. doi: 10.1086/514917. [DOI] [PubMed] [Google Scholar]
- 38.Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003;52:696–704. doi: 10.1080/10635150390235520. [DOI] [PubMed] [Google Scholar]
- 39.Shimodaira H. An approximately unbiased test of phylogenetic tree selection. Syst Biol. 2002;51:492–508. doi: 10.1080/10635150290069913. [DOI] [PubMed] [Google Scholar]
- 40.Susko E, Leigh J, Doolittle WF, Bapteste E. Visualizing and assessing phylogenetic congruence of core gene sets: a case study of the gamma-proteobacteria. Mol Biol Evol. 2006;23:1019–1030. doi: 10.1093/molbev/msj113. [DOI] [PubMed] [Google Scholar]
- 41.Martin DP, Williamson C, Posada D. RDP2: recombination detection and analysis from sequence alignments. Bioinformatics. 2005;21:260–262. doi: 10.1093/bioinformatics/bth490. [DOI] [PubMed] [Google Scholar]
- 42.Glitza IC, Ehrhard I, Muller-Pebody B, Reintjes R, Breuer T, et al. Longitudinal study of meningococcal carrier rates in teenagers. Int J Hyg Environ Health. 2008;211:263–272. doi: 10.1016/j.ijheh.2007.05.006. [DOI] [PubMed] [Google Scholar]
- 43.Bauer FJ, Rudel T, Stein M, Meyer TF. Mutagenesis of the Neisseria gonorrhoeae porin reduces invasion in epithelial cells and enhances phagocyte responsiveness. Mol Microbiol. 1999;31:903–913. doi: 10.1046/j.1365-2958.1999.01230.x. [DOI] [PubMed] [Google Scholar]
- 44.Casadevall A, Pirofski LA. Host-pathogen interactions: redefining the basic concepts of virulence and pathogenicity. Infect Immun. 1999;67:3703–3713. doi: 10.1128/iai.67.8.3703-3713.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Craig L, Pique ME, Tainer JA. Type IV pilus structure and bacterial pathogenicity. Nat Rev Microbiol. 2004;2:363–378. doi: 10.1038/nrmicro885. [DOI] [PubMed] [Google Scholar]
- 46.Howie HL, Glogauer M, So M. The N. gonorrhoeae type IV pilus stimulates mechanosensitive pathways and cytoprotection through a pilT-dependent mechanism. PLoS Biol. 2005;3:e100. doi: 10.1371/journal.pbio.0030100. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Lee SW, Higashi DL, Snyder A, Merz AJ, Potter L, et al. PilT is required for PI(3,4,5)P3-mediated crosstalk between Neisseria gonorrhoeae and epithelial cells. Cell Microbiol. 2005;7:1271–1284. doi: 10.1111/j.1462-5822.2005.00551.x. [DOI] [PubMed] [Google Scholar]
- 48.Kaplan JB, Fine DH. Biofilm dispersal of Neisseria subflava and other phylogenetically diverse oral bacteria. Appl Environ Microbiol. 2002;68:4943–4950. doi: 10.1128/AEM.68.10.4943-4950.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Aho EL, Keating AM, McGillivray SM. A comparative analysis of pilin genes from pathogenic and nonpathogenic Neisseria species. Microb Pathog. 2000;28:81–88. doi: 10.1006/mpat.1999.0325. [DOI] [PubMed] [Google Scholar]
- 50.Berger U, Husmann D. Studies on the normal occurrence of Neisseria flavescens (Branham, 1930). Med Microbiol Immunol. 1972;158:121–127. doi: 10.1007/BF02120477. [DOI] [PubMed] [Google Scholar]
- 51.Seifert HS, Ajioka RS, Paruchuri D, Heffron F, So M. Shuttle mutagenesis of Neisseria gonorrhoeae: pilin null mutations lower DNA transformation competence. J Bacteriol. 1990;172:40–46. doi: 10.1128/jb.172.1.40-46.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18:821–829. doi: 10.1101/gr.074492.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53.Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, et al. The RAST Server: rapid annotations using subsystems technology. BMC Genomics. 2008;9:75. doi: 10.1186/1471-2164-9-75. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Katoh K, Kuma K, Toh H, Miyata T. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 2005;33:511–518. doi: 10.1093/nar/gki198. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000;17:540–552. doi: 10.1093/oxfordjournals.molbev.a026334. [DOI] [PubMed] [Google Scholar]
- 56.Rice P, Longden I, Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000;16:276–277. doi: 10.1016/s0168-9525(00)02024-2. [DOI] [PubMed] [Google Scholar]
- 57.Correia FF, Inouye S, Inouye M. A 26-base-pair repetitive sequence specific for Neisseria gonorrhoeae and Neisseria meningitidis genomic DNA. J Bacteriol. 1986;167:1009–1015. doi: 10.1128/jb.167.3.1009-1015.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58.Yang Z. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci. 1997;13:555–556. doi: 10.1093/bioinformatics/13.5.555. [DOI] [PubMed] [Google Scholar]
- 59.Shimodaira H, Hasegawa M. CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics. 2001;17:1246–1247. doi: 10.1093/bioinformatics/17.12.1246. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplemental text and references.
(0.11 MB DOC)
HumanNeisseria type IV pilus (Tfp) biogenesis and pilin modification genes. Species and strain names are listed in the phylogenetic tree on the left.N. meningitidis carrier strains are denoted by asterisks. Each box represents an individual gene. Genes connected by a horizontal line are contiguous on the chromosome. A forward slash (/) indicates genes that have been confirmed as pseudogenes by sequencing. A backwards slash (\) represents hypothetical pseudogenes whose status has not been confirmed by experimentation. Genes in grey are not involved in Tfp biosynthesis or pilin modification. Tfp genes that have not yet been named are listed by their locus tag designations. The designations of commensalpilC orthologs are based on genome context and sequence homology topilC1 orpilC2 of pathogenicNeisseria.
(0.89 MB TIF)
Capsular polysaccharide genes of humanNeisseria species. TheNeisseria capsular locus is made up of five regions: A, B, C, D, and E. Some sequences also contain a duplicate of the D region, denoted as D'. A composite of the multi-species capsular locus is depicted at the bottom of the figure and includes gene names based on published nomenclature. Each box represents an individual gene, except for numbered boxes, which represent genes of the number listed. Genes of the same color are part of the same pathway; grey boxes denote genes whose functions are unknown or unrelated to the capsule. Genes connected by a horizontal line are contiguous on the chromosome. A backwards slash (\) represents hypothetical pseudogenes whose status has not been confirmed by experimentation. Note that inN. subflava, the C and E regions as well as gene NLA0370 are in an inverted orientation compared to the other sequences. Also, whetherN. elongata,N. sicca, andN. mucosa contain a D or D' region cannot be ascertained from the available data.
(0.74 MB TIF)
Genome sequences used in this study.
(0.07 MB PDF)
General characteristics ofNeisseria genomes.
(0.07 MB PDF)
Presence or absence of phase variable genes in commensalNeisseria. Targets were identified by searching the literature for genes known or hypothesized to be phase variable in pathogenicNeisseria.
(0.17 MB PDF)
Genes known or hypothesized to be involved inNeisseria virulence. The 177 targets were identified by searching the literature for genes previously identified as having a role inNeisseria virulence. Genes in bold type are present in all 19Neisseria genomes used in the study.
(0.13 MB PDF)
Copy number ofpilE,pilS andopa genes inNeisseria species. See supplemental references (Text S1) for additional information onN. meningitidis MC58[3],[20] andN. gonorrhoeae FA1090[20],[21].
(0.08 MB PDF)
Recombination of virulence genes amongNeisseria species. The RDPv3.18 program was used to test for recombination in 69 virulence genes that are shared by allNeisseria species.
(0.13 MB PDF)
Primers used for PCR amplification and sequencing of potential pseudogenes.
(0.07 MB PDF)
Common metabolic pathways present in commensalNeisseria. Genes from theN. meningitidis MC58 genome are included as a reference.
(0.18 MB XLS)
Virulence genes present in all 19Neisseria genomes. Note that theN. subflava andN. cinerea genome sequences both contain two copies ofpilE; the second copy is listed directly below the first (see rows 74–75).
(0.06 MB XLS)
Neisseria strain-specific genes. The name on each tab indicates the species included for the given table.
(0.26 MB XLS)
CommensalNeisseria-specific genes. Genes present in two or more commensal genomes but absent from allN. meningitidis andN. gonorrhoeae genomes. See supplemental reference[19] (Text S1) for abbreviations of the COG categories.
(0.22 MB XLS)
PathogenicNeisseria-specific genes. Tab “N. meningitides” includes the sevenN. meningitidis strains analyzed; tab “N. gonorrhoeae” includes the threeN. gonorrhoeae genomes studied. See supplemental reference[19] (Text S1) for abbreviations of the COG categories.
(0.04 MB XLS)