Compartive Overview of The Genomic and Genetic Differences Between The Pathogenic Neisseria Strains and Species

Plasmid 54 (2005) 191218 www.elsevier.
com/locate/yplas
Review
Comparative overview of the genomic and genetic dierences between the pathogenic Neisseria strains and species
Lori A.S. Snyder a, John K. Davies b, Catherine S. Ryan b, Nigel J. Saunders a,*
b
Bacterial Pathogenesis and Functional Genomics Group, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK Australian Bacterial Pathogenesis Research Program, Department of Microbiology, Monash University, Vic. 3800, Australia Received 1 March 2005, revised 18 April 2005 Available online 15 July 2005 Communicated by Julian I. Rood
Abstract The availability of complete genome sequences from multiple pathogenic Neisseria strains and species has enabled a comprehensive survey of the genomic and genetic dierences occurring within these species. In this review, we describe the chromosomal rearrangements that have occurred, and the genomic islands and prophages that have been identied in the various genomes. We also describe instances where specic genes are present or absent, other instances where specic genes have been inactivated, and situations where there is variation in the version of a gene that is present. We also provide an overview of mosaic genes present in these genomes, and describe the variation systems that allow the expression of particular genes to be switched ON or OFF. We have also described the presence and location of mobile non-coding elements in the various genomes. Finally, we have reviewed the incidence and properties of various extra-chromosomal elements found within these species. The overall impression is one of genomic variability and instability, resulting in increased functional exibility within these species. 2005 Elsevier Inc. All rights reserved.
Keywords: Neisseria meningiyidis; Neisseria gonorrhoeae; Comparative genomics
1. Introduction A genome sequence is a single time-point snapshot of a subculture, of a strain, of a species of

Corresponding author. Fax: +44 1865 275515. E-mail addresses: Lori.Snyder@pathology.oxford.ac.uk (L.A.S. Snyder), John.Davies@med.monash.edu.au (J.K. Davies), Nigel.Saunders@pathology.oxford.ac.uk (N.J. Saunders).
*
bacteria, and is inherently a singular example of a complete bacterial system. However, once multiple sequences become available for comparative analysis, and once the genome sequence characteristics as a whole can be considered in the light of existing experimental data for a species, a much deeper and more informative picture can be perceived.
0147-619X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2005.04.005
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L.A.S. Snyder et al. / Plasmid 54 (2005) 191218
The pathogenic Neisseria species: Neisseria meningitidis and Neisseria gonorrhoeae are important human pathogens, being the principal causes of bacterial meningitis and gonorrhoea, respectively. These species have been the subject of intense study over many years by a substantial research community, based upon their medical disease signicance. There are therefore many informative studies that can be drawn upon, tested against, and reconsidered in the light of the complete genome sequences. In this context, these sequences in addition to serving as research resources in their own right, also provide an important framework for understanding neisserial biology and for the design and construction of future experiments.
2. Large-scale genome characteristics 2.1. Neisserial sequences available There are now four complete pathogenic Neisseria spp. genome sequences publicly available. The rst two sequences were essentially completed at the same time. The rst of these to be published was of N. meningitidis serogroup B strain MC58 (Tettelin et al., 2000), composed of 2,272,351 base pairs, with 2158 annotated coding sequences. The second was of N. meningitidis serogroup A strain Z2491 (Parkhill et al., 2000), with 2,184,406 base pairs and 2121 annotated coding sequences of which 1968 are considered orthologous to those identied in N. meningitidis strain MC58 (Tettelin et al., 2000). Additionally, the complete genomes of N. gonorrhoeae strain FA1090 and N. meningitidis serogroup C strain FAM18 have been sequenced and are publicly available, but at the time of writing they are not formally published, although the N. gonorrhoeae strain FA1090 sequence and annotation is available Accession number: AE004969. In addition, the genome sequence of the non-pathogenic commensal N. lactamica is nearing completion. Both published meningococcal genomes have four rRNAs, numerous copies of insertion sequences, variably distributed Correia Repeat Enclosed Elements (CREEs), and nearly 1900 copies of the neisserial uptake signal sequence (Liu et al., 2002; Parkhill et al., 2000;
Tettelin et al., 2000). Strain MC58 has 59 tRNAs (Tettelin et al., 2000), while strain Z2491 has 58, lacking tRNA Asn (Parkhill et al., 2000). The most striking structural dierences between the genomes of these two meningococci are the presence of a major chromosomal inversion of 955 kb and a 32 kb perfect tandem duplication in strain MC58 (Tettelin et al., 2000). This duplication aects genes NMB1124 to NMB1159, which are duplicated in NMB1162 to NMB1197. The functional consequences of this duplication have yet to be investigated experimentally, but it may generate gene dose eects for the 33 genes that have been duplicated. In addition to the genome sequences, there are 1066 sequence entries in EMBL containing complete coding sequence information, totalling 1,648,127 bases (as of 25th February 2005, accessed using SRS, hosted by the Computational Biology Research Group, University of Oxford). This provides a wealth of additional sequence information. 2.2. Strains with physical maps Before whole genome sequencing, physical and metabolic maps were constructed for several neisserial genomes. Five neisserial maps were constructed, two for N. gonorrhoeae strains and three for N. meningitdis strains. Two of these were of strains that have now been sequenced: N. meningitidis strain Z2491 (Dempsey et al., 1995) and N. gonorrhoeae strain FA1090 (Dempsey et al., 1991). The gonococcal map identied 11 opa genes (Dempsey et al., 1991), which was conrmed by the genome sequence. Additionally, maps were made of N. meningitidis strain B1940 (Bautsch, 1993; Ga her et al., 1996), N. meningitidis strain 44/ 76 (Froholm et al., 2000), and N. gonorrhoeae strain MS11-N198 (Bihlmaier et al., 1991). The N. meningitidis strain B1940 identied four rRNA loci and four opa genes (Ga her et al., 1996). N. gonorrhoeae strain MS11-N198 has four rRNA loci and 11 opa genes, by its map (Bihlmaier et al., 1991). 2.3. Chromosomal rearrangements The rearrangement of sections of the chromosomes of dierent strains, relative to one another, has been noted in the genome sequencing projects
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(Tettelin et al., 2000), in the analysis of physical maps of the chromosomes (Dempsey et al., 1995; Froholm et al., 2000), and in the course of other studies (Gibbs and Meyer, 1996). There are no apparent chromosomal rearrangements when the physical map of N. meningitidis strain B1940 is compared to N. meningitidis strain Z2491 (Ga her et al., 1996; Parkhill et al., 2000), but there are rearrangements between N. meningitidis strains MC58 and Z2491 (Parkhill et al., 2000; Tettelin et al., 2000). Chromosomal rearrangements were identied in the more recent study that mapped the N. meningitidis strain 44/76 and compared it to simplied maps of 29 other meningococcal strains, also from the ET-5 complex, suggesting chromosomal rearrangements have occurred in the ET-5 complex strains during epidemic spread (Froholm et al., 2000). Chromosomal rearrangements occur over a wide size range and include both linear transpositions and inversions. Representative comparisons of the complete genomes are shown in Figs. 1A and B. The rates and functional consequences of theses rearrangements have not yet been determined, and it may be that some changes seen in genome sequences are transient and may not persist over time in the environment. However, on the basis of the currently available genome sequences, as illustrated, there are changes that could be considered to be related to genetic distance between strains, and the gonococcal and meningococcal sequences dier signicantly more than the meningococcal sequences do from each other.
3. Islands, MMEs, and prophage 3.1. Identied islands Unlike many other bacterial species, no classical Pathogenicity Islands with characteristic adjacent tRNA loci, anking direct repeats, foreign DNA signatures, and virulence genes (Blum et al., 1994; Hacker et al., 1990) have been found in the Neisseria spp. There are, however, a number of large regions with foreign DNA characteristics of divergent % GC and nucleotide signatures that dier between strains MC58 and Z2491, which
have been called Islands of Horizontal Transfer (IHTs) (Tettelin et al., 2000). Analysis of the genome sequence of N. meningitidis strain MC58 found three IHTs (IHT-A, IHT-B, IHT-C) based upon dinucleotide signatures of the regions and sequence comparisons (Tettelin et al., 2000). IHT-A has two subregions, the rst of which contains the genes responsible for biosynthesis of the serogroup B capsule, while the second contains an ABC transporter and a secreted protein. IHT-B is comprised entirely of hypothetical genes. IHT-C contains three toxin/toxin related homologues as well as genes that appear to be bacteriophage associated. The N. meningitidis strain Z2491 genome does not contain these islands, but does contain a single divergent region of its own (Tettelin et al., 2000). A large island has also been identied in N. gonorrhoeae strain MS11, the Gonococcal Genetic Island (GGI), which is the largest and most widespread in this species identied to date (GenBank Accession No.: AY803022) (Dillard and Seifert, 2001; Hamilton et al., 2005). The majority (80%) of gonococcal clinical isolates carry the GGI, which is apparently species-specic for N. gonorrhoeae (Dillard and Hamilton, 2002; Dillard and Seifert, 2001). It contains 61 coding sequences in 57 kb, including genes encoding a Type IV Secretion System capable of exporting DNA (Hamilton et al., 2005), which is believed to have a role in horizontal gene transfer, and either the traG or sac-4 gene, which are alternative alleles involved in serum resistance (Dillard and Seifert, 2001). None of the large meningococcal islands or the GGI contain CREEs, commonly found around the chromosome (Tettelin et al., 2000). There are also relatively few neisserial uptake signal sequences present in these regions, in comparison with the rest of the genome (Tettelin et al., 2000). The rareness of these general neisserial chromosomal markers is consistent with these regions being horizontally acquired from unrelated species backgrounds. 3.2. MMEs Several instances of smaller strain-specic islands have been reported. In many cases, these
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full the recently described criteria of Minimal Mobile Elements (MME) (Saunders and Snyder, 2002). MMEs are sites in which strain-specic
genes are located between anking genes with conserved sequence and chromosomal organization, such that these anking regions can serve as
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substrates for homologous recombination following natural transformation. The rst of the regions to be specically characterized, between the pheS and pheT genes of the Neisseria spp., contains nine dierent genes or combinations of genes between the conserved anking sequences in the strain set used (Saunders and Snyder, 2002). MMEs often include restriction-modication system genes. For example, the Lineage III-associated meningococcal restriction-modication system NmeSI is located between hrpA and a hypothetical protein gene in strain 800615. Meningococcal strain MC58, has a putative transcriptional regulator and two hypothetical protein genes in this same location, and in meningococcal strain Z2491, there is a dierent methylase, a putative patch repair protein, and two hypothetical genes (Bart et al., 2001). The gonococcal restriction-modication system NgoMI is located between trpE and purK, however, in meningococcal strains FAM18 and Z2491 a hypothetical protein gene is present in this location instead (Zhu et al., 1999). Although less commonly seen, virulence genes are also associated with MMEs. There are variations in the presence of genes between glyA and dedA, where the gene for the meningococcal outer membrane protein Opc is located in approximately 70% of meningococcal strains (Seiler et al., 1996; Zhu et al., 1999, 2003). The meningococcal
capsule biosynthesis and transport genes appear to have inserted between tex and galE, which are also present in gonococci. The genes in this region, including subsequently the capsule genes themselves, then have the potential to serve as regions of homology for recombination and exchange of capsule genes between meningococci (Claus et al., 2002; Dolan-Livengood et al., 2003; Petering et al., 1996). Fig. 2 shows this region in the four sequenced neisserial genomes, and other strains for which the region has been sequenced. Additionally, the gene for the iron-acquisition protein HmbR appears to be within an exchangeable genetic locus, where hmbR, exl3, or exl3A and exl2 are mutually exclusive alternatives (Kahler et al., 2001). In addition, at least seven dierent regions, that could now be considered as MMEs, containing pathogen-specic genes were identied by subtractive hybridization and comparative genome hybridization to membrane microarrays (Klee et al., 2000; Perrin et al., 2002), although whether these genes play a direct role in pathogenesis has yet to be determined. 3.3. Prophages Prior to the completion of the genome sequences, it was generally considered that although neisserial strains possessed bacteriocins (Allunans
b
Fig. 1. Comparisons of the sequenced Neisseria spp. genomes. Chromosomal rearrangements and inversions were identied using MuMmer v3.05 to align the sequenced genomes and mummerplot was used to generate dot plots comparing one neisserial genome, on the x-axis, to another, plotted on the y-axis (A). Before comparison, all neisserial genomes were edited to start at dnaA and the orientation of the N. meningitidis strain MC58 sequence was reversed with respect to the published sequence so that it was in the same orientation as the other three genomes. The genome of N. meningitidis strain MC58 (on the x-axis; panels 1, 2, and 4) was then compared to each of the other three neisserial genomes (on the y-axis) and the N. meningitidis strain Z2491 genome (x-axis; panel 3) was compared to the N. meningitidis strain FAM18 genome (y-axis). Where the genome sequences are the same a coloured dot or line appears, with forward matches being displayed in red and reverse in green. The dot plot displays multiple inversions, rearrangements, and gaps between the neisserial genome sequences. These can be readily visualized in the diagrams generated from the dot plot data (B). In these diagrams, the reference strain genome (x-axis on the dot plots) is on the left and the sequence runs from top to bottom, starting with dnaA. Corresponding segments of the genomes with the same DNA sequence are indicated by the same colour and are joined by a single line, running from middle of each segment to middle of the corresponding segment. The orientation of a segment in the comparison strain (y-axis from the dot plot) relative to its reference strain is indicated by arrowheads. Upward pointing arrowheads indicate inversions relative to the reference strain. We can see, therefore, that between N. meningitidis strains MC58 and Z2491, there is one large inversion (grey-blue to orange segments), within which is a smaller inversion and rearrangement (purple segment moves location and inverts relative to the surrounding sequence) and there is one smaller inversion (green segment). There are many inversions and chromosomal rearrangements between N. meningitidis strain MC58 (and therefore also the other meningococci) and N. gonorrhoeae strain FA1090. Large regions of strain-specic genes, to which there is no correspondence in the genome to which it is being compared, are represented by gaps in the gure.
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et al., 1998; Allunans and Bovre, 1996), they did not have bacteriophage. This is contrary to reports from the 1950s and 1960s, in which bacteriophage against Neisseria spp. were identied, although susceptibility and lysis appeared to be dependent on media, strain, and passage histories (Cary and Hunter, 1967; Stone et al., 1956). Given these early results, the ubiquity of bacteriophage, and the common LPS targets that many can use, it was not surprising that both of the published genome sequences contain strain-divergent bacteriophagederived sequences (Parkhill et al., 2000; Tettelin et al., 2000). Additional analyses of such sequences have been made following these observations. N. meningitidis strain FAM18, strains of the ET-37 complex, and strains of the A4 cluster contain a large prophage with some similarity to lambda (Claus et al., 2001). Three loci with Mulike prophages can be found in the N. meningitidis strain Z2491 genome, although only one is potentially intact (Morgan et al., 2002). A region similar to the Mu-like prophages of strain Z2491 can be found in N. meningitidis strain MC58 (Masignani et al., 2001; Morgan et al., 2002). In the case of strain MC58, the region contains 46 coding sequences, 29 with homology to prophage genes, and appears to have been inserted into an ABC transporter-encoding gene (Masignani et al., 2001). Several regions containing bacteriophagederived sequence are also evident in the genome se-
quence of N. gonorrhoeae strain FA1090. To date, there has been no demonstration of a functionally intact bacteriophage from these species. However, there is evidence that some of the associated genes are expressed and present on the cell surface (Masignani et al., 2001).
4. Dierences in the presence of specic genes Prior to the availability of complete genome sequences, a signicant amount was already known about some gene dierences between the Neisseria species and strains. Because of the predominant focus of research upon disease, these studies largely focused upon virulence determinants. 4.1. opc The outer membrane protein Opc is involved in adhesion and invasion of host cells (Virji et al., 1992) through binding to host proteoglycan receptors (de Vries et al., 1998) and is encoded by the gene opc. This gene has only been identied in strains of N. meningitidis, and it is present in approximately 70% of strains (Seiler et al., 1996). It has a lower percentage G+C than the genome average, which is consistent with its having been horizontally acquired after the separation of the
b
Fig. 2. Comparison of the galE-tex MME region in strains of N. meningitidis and N. gonorrhoeae. The capsule biosynthesis and transport genes of encapsulated N. meningitidis strains are located between galE and tex, as illustrated by strains MC58, Z2491, FAM18, a-707, M7575, and LCDC 78189. Complete contiguous sequence of this region from galE to tex is not available for strains a707, M7575, and LCDC 78189, however, the sequence from galE to the internally conserved ctrA capsule transport gene is available from EMBL (indicated by diagonal bars). In N. gonorrhoeae strains FA1090 and MS11-E1 and in unencapsulated N. meningitidis strain a-14, galE and tex are adjacent and the intergenic sequences in these strains are nearly identical. This arrangement of genes suggests that galE and tex are the conserved anking genes of this MME, which then became the location for the horizontal transfer of these capsule genes. Capsular variation in serogroup would then appear to be due to additional changes in the locus, where the anking homologous sequences are now galE and ctrA (as illustrated by the dierences in Z2491, a-707, M7575, and LCDC78189 relative to MC58 and FAM18) or NMB0065 and siaC (as illustrated by the dierences between MC58 and FAM18). Although it cannot be determined at this time the order in which these processes lead to the evolution of the various meningococcal capsular serogroups, it is evident that the acquisition of these capsule genes in the galE-tex locus is either not the dening speciation event between N. meningitidis and N. gonorrhoeae or that further horizontal exchange between the species has occurred post-speciation, leading to the loss of the capsule genes in this locus in meningococcal strains such as a-14. It is interesting to note, however, that the inverse has never been reported, to whit the acquisition of these capsule genes in the gonococcus through homologous recombination with the conserved anking genes galE and tex. Gene locus identiers are included for annotated complete genome sequences. Gene sizes are indicated and relative sizes of the genes are to scale, although the intergenic regions are not to scale. The length of the intergenic regions between galE and tex or, when not available, between galE and ctrA are indicated. Accession numbers are indicated for sequences obtained from EMBL.
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pathogenic species (Seiler et al., 1996). N. meningitidis strains of the ST-11/ET-37 complex and the ST-8/A4 cluster do not have the opc gene (Seiler et al., 1996). A gene with limited homology has been identied in N. gonorrhoeae strain FA1090, that has 59% identity to the meningococcal strain Z2491 opc (Zhu et al., 1999). However, this gonococcal gene is poorly expressed (Zhu et al., 1999) and the encoded protein should not be assumed to be the functional orthologue of the meningococcal protein. The locus containing opc in the meningococci is diverse with respect to both the genes and organizations present between glyA and dedA in dierent strains, suggestive of an MME-like element. In all, ve variants of this region have been described: (1) containing two IS1106 elements 5 0 of opc (as in N. meningitidis strain Z2491); (2) containing a single IS1106 5 0 of opc (as in N. meningitidis strain MC58); (3) containing orfX, orfY, and the coding sequence with 59% identity to opc (as in N. gonorrhoeae strain FA1090); (4) similar to that in strain FA1090, but with 699 bp, encompassing orfY, deleted (as in N. polysaccharea strain 89357); (5) no coding sequences in the intergenic region between glyA and dedA (as in N. meningitidis strain FAM18) (Zhu et al., 2003). 4.2. Restriction-modication systems Many restriction-modication systems have been identied in the Neisseria spp. and their presence within the genomes is frequently strainspecic (Bart et al., 2000, 2001; Cantalupo et al., 2001; Claus et al., 2000; Gunn and Stein, 1993, 1997; Lau et al., 1994; Morgan et al., 1996; Nolling and de Vos, 1992; Norlander et al., 1981; Piekarowicz et al., 2001; Stein et al., 1995; Sullivan and Saunders, 1989). Notably, one system, NgoPII has signicant sequence homology to the MthT1 system from Methanobacterium thermoformicium, suggesting that there has been transfer between Archea and Bacteria at some point in evolutionary history, although not necessarily directly between Archea and Neisseria. The homology does not extend beyond the coding regions, the genes relying on the incorporation into the genome in an area which places them
under promoter control appropriate for the organism, rather than importing a system that may not function in the new genetic background (Nolling and de Vos, 1992). 4.3. drg One particular dierence in DNA methylation systems between strains was highlighted because it was suggested that it was associated with virulence associated phase variation rate dierences between strains (Bucci et al., 1999). The gene encoding the Dam methylase is replaced in some strains by one encoding a restriction enzyme that cleaves the methylated Dam site. This has been called the Dam replacement gene, drg. Originally, the loss of Dam was reported to be associated with invasive strains and a mutator phenotype. Immediately following this publication, chromosomal digests were conducted on a collection of invasive strains with DpnII and Sau3A, which are, respectively, blocked by and insensitive to Dam methylation. The association between invasive disease isolates and the loss of Dam methylation was not reproduced in our collection (Saunders, unpublished), and the original report probably represented some sampling bias within the collection of strains used. A more extensive and rigorous assessment including analysis of the presence and absence of the dam and drg genes was recently reported (Jolley et al., 2004). Subsequently, it has been shown that there is no association between either invasive disease isolates or with mutator phenotypes with respect to the drg or dam status of strains (Martin et al., 2004; Richardson and Stojiljkovic, 2001). This story attests to the importance of performing studies using properly representative strain collections, and also raises interesting questions about the nature of error correction in this species and its insensitivity to the presence or absence of Dam methylation. While a proportion of mutator phenotypes have now been ascribed to deciencies in mutS, mutL, and dinB (Martin et al., 2004; Richardson and Stojiljkovic, 2001; Richardson et al., 2002), the genetic basis for these interesting phenotypes has not been completely resolved.
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4.4. Iron-acquisition genes The ability to acquire iron has often been considered to be a virulence determinant, although it is more correctly understood as a necessary means of survival within the host, where free iron is scarce and is no more essential to a pathogen than to a commensal. There are several bound forms of iron available including that associated with lactoferrin, transferrin, heme, haemoglobin and haemoglobinhaptoglobin (AlaAldeen et al., 1993; Biswas and Sparling, 1995; Chen et al., 1998; Richardson and Stojiljkovic, 1999). The Neisseria spp. have several iron-acquisition systems to utilize these, but the genes for each system are not universally present in all strains. Human transferrin, an iron-binding glycoprotein, can be bound by a transferrin receptor present on the surface of N. meningitidis and N. polysaccharea (AlaAldeen et al., 1993). The gene encoding the transferrin binding protein, tbpA, is itself divergent in hypervariable regions of the coding sequence, which are presumed to relate to exposed surface epitopes (Cornelissen et al., 2000; Legrain et al., 1998). There is minimal diversity, however, in the gene for the major iron-binding protein Fbp in the pathogenic Neisseria spp., although more diversity is seen in the commensal Neisseria spp. (Genco et al., 1994). There are also two receptors for haemoglobin, HmbR and HpuA, which are both variable in their presence between strains as well as in their expression through phase variation (Chen et al., 1998; Lewis et al., 1999; Richardson and Stojiljkovic, 1999). This variation in the repertoire, divergent regions, and phase variable expression of iron-acquisition genes probably reects diversifying immunological selection. The repertoire of potential iron donors is conserved from host-to-host so, supercially, there would appear to be no advantage to lacking any particular system, and a functional gain for the presence of each. Each protein has to be surface located to bind to its iron-carrying substrate, as such it has to be exposed to the immune system. As relatively conserved targets, these proteins are likely to generate not only antibodies that can lead to the eradication of a colonizing strain, but also anti-
bodies providing cross-immunity to unrelated strains. A probably similar process has been recognized in Salmonella enterica where the agellae are frequently antigenically conserved, stimulating cross-immunity to other agellate strains, in which phase variable expression of this antigen facilitates re-colonization through evasion of cross-immunity (Norris and Baumler, 1999). The population variations in the repertoire of these genes therefore possibly represent a selective advantage by facilitating the re-colonization of a host previously colonized by, and immune to, other strains. 4.5. LPS genes The lipopolysaccharide (LPS) of the Neisseria spp. is also called lipo-oligosaccharide (LOS) due to the absence of O-antigen in its structure. The neisserial LPS is structurally diverse, possessing variable oligosaccharide chains (Zhu et al., 2002). The genes encoding the LPS biosynthetic enzymes are variably present between dierent strains and species, particularly in N. meningitidis, and some of those that are present are phase variable in some strains (Berrington et al., 2002; Jennings et al., 1999; Shafer et al., 2002; Zhu et al., 2002). There is also evidence of variation in gene presence and its relationship with LPS structure in the commensal Neisseria spp. (Arking et al., 2001). As a result, the LPS on the surface of the bacteria is heterogeneous (Zhu et al., 2002) within and between both neisserial strains and species. The LPS biosynthesis genes, called lgt, are present in three loci: lgt-1, lgt-2, and lgt-3. In N. gonorrhoeae, the lgt-1 locus contains lgtA, lgtB, lgtC, lgtD, lgtE, lgtH, and lgtZ, although the function of the latter two genes is not yet known. The lgt-2 locus contains lgtF and rfaK, while the lgt-3 locus contains only the lgtG gene (Zhu et al., 2002). Homopolymeric tracts mediating phase variable expression have been found in four of these genes in some strains: lgtA, lgtC, lgtD, and lgtG (Zhu et al., 2002). This allows an individual gonococcal strain to synthesize a variety of LPS structures (Shafer et al., 2002). Meningococcal strains have a smaller repertoire of genes in their lgt-1 locus, most strains having only three genes, usually lgtA, lgtB, and lgtE.
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However, unlike within N. gonorrhoeae, there are variations in the meningococcal complement of lgt genes. N. meningitidis strain 126E has a large deletion in the lgt-1 locus that has inactivated lgtA and lgtB, leaving lgtC and lgtE; N. meningitidis strain 7880 has only lgtE; and N. meningitidis strain M978 has lgtA, lgtB, lgtC, and lgtE (Jennings et al., 1999). Variability in the presence of genes in the other two loci, lgt-2 and lgt-3, is less common (Zhu et al., 2002). The reasons for this diversity may be similar to those outlined for iron-acquisition genes. In addition to the dierences in complements of LPS biosynthesis genes in strains of N. meningitidis, which of these genes is capable of phase variable expression diers between strains. In general, meningococcal strains can phase vary the expression of either a-chain extensions, through lgtA and lgtC, and/or b-chain extensions, through lgtG. The phase variable expression of lgtA and lgtC can signicantly aect the ability of N. gonorrhoeae to resist the bacteriolytic action of normal human serum, with bloodstream isolates being lgtA phase OFF (Shafer et al., 2002). Phase variation of lgtA allows the Neisseria to vary between two possible terminal structures from the rst heptose of the LPS (Jennings et al., 1999). The alternative structures generated through the phase variation of lgtA also relate to whether the LPS can be sialylated, but the ability to phase vary sialylation is not necessary for meningococcal invasion, as lgtA is not phase variable in some N. meningitidis strains (Berrington et al., 2002). Additionally, lgtD, encoding another glycosyltransferase, contains a homopolymeric tract involved in the phase variation of the associated LPS structure (Zhu et al., 2002). 4.6. Capsule genes Among the pathogenic Neisseria spp., the genes for the biosynthesis of capsular polysaccharides are found only in N. meningitidis and this is one of the dierentiating characteristics between the two pathogenic species. There are at least 13 recognized capsular polysaccharides that can be generated by N. meningitidis strains, of which ve are predominantly associated with invasive disease:
A, B, C, W-135, and Y (Claus et al., 2002). The capsule is believed to be capable of protecting the meningococcus against phagocytosis (Read et al., 1996), and preventing desiccation during transmission (Virji, 1996). Strains colonizing the nasopharynx often express reduced amounts of capsule compared to invasive disease isolates from the blood (Mackinnon et al., 1993) and are frequently acapsulate due to the phase variable expression of capsule biosynthesis genes (Hammerschmidt et al., 1996a,b; Lavitola et al., 1999) or the absence of intact capsule production systems (Claus et al., 2002; Dolan-Livengood et al., 2003). The capsule synthesis gene cluster is composed of ve major regions: (1) region A contains the serogroup specic polysaccharide synthesis genes; (2) region B contains the lipid modication genes; (3) region C contains the ctr polysaccharide transport genes; (4) region D and the truncated region D 0 contain genes for LPS synthesis; and (5) region E, which contains genes of unknown function (Claus et al., 2002; Petering et al., 1996). In region A, changes in the sequence of the siaD gene determines the dierences between the capsules of serogroup B, C, Y, and W-135, while the entire region contains dierent genes in serogroup A (Claus et al., 2002). Allelic exchange can occur between meningococci of dierent serogroups due to the similarity between the capsular genes, particularly the siaD of serogroups B and C, resulting in capsule switching during outbreaks (Swartley et al., 1997). In N. meningitidis strains, the presence of dierent genes varies, the expression of the genes present can phase vary, and the order of the genes in the capsule synthesis gene cluster can dier as well. In those strains investigated, the genes are either in a B-D 0 -E-C-A-D conguration or in a B-D-A-C-E-D 0 conguration (Claus et al., 2002). Regions D and E are present in N. gonorrhoeae and N. lactamica, containing the tex and galE genes that ank the capsule synthesis (region A) and transport gene (region C) regions in meningococci (Fig. 2). It is therefore believed, supported by signatures found associated with horizontally acquired genes, that the encapsulation of the meningococci is the result of horizontal DNA transfer of the capsule biosynthesis genes into this
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region (Claus et al., 2002; Dolan-Livengood et al., 2003; Petering et al., 1996). Amongst all this variation in the capsule synthesis gene cluster, the ctrA gene in region C, encoding the outer membrane transporter of capsular polysaccharide, is conserved in most meningococcal clinical isolates and is not present in other neisserial species (Claus et al., 2002; Frosch et al., 1992; Sadler et al., 2003). 4.7. Other genes showing gene-complement dierences Other genes have been identied which are not universally present in the Neisseria spp. The N. meningitidis gene gpxA is not present in the N. gonorrhoeae or commensal Neisseria spp. strains that have been investigated (Moore and Sparling, 1995). The RTX family genes frpA and frpC appear to be limited to the meningococcal species (Thompson et al., 1993). While the pathogenic Neisseria spp. can decorate their pili with phosphorylcholine, the ability to decorate neisserial LPS is possessed only by the commensal strains, which have a homologue of the Haemophilus inuenzae licA gene (Serino and Virji, 2000). The dcwcluster associated gene dca, on the other hand, is present only in the pathogenic neisserial strains (Perrin et al., 1999; Snyder et al., 2001a), is associated with an the presence of phosphorylcholine on the pili (Warren and Jennings, 2003), and inactivation of this gene has dierent consequences for transformability in N. meningitidis and N. gonorrhoeae (Snyder et al., 2001a). The gene for IgA1 protease is also present only in the pathogenic Neisseria spp. (Koomey and Falkow, 1984; Perrin et al., 2002). It is likely that with advances in comparative genome microarray hybridization, or genomotyping, and MME analyses, many more such strain- and species-specic neisserial genes will be identied.
mutations aecting coding potential, as well as some instances of insertional inactivation. 5.1. porA The outer membrane protein PorA, a porin (Tommassen et al., 1990), is only functionally present in N. meningitidis. A porA gene is present in N. gonorrhoeae, but the gonococcal porA contains inactivating mutations in both the promoter region and the coding region of the gene (Feavers and Maiden, 1998). The porA gene appears to be absent from the commensal Neisseria spp. (Feavers and Maiden, 1998), or at least is only present in some strains of some commensal species (Wol and Stern, 1995). 5.2. Restriction-modication systems As with porA, the function of restriction-modication systems cannot be simply assumed to be equivalent on the basis of gene presence and absence alone, as other changes may also have functional consequences. In the case of the N. gonorrhoeae strain FA1090 Hsd system NgoAV, the genes hsdM and hsdR are what would be expected for a typical Type I restriction-modication system. The hsdS gene, however, has been tandemly duplicated, with the rst copy, hsdS1 being functional in site recognition for the restriction-modication system and the second, hsdS2 having no apparent function. While functional for site recognition, the gonococcal HsdS1 is also truncated and recognizes a palindromic sequence with its single remaining DNA-binding site, rather than the non-palindromic sequences typical of Type I restriction-modication systems where the site recognition protein has two DNA-binding domains (Piekarowicz et al., 2001). While present in N. meningitidis strains MC58 and Z2491, the genes for this system are divergent. The strain MC58 hsdS1 is non-functional due to a point mutation, while that of strain Z2491 is non-functional due to dierent mutations, as well as having a deletion in hsdR. All three neisserial strains also display divergence in the organization of this locus (Piekarowicz et al., 2001), therefore, even when the systems genes are present, there is a large degree
5. Degenerate genes A noticeable feature of all the completed neisserial genome sequences is the presence of numerous genes that contain frame-shifts, deletions, and
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of variability in the neisserial restriction-modication systems. 5.3. Iron-acquisition genes Neisseria gonorrhoeae and N. meningitidis both have a lactoferrin receptor, encoded by lbpA, that is capable of scavenging iron from human lactoferrin. Some clinical isolates of N. gonorrhoeae have been identied that have mutations in or near lbpA and do not express the lactoferrin receptor, therefore acquiring iron from lactoferrin is not essential (Biswas and Sparling, 1995). 5.4. Regulatory genes In comparing the annotated neisserial genome sequences, there are at least nine situations where a particular regulatory gene has been inactivated, in one or more genomes (Davies, unpublished). Perhaps the best example of pseudogene generation concerns several regulatory genes that may have once had a role in the regulation of the pilE gene, which encodes pilin. Upstream of pilE in both N. gonorrhoeae and N. meningitidis is a consensus sequence for a r54 promoter (Fyfe et al., 1995). For function, these promoters require the alternative sigma factor RpoN, and an activator protein that binds upstream of the promoter. Consensus sequences for r54 promoters have been found upstream of other gonococcal genes including pilC (Taha et al., 1996) , comA (Facius and Meyer, 1993), and pip (Albertson and Koomey, 1993). In addition, the nding of a gonococcal protein which co-puries with RNA polymerase and reacts with a monoclonal antibody raised against the Salmonella typhimurium RpoN protein (Klimpel et al., 1989), resulted in attempts to identify rpoN and the gene encoding the relevant activator in N. gonorrhoeae. However, the pilE r54 promoter has since been shown to be non-functional in N. gonorrhoeae (Fyfe et al., 1995), despite the fact that is functional in a P. aeruginosa background (Carrick et al., 1997). Subsequently, remnants of an rpoN gene have been identied in neisserial species and strains (Laskos et al., 1998). Regulators required for activation of gene expression via r54 promoters are often members
of two-component regulatory systems (Kofoid and Parkinson, 1988). Remnants of such a system that may have once controlled piliation have also been identied (Carrick et al., 2000). Standard methods such as Southern hybridization, and complementation assays in rpoN mutants of E. coli and P. aeruginosa rpoN mutants had failed to identify a rpoN gene in N. gonorrhoeae MS11A (Laskos et al., 1998). Subsequent sequence analysis of the gonococcal FA1090 genome sequence indicated a region of sequence that did not constitute a CDS, yet encoded motifs characteristic of RpoN proteins including the RpoN box unique to RpoN proteins (Laskos et al., 1998). The region of sequence was denoted RLS for r poN-like sequence. Based on a comparison of the amino acid sequences of RLS and the E. coli RpoN protein it appears a deletion of 414 bp, spanning the region encoding the DNA-binding motifs, has occurred in the gonococcus. This resulted in a frameshift mutation and rendered the gonococcal gene incapable of encoding a functional RpoN protein that could bind DNA (Laskos et al., 1998). Other N. gonorrhoeae strains and N. meningitidis and N. subava strains contain RLS [(Laskos et al., 1998); Laskos and Davies, unpublished]. In all cases, the same 414 bp deletion was present. The N. lactamica strain tested appeared to have an intact rpoN gene, but it was unable to complement a P. aeruginosa rpoN mutant (Laskos and Davies, unpublished). It was also possible to identify remnants of a two-component regulatory system in N. gonorrhoeae MS11A that shares sequence similarity with the pilS and pilR genes of P. aeruginosa (Carrick et al., 2000). A single CDS denoted rsp for regulator/sensor protein was identied (Carrick et al., 2000), that potentially encodes a protein that is similar at its amino terminus to the N terminus of histidine kinase sensor proteins. It is predicted to be a transmembrane protein and has the conserved histidine residue that is the site of autophosphorylation in the transmitter domain of these sensor proteins (Fig. 3). At its carboxy terminus it is similar to the equivalent region of regulatory proteins in that it encodes a DNA binding motif which is characteristic of the output domain of these proteins, but it lacks a complete ATP-bind-
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Fig. 3. Schematic diagram of the predicted structure of the P. aeruginosa PilS and PilR proteins and neisserial proteins including Rsp (Carrick et al., 2000) that share similarity with these P. aeruginosa proteins. Conserved domains are indicated. These are the histidine residue (H), the cytoplasmic transmitter domain (F) and one or two nucleotide binding motifs (G1/G2) in histidine kinase sensors and an aspartate residue (D), Walker Boxes A and B (WBA/WBB) of the ATP-binding site and DNA-binding motif (HTH) in response regulator proteins. Ng, N. gonorrhoeae; Nl, N. lactamica; NMB, N. meningitidis strain MC58; NMA, N. meningitidis strain Z2491; Ns, N. subava. The pale blue rectangle represents the inner membrane. The dark blue line represents sequence similar to the equivalent portion of PilS, including the transmembrane domains. The red and orange lines represent sequences similar to the equivalent portions of PilR and sensor proteins in general, respectively. The sequence represented by the purple line is not similar to any sequence in the databases, and the green dashed line represents that portion of the Ns locus for which there is no sequence information. The frameshift in the Ns sequence is indicated. Figure is not to scale.
ing site (Fig. 3). Analysis of the gonococcal strain FA1090 genome sequence shows rsp is also present in this strain. Analyses of the sequenced meningococcal genomes reveal two CDSs that could encode portions of the equivalent sensor and response regulator proteins, these being NMB1606/1607 and NMA1803/1805, respectively (Fig. 3). NMB1606 and NMA1803 putatively encode transmembrane bound proteins containing the conserved histidine residue found in sensor proteins, and appear to be
fused with a CDS encoding a product of unknown function. NMB1607 and NMA1805 appear to contain an internal deletion in that they encode conserved portions of regulatory proteins normally found at both the amino- and carboxy-terminus, without motifs normally found in between (Fig. 3). Partial sequencing of the equivalent region of the genome of the commensal N. subava has revealed a dierent arrangement (Ryan and Davies, unpublished). Sequence similarity to PilS can be found in two dierent reading frames. These CDSs
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both potentially encode a product sharing similarity to the C terminus of histidine kinase sensors, one to the region encoding the conserved histidine residue that is very similar to the sequence found in N. meningitidis, and the other to part of the nucleotide binding motif (Fig. 3). A separate CDS, where the derived amino acid sequence is similar to response regulatory proteins, appears to be identical to NMB1607 and NMA1805 from N. meningitidis (Fig. 3). Analysis of the nearly completed N. lactamica genome sequence suggests a single CDS identical to that present in N. gonorrhoeae. These ndings suggest that functional genes, equivalent to those found in P. aeruginosa, once controlled transcription of the pilE gene in an ancestor of the present Neisseria species. Given the wide but not universal occurrence of RLS, the inactivation of the rpoN gene appears to have preceded the evolution of the present species. The subsequent inactivation of the pilS and pilR genes appears to be a more recent event, that has followed dierent routes in dierent species.
to horizontal exchange between the species, a N. meningitidis isolate has been identied that pos zsesses a gonococcal PIB, encoded by porB1b (Va quez et al., 1995). Rarely, strains of N. gonorrhoeae have been identied that react to both PIA and PIB antibodies, which is presumed to be due to mosaicism within the porB allele (De La Fuente zquez, 1991; Gill et al., 1994). and Va 6.2. Class I or class II pili The expression of neisserial type IV pili is required for attachment to host cells (Swanson, 1973), high frequency transformation (Biswas et al., 1977), and twitching motility (Wolfgang et al., 1998). Strains of N. meningitidis can express either class I or class II pili. Class I pili are possessed by both N. meningitidis and N. gonorrhoeae, with the major pilin subunit being encoded by pilE, which undergoes antigenic variation through recombination with silent cassettes (Segal et al., 1985). The subunits of class II pili are slightly smaller and are encoded by a dierent gene that apparently has no silent cassette counterparts. N. meningitidis strain FAM18 and N. lactamica have class II pili (Aho and Cannon, 1988; Aho et al., 1997, 2000). 6.3. pilC
6. Variations in the versions of genes present 6.1. porB There are several known variations of the porB gene, encoding a porin capable of translocating into host cell mitochondrial membranes that has been found to both cause (Mu ller et al., 2000, 1999) and prevent apoptosis of host cells (Massari et al., 2000, 2003). These opposing observations may be due to dierences in the host cells, bacterial strains, protein purication methods, growth media, stages of infection, and/or variations in the porB sequence (Binnicker et al., 2004). In N. gonorrhoeae there are two mutually exclusive PorB proteins present, called PIA and PIB (Carbonetti et al., 1988), the genes for which have been renamed to porB1a and porB1b. In N. meningitidis, two types of PorB exist as well, class 2 and class3, encoded by porB2 and porB3 (Feavers and Maiden, 1998). Because they are surface exposed antigens, the genes themselves can be highly divergent, but cluster into these four classes. Due
Two copies of the pilC gene are present in distant locations on the pathogenic neisserial genomes (Parkhill et al., 2000; Tettelin et al., 2000). The expression of both copies is dependent on phase variation, and expression of at least one version of pilC is required for piliation of the bacterial cell for both class I and class II neisserial pili (Jonsson et al., 1995; Ryll et al., 1997). In N. gonorrhoeae, expression of pilC1 or pilC2 results in similar amounts of pili and the two PilC proteins bind equally to target host cells. In the N. meningitidis strains investigated to date, however, only PilC1 mediates binding to host cells (Jonsson et al., 1995). N. meningitidis strains MC58 and Z2491, have equivalent pilC genes. However, the sequences of both pilC genes from N. gonorrhoeae strain MS11 (Rudel et al., 1995) dier from those in gonococcal strain FA1090, and there are often
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substantial dierences between genes annotated as pilC1 and pilC2 between strains of both species. These dierences in the pilC genes make the routine annotation of pilC genes as simply pilC1 and pilC2 not only misleading with regard to their sequence homology, but also potentially with respect to their function.
regions (Bhat et al., 1992) are apparent, and were shown to relate to the three exposed loops of the protein proposed by the model of its structure (Malorny et al., 1998). 7.2. penA Penicillin-binding protein 2 (PBP2) of the Neisseria spp. is a peptidoglycan transglycosylase and transpeptidase (Spratt and Cromie, 1988) and its gene, penA, is a component of the neisserial dcw cluster (Francis et al., 2000; Snyder et al., 2001a). Alternations in the anity of PBP2 for penicillin leads to chromosomally encoded penicillin-resistance, which is synergistically augmented by chromosomal mutations in penB and the mtr eux pump system (Faruki and Sparling, 1986). It was discovered that the acquisition of lower anity PBP2 by the pathogenic Neisseria spp. was due to the horizontal transfer of blocks of N. avescens penA DNA, which homologously recombined with the native penA to generate lower penicillin anity mutants. When penA is examined from seven Neisseria spp. a complex mosaic structure generated by horizontal DNA transfer between commensal and pathogenic strains is apparent in this essential gene (Spratt et al., 1989, 1992). 7.3. pilE Mosaicism is also found within the pilE gene encoding the major pilin subunit (Hagblom et al., 1985; Hill, 1996; Seifert et al., 1988). In this case one, or in some strains two, expression cassettes undergo recombination with pilS sequences (Gibbs et al., 1989; Haas et al., 1992; HowellAdams and Seifert, 1999, 2000; Koomey et al., 1987; Meyer et al., 1984; Parkhill et al., 2000; Segal et al., 1985; Seifert et al., 1988; Swanson et al., 1987, 1990; Tettelin et al., 2000). These are sequences with homology with most of the protein encoding region of the expressed pilE gene, but which have no promoter or the 5 0 terminal portion of the pilin subunit and are therefore not capable of expression (Haas and Meyer, 1986; Haas et al., 1992; Hagblom et al., 1985; Meyer et al., 1984). Each pil gene has six variable regions where polymorphisms are primarily located, which have
7. Mosaic genes 7.1. opa The pathogenic Neisseria spp. have multiple copies of the opa gene; three or four copies in N. meningitidis, which encode what were originally called the class 5 proteins, and up to 11 copies in N. gonorrhoeae, which encode what were called the P.II proteins. These proteins are abundant outer membrane proteins that target certain host cell CEA family receptors and syndecan proteoglycan family receptors (Chen et al., 1997, 1995; Popp et al., 1999; van Putten and Paul, 1995; Virji et al., 1996a,b). Probably due to their prevalence on the outer membrane and exposure to the immune system, the expression of these proteins is both phase and antigenically variable. Antigenic diversity is generated through recombination between opa genes, which swap and re-assort between two hypervariable and one semivariable regions, generating a family of mosaic opa genes (Aho et al., 1991). In a collection of meningococci from West Africa, seven electrophoretically distinct Opa proteins were found, although each isolate contains only three opa genes; the dierences in the Opas are generated by recombination following transformation from other strains, generating mosaics (Hobbs et al., 1994). Likewise, in a collection of N. meningitidis strains of the ET-37 complex, 26 dierent Opa proteins have been identied, encoded within four genes per genome (Hobbs et al., 1998). Two opa genes have been found in N. lactamica and N. ava and one in N. sicca, N. mucosa, and N. subava (Wol and Stern, 1995). When the predicted sequences of 45 Opa proteins from N. meningitidis, N. gonorrhoeae, N. sicca, and N. ava are aligned, the divergence of the semivariable and hypervariable
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been termed minicassettes (Haas and Meyer, 1986; Haas et al., 1992). Because the location of the ends of the regions that are exchanged during the non-reciprocal recombination vary, this system provides a vast potential for generating diversity within these genes and each pilE gene in each strain can eectively be considered to be unique. 7.4. mafB and fhaB A similar expression cassette and silent cassette arrangement to that of the pilE and pilS system may exist for mafB and fhaB and their apparent silent cassettes. A lipoprotein is proposed to be encoded by mafA and a predicted secreted protein by mafB, which together are thought to have adhesin activity in N. gonorrhoeae (Parkhill et al., 2000). The region 3 0 of the mafB gene contains open reading frames lacking initiation codons, but with homology to mafB, suggesting that these reading frames are silent cassettes to mafB as the pilS cassettes are to pilE. The same may also be true for fhaB, which has homology to the lamentous haemaglutinin precursor gene from Bordetella pertussis, but was annotated as a hypothetical protein in N. meningitidis strain Z2491 and a haemaglutinin/haemolysin-related protein in N. meningitidis strain MC58. There are also apparently silent cassettes of the fhaB sequence, although these have been less extensively examined than either of the other systems. As with mafA and mafB, the gene located 5 0 of fhaB may be required for its function, as fhaA has homology to the accessory secretion protein in the B. pertussis lamentous haemaglutinin system (Klee et al., 2000; Locht et al., 1993). The proposed expression/silent cassette arrangement of both mafB and fhaB has only come to light following the assessment of the complete genome sequences (Klee et al., 2000; Parkhill et al., 2000).
are adaptive for dierent environmental conditions. In the Neisseria spp. gene switching is associated with simple sequence repeats that can be used to identify genes with phase variable potential. Further, a greater proportion of these genes dier between the sequenced strains than do other genes. There are also variations in whether the genes, when present, are phase variable. There are therefore two dierent levels at which phase variation and phase variable genes are associated with strain dierences and their dierences in behaviour. The role of phase variation in adaptation to dierent host conditions and strain tness has recently been reviewed separately (Saunders, 2003; Salau n et al., 2003,), and the phase variable genes of the pathogenic Neisseria species singly and in combination have been described in detail elsewhere (Saunders et al., 2000, 2001b). Although not addressing pathogenic Neisseria, a recent complete study of the predicted phase variable repertoire of genes in a population-based study of H. pylori illustrates the issues of variation of these genes between strains within a diverse panmictic population (Salau n et al., 2004), which closely parallels the situation in the Neisseria spp.
9. Presence and location of mobile non-coding elements In addition to dierences in coding sequences between strains, several non-coding elements are variably present or are present in dierent locations between dierent strains. One of the most clearly described IS elements in N. meningitidis is IS1301, which has been found in 29% of meningococci tested, and is present in all serogroups. It has not been found in other Neisseria spp. (Hilse et al., 2000). Disruptions of siaA and porA by IS1301 have been reported in meningococci (Hammerschmidt et al., 1996a; Newcombe et al., 1998). Other identied IS elements include members of the IS families IS3, IS5 (which includes IS1106), IS30, IS110, and an IS element related to IS1016 (Tettelin et al., 2000). In addition to typical IS elements, Neisseria spp. also contain another apparently mobile element, although the transposase responsible for
8. Variations in the genes expressed 8.1. Phase variation Phase variation describes a process of gene ON/ OFF switching that is associated with genes that
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its amplication and mobilization has yet to be determined. A 26 base pair repeat, found multiple times within the neisserial genome, was identied and found to be normally arranged as an inverted repeat with a characteristic core region (Correia et al., 1986, 1988). In this conguration, it is referred to as a CREE. The functional consequences of the local presence of a CREE and its mobility appear to be dependent on the specic sequence of the CREE, its location, and the sequences anking it. The rst function of a CREE reported was the identication of a promoter at the end of the element proximal to the gene uvrB. In this case, the CREE sequence, ending TATA, is inserted next to the chromosomal sequence CT, the CREE and native sequence combining to form the 10 element of a promoter (Black et al., 1995). In this case, the 35 is within the CREE itself. In the same report, a putative gearbox promoter was also identied within this CREE. A CREE with a similar gearbox-like sequence is located in the dcw cluster. While this gearbox promoter is active in E. coli, it is not active in the neisserial genome (Snyder et al., 2003), as would be expected in bacteria lacking rpoS (Tettelin et al., 2000). There is another means by which CREE generate promoters, as identied in N. lactamica strain L18. In this case, the promoter is generated at the end of the CREE distal to the dcaC gene. The entirety of the 10 element is contained within the CREE, while the 35 element is part of the native chromosome (Snyder et al., 2003). The CREE has also been shown to be transcriptionally active 5 0 of drg, through investigation of strains of N. lactamica with and without the CREE in this location (Cantalupo et al., 2001). It has been suggested that, since the CREE are predicted to form hairpin structures due to the terminal inverted repeats, these elements act in transcriptional termination. Additionally, it was believed that they were present only at the end of transcripts, although they are now know to be within coding sequences, between promoters and genes, and components of promoters (Black et al., 1995; Klee et al., 2000; Liu et al., 2002; Parkhill et al., 2000; Snyder et al., 2003). The CREE within the dcw cluster was claimed to be a transcriptional terminator (Francis et al., 2000), but
transcription through this region was later demonstrated (Snyder et al., 2003). More recently, CREE have been proposed to direct RNaseIII digestion of associated transcripts (De Gregorio et al., 2003). In N. meningitidis, the mtrCDE genes, encoding an eux pump system, have been uncoupled from the MtrR/MtrA regulation system of the gonococci due to the insertion of a CREE 5 0 of these genes, which now places the meningococcal mtr eux system under the regulation of IHF and RNaseIII (Rouquette-Loughlin et al., 2004). There are 102 to 270 copies of the CREE distributed throughout the genome (Liu et al., 2002). It has therefore been suggested that regions lacking CREE are more likely to be those that have been acquired by horizontal transfer relatively recently (Buisine et al., 2002).
10. Presence of extra-chromosomal elements In terms of the variety of plasmids found within each pathogenic Neisseria species, and the carriage frequency, little has changed in the 15 years since this topic was last reviewed (Dillon and Yeung, 1989; Roberts, 1989). Most isolates of N. gonorrhoeae, but not N. meningitidis, carry plasmids. Most, but not all, of these plasmids are specic to one or other of the species. While the potential impact of plasmid carriage may have changed little in those 15 years, our understanding of the function of plasmid-encoded gene products has increased, as the result of various sequencing projects. 10.1. Gonococcal cryptic plasmid Nearly all strains of N. gonorrhoeae carry a 4.2 kb plasmid (Roberts and Falkow, 1979) that is usually referred to as the gonococcal cryptic plasmid. This plasmid was rst described in 1972 (Engelkirk and Schoenhard, 1973) and was amongst the rst plasmids where the complete nucleotide sequence was determined (Korch et al., 1985). This plasmid must have been re-sequenced during the sequencing of the genome of gonococcal strain FA1090, but that sequence (as opposed to that of the chromosome) is not publicly available. The original nucleotide sequence en-
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abled the rst prediction of the genetic content and transcriptional organization of the plasmid (Korch et al., 1985), but no phenotype has ever been associated with plasmid carriage (Biswas et al., 1986). Several structural variants of this plasmid are known to occur. In some strains, a specic 54 nt deletion has occurred between a directly repeated 44 nt sequence (Foster and Foster, 1976; Hagblom et al., 1986), while in others a 156 nt insertion has occurred within one of the 44 nt repeats (Roy et al., 1988). In both cases, the genetic change has occurred within what was originally referred to as the cppB gene (Korch et al., 1985). Finally, in some gonococcal strains the cryptic plasmid is present as a stable 12.6 kb trimer, consisting of three directly-repeated copies of the cryptic plasmid (Johnson et al., 1983). The genetic processes that promote the formation of stable trimers in these strains, and the reason they do not segregate dimeric and monomeric forms of the plasmid has never been explained. The nucleotide sequence of the cryptic plasmid suggests a compact genetic organization with two transcriptional units, one of which was suggested to be composed of ORF6, ORF7, cppA, cppB, and cppC (Korch et al., 1985). ORF6 and ORF7 have GTG initiation codons, and the available evidence suggests that they are not expressed (Hagblom et al., 1986; Sarandopoulos and Davies, 1993a). The derived amino acid sequence from ORF6 shows no similarity to any sequence in the databases. As nucleotide sequences from a variety of bacterial species has accumulated, it has become clear that most of this portion of the plasmid is the remnant of a mobilization region very similar to that found in a range of bacterial plasmids that also encode bacteriocins (Sarandopoulos and Davies, 1993a; Wertz and Riley, 2004). In N. gonorrhoeae only a portion of this mobilization region is present, and even that has been inactivated by a 10 nt duplication (Sarandopoulos and Davies, 1993a). This region extends from ORF7 through cppC to part way through cppB, corresponding to parts of mobC, mobA, and mobB, respectively (Sarandopoulos and Davies, 1993a). The derived amino acid sequence of the carboxy-terminal portion of the CppB protein shows no similarity to any protein in the databases. The predicted amino
acid sequence of CppA displays similarity to hypothetical proteins in a range of species. A second transcriptional unit on the cryptic plasmid was suggested to consist of ORFs 1-5 (Korch et al., 1985). Again, sequence comparisons now enable predictions for the function of some of these putative genes. The predicted products of ORF1 and ORF2 have strong sequence similarity to the products of the RepA and RepB genes involved in the replication of plasmids in a range of bacterial species (Ankri et al., 1996; De Mot et al., 1997). Such sequence comparisons have also enabled a prediction of the origin of replication on the cryptic plasmid (De Mot et al., 1997). The predicted product from ORF3 shows no sequence similarity to any protein in the databases. The predicted protein product from ORF4 shows signicant sequence similarity to a putative specialized RNA polymerase sigma factor from Haemophilus inuenzae, and production of this protein has been correlated with resistance to rifampicin in Escherichia coli (Sarandopoulos and Davies, 1993a). ORF5 appears to encode a protein of the VapD (virulence associated protein D) family (Katz et al., 1992). Despite the name of the family, none of the proteins it contains appears to have a known function. 10.2. Sequence similarity between the cryptic plasmid and neisserial genomes There are many reports suggesting nucleotide sequence similarity between the gonococcal cryptic plasmid and the chromosomes of N. gonorrhoeae (Biswas et al., 1986; Hagblom et al., 1986) and serogroup B N. meningitidis (Grimholt et al., 1993), and cryptic plasmids found in the meningococcus (Ison et al., 1986). For the most part, these claims have been made on the basis of Southern hybridization experiments that employed varying levels of stringency, and therefore have to be viewed with some caution. One report suggests that the hybridization between the cryptic plasmid and gonococcal chromosome was the result of short repetitive, rather than any contiguous, nucleotide sequence (Sarandopoulos and Davies, 1993b). As far as we are aware none of the meningococcal cryptic plasmids have been sequenced, so direct sequence comparisons are not yet possible.
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However, the availability of a gonococcal and several meningococcal genome sequences enables other suggestions of sequence similarity to be directly tested. A simple BLASTn search (Altschul et al., 1990) suggests that there is no substantial sequence similarity between the gonococcal cryptic plasmid and the genomes of N. gonorrhoeae strain FA1090, N. meningitidis strain Z2491, or N. meningitidis strain FAM18. However, there are three short (50-80 nt) regions with 86-95% nucleotide sequence identity shared between the gonococcal cryptic plasmid and the chromosome of N. meningitidis strain MC58. These regions of nucleotide sequence identity are clustered on both DNA molecules, and may explain why gonococcal cryptic plasmid probes appear to hybridize more strongly with the serogroup B, as opposed to other meningococcal genomes (Grimholt et al., 1993). One of these regions is contained within the cryptic plasmid cppA gene and overlaps two contiguous restriction fragments identied by Grimholt et al. (1993) as hybridizing to the serogroup B meningococcal genome. The equivalent portion of the meningococcal genome has been named NMB1754, and annotated as producing a cryptic plasmid protein A-related protein. This CDS has a TTG initiation codon, and the predicted amino acid sequence shows similarity to just the carboxy-terminal portion of CppA. The additional two sequences that show sequence identity to the serogroup B meningococcal genome overlap, and are situated within the repB gene (ORF2). One sequence overlaps, and the other is contained within, a 66 nt HhaI fragment identied by Grimholt et al. (1993) as hybridizing to the serogroup B meningococcal genome. The equivalent portions of the strain MC58 genome are located between NMB1752 and NMB1753, and overlapping the start of NMB1755. Between NMB1752 and NMB1753, the derived amino sequence similarity to the cryptic plasmid RepB protein extends out in both directions from the region of nucleotide sequence identity. This might suggest that a segment derived from a related plasmid, rather than gonococcal cryptic plasmid is integrated at this site. It should also be noted that the derived amino acid sequence of NMB1753, also located in this region, appears to be a member of the VapD family, and
shows a high degree of sequence identity to the predicted product of ORF5. However, there is no high nucleotide sequence identity between the cryptic plasmid ORF5 and NMB1753. The second site, overlaps the start of NMB1755. This CDS has a GTG initiation codon, and is suggested to express a hypothetical protein. This site is notable for the presence of a neisserial uptake sequence (5 0 -GCCGTCTGAA-3 0 ), which is specic to this genus (Goodman and Scocca, 1988). The region of sequence identity overlaps the only copy of this sequence on the cryptic plasmid. The presence of the uptake sequence is signicant in that this segment of the meningococcal genome is in the middle of IHT-C (Tettelin et al., 2000). The presence of the uptake sequence suggests that this segment of IHTC is of neisserial origin, but the sequence similarity seems to be more at the derived amino acid, rather than nucleotide sequence, level. Again one explanation is that segments of those meningococcal plasmids that show sequence similarity to the gonococcal cryptic plasmid (Ison et al., 1986) have been integrated at this site. 10.3. b-Lactamase producing plasmids An increasing proportion of gonococcal strains are resistant to penicillin, resulting from the presence of a plasmid encoding a TEM b-lactamase (Elwell et al., 1977). Plasmids from dierent strains vary in size, and were originally named for the geographic region (Asia, Africa, Rio, etc.) where they were rst found (Dillon and Yeung, 1989). Several of these plasmids have had their nucleotide sequence determined (Pagotto et al., 2000), and it is clear that they are all structural variants of an ancestral plasmid that probably originated in Haemophilus spp. (Brunton et al., 1986). 10.4. Conjugative plasmids Many gonococcal strains harbour a 39 kb conjugative plasmid, that was rst discovered in 1974 (Elwell and Falkow, 1975), but has been found in strains stored since the 1940s (Roberts et al., 1979). This plasmid has not been sequenced, but has been demonstrated to promote its own transfer to recipient gonococcal cells, and also to
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mobilize the b-lactamase plasmids (Eisenstein et al., 1977; Roberts and Falkow, 1979). In contrast to early reports (Roberts and Falkow, 1978), this plasmid does not promote transfer of chromosomal DNA to recipient cells (Norlander et al., 1979). The recombination events detected in these early experiments appear to have resulted from transformation of the plasmid-containing cell by recipient cell DNA, perhaps suggesting that the conjugative plasmid endows non-piliated host cells with a low level of competence for natural transformation (Norlander et al., 1979). This plasmid appears to be only stably maintained in N. gonorrhoeae and certain strains of the closely related N. cinerea (Genco et al., 1984). More recently, tetracycline-resistance strains of N. gonorrhoeae have emerged (Morse et al., 1986). Tetracycline resistance results from the presence of an approximately 40 kb plasmid carrying the tetM determinant (Morse et al., 1986). There was initially some debate as to the origin of these plasmids, and in particular whether they were the result of the insertion of a tetM determinant into the 39 kb gonococcal conjugative plasmid (Gascoyne et al., 1990; Morse et al., 1986). It is now clear that some plasmids (referred to as the Dutch type) are indeed based on the gonococcal conjugative plasmid, while others (referred to as the American type) dier in their physical map suggesting that they at least result from a dierent transposition event (Gascoyne et al., 1991). It now seems clear that at least the Dutch TetM plasmid is the result of a class II transposition event involving the gonococcal conjugative plasmid (Swartley et al., 1993). Transposition appears to have been accompanied by a deletion event that removed part of the transposon and a segment of DNA from the original conjugative plasmid (Swartley et al., 1993). Despite its origin, the TetM plasmid appears to have a host range that is broader than the conjugative plasmid, as it can be transferred to, and stably maintained in, a range of species (Roberts and Knapp, 1988). 10.5. Meningococcal plasmids In contrast to those found in N. gonorrhoeae, the plasmids of N. meningitidis have been poorly
characterized. Cryptic plasmids of various sizes have been described in some strains (Verschueren et al., 1982), but all that is known about them is that some appear to share sequence similarity with the gonococcal cryptic plasmid (Ison et al., 1986). The gonococcal b-lactamase and TetM plasmids can both be transferred to, and maintained, in a meningococcal background (Roberts and Knapp, 1988). Finally a series of plasmids that appear related to RSF1010 have been reported in meningococcal strains (Facinelli and Varaldo, 1987; Rotger et al., 1986). Dierent variants of these plasmids encode various combinations of resistance to penicillin, streptomycin and sulphonamide (Rotger et al., 1986). These RSF1010-related plasmids have not been reported in N. gonorrhoeae.
11. Conclusions Above all, the key messages from this review are ones of change and exibility. These species were known to have relatively panmictic population structures (Smith et al., 1993) due to frequent intra-species recombination events facilitated by a common uptake signal sequence (Elkins et al., 1991; Goodman and Scocca, 1988). In addition, these species have been recognized to generate diversity through a silent cassette system for the generation of pilin variants (Swanson et al., 1987), and many phase variable genes (Saunders et al., 2000; Snyder et al., 2001b). However, the sheer scale of the impact of these processes, as well as dierences in gene complements and other forms of rearrangement and re-organization, could not have been fully appreciated in the absence of multiple complete genomes. Additional silent-expressed cassette systems (Parkhill et al., 2000), the location of repeated intergenic elements (Elkins et al., 1991; Goodman and Scocca, 1988; Liu et al., 2002), as well as the generation of variant proteins through alterations in coding tandem repeats (Jordan et al., 2003), have been dened in studies founded upon analysis and comparison of the genomes. Comparative study of these strains and species oers a perspective from which a new holistic understanding of these dynamic systems
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and their biology can be appreciated. In this review, we have highlighted some of the main areas that illustrate aspects of this exibility and the systems in which genomic variability and instability provide a basis for increased functional exibility and population tness.
Acknowledgments LAS is supported by a Wellcome Trust Project Grant. The authors would like to thank Dr Simon McGowan, of the Dunn School/WIMM Computational Biology Research Group, for the whole genome comparisons presented in Fig. 1, and Dr Charlene Kahler for a comparison of regulatory genes found in the sequenced genomes. Work in the Davies laboratory was supported by the Australian Bacterial Pathogenesis Program, funded by a Program Grant from the Australian National Health and Medical Research Council. The N. gonorrhoeae sequence was obtained from the University of Oklahoma, the Gonococcal Genome Sequencing Project which was supported by USPHS/NIH grant #AI-38399 (Accession number: AE004969). The N. meningitidis strain FAM18 and N. lactamica genome sequence data were produced by the Sanger Institute and can be obtained from ftp://ftp.sanger.ac.uk/pub/ pathogens/nm/ and ftp://ftp.sanger.ac.uk/pub/ pathogens/nm/.
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Plasmid 54 (2005) 191218 www.elsevier.

1. Introduction A genome sequence is a single time-point snapshot of a subculture, of a strain, of a species of

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

L.A.S. Snyder et al. / Plasmid 54 (2005) 191218

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