compartive overview of the genomic and genetic differences between the pathogenic neisseria strains...

8/13/2019 Compartive Overview of the Genomic and Genetic Differences Between the Pathogenic Neisseria Strains and Species

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Review

Comparative overview of the genomic and genetic differencesbetween the pathogenic Neisseria strains and species

Lori A.S. Snyder a, John K. Davies b, Catherine S. Ryan b, Nigel J. Saunders a,*

a Bacterial Pathogenesis and Functional Genomics Group, Sir William Dunn School of Pathology,

University of Oxford, South Parks Road, Oxford OX1 3RE, UKb Australian Bacterial Pathogenesis Research Program, Department of Microbiology, Monash University, Vic. 3800, Australia

Received 1 March 2005, revised 18 April 2005Available online 15 July 2005

Communicated by Julian I. Rood

Abstract

The availability of complete genome sequences from multiple pathogenic Neisseriastrains and species has enabled acomprehensive survey of the genomic and genetic differences occurring within these species. In this review, we describethe chromosomal rearrangements that have occurred, and the genomic islands and prophages that have been identified

in the various genomes. We also describe instances where specific genes are present or absent, other instances wherespecific 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 allowthe expression of particular genes to be switched ON or OFF. We have also described the presence and location ofmobile non-coding elements in the various genomes. Finally, we have reviewed the incidence and properties of variousextra-chromosomal elements found within these species. The overall impression is one of genomic variability and insta-bility, resulting in increased functional flexibility 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 snap-shot of a subculture, of a strain, of a species of

bacteria, and is inherently a singular example ofa complete bacterial system. However, once multi-ple sequences become available for comparativeanalysis, and once the genome sequence character-istics as a whole can be considered in the light ofexisting experimental data for a species, a muchdeeper and more informative picture can beperceived.

0147-619X/$ - see front matter 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.plasmid.2005.04.005

* Corresponding author. Fax: +44 1865 275515.E-mail addresses: [email protected]

(L.A.S. Snyder),[email protected](J.K. Davies),[email protected](N.J. Saunders).

Plasmid 54 (2005) 191218

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The pathogenicNeisseriaspecies:Neisseria men-ingitidis and Neisseria gonorrhoeae are importanthuman pathogens, being the principal causes of

bacterial meningitis and gonorrhoea, respectively.These species have been the subject of intensestudy over many years by a substantial researchcommunity, based upon their medical disease sig-nificance. There are therefore many informativestudies that can be drawn upon, tested against,and reconsidered in the light of the complete gen-ome sequences. In this context, these sequences inaddition to serving as research resources in theirown right, also provide an important frameworkfor understanding neisserial biology and for thedesign and construction of future experiments.

2. Large-scale genome characteristics

2.1. Neisserial sequences available

There are now four complete pathogenic Neis-seria spp. genome sequences publicly available.The first two sequences were essentially completedat the same time. The first of these to be publishedwas of N. meningitidis serogroup B strain MC58

(Tettelin et al., 2000), composed of 2,272,351 basepairs, with 2158 annotated coding sequences. Thesecond was ofN. meningitidis serogroup A strainZ2491 (Parkhill et al., 2000), with 2,184,406 basepairs and 2121 annotated coding sequences ofwhich 1968 are considered orthologous to thoseidentified in N. meningitidis strain MC58 (Tettelinet al., 2000). Additionally, the complete genomesofN. gonorrhoeaestrain FA1090 and N. meningit-idis serogroup C strain FAM18 have been se-quenced and are publicly available, but at the

time of writing they are not formally published,although the N. gonorrhoeae strain FA1090 se-quence and annotation is available Accessionnumber: AE004969. In addition, the genome se-quence of the non-pathogenic commensal N. lact-amica is nearing completion. Both publishedmeningococcal genomes have four rRNAs, numer-ous copies of insertion sequences, variably distrib-uted Correia Repeat Enclosed Elements (CREEs),and nearly 1900 copies of the neisserial uptake sig-nal 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 differences between the gen-omes of these two meningococci are the presence ofa major chromosomal inversion of 955 kb and a32 kb perfect tandem duplication in strain MC58(Tettelin et al., 2000). This duplication affects genesNMB1124 to NMB1159, which are duplicated inNMB1162 to NMB1197. The functional conse-quences of this duplication have yet to be investigat-ed experimentally, but it may generate gene doseeffects for the 33 genes that have been duplicated.

In addition to the genome sequences, there are1066 sequence entries in EMBL containing com-plete coding sequence information, totalling1,648,127 bases (as of 25th February 2005, accessedusing SRS, hosted by the Computational BiologyResearch Group, University of Oxford). This pro-vides a wealth of additional sequence information.

2.2. Strains with physical maps

Before whole genome sequencing, physical andmetabolic maps were constructed for several neis-serial genomes. Five neisserial maps were con-

structed, two for N. gonorrhoeae strains andthree forN. meningitdisstrains. Two of these wereof strains that have now been sequenced: N. men-ingitidis strain Z2491 (Dempsey et al., 1995) andN. gonorrhoeae strain FA1090 (Dempsey et al.,1991). The gonococcal map identified 11opa genes(Dempsey et al., 1991), which was confirmed bythe genome sequence. Additionally, maps weremade of N. meningitidis strain B1940 (Bautsch,1993; Gaher et al., 1996),N. meningitidisstrain 44/76 (Froholm et al., 2000), and N. gonorrhoeae strain

MS11-N198 (Bihlmaier et al., 1991). TheN. menin-gitidis strain B1940 identified four rRNA loci andfouropa genes (Gaher et al., 1996).N. gonorrhoeaestrain MS11-N198 has four rRNA loci and 11 opagenes, by its map (Bihlmaier et al., 1991).

2.3. Chromosomal rearrangements

The rearrangement of sections of the chromo-somes of different 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 physicalmaps of the chromosomes (Dempsey et al., 1995;Froholm et al., 2000), and in the course of other

studies (Gibbs and Meyer, 1996). There are noapparent chromosomal rearrangements when thephysical map of N. meningitidis strain B1940 iscompared to N. meningitidis strain Z2491 (Gaheret al., 1996; Parkhill et al., 2000), but there arerearrangements between N. meningitidis strainsMC58 and Z2491 (Parkhill et al., 2000; Tettelinet al., 2000). Chromosomal rearrangements wereidentified in the more recent study that mappedthe N. meningitidis strain 44/76 and compared itto simplified maps of 29 other meningococcalstrains, also from the ET-5 complex, suggestingchromosomal rearrangements have occurred inthe ET-5 complex strains during epidemic spread(Froholm et al., 2000).

Chromosomal rearrangements occur over a widesize range and include both linear transpositionsand inversions. Representative comparisons of thecomplete genomes are shown in Figs. 1A and B.The rates and functional consequences of thesesrearrangements have not yet been determined,and it may be that some changes seen in genomesequences are transient and may not persist over

time in the environment. However, on the basis ofthe currently available genome sequences, as illus-trated, there are changes that could be consideredto be related to genetic distance between strains,and the gonococcal and meningococcal sequencesdiffer significantly more than the meningococcalsequences do from each other.

3. Islands, MMEs, and prophage

3.1. Identified islands

Unlike many other bacterial species, no classi-cal Pathogenicity Islands with characteristic adja-cent tRNA loci, flanking direct repeats, foreignDNA signatures, and virulence genes (Blumet al., 1994; Hacker et al., 1990) have been foundin theNeisseriaspp. There are, however, a numberof large regions with foreign DNA characteristicsof divergent % GC and nucleotide signatures thatdiffer between strains MC58 and Z2491, which

have been called Islands of Horizontal Transfer(IHTs) (Tettelin et al., 2000).

Analysis of the genome sequence ofN. meningit-

idis strain MC58 found three IHTs (IHT-A, IHT-B,IHT-C) based upon dinucleotide signatures of theregions and sequence comparisons (Tettelin et al.,2000). IHT-A has two subregions, the first of whichcontains the genes responsible for biosynthesis ofthe serogroup B capsule, while the second containsan ABC transporter and a secreted protein. IHT-Bis comprised entirely of hypothetical genes. IHT-Ccontains three toxin/toxin related homologues aswell as genes that appear to be bacteriophage asso-ciated. The N. meningitidis strain Z2491 genomedoes not contain these islands, but does contain asingle divergent region of its own (Tettelin et al.,2000).

A large island has also been identified inN. gon-orrhoeae strain MS11, the Gonococcal Genetic Is-land (GGI), which is the largest and mostwidespread in this species identified to date (Gen-Bank Accession No.: AY803022) (Dillard andSeifert, 2001; Hamilton et al., 2005). The majority(80%) of gonococcal clinical isolates carry theGGI, which is apparently species-specific for N.gonorrhoeae (Dillard and Hamilton, 2002; Dillard

and Seifert, 2001). It contains 61 coding sequencesin 57 kb, including genes encoding a Type IVSecretion System capable of exporting DNA(Hamilton et al., 2005), which is believed to havea role in horizontal gene transfer, and either thetraGor sac-4 gene, which are alternative alleles in-volved in serum resistance (Dillard and Seifert,2001).

None of the large meningococcal islands or theGGI contain CREEs, commonly found aroundthe chromosome (Tettelin et al., 2000). There are

also relatively few neisserial uptake signal sequencespresent in these regions, in comparison with the restof the genome (Tettelin et al., 2000). The rareness ofthese general neisserial chromosomal markers isconsistent with these regions being horizontallyacquired from unrelated species backgrounds.

3.2. MMEs

Several instances of smaller strain-specificislands have been reported. In many cases, these

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fulfil the recently described criteria of MinimalMobile Elements (MME) (Saunders and Snyder,2002). MMEs are sites in which strain-specific

genes are located between flanking genes with con-served sequence and chromosomal organization,such that these flanking regions can serve as



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substrates for homologous recombination follow-ing natural transformation. The first of the regionsto be specifically characterized, between the pheS

andpheTgenes of theNeisseriaspp., contains ninedifferent genes or combinations of genes betweenthe conserved flanking sequences in the strain setused (Saunders and Snyder, 2002).

MMEs often include restriction-modificationsystem genes. For example, the Lineage III-associ-ated meningococcal restriction-modification sys-tem NmeSI is located between hrpA and ahypothetical protein gene in strain 800615. Menin-gococcal strain MC58, has a putative transcrip-tional regulator and two hypothetical proteingenes in this same location, and in meningococcalstrain Z2491, there is a different methylase, a puta-tive patch repair protein, and two hypotheticalgenes (Bart et al., 2001). The gonococcal restric-tion-modification system NgoMI is located be-tween trpEand purK, however, in meningococcalstrains FAM18 and Z2491 a hypothetical proteingene is present in this location instead (Zhuet al., 1999).

Although less commonly seen, virulence genesare also associated with MMEs. There are varia-tions in the presence of genes between glyA and

dedA, where the gene for the meningococcal outermembrane protein Opc is located in approximate-ly 70% of meningococcal strains (Seiler et al.,1996; Zhu et al., 1999, 2003). The meningococcal

capsule biosynthesis and transport genes appearto have inserted between tex and galE, whichare also present in gonococci. The genes in this

region, including subsequently the capsule genesthemselves, then have the potential to serve as re-gions of homology for recombination and ex-change of capsule genes between meningococci(Claus et al., 2002; Dolan-Livengood et al.,2003; Petering et al., 1996). Fig. 2shows this re-gion in the four sequenced neisserial genomes,and other strains for which the region has beensequenced.

Additionally, the gene for the iron-acquisitionprotein HmbR appears to be within an exchange-able genetic locus, wherehmbR,exl3, orexl3A andexl2 are mutually exclusive alternatives (Kahleret al., 2001). In addition, at least seven differentregions, that could now be considered as MMEs,containing pathogen-specific genes were identifiedby subtractive hybridization and comparativegenome hybridization to membrane microarrays(Klee et al., 2000; Perrin et al., 2002), althoughwhether these genes play a direct role in pathogen-esis has yet to be determined.

3.3. Prophages

Prior to the completion of the genome sequenc-es, it was generally considered that although neis-serial strains possessed bacteriocins (Allunans

Fig. 1. Comparisons of the sequenced Neisseria spp. genomes. Chromosomal rearrangements and inversions were identified usingMuMmer v3.05 to align the sequenced genomes and mummerplot was used to generate dot plots comparing one neisserial genome, onthe x-axis, to another, plotted on the y-axis (A). Before comparison, all neisserial genomes were edited to start at dnaA and theorientation of theN. meningitidisstrain MC58 sequence was reversed with respect to the published sequence so that it was in the sameorientation as the other three genomes. The genome of N. meningitidis strain MC58 (on the x-axis; panels 1, 2, and 4) was thencompared to each of the other three neisserial genomes (on the y-axis) and the N. meningitidisstrain Z2491 genome (x-axis; panel 3)

was compared to the N. meningitidisstrain FAM18 genome (y-axis). Where the genome sequences are the same a coloured dot or lineappears, 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 arejoined by a single line, running from middle of each segment to middle of the corresponding segment. The orientation of a segment inthe comparison strain (y-axis from the dot plot) relative to its reference strain is indicated by arrowheads. Upward pointing arrowheadsindicate inversions relative to the reference strain. We can see, therefore, that between N. meningitidisstrains MC58 and Z2491, there isone large inversion (grey-blue to orange segments), within which is a smaller inversion and rearrangement (purple segment moveslocation and inverts relative to the surrounding sequence) and there is one smaller inversion (green segment). There are manyinversions and chromosomal rearrangements between N. meningitidis strain MC58 (and therefore also the other meningococci) andN. gonorrhoeaestrain FA1090. Large regions of strain-specific genes, to which there is no correspondence in the genome to which it isbeing compared, are represented by gaps in the figure.

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et al., 1998; Allunans and Bovre, 1996), they didnot have bacteriophage. This is contrary to reportsfrom the 1950s and 1960s, in which bacteriophage

against Neisseria spp. were identified, althoughsusceptibility and lysis appeared to be dependenton media, strain, and passage histories (Cary andHunter, 1967; Stone et al., 1956). Given these earlyresults, the ubiquity of bacteriophage, and thecommon LPS targets that many can use, it wasnot surprising that both of the published genomesequences contain strain-divergent bacteriophage-derived sequences (Parkhill et al., 2000; Tettelinet al., 2000). Additional analyses of such sequenceshave been made following these observations.N. meningitidis strain FAM18, strains of theET-37 complex, and strains of the A4 cluster con-tain a large prophage with some similarity tolambda (Claus et al., 2001). Three loci with Mu-like prophages can be found in the N. meningitidisstrain Z2491 genome, although only one is poten-tially intact (Morgan et al., 2002). A region similarto the Mu-like prophages of strain Z2491 can befound in N. meningitidis strain MC58 (Masignaniet al., 2001; Morgan et al., 2002). In the case ofstrain MC58, the region contains 46 codingsequences, 29 with homology to prophage genes,

and appears to have been inserted into an ABCtransporter-encoding gene (Masignani et al.,2001). Several regions containing bacteriophage-derived sequence are also evident in the genome se-

quence ofN. gonorrhoeae strain FA1090. To date,there has been no demonstration of a functionallyintact bacteriophage from these species. However,

there is evidence that some of the associated genesare expressed and present on the cell surface(Masignani et al., 2001).

4. Differences in the presence of specific genes

Prior to the availability of complete genomesequences, a significant amount was alreadyknown about some gene differences between theNeisseria species and strains. Because of the pre-dominant focus of research upon disease, thesestudies largely focused upon virulencedeterminants.

4.1. opc

The outer membrane protein Opc is involved inadhesion and invasion of host cells (Virji et al.,1992) through binding to host proteoglycan recep-tors (de Vries et al., 1998) and is encoded by thegene opc. This gene has only been identified instrains 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 genomeaverage, which is consistent with its having beenhorizontally acquired after the separation of the

Fig. 2. Comparison of the galE-tex MME region in strains of N. meningitidis and N. gonorrhoeae. The capsule biosynthesis andtransport genes of encapsulated N. meningitidis strains are located between galEand tex, as illustrated by strains MC58, Z2491,FAM18,a-707, M7575, and LCDC 78189. Complete contiguous sequence of this region fromgalEto tex is not available for strainsa-707, M7575, and LCDC 78189, however, the sequence from galEto the internally conservedctrAcapsule transport gene is availablefrom EMBL (indicated by diagonal bars). In N. gonorrhoeae strains FA1090 and MS11-E1 and in unencapsulated N. meningitidisstrain a-14, galEand tex are adjacent and the intergenic sequences in these strains are nearly identical. This arrangement of genes

suggests thatgalEandtex are the conserved flanking genes of this MME, which then became the location for the horizontal transfer ofthese capsule genes. Capsular variation in serogroup would then appear to be due to additional changes in the locus, where the flankinghomologous sequences are now galEand ctrA (as illustrated by the differences in Z2491, a-707, M7575, and LCDC78189 relative toMC58 and FAM18) or NMB0065 and siaC(as illustrated by the differences between MC58 and FAM18). Although it cannot bedetermined at this time the order in which these processes lead to the evolution of the various meningococcal capsular serogroups, it isevident that the acquisition of these capsule genes in the galE-tex locus is either not the defining speciation event between N.meningitidisand N. gonorrhoeaeor that further horizontal exchange between the species has occurred post-speciation, leading to theloss of the capsule genes in this locus in meningococcal strains such as a-14. It is interesting to note, however, that the inverse has neverbeen reported, to whit the acquisition of these capsule genes in the gonococcus through homologous recombination with the conservedflanking genesgalEandtex. Gene locus identifiers are included for annotated complete genome sequences. Gene sizes are indicated andrelative sizes of the genes are to scale, although the intergenic regions are not to scale. The length of the intergenic regions between galEand tex or, when not available, between galEand ctrA are indicated. Accession numbers are indicated for sequences obtained fromEMBL.

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pathogenic species (Seiler et al., 1996).N. meningit-idis strains of the ST-11/ET-37 complex and theST-8/A4 cluster do not have the opc gene (Seiler

et al., 1996). A gene with limited homology hasbeen identified in N. gonorrhoeae strain FA1090,that has 59% identity to the meningococcal strainZ2491 opc (Zhu et al., 1999). However, this gono-coccal gene is poorly expressed (Zhu et al., 1999)and the encoded protein should not be assumedto be the functional orthologue of the meningococ-cal protein. The locus containing opc in the men-ingococci is diverse with respect to both thegenes and organizations present between glyAand dedA in different strains, suggestive of anMME-like element. In all, five variants of this re-gion have been described: (1) containing twoIS1106 elements 50 of opc (as in N. meningitidisstrain Z2491); (2) containing a single IS1106 50

ofopc (as inN. meningitidisstrain MC58); (3) con-taining orfX, orfY, and the coding sequence with59% identity to opc (as in N. gonorrhoeae strainFA1090); (4) similar to that in strain FA1090,but with 699 bp, encompassing orfY, deleted (asin N. polysaccharea strain 89357); (5) no codingsequences in the intergenic region between glyAand dedA (as in N. meningitidis strain FAM18)

(Zhu et al., 2003).

4.2. Restriction-modification systems

Many restriction-modification systems havebeen identified in the Neisseriaspp. and their pres-ence within the genomes is frequently strain-specific (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 sys-tem, NgoPII has significant sequence homologyto the MthT1 system from Methanobacteriumthermoformicium, suggesting that there has beentransfer between Archea and Bacteria at somepoint in evolutionary history, although not neces-sarily directly between Archea and Neisseria. Thehomology does not extend beyond the codingregions, the genes relying on the incorporationinto the genome in an area which places them

under promoter control appropriate for theorganism, rather than importing a system thatmay not function in the new genetic background

(Nolling and de Vos, 1992).

4.3. drg

One particular difference in DNA methylationsystems between strains was highlighted becauseit was suggested that it was associated with viru-lence associated phase variation rate differencesbetween strains (Bucci et al., 1999). The geneencoding the Dam methylase is replaced in somestrains by one encoding a restriction enzyme thatcleaves the methylated Dam site. This has beencalled the Dam replacement gene, drg. Originally,the loss of Dam was reported to be associatedwith invasive strains and a mutator phenotype.Immediately following this publication, chromo-somal digests were conducted on a collection ofinvasive strains with DpnII and Sau3A, whichare, respectively, blocked by and insensitive toDam methylation. The association between inva-sive disease isolates and the loss of Dam methyl-ation was not reproduced in our collection(Saunders, unpublished), and the original report

probably represented some sampling bias withinthe collection of strains used. A more extensiveand rigorous assessment including analysis ofthe presence and absence of the dam and drggenes was recently reported (Jolley et al., 2004).Subsequently, it has been shown that there is noassociation between either invasive disease iso-lates or with mutator phenotypes with respectto the drg or dam status of strains (Martinet al., 2004; Richardson and Stojiljkovic, 2001).This story attests to the importance of perform-

ing studies using properly representative straincollections, and also raises interesting questionsabout the nature of error correction in thisspecies and its insensitivity to the presence or ab-sence of Dam methylation. While a proportion ofmutator phenotypes have now been ascribed todeficiencies in mutS, mutL, and dinB (Martin etal., 2004; Richardson and Stojiljkovic, 2001;Richardson et al., 2002), the genetic basis forthese interesting phenotypes has not been com-pletely resolved.



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4.4. Iron-acquisition genes

The ability to acquire iron has often been con-

sidered to be a virulence determinant, although itis more correctly understood as a necessary meansof survival within the host, where free iron isscarce and is no more essential to a pathogen thanto a commensal. There are several bound forms ofiron available including that associated with lacto-ferrin, transferrin, heme, haemoglobin and haemo-globinhaptoglobin (AlaAldeen et al., 1993;Biswas and Sparling, 1995; Chen et al., 1998; Rich-ardson and Stojiljkovic, 1999). The Neisseria spp.have several iron-acquisition systems to utilizethese, but the genes for each system are not univer-sally present in all strains. Human transferrin, aniron-binding glycoprotein, can be bound by atransferrin receptor present on the surface ofN. meningitidis and N. polysaccharea (AlaAldeenet al., 1993). The gene encoding the transferrinbinding protein, tbpA, is itself divergent in hyper-variable regions of the coding sequence, whichare presumed to relate to exposed surface epitopes(Cornelissen et al., 2000; Legrain et al., 1998).There is minimal diversity, however, in the genefor the major iron-binding protein Fbp in the

pathogenicNeisseriaspp., although more diversityis seen in the commensal Neisseria spp. (Gencoet al., 1994). There are also two receptors forhaemoglobin, HmbR and HpuA, which are bothvariable in their presence between strains as wellas in their expression through phase variation(Chen et al., 1998; Lewis et al., 1999; Richardsonand Stojiljkovic, 1999).

This variation in the repertoire, divergent re-gions, and phase variable expression of iron-acqui-sition genes probably reflects diversifying

immunological selection. The repertoire of poten-tial iron donors is conserved from host-to-hostso, superficially, there would appear to be noadvantage to lacking any particular system, anda functional gain for the presence of each. Eachprotein has to be surface located to bind to itsiron-carrying substrate, as such it has to be ex-posed to the immune system. As relatively con-served targets, these proteins are likely togenerate not only antibodies that can lead to theeradication of a colonizing strain, but also anti-

bodies providing cross-immunity to unrelatedstrains. A probably similar process has been recog-nized inSalmonella entericawhere the flagellae are

frequently antigenically conserved, stimulatingcross-immunity to other flagellate strains, in whichphase variable expression of this antigen facilitatesre-colonization through evasion of cross-immunity(Norris and Baumler, 1999). The population vari-ations in the repertoire of these genes thereforepossibly represent a selective advantage by facili-tating the re-colonization of a host previously col-onized by, and immune to, other strains.

4.5. LPS genes

The lipopolysaccharide (LPS) of the Neisseriaspp. is also called lipo-oligosaccharide (LOS) dueto the absence of O-antigen in its structure. Theneisserial LPS is structurally diverse, possessingvariable oligosaccharide chains (Zhu et al., 2002).The genes encoding the LPS biosynthetic enzymesare variably present between different strains andspecies, particularly in N. meningitidis, and someof those that are present are phase variable in somestrains (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 andits relationship with LPS structure in the commen-salNeisseriaspp. (Arking et al., 2001). As a result,the LPS on the surface of the bacteria is heteroge-neous (Zhu et al., 2002) within and between bothneisserial strains and species.

The LPS biosynthesis genes, called lgt, are pres-ent in three loci:lgt-1,lgt-2, andlgt-3. InN. gonor-rhoeae, the lgt-1 locus contains lgtA, lgtB, lgtC,lgtD, lgtE, lgtH, and lgtZ, although the functionof the latter two genes is not yet known. The

lgt-2 locus contains lgtFand rfaK, while the lgt-3locus contains only the lgtG gene (Zhu et al.,2002). Homopolymeric tracts mediating phasevariable expression have been found in four ofthese genes in some strains: lgtA, lgtC, lgtD, andlgtG (Zhu et al., 2002). This allows an individualgonococcal strain to synthesize a variety of LPSstructures (Shafer et al., 2002).

Meningococcal strains have a smaller repertoireof genes in their lgt-1 locus, most strains havingonly three genes, usually lgtA, lgtB, and lgtE.



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However, unlike within N. gonorrhoeae, there arevariations in the meningococcal complement oflgt genes. N. meningitidis strain 126E has a large

deletion in thelgt-1 locus that has inactivated lgtAand lgtB, leaving lgtC and lgtE; N. meningitidisstrain 7880 has only lgtE; and N. meningitidisstrain M978 has lgtA, lgtB, lgtC, and lgtE (Jen-nings et al., 1999). Variability in the presence ofgenes in the other two loci, lgt-2 and lgt-3, is lesscommon (Zhu et al., 2002). The reasons for thisdiversity may be similar to those outlined foriron-acquisition genes.

In addition to the differences in complements ofLPS biosynthesis genes in strains ofN. meningiti-dis, which of these genes is capable of phase vari-able expression differs between strains. Ingeneral, meningococcal strains can phase varythe expression of either a-chain extensions,through lgtAand lgtC, and/or b-chain extensions,through lgtG. The phase variable expression oflgtA and lgtC can significantly affect the abilityofN. gonorrhoeaeto resist the bacteriolytic actionof normal human serum, with bloodstream isolatesbeing lgtAphase OFF (Shafer et al., 2002). Phasevariation of lgtA allows the Neisseria to vary be-tween two possible terminal structures from the

first heptose of the LPS (Jennings et al., 1999).The alternative structures generated through thephase variation of lgtA also relate to whether theLPS can be sialylated, but the ability to phase varysialylation is not necessary for meningococcalinvasion, as lgtA is not phase variable in someN. meningitidis strains (Berrington et al., 2002).Additionally, lgtD, encoding another glycosyl-transferase, contains a homopolymeric tract in-volved in the phase variation of the associatedLPS structure (Zhu et al., 2002).

4.6. Capsule genes

Among the pathogenicNeisseriaspp., the genesfor the biosynthesis of capsular polysaccharidesare found only in N. meningitidis and this is oneof the differentiating characteristics between thetwo pathogenic species. There are at least 13 recog-nized capsular polysaccharides that can be gener-ated by N. meningitidis strains, of which five arepredominantly associated with invasive disease:

A, B, C, W-135, and Y (Claus et al., 2002). Thecapsule is believed to be capable of protectingthe meningococcus against phagocytosis (Read

et al., 1996), and preventing desiccation duringtransmission (Virji, 1996). Strains colonizing thenasopharynx often express reduced amounts ofcapsule compared to invasive disease isolates fromthe blood (Mackinnon et al., 1993) and are fre-quently acapsulate due to the phase variableexpression of capsule biosynthesis genes (Ham-merschmidt et al., 1996a,b; Lavitola et al., 1999)or the absence of intact capsule production sys-tems (Claus et al., 2002; Dolan-Livengood et al.,2003).

The capsule synthesis gene cluster is composedof five major regions: (1) region A contains theserogroup specific polysaccharide synthesis genes;(2) region B contains the lipid modification genes;(3) region C contains the ctr polysaccharide trans-port genes; (4) region D and the truncated regionD0 contain genes for LPS synthesis; and (5) regionE, which contains genes of unknown function(Claus et al., 2002; Petering et al., 1996). In regionA, changes in the sequence of the siaDgene deter-mines the differences between the capsules of sero-group B, C, Y, and W-135, while the entire region

contains different genes in serogroup A (Clauset al., 2002). Allelic exchange can occur betweenmeningococci of different serogroups due to thesimilarity between the capsular genes, particularlythe siaD of serogroups B and C, resulting in cap-sule switching during outbreaks (Swartley et al.,1997). In N. meningitidis strains, the presence ofdifferent genes varies, the expression of the genespresent can phase vary, and the order of the genesin the capsule synthesis gene cluster can differ aswell. In those strains investigated, the genes are

either in a B-D0

-E-C-A-D configuration or in aB-D-A-C-E-D0 configuration (Claus et al., 2002).Regions D and E are present in N. gonorrhoeaeand N. lactamica, containing the tex and galEgenes that flank the capsule synthesis (region A)and transport gene (region C) regions in meningo-cocci (Fig. 2). It is therefore believed, supported bysignatures found associated with horizontally ac-quired genes, that the encapsulation of the men-ingococci is the result of horizontal DNAtransfer 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 syn-

thesis gene cluster, the ctrA gene in region C,encoding the outer membrane transporter of cap-sular polysaccharide, is conserved in most menin-gococcal clinical isolates and is not present inother neisserial species (Claus et al., 2002; Froschet al., 1992; Sadler et al., 2003).

4.7. Other genes showing gene-complement

differences

Other genes have been identified which are notuniversally present in the Neisseria spp. TheN. meningitidis gene gpxA is not present in theN. gonorrhoeae or commensal Neisseria spp.strains that have been investigated (Moore andSparling, 1995). The RTX family genes frpA andfrpC appear to be limited to the meningococcalspecies (Thompson et al., 1993). While the patho-genic Neisseria spp. can decorate their pili withphosphorylcholine, the ability to decorate neisseri-al LPS is possessed only by the commensal strains,which have a homologue of the Haemophilus influ-enzae licA gene (Serino and Virji, 2000). The dcw-

cluster associated gene dca, on the other hand, ispresent only in the pathogenic neisserial strains(Perrin et al., 1999; Snyder et al., 2001a), is associ-ated with an the presence of phosphorylcholine onthe pili (Warren and Jennings, 2003), and inactiva-tion of this gene has different consequences fortransformability in N. meningitidis and N. gonor-rhoeae (Snyder et al., 2001a). The gene for IgA1protease is also present only in the pathogenicNeisseria spp. (Koomey and Falkow, 1984; Perrinet al., 2002). It is likely that with advances in com-

parative genome microarray hybridization, orgenomotyping, and MME analyses, many moresuch strain- and species-specific neisserial geneswill be identified.

5. Degenerate genes

A noticeable feature of all the completed neisse-rial genome sequences is the presence of numerousgenes that contain frame-shifts, deletions, and

mutations affecting coding potential, as well assome instances of insertional inactivation.

5.1. porA

The outer membrane protein PorA, a porin(Tommassen et al., 1990), is only functionally pres-ent in N. meningitidis. A porA gene is present inN. gonorrhoeae, but the gonococcalporA containsinactivating mutations in both the promoterregion and the coding region of the gene (Feaversand Maiden, 1998). The porA gene appears to beabsent from the commensal Neisseria spp. (Fea-vers and Maiden, 1998), or at least is only presentin some strains of some commensal species (Wolffand Stern, 1995).

5.2. Restriction-modification systems

As with porA, the function of restriction-modi-fication systems cannot be simply assumed to beequivalent on the basis of gene presence and ab-sence alone, as other changes may also havefunctional consequences. In the case of the N. gon-orrhoeae strain FA1090 Hsd system NgoAV, thegenes hsdMand hsdRare what would be expected

for a typical Type I restriction-modification sys-tem. The hsdSgene, however, has been tandemlyduplicated, with the first copy, hsdS1 being func-tional in site recognition for the restriction-modifi-cation system and the second, hsdS2 having noapparent function. While functional for site recog-nition, the gonococcal HsdS1 is also truncated andrecognizes a palindromic sequence with its singleremaining DNA-binding site, rather than thenon-palindromic sequences typical of Type Irestriction-modification systems where the site rec-

ognition protein has two DNA-binding domains(Piekarowicz et al., 2001). While present in N.meningitidis strains MC58 and Z2491, the genesfor this system are divergent. The strain MC58hsdS1 is non-functional due to a point mutation,while that of strain Z2491 is non-functional dueto different mutations, as well as having a deletionin hsdR. All three neisserial strains also displaydivergence in the organization of this locus (Pie-karowicz et al., 2001), therefore, even when thesystems genes are present, there is a large degree



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of variability in the neisserial restriction-modifica-tion systems.

5.3. Iron-acquisition genes

Neisseria gonorrhoeaeand N. meningitidis bothhave a lactoferrin receptor, encoded by lbpA, thatis capable of scavenging iron from human lactofer-rin. Some clinical isolates ofN. gonorrhoeae havebeen identified that have mutations in or nearlbpAand do not express the lactoferrin receptor, there-fore acquiring iron from lactoferrin is not essential(Biswas and Sparling, 1995).

5.4. Regulatory genes

In comparing the annotated neisserial genomesequences, there are at least nine situations wherea particular regulatory gene has been inactivated,in one or more genomes (Davies, unpublished).Perhaps the best example of pseudogene genera-tion concerns several regulatory genes that mayhave once had a role in the regulation of the pilEgene, which encodes pilin. Upstream of pilE inbothN. gonorrhoeae and N. meningitidis is a con-sensus sequence for a r54 promoter (Fyfe et al.,

1995). For function, these promoters require thealternative sigma factor RpoN, and an activatorprotein that binds upstream of the promoter. Con-sensus sequences for r54 promoters have beenfound upstream of other gonococcal genes includ-ing pilC (Taha et al., 1996) , comA (Facius andMeyer, 1993), and pip (Albertson and Koomey,1993). In addition, the finding of a gonococcalprotein which co-purifies with RNA polymeraseand reacts with a monoclonal antibody raisedagainst the Salmonella typhimurium RpoN protein

(Klimpel et al., 1989), resulted in attempts to iden-tify rpoNand the gene encoding the relevant acti-vator in N. gonorrhoeae. However, the pilE r54

promoter has since been shown to be non-func-tional inN. gonorrhoeae(Fyfe et al., 1995), despitethe fact that is functional in a P. aeruginosaback-ground (Carrick et al., 1997). Subsequently, rem-nants of an rpoN gene have been identified inneisserial species and strains (Laskos et al.,1998). Regulators required for activation of geneexpression via r54 promoters are often members

of two-component regulatory systems (Kofoidand Parkinson, 1988). Remnants of such a systemthat may have once controlled piliation have also

been identified (Carrick et al., 2000).Standard methods such as Southern hybridiza-tion, and complementation assays inrpoNmutantsof E. coli and P. aeruginosa rpoN mutants hadfailed to identify a rpoN gene in N. gonorrhoeaeMS11A (Laskos et al., 1998). Subsequent sequenceanalysis of the gonococcal FA1090 genome se-quence indicated a region of sequence that didnot constitute a CDS, yet encoded motifs charac-teristic of RpoN proteins including the RpoNbox unique to RpoN proteins (Laskos et al.,1998). The region of sequence was denoted RLSfor r poN-like sequence. Based on a comparisonof the amino acid sequences ofRLSand theE. coliRpoN protein it appears a deletion of 414 bp,spanning the region encoding the DNA-bindingmotifs, has occurred in the gonococcus. Thisresulted in a frameshift mutation and renderedthe gonococcal gene incapable of encoding a func-tional RpoN protein that could bind DNA (Las-kos et al., 1998). Other N. gonorrhoeae strainsandN. meningitidisandN. subflavastrains containRLS [(Laskos et al., 1998); Laskos and Davies,

unpublished]. In all cases, the same 414 bp deletionwas present. The N. lactamica strain tested ap-peared to have an intact rpoNgene, but it was un-able to complement a P. aeruginosa rpoN mutant(Laskos and Davies, unpublished).

It was also possible to identify remnants of atwo-component regulatory system in N. gonor-rhoeaeMS11A that shares sequence similarity withthe pilSand pilR genes ofP. aeruginosa (Carricket al., 2000). A single CDS denotedrsp for regula-tor/sensor protein was identified (Carrick et al.,

2000), that potentially encodes a protein that issimilar at its amino terminus to the N terminusof histidine kinase sensor proteins. It is predictedto be a transmembrane protein and has the con-served histidine residue that is the site of autophos-phorylation in the transmitter domain of thesesensor proteins (Fig. 3). At its carboxy terminusit is similar to the equivalent region of regulatoryproteins in that it encodes a DNA binding motifwhich is characteristic of the output domain ofthese proteins, but it lacks a complete ATP-bind-



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ing site (Fig. 3). Analysis of the gonococcal strainFA1090 genome sequence showsrspis also presentin this strain.

Analyses of the sequenced meningococcal gen-omes reveal two CDSs that could encode portionsof the equivalent sensor and response regulatorproteins, these being NMB1606/1607 andNMA1803/1805, respectively (Fig. 3). NMB1606and NMA1803 putatively encode transmembranebound proteins containing the conserved histidineresidue found in sensor proteins, and appear to be

fused with a CDS encoding a product of unknownfunction. NMB1607 and NMA1805 appear tocontain an internal deletion in that they encodeconserved portions of regulatory proteins normal-ly found at both the amino- and carboxy-terminus,without motifs normally found in between (Fig. 3).

Partial sequencing of the equivalent region ofthe genome of the commensal N. subflava has re-vealed a different arrangement (Ryan and Davies,unpublished). Sequence similarity to PilS can befound in two different reading frames. These CDSs

Fig. 3. Schematic diagram of the predicted structure of theP. aeruginosaPilS and PilR proteins and neisserial proteins including Rsp(Carrick et al., 2000) that share similarity with these P. aeruginosaproteins. Conserved domains are indicated. These are the histidineresidue (H), the cytoplasmic transmitter domain (F) and one or two nucleotide binding motifs (G1/G2) in histidine kinase sensors andan aspartate residue (D), Walker Boxes A and B (WBA/WBB) of the ATP-binding site and DNA-binding motif (HTH) in responseregulator proteins. Ng,N. gonorrhoeae; Nl,N. lactamica; NMB,N. meningitidisstrain MC58; NMA,N. meningitidisstrain Z2491; Ns,N. subflava. The pale blue rectangle represents the inner membrane. The dark blue line represents sequence similar to the equivalentportion of PilS, including the transmembrane domains. The red and orange lines represent sequences similar to the equivalent portionsof PilR and sensor proteins in general, respectively. The sequence represented by the purple line is not similar to any sequence in thedatabases, and the green dashed line represents that portion of the Ns locus for which there is no sequence information. The frameshiftin the Ns sequence is indicated. Figure is not to scale.



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both potentially encode a product sharing similar-ity to the C terminus of histidine kinase sensors,one to the region encoding the conserved histidine

residue that is very similar to the sequence foundin N. meningitidis, and the other to part of thenucleotide binding motif (Fig. 3). A separateCDS, where the derived amino acid sequence issimilar to response regulatory proteins, appearsto be identical to NMB1607 and NMA1805from N. meningitidis (Fig. 3). Analysis of thenearly completed N. lactamica genome sequencesuggests a single CDS identical to that present inN. gonorrhoeae.

These findings suggest that functional genes,equivalent to those found in P. aeruginosa, oncecontrolled transcription of the pilE gene in anancestor of the present Neisseria species. Giventhe wide but not universal occurrence of RLS,the inactivation of the rpoNgene appears to havepreceded the evolution of the present species.The subsequent inactivation of the pilSand pilRgenes appears to be a more recent event, that hasfollowed different routes in different species.

6. Variations in the versions of genes present

6.1. porB

There are several known variations of the porBgene, encoding a porin capable of translocatinginto host cell mitochondrial membranes that hasbeen found to both cause (Muller et al., 2000,1999) and prevent apoptosis of host cells (Massariet al., 2000, 2003). These opposing observationsmay be due to differences in the host cells, bacterialstrains, protein purification methods, growth med-

ia, stages of infection, and/or variations in theporBsequence (Binnicker et al., 2004). InN. gonor-rhoeaethere are two mutually exclusive PorB pro-teins present, called PIA and PIB (Carbonettiet al., 1988), the genes for which have been re-named 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 Maid-en, 1998). Because they are surface exposed anti-gens, the genes themselves can be highlydivergent, but cluster into these four classes. Due

to horizontal exchange between the species, a N.meningitidis isolate has been identified that pos-sesses a gonococcal PIB, encoded byporB1b (Vaz-

quez et al., 1995). Rarely, strains ofN. gonorrhoeaehave been identified that react to both PIA andPIB antibodies, which is presumed to be due tomosaicism within the porB allele (De La Fuenteand Vazquez, 1991; Gill et al., 1994).

6.2. Class I or class II pili

The expression of neisserial type IV pili is re-quired for attachment to host cells (Swanson,1973), high frequency transformation (Biswaset al., 1977), and twitching motility (Wolfganget al., 1998). Strains ofN. meningitidiscan expresseither class I or class II pili. Class I pili are pos-sessed by bothN. meningitidisandN. gonorrhoeae,with the major pilin subunit being encoded bypilE, which undergoes antigenic variation throughrecombination with silent cassettes (Segal et al.,1985). The subunits of class II pili are slightlysmaller and are encoded by a different gene thatapparently has no silent cassette counterparts.N. meningitidis strain FAM18 and N. lactamicahave class II pili (Aho and Cannon, 1988; Aho

et al., 1997, 2000).

6.3. pilC

Two copies of the pilCgene are present in dis-tant locations on the pathogenic neisserial gen-omes (Parkhill et al., 2000; Tettelin et al., 2000).The expression of both copies is dependent onphase variation, and expression of at least one ver-sion ofpilCis required for piliation of the bacterialcell for both class I and class II neisserial pili

(Jonsson et al., 1995; Ryll et al., 1997). In N. gon-orrhoeae, expression of pilC1 or pilC2 results insimilar amounts of pili and the two PilC proteinsbind equally to target host cells. In the N. menin-gitidis strains investigated to date, however, onlyPilC1 mediates binding to host cells (Jonssonet al., 1995). N. meningitidis strains MC58 andZ2491, have equivalent pilC genes. However, thesequences of both pilCgenes from N. gonorrhoeaestrain MS11 (Rudel et al., 1995) differ from thosein gonococcal strain FA1090, and there are often



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substantial differences between genes annotated aspilC1 and pilC2 between strains of both species.These differences in the pilCgenes make the rou-

tine annotation ofpilCgenes as simply pilC1 andpilC2 not only misleading with regard to their se-quence homology, but also potentially with respectto their function.

7. Mosaic genes

7.1. opa

The pathogenic Neisseria spp. have multiplecopies of the opa gene; three or four copies inN. meningitidis, which encode what were originallycalled the class 5 proteins, and up to 11 copies inN. gonorrhoeae, which encode what were calledthe P.II proteins. These proteins are abundant out-er membrane proteins that target certain host cellCEA family receptors and syndecan proteoglycanfamily receptors (Chen et al., 1997, 1995; Poppet al., 1999; van Putten and Paul, 1995; Virjiet al., 1996a,b). Probably due to their prevalenceon the outer membrane and exposure to the im-mune system, the expression of these proteins is

both phase and antigenically variable. Antigenicdiversity is generated through recombination be-tween opa genes, which swap and re-assort be-tween two hypervariable and one semivariableregions, generating a family of mosaic opa genes(Aho et al., 1991). In a collection of meningococcifrom West Africa, seven electrophoretically dis-tinct Opa proteins were found, although each iso-late contains only three opa genes; the differencesin the Opas are generated by recombination fol-lowing transformation from other strains, generat-

ing mosaics (Hobbs et al., 1994). Likewise, in acollection of N. meningitidis strains of the ET-37complex, 26 different Opa proteins have been iden-tified, encoded within four genes per genome(Hobbs et al., 1998). Two opa genes have beenfound in N. lactamica and N. flava and one inN. sicca, N. mucosa, and N. subflava (Wolff andStern, 1995). When the predicted sequences of 45Opa proteins from N. meningitidis, N. gonor-rhoeae, N. sicca, and N. flava are aligned, thedivergence of the semivariable and hypervariable

regions (Bhat et al., 1992) are apparent, and wereshown to relate to the three exposed loops of theprotein proposed by the model of its structure

(Malorny et al., 1998).

7.2. penA

Penicillin-binding protein 2 (PBP2) of the Neis-seria spp. is a peptidoglycan transglycosylase andtranspeptidase (Spratt and Cromie, 1988) and itsgene, penA, is a component of the neisserial dcwcluster (Francis et al., 2000; Snyder et al., 2001a).Alternations in the affinity of PBP2 for penicillinleads to chromosomally encoded penicillin-resis-tance, which is synergistically augmented by chro-mosomal mutations in penB and the mtr effluxpump system (Faruki and Sparling, 1986). It wasdiscovered that the acquisition of lower affinityPBP2 by the pathogenic Neisseria spp. was dueto the horizontal transfer of blocks ofN. flavescenspenADNA, which homologously recombined withthe nativepenAto generate lower penicillin affinitymutants. WhenpenAis examined from sevenNeis-seriaspp. a complex mosaic structure generated byhorizontal DNA transfer between commensal andpathogenic strains is apparent in this essential gene

(Spratt et al., 1989, 1992).

7.3. pilE

Mosaicism is also found within the pilE geneencoding the major pilin subunit (Hagblomet al., 1985; Hill, 1996; Seifert et al., 1988). In thiscase one, or in some strains two, expression cas-settes undergo recombination with pilS sequences(Gibbs et al., 1989; Haas et al., 1992; Howell-Adams and Seifert, 1999, 2000; Koomey et al.,

1987; Meyer et al., 1984; Parkhill et al., 2000; Segalet al., 1985; Seifert et al., 1988; Swanson et al.,1987, 1990; Tettelin et al., 2000). These aresequences with homology with most of the proteinencoding region of the expressed pilE gene, butwhich have no promoter or the 50 terminal portionof the pilin subunit and are therefore not capableof expression (Haas and Meyer, 1986; Haaset al., 1992; Hagblom et al., 1985; Meyer et al.,1984). Eachpilgene has six variable regions wherepolymorphisms are primarily located, which have



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been termed minicassettes (Haas and Meyer, 1986;Haas et al., 1992). Because the location of the endsof the regions that are exchanged during the

non-reciprocal recombination vary, this systemprovides a vast potential for generating diversitywithin these genes and each pilE gene in eachstrain can effectively be considered to be unique.

7.4. mafB and fhaB

A similar expression cassette and silent cassettearrangement to that of the pilE and pilS systemmay exist formafBandfhaBand their apparent si-lent cassettes. A lipoprotein is proposed to beencoded bymafAand a predicted secreted proteinbymafB, which together are thought to have adhe-sin activity in N. gonorrhoeae (Parkhill et al.,2000). The region 3 0 of the mafBgene containsopen reading frames lacking initiation codons,but with homology to mafB, suggesting that thesereading frames are silent cassettes to mafBas thepilS cassettes are to pilE. The same may also betrue for fhaB, which has homology to the filamen-tous haemaglutinin precursor gene fromBordetellapertussis, but was annotated as a hypothetical pro-tein in N. meningitidis strain Z2491 and a haema-

glutinin/haemolysin-related protein in N.meningitidisstrain MC58. There are also apparent-ly silent cassettes of the fhaB sequence, althoughthese have been less extensively examined thaneither of the other systems. As with mafA andmafB, the gene located 50 offhaBmay be requiredfor its function, as fhaA has homology to theaccessory secretion protein in the B. pertussis fila-mentous haemaglutinin system (Klee et al., 2000;Locht et al., 1993). The proposed expression/silentcassette arrangement of both mafBand fhaBhas

only come to light following the assessment ofthe complete genome sequences (Klee et al.,2000; Parkhill et al., 2000).

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

are adaptive for different environmental condi-tions. In theNeisseriaspp. gene switching is asso-ciated with simple sequence repeats that can be

used to identify genes with phase variable poten-tial. Further, a greater proportion of these genesdiffer between the sequenced strains than do othergenes. There are also variations in whether thegenes, when present, are phase variable. Thereare therefore two different levels at which phasevariation and phase variable genes are associatedwith strain differences and their differences inbehaviour. The role of phase variation in adapta-tion to different host conditions and strain fitnesshas recently been reviewed separately (Saunders,2003; Salaun et al., 2003,), and the phase variablegenes of the pathogenic Neisseria species singlyand in combination have been described in detailelsewhere (Saunders et al., 2000, 2001b). Althoughnot addressing pathogenic Neisseria, a recentcomplete study of the predicted phase variablerepertoire of genes in a population-based studyof H. pylori illustrates the issues of variation ofthese genes between strains within a diverse pan-mictic population (Salaun et al., 2004), whichclosely parallels the situation in the Neisseria spp.

9. Presence and location of mobile non-coding

elements

In addition to differences in coding sequencesbetween strains, several non-coding elements arevariably present or are present in different loca-tions between different strains.

One of the most clearly described IS elements inN. meningitidisis IS1301, which has been found in29% of meningococci tested, and is present in all

serogroups. It has not been found in other Neisse-ria spp. (Hilse et al., 2000). Disruptions of siaAand porA by IS1301 have been reported inmeningococci (Hammerschmidt et al., 1996a;Newcombe et al., 1998). Other identified IS ele-ments include members of the IS families IS3,IS5 (which includes IS1106), IS30, IS110, and anIS element related to IS1016 (Tettelin et al., 2000).

In addition to typical IS elements, Neisseriaspp. also contain another apparently mobile ele-ment, although the transposase responsible for



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its amplification and mobilization has yet to bedetermined. A 26 base pair repeat, found multipletimes within the neisserial genome, was identified

and found to be normally arranged as an invertedrepeat with a characteristic core region (Correiaet al., 1986, 1988). In this configuration, it is re-ferred to as a CREE. The functional consequencesof the local presence of a CREE and its mobilityappear to be dependent on the specific sequenceof the CREE, its location, and the sequences flank-ing it. The first function of a CREE reported wasthe identification of a promoter at the end of theelement proximal to the gene uvrB. In this case,the CREE sequence, ending TATA, is insertednext to the chromosomal sequence CT, the CREEand native sequence combining to form the 10element of a promoter (Black et al., 1995). In thiscase, the 35 is within the CREE itself. In thesame report, a putative gearbox promoter was alsoidentified within this CREE. A CREE with a sim-ilar gearbox-like sequence is located in the dcwcluster. While this gearbox promoter is active inE. coli, it is not active in the neisserial genome(Snyder et al., 2003), as would be expected in bac-teria lacking rpoS (Tettelin et al., 2000). There isanother means by which CREE generate promot-

ers, as identified in N. lactamicastrain L18. In thiscase, the promoter is generated at the end of theCREE distal to the dcaC gene. The entirety ofthe 10 element is contained within the CREE,while the 35 element is part of the native chro-mosome (Snyder et al., 2003). The CREE has alsobeen shown to be transcriptionally active 5 0 ofdrg,through investigation of strains of N. lactamicawith and without the CREE in this location (Can-talupo et al., 2001).

It has been suggested that, since the CREE are

predicted to form hairpin structures due to the ter-minal inverted repeats, these elements act in tran-scriptional termination. Additionally, it wasbelieved that they were present only at the end oftranscripts, although they are now know to bewithin coding sequences, between promoters andgenes, and components of promoters (Blacket al., 1995; Klee et al., 2000; Liu et al., 2002; Park-hill et al., 2000; Snyder et al., 2003). The CREEwithin the dcw cluster was claimed to be a tran-scriptional terminator (Francis et al., 2000), but

transcription through this region was later demon-strated (Snyder et al., 2003). More recently, CREEhave been proposed to direct RNaseIII digestion

of associated transcripts (De Gregorio et al.,2003). In N. meningitidis, the mtrCDE genes,encoding an efflux pump system, have been uncou-pled from the MtrR/MtrA regulation system ofthe gonococci due to the insertion of a CREE 5 0

of these genes, which now places the meningococ-calmtr efflux system under the regulation of IHFand RNaseIII (Rouquette-Loughlin et al., 2004).There are 102 to 270 copies of the CREE distribut-ed throughout the genome (Liu et al., 2002). It hastherefore been suggested that regions lackingCREE are more likely to be those that have beenacquired by horizontal transfer relatively recently(Buisine et al., 2002).

10. Presence of extra-chromosomal elements

In terms of the variety of plasmids found withineach pathogenicNeisseriaspecies, and the carriagefrequency, little has changed in the 15 years sincethis topic was last reviewed (Dillon and Yeung,1989; Roberts, 1989). Most isolates of N. gonor-

rhoeae, but not N. meningitidis, carry plasmids.Most, but not all, of these plasmids are specific toone or other of the species. While the potential im-pact of plasmid carriage may have changed little inthose 15 years, our understanding of the functionof 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) thatis usually referred to as the gonococcal crypticplasmid. This plasmid was first described in 1972(Engelkirk and Schoenhard, 1973) and wasamongst the first plasmids where the completenucleotide sequence was determined (Korchet al., 1985). This plasmid must have been re-se-quenced during the sequencing of the genome ofgonococcal strain FA1090, but that sequence (asopposed to that of the chromosome) is not public-ly available. The original nucleotide sequence en-



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abled the first prediction of the genetic content andtranscriptional organization of the plasmid (Korchet al., 1985), but no phenotype has ever been asso-

ciated with plasmid carriage (Biswas et al., 1986).Several structural variants of this plasmid areknown to occur. In some strains, a specific 54 ntdeletion has occurred between a directly repeated44 nt sequence (Foster and Foster, 1976; Hagblomet al., 1986), while in others a 156 nt insertion hasoccurred within one of the 44 nt repeats (Roy etal., 1988). In both cases, the genetic change has oc-curred within what was originally referred to as thecppB gene (Korch et al., 1985). Finally, in somegonococcal strains the cryptic plasmid is presentas a stable 12.6 kb trimer, consisting of threedirectly-repeated copies of the cryptic plasmid(Johnson et al., 1983). The genetic processes thatpromote the formation of stable trimers in thesestrains, and the reason they do not segregatedimeric and monomeric forms of the plasmid hasnever been explained.

The nucleotide sequence of the cryptic plasmidsuggests a compact genetic organization with twotranscriptional units, one of which was suggestedto be composed of ORF6, ORF7, cppA, cppB,and cppC (Korch et al., 1985). ORF6 and ORF7

have GTG initiation codons, and the available evi-dence suggests that they are not expressed (Hagb-lom et al., 1986; Sarandopoulos and Davies,1993a). The derived amino acid sequence fromORF6 shows no similarity to any sequence in thedatabases. As nucleotide sequences from a varietyof bacterial species has accumulated, it has becomeclear that most of this portion of the plasmid is theremnant of a mobilization region very similar tothat found in a range of bacterial plasmids thatalso encode bacteriocins (Sarandopoulos and Da-

vies, 1993a; Wertz and Riley, 2004). In N. gonor-rhoeae only a portion of this mobilization regionis present, and even that has been inactivated bya 10 nt duplication (Sarandopoulos and Davies,1993a). This region extends from ORF7 throughcppC to part way through cppB, correspondingto parts of mobC, mobA, and mobB, respectively(Sarandopoulos and Davies, 1993a). The derivedamino acid sequence of the carboxy-terminal por-tion of the CppB protein shows no similarity toany protein in the databases. The predicted amino

acid sequence of CppA displays similarity to hypo-thetical proteins in a range of species. A secondtranscriptional unit on the cryptic plasmid was

suggested to consist of ORFs 1-5 (Korch et al.,1985). Again, sequence comparisons now enablepredictions for the function of some of these puta-tive genes. The predicted products of ORF1 andORF2 have strong sequence similarity to the prod-ucts of the RepA and RepB genes involved in thereplication of plasmids in a range of bacterial spe-cies (Ankri et al., 1996; De Mot et al., 1997). Suchsequence comparisons have also enabled a predic-tion of the origin of replication on the cryptic plas-mid (De Mot et al., 1997). The predicted productfrom ORF3 shows no sequence similarity to anyprotein in the databases. The predicted proteinproduct from ORF4 shows significant sequencesimilarity to a putative specialized RNA polymer-ase sigma factor fromHaemophilus influenzae, andproduction of this protein has been correlated withresistance to rifampicin in Escherichia coli(Sarandopoulos and Davies, 1993a). ORF5 ap-pears to encode a protein of the VapD (virulenceassociated protein D) family (Katz et al., 1992).Despite the name of the family, none of the pro-teins it contains appears to have a known function.

10.2. Sequence similarity between the cryptic

plasmid and neisserial genomes

There are many reports suggesting nucleotidesequence similarity between the gonococcal crypticplasmid and the chromosomes ofN. gonorrhoeae(Biswas et al., 1986; Hagblom et al., 1986) andserogroup B N. meningitidis (Grimholt et al.,1993), and cryptic plasmids found in the meningo-coccus (Ison et al., 1986). For the most part, these

claims have been made on the basis of Southernhybridization experiments that employed varyinglevels of stringency, and therefore have to beviewed with some caution. One report suggeststhat the hybridization between the cryptic plasmidand gonococcal chromosome was the result ofshort repetitive, rather than any contiguous, nucle-otide sequence (Sarandopoulos and Davies,1993b). As far as we are aware none of the menin-gococcal cryptic plasmids have been sequenced, sodirect sequence comparisons are not yet possible.



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However, the availability of a gonococcal and sev-eral meningococcal genome sequences enablesother suggestions of sequence similarity to be

directly tested. A simple BLASTn search (Altschulet al., 1990) suggests that there is no substantial se-quence similarity between the gonococcal crypticplasmid and the genomes ofN. gonorrhoeaestrainFA1090,N. meningitidisstrain Z2491, orN. menin-gitidis strain FAM18. However, there are threeshort (50-80 nt) regions with 86-95% nucleotide se-quence identity shared between the gonococcalcryptic plasmid and the chromosome ofN. menin-gitidis strain MC58.

These regions of nucleotide sequence identityare clustered on both DNA molecules, and mayexplain why gonococcal cryptic plasmid probesappear to hybridize more strongly with the sero-group B, as opposed to other meningococcal gen-omes (Grimholt et al., 1993). One of these regionsis contained within the cryptic plasmid cppA geneand overlaps two contiguous restriction fragmentsidentified byGrimholt et al. (1993)as hybridizingto the serogroup B meningococcal genome. Theequivalent portion of the meningococcal genomehas been named NMB1754, and annotated as pro-ducing a cryptic plasmid protein A-related pro-

tein. This CDS has a TTG initiation codon, andthe predicted amino acid sequence shows similarityto just the carboxy-terminal portion of CppA.

The additional two sequences that show se-quence identity to the serogroup B meningococcalgenome overlap, and are situated within the repBgene (ORF2). One sequence overlaps, and the otheris contained within, a 66 nt HhaI fragment identifiedbyGrimholt et al. (1993)as hybridizing to the sero-group B meningococcal genome. The equivalentportions of the strain MC58 genome are located be-

tween NMB1752 and NMB1753, and overlappingthe start of NMB1755. Between NMB1752 andNMB1753, the derived amino sequence similarityto the cryptic plasmid RepB protein extends out inboth directions from the region of nucleotide se-quence identity. This might suggest that a segmentderived from a related plasmid, rather than gono-coccal cryptic plasmid is integrated at this site. Itshould also be noted that the derived amino acid se-quence of NMB1753, also located in this region, ap-pears to be a member of the VapD family, and

shows a high degree of sequence identity to the pre-dicted product of ORF5. However, there is no highnucleotide 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 sug-gested to express a hypothetical protein. This site isnotable for the presence of a neisserial uptake se-quence (50-GCCGTCTGAA-3 0), which is specificto this genus (Goodman and Scocca, 1988). The re-gion of sequence identity overlaps the only copy ofthis sequence on the cryptic plasmid. The presenceof the uptake sequence is significant in that this seg-ment of the meningococcal genome is in the middleof IHT-C (Tettelin et al., 2000). The presence of theuptake sequence suggests that this segment of IHT-C is of neisserial origin, but the sequence similarityseems to be more at the derived amino acid, ratherthan nucleotide sequence, level. Again one expla-nation is that segments of those meningococcalplasmids that show sequence similarity to the gon-ococcal cryptic plasmid (Ison et al., 1986) havebeen integrated at this site.

10.3. b-Lactamase producing plasmids

An increasing proportion of gonococcal strainsare resistant to penicillin, resulting from the pres-ence of a plasmid encoding a TEM b-lactamase(Elwell et al., 1977). Plasmids from different strainsvary in size, and were originally named for the geo-graphic region (Asia, Africa, Rio, etc.) where theywere first found (Dillon and Yeung, 1989). Severalof these plasmids have had their nucleotide se-quence determined (Pagotto et al., 2000), and itis clear that they are all structural variants of anancestral plasmid that probably originated in Hae-

mophilus spp. (Brunton et al., 1986).

10.4. Conjugative plasmids

Many gonococcal strains harbour a 39 kb con-jugative plasmid, that was first discovered in1974 (Elwell and Falkow, 1975), but has beenfound in strains stored since the 1940s (Robertset al., 1979). This plasmid has not been sequenced,but has been demonstrated to promote its owntransfer to recipient gonococcal cells, and also to



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mobilize the b-lactamase plasmids (Eisensteinet al., 1977; Roberts and Falkow, 1979). In con-trast to early reports (Roberts and Falkow,

1978), this plasmid does not promote transfer ofchromosomal DNA to recipient cells (Norlanderet al., 1979). The recombination events detectedin these early experiments appear to have resultedfrom transformation of the plasmid-containingcell by recipient cell DNA, perhaps suggesting thatthe conjugative plasmid endows non-piliated hostcells with a low level of competence for naturaltransformation (Norlander et al., 1979). Thisplasmid appears to be only stably maintained inN. gonorrhoeae and certain strains of the closelyrelated N. cinerea (Genco et al., 1984).

More recently, tetracycline-resistance strains ofN. gonorrhoeaehave emerged (Morse et al., 1986).Tetracycline resistance results from the presence ofan approximately 40 kb plasmid carrying the tetMdeterminant (Morse et al., 1986). There was initial-ly some debate as to the origin of these plasmids,and in particular whether they were the result ofthe insertion of a tetM determinant into the39 kb gonococcal conjugative plasmid (Gascoyneet al., 1990; Morse et al., 1986). It is now clear thatsome plasmids (referred to as the Dutch type)

are indeed based on the gonococcal conjugativeplasmid, while others (referred to as the Ameri-can type) differ in their physical map suggestingthat they at least result from a different transposi-tion event (Gascoyne et al., 1991). It now seemsclear that at least the Dutch TetM plasmid isthe result of a class II transposition event involvingthe gonococcal conjugative plasmid (Swartleyet al., 1993). Transposition appears to have beenaccompanied by a deletion event that removedpart of the transposon and a segment of DNA

from the original conjugative plasmid (Swartleyet al., 1993). Despite its origin, the TetM plasmidappears to have a host range that is broader thanthe conjugative plasmid, as it can be transferredto, 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 ofN. meningitidis have been poorly

characterized. Cryptic plasmids of various sizeshave been described in some strains (Verschuerenet al., 1982), but all that is known about them is

that some appear to share sequence similarity withthe gonococcal cryptic plasmid (Ison et al., 1986).The gonococcal b-lactamase and TetM plasmidscan both be transferred to, and maintained, in ameningococcal background (Roberts and Knapp,1988). Finally a series of plasmids that appearrelated to RSF1010 have been reported in menin-gococcal strains (Facinelli and Varaldo, 1987;Rotger et al., 1986). Different variants of theseplasmids encode various combinations ofresistance to penicillin, streptomycin and sulph-onamide (Rotger et al., 1986). These RSF1010-re-lated plasmids have not been reported inN. gonorrhoeae.

11. Conclusions

Above all, the key messages from this revieware ones of change and flexibility. These specieswere known to have relatively panmictic popula-tion structures (Smith et al., 1993) due to frequentintra-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 generatediversity through a silent cassette system for thegeneration of pilin variants (Swanson et al.,1987), and many phase variable genes (Saunderset al., 2000; Snyder et al., 2001b). However, thesheer scale of the impact of these processes, as wellas differences in gene complements and otherforms of rearrangement and re-organization,could not have been fully appreciated in the ab-

sence of multiple complete genomes. Additional si-lent-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 vari-ant proteins through alterations in coding tandemrepeats (Jordan et al., 2003), have been defined instudies founded upon analysis and comparison ofthe genomes. Comparative study of these strainsand species offers a perspective from which a newholistic understanding of these dynamic systems



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and their biology can be appreciated. In this re-view, we have highlighted some of the main areasthat illustrate aspects of this flexibility and the sys-

tems in which genomic variability and instabilityprovide a basis for increased functional flexibilityand population fitness.

Acknowledgments

LAS is supported by a Wellcome Trust ProjectGrant. The authors would like to thank Dr SimonMcGowan, of the Dunn School/WIMM Compu-tational Biology Research Group, for the wholegenome comparisons presented in Fig. 1, and DrCharlene Kahler for a comparison of regulatorygenes found in the sequenced genomes. Work inthe Davies laboratory was supported by the Aus-tralian Bacterial Pathogenesis Program, fundedby a Program Grant from the Australian NationalHealth and Medical Research Council.

The N. gonorrhoeae sequence was obtainedfrom the University of Oklahoma, the GonococcalGenome Sequencing Project which was supportedby USPHS/NIH grant #AI-38399 (Accessionnumber: AE004969). The N. meningitidis strain

FAM18 and N. lactamica genome sequence datawere produced by the Sanger Institute and canbe obtained from ftp://ftp.sanger.ac.uk/pub/pathogens/nm/ and ftp://ftp.sanger.ac.uk/pub/pathogens/nm/.

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