1 Bacterial Pathogenesis and Functional Genomics Group, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK
2 Department of Computer Science, University of Warwick, Coventry CV4 7AL, UK
Correspondence
Nigel J. Saunders
Nigel.Saunders{at}path.ox.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are DQ171942, DQ171943, DQ171944, and DQ171945.
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INTRODUCTION |
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The original pan-Neisseria microarray (Snyder et al., 2004) was designed, constructed, and tested by an international consortium and contains probes representing all coding regions from three neisserial genomes (N. gonorrhoeae strain FA1090, and N. meningitidis strains MC58 and Z2491) and our predicted gene sequences from the GGI of N. gonorrhoeae strain MS11. Probes were designed based upon a comparative analysis of the genomes, so that common probes could be used to assess orthologous genes between the neisserial strains and species. The use of the pan-Neisseria microarray to assess gene complements in strains other than those used for its design has been published previously for N. gonorrhoeae (Snyder et al., 2004
). An extended version of the pan-Neisseria microarray (-v2) has been constructed, which includes additional gene probes based on the N. meningitidis strain FAM18 genome sequence, neisserial genes from GenBank/EMBL that are not in any of the genome sequences currently available, and genes identified through our ongoing investigations of minimal mobile elements (MMEs) (Saunders & Snyder, 2002
; Snyder et al., 2004
).
We previously reported the presence of the GGI in N. gonorrhoeae strain FA19, identified by comparative genome hybridization (CGH) using the pan-Neisseria microarray (Snyder et al., 2004). During a test of the ability of the pan-Neisseria microarray-v2 to generate CGH data from diverse strains of N. meningitidis, hybridization to the GGI-associated probes was identified in serogroup H strain 98/250521. Due to previous reports suggesting that GGI genes were restricted to N. gonorrhoeae (Dillard & Seifert, 2001
; Dillard & Hamilton, 2002
), these results were repeated and expanded to include other serogroup H strains, as well as strains from other serogroups. The microarray hybridization data suggest that these different N. meningitidis capsular serogroup strains contain both complete and partial versions of the GGI.
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METHODS |
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Bacterial strains used.
Representative strains from serogroups C (strains SB25 and 2120), W-135 (strain A22), X (strain 860060), Y (strain 860800), and Z (strain E32) were obtained from the MLST (multilocus sequence typing) meningococcal strain collection (http://pubmlst.org/neisseria/). To supplement these, additional strains of atypical serogroups were obtained from the Meningococcal Reference Laboratory [Manchester Public Health Laboratory Service (PHLS), UK]. In all, the CGH data analysed were from N. meningitidis strains MC58 (serogroup B, sequence strain), Z2491 (serogroup A, sequence strain), FAM18 (serogroup C, sequence strain), SB25 (serogroup C, MLST strain), 2120 (serogroup C, MLST strain), A22 (serogroup W-135, MLST strain), 860060 (serogroup X, MLST strain), 860800 (serogroup Y, MLST strain), E32 (serogroup Z, MLST strain), 00/240794 (serogroup D, reference strain NCTC 009714), 01/241825 (serogroup 29E, PHLS strain), 98/250521 (serogroup H, PHLS strain), 97/252572 (serogroup H, PHLS strain), 97/252675 (serogroup H, PHLS strain), 01/241809 (serogroup X, PHLS strain), 01/241693 (serogroup X, PHLS strain), 00/240868 (serogroup Z, PHLS strain), 01/241422 (serogroup Z, PHLS strain), and 01/241471 (serogroup Z, PHLS strain). N. gonorrhoeae strains MS11 and FA19, as strains known to contain the GGI (Snyder et al., 2004), were hybridized to the pan-Neisseria microarray-v2 as controls.
Comparative genome microarray hybridization.
Chromosomal DNA was extracted (McAllister & Stephens, 1993) from the neisserial strains following growth on GC medium (BD) with Kellogg and ferric nitrate supplements (Kellogg et al., 1963
) at 37 °C, in 5 % (v/v) CO2 overnight. DNA (20 µg) was fluorescently labelled through direct incorporation of FluoroLink Cy3dCTP or FluoroLink Cy5dCTP (Amersham Biosciences) using 5 units DNA polymerase I Klenow fragment (Bioline) and 3 µg random hexamer primers (Invitrogen). Unincorporated nucleotides and random primers were removed using QIAquick Nucleotide Removal columns (Qiagen) according to the manufacturer's instructions. Labelled DNADNA probe microarray hybridizations were conducted in 4x SSC, 0·29 % SDS under LifterSlips (Erie Scientific) at 65 °C overnight. The slides were washed at 65 °C in 1x SSC, 0·05 % SDS for 2 min, at room temperature in 0·06x SSC for 2 min, and in fresh 0·06x SSC for 2 min before drying by airbrush (Paasche, with SimAir compressor). Microarray slides were scanned using a ScanArray Express HT Microarray Scanner (Perkin Elmer) and analysed using ScanArray Express software v2.1 (Perkin Elmer).
Each strain was compared against two other strains in a three-way experimental design, such that strains A, B, and C were compared as A vs B, B vs C, and C vs A. The presence or absence of any gene in one pairwise analysis was validated by comparison to the results of the unrelated comparative hybridization. If necessary, hybridizations were repeated to resolve ambiguities.
Dinucleotide signature analysis and G+C content determination.
Gene-by-gene dinucleotide signature analysis has previously been applied to the complete genome sequence of N. meningitidis strain MC58 (Tettelin et al., 2000) and to the genes of the neisserial dcw cluster in N. gonorrhoeae (Snyder et al., 2001
). A revised version of this methodology was used for this study, which compensates for the sampling effects of addressing variable gene lengths that tend to artefactually increase the divergence of shorter sequences (Saunders et al., 2005
). The variation in the signature of each individual coding region, in comparison to the signature of the genome as a whole, can indicate genes that have been acquired from other species with different compositional biases. Dinucleotide signatures were determined for the complete genome of N. gonorrhoeae strain FA1090 for reference, for each of the coding regions of this genome, and for the N. gonorrhoeae strain MS11 GGI (GenBank accession no. AY803022). The deviation from the mean genome signature of each coding sequence was determined and compared against the mean divergence of all annotated coding sequences in the genome.
The percentage of G and C bases within each gene was determined and compared with the G+C content of the N. gonorrhoeae strain FA1090 genome sequence.
GGI location determination using microarrays.
The determination of the location of the GGI was attempted using a modification of the CGH protocol described above. Specific primers that pointed out from the GGI (Table 1) were used instead of random primers. Primers at the GGI01 end of the GGI were used with FluoroLink Cy3dCTP, and primers at the GGI56/GGI57 end of the GGI were used with FluoroLink Cy5dCTP (Amersham Biosciences). All other conditions and methods were identical to those used for CGH.
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RESULTS |
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To verify that each of the tested strains was correctly identified as N. meningitidis, the microarray results were assessed for the presence of genes such as the ctr (NMB0071, NMB0072, NMB0073, and NMB0074) and lip (NMB0082 and NMB0083) capsule biosynthesis genes and opc (NMB1053). In all cases, these characteristic meningococcal genes were present in the tested meningococcal strains.
Serogroup H.
A near complete version of the GGI, with 47 of the 50 probed GGI annotated coding regions, is present in N. meningitidis strain 98/250521 (Table 2). Two other serogroup H strains were assessed: N. meningitidis strains 97/252572 and 97/252675 (Table 2
). Strain 97/252572 did not hybridize to the GGI probes. Strain 97/252675 hybridized to some of the probes, indicating that portions of the GGI (GGI01GGI07 and GGI29GGI41) are present, but large contiguous sections appear to be absent.
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Serogroup Z.
Four strains of N. meningitidis serogroup Z were assessed: E32, 01/241471, 00/240868, and 01/241422 (Table 2). Strain E32 did not hybridize to the GGI probes. Strains 00/240868 and 01/241422 have identical GGI probe hybridization profiles, which are similar to that of serogroup H strain 97/282675, containing GGI01GGI08 and GGI29GGI41. The most complete GGI of those identified in the serogroup Z strains is in strain 01/241471, although GGI42GGI56 are apparently absent. Other GGI genes are also absent or divergent in this strain, while one of the hybridized probes is not hybridized by any of the other meningococci assessed in this study (GGI57).
Other meningococcal strains.
Of the combined MLST/PHLS strain set, strains of serogroups C (strains SB25 and 2120), D (strain 00/240794), 29E (strain 01/241825), X (strains 860060, 01/241809, and 01/241693), and Y (strains O929 and 860800) did not hybridize with GGI-associated probes.
Dinucleotide signature analysis and G+C content
The dinucleotide signatures and G+C content of the predicted coding regions of the GGI are supportive of the horizontal acquisition origin of some genes and segments of the GGI. The weighted deviation of the dinucleotide signature and the G+C content were calculated for each of the 57 coding regions (Fig. 3). The mean divergence of the gonococcal genes in the FA1090 genome is 0·062 %, with an SD of 0·021 and 95 % confidence intervals of 0·0009. Compared with the FA1090 genes, GGI23 (traG) is the second most divergent gene not encoded by a repeat (a different source of signature divergence) assessed. These results show that several components of the GGI have sequence characteristics suggesting that they are not neisserial in origin. However, some regions may not have a foreign source, and/or have been in the neisseriae sufficiently long to have ameliorated to a typical neisserial composition.
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Knock-out mutants of the GGI
N. gonorrhoeae strains FA1090 and FA19 and N. meningitidis strain 98/250521 were transformed with PCR products generated using specific primers (GGIdelF and GGIdelR, Table 1) and pGGIdelPrecAkan or pGGIdelaphA-3 as the templates. Kanamycin-resistant colonies were obtained for all three strains following transformation, and colony PCR using kanamycin cassette directed primers produced amplicons of the expected sizes. Transformants of strains FA19 and 98/250521 were grown overnight without selection for DNA preparation. Hybridization to the pan-Neisseria microarray-v2 indicated that the GGI genes are still present in these strains, further suggesting that the GGI genes were present in more than one location.
Sequencing demonstrates the chromosomal and extrachromosomal nature of the GGI
Given the observations suggesting both chromosomal and additional extrachromosomal locations for the GGI, primers were designed to generate products that would be specific and confirm the presence of this element in both states. Sequencing from N. gonorrhoeae strain MS11 (GenBank accession nos DQ171943 and DQ171944) joined both ends of the established GGI sequence (AY803022) to the N. gonorrhoeae strain FA1090 genome sequence (AE004969) at the dif site (Figs 1 and 2) identified previously (Hamilton et al., 2005
). In contrast to our previous observations, these sequence data (GenBank accession no. DQ171943) revealed that the strain FA1090 hypothetical gene XNG0741 is present in strain MS11 and that the GGI has inserted within the MMEung at the dif site (Fig. 1
), rather than according to the MME model (Snyder et al., 2004
). However, the variation in gene presence within this MME between strains FA1090, MC58, and FAM18 would still classify this location as an MME.
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Sequencing of the strain MS11 amplicon generated using primers GGI01out1 and GGI57out3 (GenBank accession no. DQ171945) confirmed the existence of a circularized form of the GGI in which the two outward ends are joined at the dif site (Fig. 2).
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DISCUSSION |
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Differential presence of genes within the GGI has been previously noted and is the basis for the defined classes of GGI (Dillard & Seifert, 2001). Class I GGIs contain the genes that have been sequenced from N. gonorrhoeae strain MS11 (GenBank accession no. AY803022) (Dillard & Seifert, 2001
; Dillard & Hamilton, 2002
; Hamilton et al., 2005
). Class II GGIs contain the sac-4 allele of traG. The absence of atlA from the GGI of some gonococcal strains has been reported previously as class III GGIs and is associated with an alternative sequence present in the equivalent location, although its nature has not been determined (Dillard & Seifert, 2001
). The absence of atlA in these meningococcal versions of the GGI suggests that they belong to class III. It would be interesting, therefore, to compare a class III gonococcal GGI with those from N. meningitidis strains 98/250521 and A22. In addition, all but one of the strains lacked hybridization to the GGI parA probe; therefore, this may indicate either a meningococcal or a GGI class III-specific variation in this sequence, particularly since the associated GGI parB gene is present in these strains and sequencing from strain A22 indicated that at least a portion of parA is present (GenBank accession no. DQ171942).
The previous classification of the GGI as specific to N. gonorrhoeae was based on Southern blots conducted using just two hybridization probes: to atlA and traG, both of which were known to be variably present in the GGIs (Dillard & Seifert, 2001). The absence of the atlA gene in class III GGIs of N. gonorrhoeae is also seen in the meningococcal strains; therefore, the use of this gene as a probe would have prevented identification of any class III GGIs in the strains tested. Further, class II GGIs contain a divergent allele of traG (sac-4), therefore a traG probe would not necessarily have identified strains with class II GGIs. The N. meningitidis strains tested previously were of serogroups A, B, C, and D (Dillard & Seifert, 2001
). Our microarray hybridizations with DNA from these serogroups also did not reveal GGI sequences.
Although probes on the pan-Neisseria microarray are designed to the most conserved regions of the genes where multiple sequences of individual genes are available, there was only one GGI sequence available for design, from N. gonorrhoeae strain MS11. If there are differences (probably greater than at least 20 % divergence) between different allelic versions of genes, then this would lead to the lack of hybridization to the probe, and detection in this system. Specific primer pairs designed using the N. gonorrhoeae strain MS11 island sequence frequently failed to generate specific PCR products from meningococcal templates, further indicating divergence of these sequences.
If the GGI is contiguous and present in the same location in the N. meningitidis strains as it is in N. gonorrhoeae strain MS11, then generation of knock-out mutants of the entire GGI would make this clear when the parent and mutant DNA are hybridized to the microarray. However, after preparation of DNA from the transformants and hybridization to the microarray, it was revealed that the genes of the GGI are still present. The same was true for the N. gonorrhoeae strain FA19 transformants; therefore, sequencing was conducted to confirm the chromosomal location of the GGI. It was noted previously that an artificial construct containing the dif site readily excised from the chromosome (Hamilton et al., 2005). If the reverse is true, it may be that the GGI reintegrated at the dif site that is still present in the mutant constructs.
Sequence data revealed that, in strain MS11, the GGI is present both within the chromosome and as an extrachromosomal circular DNA element (Fig. 2). The genes of the GGI share significant similarity with the genes of the F plasmid (Hamilton et al., 2005
), therefore its presence as a free and as a chromosomally integrated plasmid is, on this basis, to be expected. The type IV secretion system encoded by the GGI has been shown to secrete chromosomal DNA, much the same as other integrated conjugative plasmids. Unlike most conjugation systems, cellcell contact is not required, although this may be because it is not necessary in the naturally competent Neisseria species. It has additionally been shown that this secreted DNA is capable of neisserial transformation (Hamilton et al., 2005
). Indeed, a neisserial conjugative plasmid was identified previously, but while the GGI is 57 kb, the conjugative plasmid was reported as being 37 kb (Elwell & Falkow, 1977
). These may represent different unrelated plasmids, or it is possible that these are variants of the GGI plasmid.
In its circularized form, the adjacent coding regions of the GGI are now orientated in the same direction. Strand switching for the coding regions occurs only between GGI03 and iagB (GGI04) and between atlA (GGI24) and cspA (GGI26) (Fig. 2). While as an integrated island, the organization is less structured, in the circularized form, the global organization of the genes and potential transcriptional units is more coherent.
The absence of some of the GGI genes in different strains suggests either that the complete GGI has developed from the combination of distinct segments, or that portions of it have been deleted or added. Compared with the genome sequence, there are distinct differences in the G+C content and dinucleotide signatures of the genes of the GGI of N. gonorrhoeae strain MS11, which suggests that these genes may have had a foreign origin (Fig. 3). However, it also shows features of considerable amelioration, or having origins not greatly divergent in composition, that suggest that it has been present within the Neisseria species for a relatively long time. A foreign origin is also consistent with the absence of the common neisserial intergenic Correia repeat (Correia et al., 1986
, 1988
), but it may also be that these elements are not maintained on neisserial plasmids.
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ACKNOWLEDGEMENTS |
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Received 27 January 2005;
revised 24 August 2005;
accepted 25 August 2005.
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