Background DNA adenine methyltransferase (Dam) activity is absent in many, but not all, disease isolates of  Neisseria meningitidis , as a consequence of the insertion of a restriction endonuclease-encoding gene, the ' dam  replacing gene' ( drg ) at the  dam  locus. Here, we report the results of a survey to assess the prevalence of  drg  in a globally representative panel of disease-associated meningococci.
Background
DNA adenine methyltransferase (Dam) activity is absent in many, but not all, disease isolates of  Neisseria meningitidis , as a consequence of the insertion of a restriction endonuclease-encoding gene, the ' dam  replacing gene' ( drg ) at the  dam  locus. Here, we report the results of a survey to assess the prevalence of  drg  in a globally representative panel of disease-associated meningococci.Neisseria meningitidisdamdrgdamdrgResults Of the known meningococcal hyper-invasive lineages investigated,  drg  was absent in all representatives of the ST-8 and ST-11 clonal complexes tested, but uniformly present in the representatives of the other hyper-invasive lineages present in the isolate collection (the ST-1, ST-4, ST-5, ST-32 and ST-41/44 clonal complexes). The patterns of sequence diversity observed in  drg  were consistent with acquisition of this gene from a source organism with a different G+C content, at some time prior to the emergence of present-day meningococcal clonal complexes, followed by spread through the meningococcal population by horizontal genetic exchange. During this spread a number of alleles have arisen by mutation and intragenic recombination.
Results
Of the known meningococcal hyper-invasive lineages investigated,  drg  was absent in all representatives of the ST-8 and ST-11 clonal complexes tested, but uniformly present in the representatives of the other hyper-invasive lineages present in the isolate collection (the ST-1, ST-4, ST-5, ST-32 and ST-41/44 clonal complexes). The patterns of sequence diversity observed in  drg  were consistent with acquisition of this gene from a source organism with a different G+C content, at some time prior to the emergence of present-day meningococcal clonal complexes, followed by spread through the meningococcal population by horizontal genetic exchange. During this spread a number of alleles have arisen by mutation and intragenic recombination.drgdrgConclusion These findings are consistent with the idea that possession of the  drg  gene may contribute to the divergence observed among meningococcal clonal complexes, but does not have a direct mechanistic involvement in virulence.
Conclusion
These findings are consistent with the idea that possession of the  drg  gene may contribute to the divergence observed among meningococcal clonal complexes, but does not have a direct mechanistic involvement in virulence.drg

Background Neisseria meningitidis , the causative agent of meningococcal meningitis and septicaemia, is a common inhabitant of the human nasopharynx, being asymptomatically carried by approximately 10% of the population [ 1 , 2 ]. There is evidence for extensive horizontal genetic exchange in populations of this antigenically and genetically variable Gram-negative bacterium [ 3 - 7 ] but, despite the diversity of carried meningococci, only a limited number of genotypes – the hyper-invasive lineages – are responsible for most reported disease [ 8 ]. These lineages have been identified by the techniques of multilocus enzyme electrophoresis [ 9 ] and multilocus sequence typing (MLST) [ 10 ] as clonal complexes. In recent years, the ST-1 complex (formerly subgroup I), ST-4 complex (subgroup IV), and ST-5 complex (subgroup III) have dominated meningococcal disease in Africa and Asia, while members of the ST-11 (ET-37) complex, ST-8 complex (cluster A4), ST-41/44 complex (lineage 3), and ST-32 (ET-5) complex have caused most disease in other parts of the world. Meningococci occasionally cause epidemic outbreaks of varying scale up to global pandemics, although at a given point in time disease in a given geographical locale is often dominated by a limited number of clonal complexes [ 8 ]. DNA adenine methyltransferase (Dam) is an enzyme involved in the mismatch repair system of bacteria. During DNA replication, only the parental strand is fully methylated by the enzyme, since methylation is not immediate, allowing a mismatch on the newly synthesised strand to be excised and replaced [ 11 ]. Disruption of Dam activity suggests that mismatch repair will be less effective, potentially allowing the creation of frameshift mutations in the homopolymeric or simple repeat motifs within the promoter or coding regions of surface antigen genes leading to the reversible switching of their expression state. This is an important mechanism found at bacterial loci that encode gene products that are advantageous under certain conditions but not others; such genes have been termed contingency loci [ 12 ]. There is extensive evidence for such loci in the meningococcal genome [ 13 ]. In particular, the ability to vary the expression state of surface-exposed cellular components is important for within host adaptation, for example by allowing evasion of the immune response in the nasopharynx. These mechanisms are also important in the occasional transition from harmless colonisation to invasive disease [ 14 - 16 ]. Examples of such loci described in the meningococcus to date include: the capsular polysaccharide [ 17 ]; the Opc outer membrane protein [ 18 - 20 ]; pili [ 21 ]; the PorA protein [ 22 ]; and opacity proteins [ 19 , 23 ]. Dam methylation has been implicated in modifying the virulence of a number of bacterial pathogens [ 24 - 26 ], but its role in  N. meningitidis  has been contentious. Bucci  et al ., 1999 [ 27 ] suggested that the absence of Dam activity was responsible for high rates of phase variation in the  siaD  capsule gene, resulting in an increased virulence of some strains. In addition, the latter work suggested that all pathogenic isolates lacked Dam activity, the  dam  gene inactivated by the insertion of a putative restriction endonuclease, named ' dam  replacing gene' ( drg ), with the genotypes  dam + / drg -  and  dam - / drg +  being mutually exclusive. However, two subsequent studies [ 28 , 29 ] have found no effect. The  drg  gene encodes a restriction enzyme,  Nme BII that is similar to the  Streptococcus pneumoniae Dpn I restriction endonuclease which cleaves at GmeATC, but not at GATC, sequences [ 30 , 31 ]. In the present study the prevalence of  drg  and its association with disease-associated isolates of  N. meningitidis  was examined by a survey of its distribution in a collection of isolates chosen to represent the known diversity of pathogenic meningococci [ 10 ]. The results suggest that, while  drg  is present in some lineages but not others, this gene has spread in the meningococcal population by horizontal genetic exchange, possibly after the introduction of the  drg  gene from an exogenous source.
Background
Neisseria meningitidis , the causative agent of meningococcal meningitis and septicaemia, is a common inhabitant of the human nasopharynx, being asymptomatically carried by approximately 10% of the population [ 1 , 2 ]. There is evidence for extensive horizontal genetic exchange in populations of this antigenically and genetically variable Gram-negative bacterium [ 3 - 7 ] but, despite the diversity of carried meningococci, only a limited number of genotypes – the hyper-invasive lineages – are responsible for most reported disease [ 8 ]. These lineages have been identified by the techniques of multilocus enzyme electrophoresis [ 9 ] and multilocus sequence typing (MLST) [ 10 ] as clonal complexes. In recent years, the ST-1 complex (formerly subgroup I), ST-4 complex (subgroup IV), and ST-5 complex (subgroup III) have dominated meningococcal disease in Africa and Asia, while members of the ST-11 (ET-37) complex, ST-8 complex (cluster A4), ST-41/44 complex (lineage 3), and ST-32 (ET-5) complex have caused most disease in other parts of the world. Meningococci occasionally cause epidemic outbreaks of varying scale up to global pandemics, although at a given point in time disease in a given geographical locale is often dominated by a limited number of clonal complexes [ 8 ].Neisseria meningitidisDNA adenine methyltransferase (Dam) is an enzyme involved in the mismatch repair system of bacteria. During DNA replication, only the parental strand is fully methylated by the enzyme, since methylation is not immediate, allowing a mismatch on the newly synthesised strand to be excised and replaced [ 11 ]. Disruption of Dam activity suggests that mismatch repair will be less effective, potentially allowing the creation of frameshift mutations in the homopolymeric or simple repeat motifs within the promoter or coding regions of surface antigen genes leading to the reversible switching of their expression state. This is an important mechanism found at bacterial loci that encode gene products that are advantageous under certain conditions but not others; such genes have been termed contingency loci [ 12 ]. There is extensive evidence for such loci in the meningococcal genome [ 13 ]. In particular, the ability to vary the expression state of surface-exposed cellular components is important for within host adaptation, for example by allowing evasion of the immune response in the nasopharynx. These mechanisms are also important in the occasional transition from harmless colonisation to invasive disease [ 14 - 16 ]. Examples of such loci described in the meningococcus to date include: the capsular polysaccharide [ 17 ]; the Opc outer membrane protein [ 18 - 20 ]; pili [ 21 ]; the PorA protein [ 22 ]; and opacity proteins [ 19 , 23 ].Dam methylation has been implicated in modifying the virulence of a number of bacterial pathogens [ 24 - 26 ], but its role in  N. meningitidis  has been contentious. Bucci  et al ., 1999 [ 27 ] suggested that the absence of Dam activity was responsible for high rates of phase variation in the  siaD  capsule gene, resulting in an increased virulence of some strains. In addition, the latter work suggested that all pathogenic isolates lacked Dam activity, the  dam  gene inactivated by the insertion of a putative restriction endonuclease, named ' dam  replacing gene' ( drg ), with the genotypes  dam + / drg -  and  dam - / drg +  being mutually exclusive. However, two subsequent studies [ 28 , 29 ] have found no effect. The  drg  gene encodes a restriction enzyme,  Nme BII that is similar to the  Streptococcus pneumoniae Dpn I restriction endonuclease which cleaves at GmeATC, but not at GATC, sequences [ 30 , 31 ].N. meningitidiset alsiaDdamdamdrgdamdrgdamdrgdrgNmeStreptococcus pneumoniae DpnIn the present study the prevalence of  drg  and its association with disease-associated isolates of  N. meningitidis  was examined by a survey of its distribution in a collection of isolates chosen to represent the known diversity of pathogenic meningococci [ 10 ]. The results suggest that, while  drg  is present in some lineages but not others, this gene has spread in the meningococcal population by horizontal genetic exchange, possibly after the introduction of the  drg  gene from an exogenous source.drgN. meningitidisdrgdrgResults Distribution of  dam  and  drg  among invasive meningococci In all cases, there was an exact correlation with the presence of  dam  and the absence of  drg , and vice-versa. Of the 84 isolates tested, 23 were indicated to be  dam + / drg -  and 61 to be  dam - / drg +  by restriction analysis and PCR amplification (see  Additional file 1 ). The presence of  dam  or  drg  was associated with particular clonal complexes. Of the hyper-invasive lineages included in the analysis, all ST-8 complex and ST-11 complex isolates were  dam + / drg - , while all of the representatives of the ST-1, ST-4, ST-5, ST-41/44, and ST-32 complexes were  dam - / drg + . Diversity of  drg  gene fragment sequences The level of nucleotide diversity, as represented by number of alleles and polymorphic sites, along with the p-distances seen within  drg  gene fragments was similar to that seen with the housekeeping genes used for MLST (Table  1 ), with a total of 19 different fragment sequences ( drg-1  –  drg-19 ) present in this collection. The ratio of non-synonymous to synonymous substitutions ( d N / d S , 0.11) was also similar to that seen in the MLST gene fragments. Three  drg  gene fragments ( drg-13 ,  drg-14  and  drg-15 ) exhibited polymorphisms resulting in single base frameshifts, two resulting in stop codons (Figure  1 ) and one fragment ( drg-16 ) had an insertion corresponding to a 24-base direct repeat. The G+C content of  drg  at 42.4% was lower than that of the housekeeping genes, which were in the range 51.1–57.4%. Split decomposition analysis of the  drg  fragment sequences generated a star phylogeny with some net-like phylogenetic structure involving the  drg-2  allele (Figure  2 ). The distribution of the different  drg  sequences amongst the clonal complexes is also shown in Figure  2 .
Results
Distribution of  dam  and  drg  among invasive meningococci In all cases, there was an exact correlation with the presence of  dam  and the absence of  drg , and vice-versa. Of the 84 isolates tested, 23 were indicated to be  dam + / drg -  and 61 to be  dam - / drg +  by restriction analysis and PCR amplification (see  Additional file 1 ). The presence of  dam  or  drg  was associated with particular clonal complexes. Of the hyper-invasive lineages included in the analysis, all ST-8 complex and ST-11 complex isolates were  dam + / drg - , while all of the representatives of the ST-1, ST-4, ST-5, ST-41/44, and ST-32 complexes were  dam - / drg + .
Distribution of  dam  and  drg  among invasive meningococci
damdrgIn all cases, there was an exact correlation with the presence of  dam  and the absence of  drg , and vice-versa. Of the 84 isolates tested, 23 were indicated to be  dam + / drg -  and 61 to be  dam - / drg +  by restriction analysis and PCR amplification (see  Additional file 1 ). The presence of  dam  or  drg  was associated with particular clonal complexes. Of the hyper-invasive lineages included in the analysis, all ST-8 complex and ST-11 complex isolates were  dam + / drg - , while all of the representatives of the ST-1, ST-4, ST-5, ST-41/44, and ST-32 complexes were  dam - / drg + .damdrgdamdrgdamdrgdamdrgdamdrgdamdrgDiversity of  drg  gene fragment sequences The level of nucleotide diversity, as represented by number of alleles and polymorphic sites, along with the p-distances seen within  drg  gene fragments was similar to that seen with the housekeeping genes used for MLST (Table  1 ), with a total of 19 different fragment sequences ( drg-1  –  drg-19 ) present in this collection. The ratio of non-synonymous to synonymous substitutions ( d N / d S , 0.11) was also similar to that seen in the MLST gene fragments. Three  drg  gene fragments ( drg-13 ,  drg-14  and  drg-15 ) exhibited polymorphisms resulting in single base frameshifts, two resulting in stop codons (Figure  1 ) and one fragment ( drg-16 ) had an insertion corresponding to a 24-base direct repeat. The G+C content of  drg  at 42.4% was lower than that of the housekeeping genes, which were in the range 51.1–57.4%. Split decomposition analysis of the  drg  fragment sequences generated a star phylogeny with some net-like phylogenetic structure involving the  drg-2  allele (Figure  2 ). The distribution of the different  drg  sequences amongst the clonal complexes is also shown in Figure  2 .
Diversity of  drg  gene fragment sequences
drgThe level of nucleotide diversity, as represented by number of alleles and polymorphic sites, along with the p-distances seen within  drg  gene fragments was similar to that seen with the housekeeping genes used for MLST (Table  1 ), with a total of 19 different fragment sequences ( drg-1  –  drg-19 ) present in this collection. The ratio of non-synonymous to synonymous substitutions ( d N / d S , 0.11) was also similar to that seen in the MLST gene fragments. Three  drg  gene fragments ( drg-13 ,  drg-14  and  drg-15 ) exhibited polymorphisms resulting in single base frameshifts, two resulting in stop codons (Figure  1 ) and one fragment ( drg-16 ) had an insertion corresponding to a 24-base direct repeat. The G+C content of  drg  at 42.4% was lower than that of the housekeeping genes, which were in the range 51.1–57.4%.drgdrg-1drg-19dNdSdrgdrg-13drg-14drg-15drg-16drgSplit decomposition analysis of the  drg  fragment sequences generated a star phylogeny with some net-like phylogenetic structure involving the  drg-2  allele (Figure  2 ). The distribution of the different  drg  sequences amongst the clonal complexes is also shown in Figure  2 .drgdrg-2drgDiscussion The meningococcus is an example of an accidental pathogen, a commensal organism that rarely causes disease and which gains no evolutionary benefit from this process [ 32 ]. A complete understanding of the diseases caused by such organisms is dependent upon an appreciation of the mechanisms by which harmless carriage develops into invasive disease [ 33 ]. The observation that some meningococcal genotypes, the hyper-invasive lineages, are more likely to cause disease than others [ 8 , 10 ] suggests that comparative studies of disease-associated and carried meningococci may identify genetic factors responsible for disease [ 34 ]. In this context, the suggestion that the possession of a single genetic change, the insertion of the  drg  gene at the  dam  locus, was essential for virulence in meningococci [ 27 ] was attractive as it provided a single characteristic associated with the disease phenotype. Further, this proposal provided a plausible mechanistic explanation, namely the promotion of rapid switching of expression of contingency genes, for example those encoding the capsule which represents the best defined meningococcal virulence determinant [ 35 ]. This is consistent with the evidence that inactivation of Dam has been shown to affect rates of recombination and phase change in other species [ 25 , 36 ]. The data presented here, however, do not support the contention that the  drg  insertion is preferentially associated with disease-associated meningococcal isolates, even those expressing serogroup B capsular polysaccharide. There was no evidence of a difference between disease-associated and carried meningococcal isolates with 76% (45/59) of disease-associated isolates and 75% (11/15) of carrier isolates possessing the  drg  insertion. The isolate collection employed by the present investigation was chosen to be globally representative of meningococcal disease in the latter part of the 20 th  century, with multiple examples of each of the major hyper-invasive meningococci identified over this period. When analysed by clonal complex, which identifies hyper-invasive lineages, it was apparent that the  drg  insertion was absent from all of the isolates representing the ST-8 and ST-11 clonal complexes, both major hyper-invasive lineages but, conversely,  drg  was present in all representatives of the hyper invasive lineages represented by the ST-1, ST-4, ST-5, ST-41/44, and ST-32 complexes. This association was independent of the serogroup expressed by the isolates and provided a likely explanation of the earlier observations [ 27 ]. As meningococcal disease in a given locale at a given time tends to be dominated by a limited number of clonal complexes [ 8 ], it is likely from the results provided here that a given sample of disease-associated isolates collected from a given locale will be uniform at the  dam  locus. At a given point in time they may be dominated by  drg  containing meningococci, while at other times isolates without this insertion might dominate. For example, as both the ST-1 and ST 32 complexes contain the  drg  insertion, this insertion would be prevalent among the disease isolates recovered by the pandemic outbreaks caused by these clonal complexes [ 37 , 38 ]; conversely, the spread of ST-11 meningococci [ 39 , 40 ] would result in an increased prevalence of disease-associated meningococci without the  drg  insertion. This observation highlights the importance of assembling genetically defined isolate collections with a prescribed sampling frame if comparisons of meningococci with potential for virulence are to be undertaken. It has been suggested that the  drg  gene has been introduced into  Neisseria  from an exogenous source [ 30 ]. The difference in the G+C% content of the  drg  gene compared to the meningococcal housekeeping genes was consistent with this idea; however, the levels of diversity among the  drg  alleles, which was similar to that observed in the meningococcal housekeeping genes, along with the reported occurrence of  drg  in other  Neisseria  species [ 30 ], suggested that this was a relatively old event predating the emergence of present-day clonal complexes. The ratio of non-synonymous to synonymous substitutions ( d N / d S ), which was also similar to that observed in meningococcal housekeeping genes, suggested that the  drg  gene had been subject to stabilising selection for conservation of function since its putative introduction into the meningococcal population, although the possession of frame shift mutations in some of the  drg  alleles demonstrated that, as with  N. lactamica , not all meningococci possessing the  drg  insertion expressed the endonuclease [ 30 ]. In other respects the distribution of the gene among clonal complexes and the patterns of nucleotide sequence variation observed were similar to those seen in meningococcal housekeeping genes, suggesting that it is subject to similar selection pressures [ 41 ]. The presence of multiple methylation systems is a characteristic of the  Neisseria  and may be related to the transformable nature of these organisms [ 42 ]. Whole genome comparisons of meningococci belonging to different clonal complexes frequently yield different restriction modification systems as the principal detectable genetic differences [ 43 , 44 ]. It is attractive to speculate that the genetic isolation of clonal complexes may be promoted by such differences [ 45 ] and in this respect the insertion of  drg  in the  dam  locus, replacing a methylase with an endonuclease, may provide a potent barrier to genetic exchange from  dam +  to  drg +  meningococci; indeed, while there was evidence for the occasional horizontal genetic exchange of  drg  alleles among the genetically diverse meningococci that possess the insertion, there was no evidence for the transformational loss of  drg  from a given clonal complex among the isolates examined here. This concept of genetic isolation among lineages receives some support from the possession of  porB2  alleles by members of the  dam +  ST-8 and ST-11 complexes and  porB3  alleles by the clonal complexes with the  drg  insertion [ 9 , 39 ], but the evidence for limitations in gene flow between these subgroups of meningococci is not at present conclusive.
Discussion
The meningococcus is an example of an accidental pathogen, a commensal organism that rarely causes disease and which gains no evolutionary benefit from this process [ 32 ]. A complete understanding of the diseases caused by such organisms is dependent upon an appreciation of the mechanisms by which harmless carriage develops into invasive disease [ 33 ]. The observation that some meningococcal genotypes, the hyper-invasive lineages, are more likely to cause disease than others [ 8 , 10 ] suggests that comparative studies of disease-associated and carried meningococci may identify genetic factors responsible for disease [ 34 ]. In this context, the suggestion that the possession of a single genetic change, the insertion of the  drg  gene at the  dam  locus, was essential for virulence in meningococci [ 27 ] was attractive as it provided a single characteristic associated with the disease phenotype. Further, this proposal provided a plausible mechanistic explanation, namely the promotion of rapid switching of expression of contingency genes, for example those encoding the capsule which represents the best defined meningococcal virulence determinant [ 35 ]. This is consistent with the evidence that inactivation of Dam has been shown to affect rates of recombination and phase change in other species [ 25 , 36 ].drgdamThe data presented here, however, do not support the contention that the  drg  insertion is preferentially associated with disease-associated meningococcal isolates, even those expressing serogroup B capsular polysaccharide. There was no evidence of a difference between disease-associated and carried meningococcal isolates with 76% (45/59) of disease-associated isolates and 75% (11/15) of carrier isolates possessing the  drg  insertion. The isolate collection employed by the present investigation was chosen to be globally representative of meningococcal disease in the latter part of the 20 th  century, with multiple examples of each of the major hyper-invasive meningococci identified over this period. When analysed by clonal complex, which identifies hyper-invasive lineages, it was apparent that the  drg  insertion was absent from all of the isolates representing the ST-8 and ST-11 clonal complexes, both major hyper-invasive lineages but, conversely,  drg  was present in all representatives of the hyper invasive lineages represented by the ST-1, ST-4, ST-5, ST-41/44, and ST-32 complexes. This association was independent of the serogroup expressed by the isolates and provided a likely explanation of the earlier observations [ 27 ]. As meningococcal disease in a given locale at a given time tends to be dominated by a limited number of clonal complexes [ 8 ], it is likely from the results provided here that a given sample of disease-associated isolates collected from a given locale will be uniform at the  dam  locus. At a given point in time they may be dominated by  drg  containing meningococci, while at other times isolates without this insertion might dominate. For example, as both the ST-1 and ST 32 complexes contain the  drg  insertion, this insertion would be prevalent among the disease isolates recovered by the pandemic outbreaks caused by these clonal complexes [ 37 , 38 ]; conversely, the spread of ST-11 meningococci [ 39 , 40 ] would result in an increased prevalence of disease-associated meningococci without the  drg  insertion. This observation highlights the importance of assembling genetically defined isolate collections with a prescribed sampling frame if comparisons of meningococci with potential for virulence are to be undertaken.drgdrgdrgdrgdamdrgdrgdrgIt has been suggested that the  drg  gene has been introduced into  Neisseria  from an exogenous source [ 30 ]. The difference in the G+C% content of the  drg  gene compared to the meningococcal housekeeping genes was consistent with this idea; however, the levels of diversity among the  drg  alleles, which was similar to that observed in the meningococcal housekeeping genes, along with the reported occurrence of  drg  in other  Neisseria  species [ 30 ], suggested that this was a relatively old event predating the emergence of present-day clonal complexes. The ratio of non-synonymous to synonymous substitutions ( d N / d S ), which was also similar to that observed in meningococcal housekeeping genes, suggested that the  drg  gene had been subject to stabilising selection for conservation of function since its putative introduction into the meningococcal population, although the possession of frame shift mutations in some of the  drg  alleles demonstrated that, as with  N. lactamica , not all meningococci possessing the  drg  insertion expressed the endonuclease [ 30 ]. In other respects the distribution of the gene among clonal complexes and the patterns of nucleotide sequence variation observed were similar to those seen in meningococcal housekeeping genes, suggesting that it is subject to similar selection pressures [ 41 ].drgNeisseriadrgdrgdrgNeisseriadddrgdrgN. lactamicadrgThe presence of multiple methylation systems is a characteristic of the  Neisseria  and may be related to the transformable nature of these organisms [ 42 ]. Whole genome comparisons of meningococci belonging to different clonal complexes frequently yield different restriction modification systems as the principal detectable genetic differences [ 43 , 44 ]. It is attractive to speculate that the genetic isolation of clonal complexes may be promoted by such differences [ 45 ] and in this respect the insertion of  drg  in the  dam  locus, replacing a methylase with an endonuclease, may provide a potent barrier to genetic exchange from  dam +  to  drg +  meningococci; indeed, while there was evidence for the occasional horizontal genetic exchange of  drg  alleles among the genetically diverse meningococci that possess the insertion, there was no evidence for the transformational loss of  drg  from a given clonal complex among the isolates examined here. This concept of genetic isolation among lineages receives some support from the possession of  porB2  alleles by members of the  dam +  ST-8 and ST-11 complexes and  porB3  alleles by the clonal complexes with the  drg  insertion [ 9 , 39 ], but the evidence for limitations in gene flow between these subgroups of meningococci is not at present conclusive.NeisseriadrgdamdamdrgdrgdrgporB2damporB3drgConclusions While it is possible that the possession of the  drg  insertion may influence meningococcal population structure, the data presented here do not support a direct association of this genotype with meningococcal virulence.
Conclusions
While it is possible that the possession of the  drg  insertion may influence meningococcal population structure, the data presented here do not support a direct association of this genotype with meningococcal virulence.drgMethods Bacterial isolates The 84 isolates used in this study (see  Additional file 1 ) were a subset of a collection of 107 diverse meningococci assembled to develop and validate the MLST scheme for  Neisseria meningitidis  [ 10 ]. The collection included representatives of the major hyper-invasive lineages and has been characterised at many genetic loci (full details of this isolate collection are available at  ). Restriction digestion of chromosomal DNA The methylation status of the chromosomal DNA of each isolate was determined by restriction digestion with the restriction endonucleases  Dpn I,  Dpn II, and  Sau 3AI, followed by separation of the digestion products by agarose gel electrophoresis. Each enzyme specifically recognises a cognate target site of GATC and cleaves this site depending on whether it is methylated or not:  Dpn I cleaves this target sequence only when N 6 -methyladenine is present within the recognition sequence;  Dpn II cleaves only unmethylated sites, and  Sau 3AI cleaves both methylated and unmethylated target sequences. PCR amplification and nucleotide sequence determination The presence of the  dam  gene was determined by polymerase chain reaction (PCR) performed using  dam  specific primers, DamF1 (5' – TAAAATGGGCAGGCGGCA – 3') and DamB2 (5' – CGTAAGGGGGATCGCAAT – 3'). These amplify a 534 bp fragment from the 5' end of the  dam  gene in  dam + strains but not from  dam -strains. The presence of  drg  was determined by PCR using primers DrgF1 (5' – CATGAATTTATTTTTCGATA – 3') and DrgB2 (5' – AATTTGCAACTGTTGGCG – 3') that bind to  drg  internal sites and produce a 705 bp fragment in isolates containing the  drg  gene. PCR amplification was also performed using primer pairs Drg5F (5' – TGTCTAAAGAACTCAAAG – 3') / DrgB3 (5' – CGGTATCGAAAAATAAAT – 3') and Drg3F (5' – ATCCATCCAATTTCCCCA – 3') / DamB5 (5' – AAATGCCGTCTGAA – 3') based on  dam  and  drg  coding regions in order to confirm that dam inactivation was due to  drg  insertion. Amplicons corresponding to the  drg  gene were purified by precipitation with poylethylene glycol and sodium chloride as described previously [ 46 ] and their nucleotide sequences determined on both strands by cycle sequencing. BigDye™ terminators (ABI, Foster city, California) and the same primers as used for amplification were employed in the extension reactions and the labelled reaction products separated on an ABI 3700 automated DNA sequencer. Each fragment was sequenced at least once on each strand and the sequences assembled with the STADEN suite of computer programs. Each unique  drg  sequence was assigned an arbitrary allele number in order of discovery. Data analysis Percentage G+C was calculated using the program START [ 47 ]. Split decomposition analysis of the  drg  gene fragments was performed using SPLITSTREE, version 3.1 [ 48 ]. The proportion of non-synonymous to synonymous substitutions was calculated by the method of Nei and Gojobori [ 49 ] using the program MEGA2 [ 50 ].
Methods
Bacterial isolates The 84 isolates used in this study (see  Additional file 1 ) were a subset of a collection of 107 diverse meningococci assembled to develop and validate the MLST scheme for  Neisseria meningitidis  [ 10 ]. The collection included representatives of the major hyper-invasive lineages and has been characterised at many genetic loci (full details of this isolate collection are available at  ).
Bacterial isolates
The 84 isolates used in this study (see  Additional file 1 ) were a subset of a collection of 107 diverse meningococci assembled to develop and validate the MLST scheme for  Neisseria meningitidis  [ 10 ]. The collection included representatives of the major hyper-invasive lineages and has been characterised at many genetic loci (full details of this isolate collection are available at  ).Neisseria meningitidisRestriction digestion of chromosomal DNA The methylation status of the chromosomal DNA of each isolate was determined by restriction digestion with the restriction endonucleases  Dpn I,  Dpn II, and  Sau 3AI, followed by separation of the digestion products by agarose gel electrophoresis. Each enzyme specifically recognises a cognate target site of GATC and cleaves this site depending on whether it is methylated or not:  Dpn I cleaves this target sequence only when N 6 -methyladenine is present within the recognition sequence;  Dpn II cleaves only unmethylated sites, and  Sau 3AI cleaves both methylated and unmethylated target sequences.
Restriction digestion of chromosomal DNA
The methylation status of the chromosomal DNA of each isolate was determined by restriction digestion with the restriction endonucleases  Dpn I,  Dpn II, and  Sau 3AI, followed by separation of the digestion products by agarose gel electrophoresis. Each enzyme specifically recognises a cognate target site of GATC and cleaves this site depending on whether it is methylated or not:  Dpn I cleaves this target sequence only when N 6 -methyladenine is present within the recognition sequence;  Dpn II cleaves only unmethylated sites, and  Sau 3AI cleaves both methylated and unmethylated target sequences.DpnDpnSauDpnDpnSauPCR amplification and nucleotide sequence determination The presence of the  dam  gene was determined by polymerase chain reaction (PCR) performed using  dam  specific primers, DamF1 (5' – TAAAATGGGCAGGCGGCA – 3') and DamB2 (5' – CGTAAGGGGGATCGCAAT – 3'). These amplify a 534 bp fragment from the 5' end of the  dam  gene in  dam + strains but not from  dam -strains. The presence of  drg  was determined by PCR using primers DrgF1 (5' – CATGAATTTATTTTTCGATA – 3') and DrgB2 (5' – AATTTGCAACTGTTGGCG – 3') that bind to  drg  internal sites and produce a 705 bp fragment in isolates containing the  drg  gene. PCR amplification was also performed using primer pairs Drg5F (5' – TGTCTAAAGAACTCAAAG – 3') / DrgB3 (5' – CGGTATCGAAAAATAAAT – 3') and Drg3F (5' – ATCCATCCAATTTCCCCA – 3') / DamB5 (5' – AAATGCCGTCTGAA – 3') based on  dam  and  drg  coding regions in order to confirm that dam inactivation was due to  drg  insertion. Amplicons corresponding to the  drg  gene were purified by precipitation with poylethylene glycol and sodium chloride as described previously [ 46 ] and their nucleotide sequences determined on both strands by cycle sequencing. BigDye™ terminators (ABI, Foster city, California) and the same primers as used for amplification were employed in the extension reactions and the labelled reaction products separated on an ABI 3700 automated DNA sequencer. Each fragment was sequenced at least once on each strand and the sequences assembled with the STADEN suite of computer programs. Each unique  drg  sequence was assigned an arbitrary allele number in order of discovery.
PCR amplification and nucleotide sequence determination
The presence of the  dam  gene was determined by polymerase chain reaction (PCR) performed using  dam  specific primers, DamF1 (5' – TAAAATGGGCAGGCGGCA – 3') and DamB2 (5' – CGTAAGGGGGATCGCAAT – 3'). These amplify a 534 bp fragment from the 5' end of the  dam  gene in  dam + strains but not from  dam -strains. The presence of  drg  was determined by PCR using primers DrgF1 (5' – CATGAATTTATTTTTCGATA – 3') and DrgB2 (5' – AATTTGCAACTGTTGGCG – 3') that bind to  drg  internal sites and produce a 705 bp fragment in isolates containing the  drg  gene. PCR amplification was also performed using primer pairs Drg5F (5' – TGTCTAAAGAACTCAAAG – 3') / DrgB3 (5' – CGGTATCGAAAAATAAAT – 3') and Drg3F (5' – ATCCATCCAATTTCCCCA – 3') / DamB5 (5' – AAATGCCGTCTGAA – 3') based on  dam  and  drg  coding regions in order to confirm that dam inactivation was due to  drg  insertion.damdamdamdamdamdrgdrgdrgdamdrgdrgAmplicons corresponding to the  drg  gene were purified by precipitation with poylethylene glycol and sodium chloride as described previously [ 46 ] and their nucleotide sequences determined on both strands by cycle sequencing. BigDye™ terminators (ABI, Foster city, California) and the same primers as used for amplification were employed in the extension reactions and the labelled reaction products separated on an ABI 3700 automated DNA sequencer. Each fragment was sequenced at least once on each strand and the sequences assembled with the STADEN suite of computer programs. Each unique  drg  sequence was assigned an arbitrary allele number in order of discovery.drgdrgData analysis Percentage G+C was calculated using the program START [ 47 ]. Split decomposition analysis of the  drg  gene fragments was performed using SPLITSTREE, version 3.1 [ 48 ]. The proportion of non-synonymous to synonymous substitutions was calculated by the method of Nei and Gojobori [ 49 ] using the program MEGA2 [ 50 ].
Data analysis
Percentage G+C was calculated using the program START [ 47 ]. Split decomposition analysis of the  drg  gene fragments was performed using SPLITSTREE, version 3.1 [ 48 ]. The proportion of non-synonymous to synonymous substitutions was calculated by the method of Nei and Gojobori [ 49 ] using the program MEGA2 [ 50 ].drgAuthors' contributions KAJ carried out the nucleotide sequencing and analysis of the data. LS performed the PCR screening of the isolates. ERM and MCJM conceived of the study and participated in its design and coordination.
Authors' contributions
KAJ carried out the nucleotide sequencing and analysis of the data. LS performed the PCR screening of the isolates. ERM and MCJM conceived of the study and participated in its design and coordination.Supplementary Material Additional File 1 Bacterial isolates used in the study Click here for file
Supplementary Material
Additional File 1
Bacterial isolates used in the studyClick here for file


The book "Understanding the Human Machine, A Primer for Bioengineering" embraces various aspects of biomedical engineering as an essential resource book for physical scientists, engineers and biomedical students. The book interfaces physiologic systems with engineering principles to capture the important concepts from a plethora of facts in field of biomedical sciences. Humors, exercise, history of biomedical discovery and elements of humility continuously emerge throughout the book; thereby, enhancing the self-taught approach for the new comers.The Introduction reflects the author's philosophy and advice for the readers from his accumulated wisdom and experience in teaching bioengineering. In Chapter 2, the author used the system approach to intertwine cardiovascular, renal, respiratory, gastrointestinal, endocrine, nervous and muscular systems. The organization is timely and effective in the era of systems biology and the emerging research towards predictive and preventive medicine.The system approach in Chapter 2 paves the way for detecting signal output from the physiologic systems. Biosensors are illustrated to measure electric signals from the visual, auditory and olfactory systems as well as the heart, brain, and muscle. The integration of electric circuits using the classic examples such as the Wheatstone bridge and operational amplifiers provides the frame work for signal acquisition and processing. Finally, the author completed the book with a touch of bioinformatics in the post genomic era.In general, the book is comprehensive and succinct, readable and interesting. The presented material spans from the fundamental principles to the applications of bioengineering.Certain areas will improve the quality of this book for the readership of undergraduates and new comers to bioengineering. Some figures are difficult to understand as a result of small captions (eg. figs 2.11 and 3.19). Others are old and lack clear labeling (eg. Figs. 2.52 and 3.15). Tissue engineering deserves a paragraph. Analogous to nanotechnology, a link to a tissue engineering website would be helpful for the readers. A paragraph on molecular engineering involving proteins, DNA and RNA molecules would enhance the balance between classic bioengineering and the emerging fields. The readers would be intrigued if the book ends with a paragraph introducing Bio-MEMS (Micro Electrical Mechanical Systems), bionanotechnology, molecular imaging, and surface chemistry.Overall, "Understanding the Human Machine, A Primer for Bioengineering" is a very useful book that highlights the fusion between organ systems and engineering principles. I would recommend this book as an introduction to bioengineering and a reference for students from physical science, engineering and life science.