BACKGROUND
the genus neisseria contains  <dig> species and biovars colonising humans most of which are non-pathogenic colonisers of the upper respiratory tract  <cit> .
only two species, neisseria gonorrhoeae, the etiological agent of gonorrhoea and neisseria meningitidis, a major cause of meningitis and septicaemia worldwide, regularly cause disease in humans  <cit> .
a major determinant in the survival of neisseria within the human host is the ability to acquire iron, the majority of which is not circulating freely in the human body but is stored in ferritin and haemoglobin or is complexed with the glycoproteins transferrin and lactoferrin  <cit> .
neisseria have devised ways to counteract this iron limitation through the evolution of several high-affinity receptor systems including the lactoferrin binding proteins a and b, the transferrin binding proteins a and b, and the haptoglobin-haemoglobin receptor hpuab, each composed of an accessory lipoprotein subunit and a tonb-dependent gated porin  <cit> .
in addition, neisseria can obtain iron through the expression of the surface-exposed haemoglobin receptor hmbr  <cit> .
based on antigenic and genomic features of tbpb and tbpb, n. meningitidis isolates can be classified into two major families: isotype i  and isotype ii   <cit> .
isotype ii tbpb genes have been documented in several n. meningitidis clonal complexes including st- <dig>  st- <dig>  st- <dig> and st-41/ <dig> as well as non-pathogenic neisseria  <cit> .
isotype i tbpb genes, on the other hand, have solely been identified among n. meningitidis isolates belonging to the st- <dig> clonal complex and have not been detected among other neisseria species.
four tbpb families were recently described based on partial nucleotide sequences from serogroup a clonal complex st- <dig> n. meningitidis and n. lactamica isolates collected in the gambia  <cit> .
families one and four contained diverse isotype ii tbpb alleles from n. meningitidis and n. lactamica isolates and families two and three included isotype i tbpb alleles.
importantly, among the  <dig> isolates analysed  only three isotype i tbpb alleles were found, all of which belong to meningococci  <cit> .
meningococci from the st- <dig> clonal complex have been a major cause of meningococcal disease worldwide throughout the last century and despite low carriage rates continue to be associated with disease  <cit> .
in addition to the isotype i tbpb gene, st- <dig> meningococci can be distinguished from other hyper-virulent clonal complexes by the absence of an opca gene and the possession of a class  <dig> porb gene  <cit> .
furthermore, similarities between the st- <dig> clonal complex isolate fam <dig> and non-pathogenic neisseria spp.
have been reported including the clustering of pile sequences  <cit>  and the comparable genetic organisation of the opca negative locus in two n. polysaccharea isolates  <cit> .
taken together, these observations indicate the occurrence of specific horizontal genetic exchange events between commensal neisseria and st- <dig> meningococci which may have contributed to the described genetic isolation of this clonal complex  <cit> .
the origin of the isotype i tbpb gene is unknown.
consequently, the distribution of the gene in a defined collection of neisseria spp.
was investigated with the hypothesis that the isotype i tbpb gene was present in other neisseria spp.
RESULTS
identification of the tbpb gene
isotype i tbpb genes were isolated from the non-pathogenic neisseria spp.
two families of the gene became apparent.
the first contained sequences closely related to meningococcal st- <dig> tbpb genes belonging to three n. polysaccharea and two n. lactamica isolates.
the second included more divergent isotype i tbpb genes from the non-pathogenic neisseria spp.
n. sicca, n. mucosa, n. flava, n. subflava, n. cinerea, n. flavescens as well as from another three n. polysaccharea isolates .
isotype ii tbpb genes were obtained from the remaining six n. lactamica and two n. polysaccharea isolates, while in agreement with previous studies, n. gonorrhoeae isolates contained isotype ii tbpb genes   <cit> .
a further  <dig> non-pathogenic neisseria isolates were analysed and found to be negative for the tbpb gene.
among these were n. polysaccharea, n. perflava, n. sicca, n. subflava, n. flava and n. mucosa isolates.
these isolates may contain divergent or truncated tbpb genes unable to be amplified with the primers used, however the remainder of this study will focus on the tbpb genes that were sequenced.
functional assessment of the protein was beyond the scope of the present study.
nevertheless, previously documented putative transferrin binding sites were observed based on predicted translations of the nucleotide sequences  <cit> .
in particular, the highly conserved domains n <dig>  n <dig> and c <dig>  critical for efficient iron uptake and located in both the n- and c- terminal segments among isolates n. gonorrhoeae fa <dig>  n. meningitidis m <dig>  moraxella catarrhalis  <dig> and acinetobacter pleuropneumoniae serotype  <dig>  were also identified  <cit> .
domain n <dig>  displayed 100% sequence identity in both isotype i tbpb families, whereas six non-synonymous changes were observed among isotype ii tbpb.
domain n <dig>  was also highly conserved among isotype i tbpb with the occurrence of three non-synonymous substitutions.
five variable sites were present among isotype ii tbpb.
domain c <dig>  showed the most diversity with the occurrence of four non-synonymous substitutions among both tbpb isotypes.
phylogenetic relationships inferred from novel neisseria tbpb sequences
all of the sequences were aligned manually with sequences starting from and ending at the same amino acid residue in each isolate.
published isotype i and ii tbpb sequences from isolates b16b <dig>  m <dig>   <dig>   <dig>   <dig>   <dig> and fa <dig>  used in previous analyses  <cit> , were also included in the alignment as well as those from the sequenced genomes of n. meningitidis isolates fam <dig>  z <dig>  mc <dig> and n. gonorrhoeae fa <dig>
phylogenetic analysis was undertaken using the software package clonalframe version  <dig> , which is a statistical model-based method initially described for inferring bacterial clonal relationships using multilocus sequence data  <cit> .
inference is performed in a bayesian framework and a neutral coalescent model is assumed based on the hypothesis that the bacteria in the sample come from a constant-sized population in which each bacterium is equally likely to reproduce, irrespective of its previous history.
the key assumption of clonalframe is that recombination events introduce a constant rate of substitutions to a contiguous region of sequence with the end result that a clonal frame can be inferred.
in the present study, over  <dig>  iterations were performed with every hundredth tree sampled after which a 95% majority-rule consensus tree was derived.
annotation was then undertaken by importing the tree into the molecular evolutionary genetics analysis software package   <cit> .
the two major isotype families were evident with each family containing a distinct cluster of genes .
the shortness of the branches for isotype i tbpb genes indicated that these were a closely related group of sequences compared with the depth of the branches seen for isotype ii tbpb genes where greater diversity is known to occur  <cit> .
closer inspection of the tree revealed the two families of isotype i tbpb genes observed by sequence analysis as well as three clusters of isotype ii tbpb genes.
for ease of interpretation, the two isotype i tbpb families have been named tbpba and tbpbb with the isotype ii clusters named tbpbc through to tbpbe .
this nomenclature is proposed according to published guidelines in bacterial genetics  <cit>  and is recommended in light of the emergence of the new families revealed in this study.
hitherto, studies in tbpb genetic diversity have focussed on a specific neisseria spp.
or meningococcal clonal complex and have not encompassed all of the neisseria species included in the present work.
this inclusion has provided a more detailed analysis of tbpb diversity and will allow a more flexible nomenclature for tbpb genes.
family tbpba was comprised of genes most closely related to those of clonal complex st- <dig> meningococci with four of these belonging to n. lactamica and n. polysaccharea isolates.
family tbpbb included a divergent cluster of isotype i genes  belonging to a variety of neisseria spp.
as well as containing a subset of n. polysaccharea isolates .
three distinct isotype ii tbpb families were apparent .
several gonococcal genes have clustered together and can be found in family tbpbe with families tbpbc and tbpbd containing genes belonging to n. lactamica, n. polysaccharea, n. gonorrhoeae and n. meningitidis isolates.
throughout the tree isolates have clustered by neisseria species indicative of within species conservation of tbpb genes.
the genbank accession numbers for new tbpb genes sequenced in this study are listed in table  <dig> as well as those belonging to previously submitted tbpb sequences.
genetic diversity of the tbpb genes
genes belonging to family tbpba were the least diverse  with  <dig> polymorphic sites, the majority of which occur among n. polysaccharea and n. lactamica isolates.
overall, six fixed differences were observed between the genes of st- <dig> meningococci and those of n. lactamica and n. polysaccharea with no shared polymorphisms between the two populations.
family b tbpb genes were more diverse  with  <dig> polymorphic sites.
in a comparison of both gene families, there were  <dig> fixed differences and  <dig> shared mutations.
as expected, families tbpbc, d and e were more diverse .
genes belonging to the n. polysaccharea isolates shared 99% identity with  <dig> segregating sites, seven of which encoded non-synonymous changes.
n. lactamica tbpb genes were more diverse with  <dig> polymorphic sites while  <dig> polymorphisms were observed among n. gonorrhoeae tbpb genes.
a total of  <dig> shared mutations were observed between n. lactamica and n. meningitidis tbpbc genes indicative of recombination, while only six were apparent between n. polysaccharea and n. meningitidis.
very little recombination was noticeable among tbpba genes or between these and family tbpbb, however a mosaic gene structure was present among the latter indicating that recombination occurred frequently among tbpbb genes from n. sicca, n. flava, n. subflava, n. mucosa, n. flavescens, n. cinerea and n. polysaccharea .
as expected, tbpb genes from families tbpbc, d and e recombined often  with most of this occurring from bases  <dig> to  <dig>
discussion
the aim of this study was to identify the origin of the isotype i tbpb gene.
previous observations have determined that these were confined to meningococci belonging to the st- <dig> clonal complex  <cit> .
in contrast, isotype ii genes were widely distributed among n. meningitidis clonal complexes and n. lactamica isolates  <cit> .
the results presented here reveal the existence of isotype i tbpb genes among diverse neisseria spp.
based on phylogenetic analysis these could be divided into two families: tbpba containing genes homologous to st- <dig> meningococci and tbpbb including more distantly related isotype i genes belonging to diverse non-pathogenic neisseria spp.
.
n. lactamica and n. polysaccharea isolates were found with both tbpb isotypes while, in agreement with previous studies, n. gonorrhoeae isolates solely contained isotype ii tbpb genes  <cit> .
phylogenetic analysis demonstrated the presence of three isotype ii families, named tbpbc through to tbpbe.
family c contained genes belonging to n. lactamica, n. polysaccharea, n. meningitidis and n. gonorrhoeae isolates, family d included another subset of isotype ii tbpb genes belonging to n. lactamica and n. meningitidis isolates and finally, family e comprised n. gonorrhoeae genes .
in light of the tbpb families now present a new nomenclature is proposed according to published guidelines in bacterial genetics  <cit> .
previously, studies in tbpb genetic diversity focussed on a specific neisseria spp.
or meningococcal clonal complex and did not encompass all of the neisseria spp.
included in the present work  <cit> .
this inclusion has provided a more detailed analysis of tbpb diversity with the proposed nomenclature allowing more flexibility for future tbpb genes.
using this scheme genes can be grouped according to the family they belong to followed by an allele number.
a number of features are shared between clonal complex st- <dig> n. meningitidis isolates and non-pathogenic neisseria.
sequences upstream of the pile gene from the class ii pilin-producing n. meningitidis strain fam <dig> were identical to the short region characterised upstream from n. polysaccharea pile  <cit> .
the n. polysaccharea isolate analysed  was included in the present study and harboured a tbpb gene similar to that of n. meningitidis isolate fam <dig>  furthermore, opca genes are absent among meningococci belonging the st- <dig> clonal complex and were also undetectable among n. polysaccharea isolates  <dig> and  <dig>  <cit> , which were found in this study with isotype i tbpb genes.
the identification of isotype i family a and b genes among neisseria spp.
is another characteristic shared with n. meningitidis isolates belonging to clonal complex st- <dig> and is indicative of the occurrence of several horizontal genetic transfer events between non-pathogenic neisseria, in particular n. polysaccharea and meningococci belonging to this clonal complex.
the evolutionary reasons leading to the existence of two tbpb isotypes among neisseria are unknown.
however, seclusion of isotype i tbpb to st- <dig> clonal complex meningococci may be due to clonal expansion or selection for this isotype.
indeed, the isotype i tbpb protein has been shown to play an essential role in iron acquisition from human transferrin with isogenic mutants deficient in tbpb failing to grow on htf as a sole iron source  <cit> .
thus, both the tbpa and tbpb parts of the transferrin complex are critical.
this was reflected in the lower diversity observed among tbpb genes belonging to families a  and b , highlighting the importance of this gene in contributing to the fitness of the organism.
there has been selection for isotype i tbpb among meningococci belonging to the st- <dig> clonal complex such that it has become restricted to this clonal complex.
in contrast, isotype ii tbpb genes have been found to provide a purely facilitating role such that tbpb-deficient mutants were only incapacitated with slower growth  <cit> .
this has been confirmed in isogenic mutagenesis studies of both tbpa and tbpb in n. gonorrhoeae, h. influenzae and m. catarrhalis   <cit> .
the non-essential role the isotype ii tbpb gene has in iron acquisition may contribute to its hyper-variability.
indeed, zhu et al found that the high rate of import among isotype ii tbpb genes, although providing a temporary advantage because of antigenic composition, resulted in reduced fitness of the isolates  <cit> .
the higher recombination patterns observed in the present study among isotype ii tbpb genes  combined with the deeper phylogeny seen  support this.
CONCLUSIONS
this work investigated the distribution of the two tbpb variants among neisseria spp.
and aimed to discover the origin of the isotype i tbpb gene.
results revealed this gene was found among diverse neisseria spp.
indicating the occurrence of a horizontal genetic transfer event between n. meningitidis and non-pathogenic neisseria.
three features shared between st- <dig> meningococci and non-pathogenic neisseria have now been described:  the presence of isotype i tbpb genes  the identical sequences upstream of the pile gene and  the analogous genetic organisation of the opca negative locus.
a revised nomenclature was proposed according to the published guidelines  <cit> .
the scheme now distinguishes isotype i tbpb genes into two new families: tbpba and tbpbb the former contained tbpb genes closely related to st- <dig> clonal complex meningococci, the latter included the more distantly related tbpb genes belonging to many non-pathogenic neisseria species.
the scheme also separates isotype ii tbpb genes into three new families: tbpbc comprising tbpb genes from n. meningitidis, n. lactamica, n. polysaccharea and n. gonorrhoeae isolates, tbpbd consisting of tbpb genes from n. lactamica and n. meningitidis isolates and finally, tbpbe containing n. gonorrhoeae tbpb genes.

 the genus neisseria contains species and biovars colonising humans most of which are non-pathogenic colonisers of the upper respiratory tract. only two species, neisseria gonorrhoeae, the etiological agent of gonorrhoea and neisseria meningitidis, a major cause of meningitis and septicaemia worldwide, regularly cause disease in humans. a major determinant in the survival of neisseria within the human host is the ability to acquire iron, the majority of which is not circulating freely in the human body but is stored in ferritin and haemoglobin or is complexed with the glycoproteins transferrin and lactoferrin. neisseria have devised ways to counteract this iron limitation through the evolution of several high-affinity receptor systems including the lactoferrin binding proteins a and b, the transferrin binding proteins a and b, and the haptoglobin-haemoglobin receptor hpuab, each composed of an accessory lipoprotein subunit and a tonb-dependent gated porin. in addition, neisseria can obtain iron through the expression of the surface-exposed haemoglobin receptor hmbr. based on antigenic and genomic features of tbpb and tbpb, n. meningitidis isolates can be classified into two major families: isotype i and isotype ii. isotype ii tbpb genes have been documented in several n. meningitidis clonal complexes including st- st- st- and st-41/ as well as non-pathogenic neisseria. isotype i tbpb genes, on the other hand, have solely been identified among n. meningitidis isolates belonging to the st- clonal complex and have not been detected among other neisseria species. four tbpb families were recently described based on partial nucleotide sequences from serogroup a clonal complex st- n. meningitidis and n. lactamica isolates collected in the gambia. families one and four contained diverse isotype ii tbpb alleles from n. meningitidis and n. lactamica isolates and families two and three included isotype i tbpb alleles. importantly, among the isolates analysed only three isotype i tbpb alleles were found, all of which belong to meningococci. meningococci from the st- clonal complex have been a major cause of meningococcal disease worldwide throughout the last century and despite low carriage rates continue to be associated with disease. in addition to the isotype i tbpb gene, st- meningococci can be distinguished from other hyper-virulent clonal complexes by the absence of an opca gene and the possession of a class porb gene. furthermore, similarities between the st- clonal complex isolate fam and non-pathogenic neisseria spp. have been reported including the clustering of pile sequences and the comparable genetic organisation of the opca negative locus in two n. polysaccharea isolates. taken together, these observations indicate the occurrence of specific horizontal genetic exchange events between commensal neisseria and st- meningococci which may have contributed to the described genetic isolation of this clonal complex. the origin of the isotype i tbpb gene is unknown. consequently, the distribution of the gene in a defined collection of neisseria spp. was investigated with the hypothesis that the isotype i tbpb gene was present in other neisseria spp.  identification of the tbpb gene isotype i tbpb genes were isolated from the non-pathogenic neisseria spp. two families of the gene became apparent. the first contained sequences closely related to meningococcal st- tbpb genes belonging to three n. polysaccharea and two n. lactamica isolates. the second included more divergent isotype i tbpb genes from the non-pathogenic neisseria spp. n. sicca, n. mucosa, n. flava, n. subflava, n. cinerea, n. flavescens as well as from another three n. polysaccharea isolates. isotype ii tbpb genes were obtained from the remaining six n. lactamica and two n. polysaccharea isolates, while in agreement with previous studies, n. gonorrhoeae isolates contained isotype ii tbpb genes. a further non-pathogenic neisseria isolates were analysed and found to be negative for the tbpb gene. among these were n. polysaccharea, n. perflava, n. sicca, n. subflava, n. flava and n. mucosa isolates. these isolates may contain divergent or truncated tbpb genes unable to be amplified with the primers used, however the remainder of this study will focus on the tbpb genes that were sequenced. functional assessment of the protein was beyond the scope of the present study. nevertheless, previously documented putative transferrin binding sites were observed based on predicted translations of the nucleotide sequences. in particular, the highly conserved domains n n and c critical for efficient iron uptake and located in both the n- and c- terminal segments among isolates n. gonorrhoeae fa n. meningitidis m moraxella catarrhalis and acinetobacter pleuropneumoniae serotype were also identified. domain n displayed 100% sequence identity in both isotype i tbpb families, whereas six non-synonymous changes were observed among isotype ii tbpb. domain n was also highly conserved among isotype i tbpb with the occurrence of three non-synonymous substitutions. five variable sites were present among isotype ii tbpb. domain c showed the most diversity with the occurrence of four non-synonymous substitutions among both tbpb isotypes. phylogenetic relationships inferred from novel neisseria tbpb sequences all of the sequences were aligned manually with sequences starting from and ending at the same amino acid residue in each isolate. published isotype i and ii tbpb sequences from isolates b16b m and fa used in previous analyses, were also included in the alignment as well as those from the sequenced genomes of n. meningitidis isolates fam z mc and n. gonorrhoeae fa phylogenetic analysis was undertaken using the software package clonalframe version, which is a statistical model-based method initially described for inferring bacterial clonal relationships using multilocus sequence data. inference is performed in a bayesian framework and a neutral coalescent model is assumed based on the hypothesis that the bacteria in the sample come from a constant-sized population in which each bacterium is equally likely to reproduce, irrespective of its previous history. the key assumption of clonalframe is that recombination events introduce a constant rate of substitutions to a contiguous region of sequence with the end result that a clonal frame can be inferred. in the present study, over iterations were performed with every hundredth tree sampled after which a 95% majority-rule consensus tree was derived. annotation was then undertaken by importing the tree into the molecular evolutionary genetics analysis software package. the two major isotype families were evident with each family containing a distinct cluster of genes. the shortness of the branches for isotype i tbpb genes indicated that these were a closely related group of sequences compared with the depth of the branches seen for isotype ii tbpb genes where greater diversity is known to occur. closer inspection of the tree revealed the two families of isotype i tbpb genes observed by sequence analysis as well as three clusters of isotype ii tbpb genes. for ease of interpretation, the two isotype i tbpb families have been named tbpba and tbpbb with the isotype ii clusters named tbpbc through to tbpbe. this nomenclature is proposed according to published guidelines in bacterial genetics and is recommended in light of the emergence of the new families revealed in this study. hitherto, studies in tbpb genetic diversity have focussed on a specific neisseria spp. or meningococcal clonal complex and have not encompassed all of the neisseria species included in the present work. this inclusion has provided a more detailed analysis of tbpb diversity and will allow a more flexible nomenclature for tbpb genes. family tbpba was comprised of genes most closely related to those of clonal complex st- meningococci with four of these belonging to n. lactamica and n. polysaccharea isolates. family tbpbb included a divergent cluster of isotype i genes belonging to a variety of neisseria spp. as well as containing a subset of n. polysaccharea isolates. three distinct isotype ii tbpb families were apparent. several gonococcal genes have clustered together and can be found in family tbpbe with families tbpbc and tbpbd containing genes belonging to n. lactamica, n. polysaccharea, n. gonorrhoeae and n. meningitidis isolates. throughout the tree isolates have clustered by neisseria species indicative of within species conservation of tbpb genes. the genbank accession numbers for new tbpb genes sequenced in this study are listed in table as well as those belonging to previously submitted tbpb sequences. genetic diversity of the tbpb genes genes belonging to family tbpba were the least diverse with polymorphic sites, the majority of which occur among n. polysaccharea and n. lactamica isolates. overall, six fixed differences were observed between the genes of st- meningococci and those of n. lactamica and n. polysaccharea with no shared polymorphisms between the two populations. family b tbpb genes were more diverse with polymorphic sites. in a comparison of both gene families, there were fixed differences and shared mutations. as expected, families tbpbc, d and e were more diverse. genes belonging to the n. polysaccharea isolates shared 99% identity with segregating sites, seven of which encoded non-synonymous changes. n. lactamica tbpb genes were more diverse with polymorphic sites while polymorphisms were observed among n. gonorrhoeae tbpb genes. a total of shared mutations were observed between n. lactamica and n. meningitidis tbpbc genes indicative of recombination, while only six were apparent between n. polysaccharea and n. meningitidis. very little recombination was noticeable among tbpba genes or between these and family tbpbb, however a mosaic gene structure was present among the latter indicating that recombination occurred frequently among tbpbb genes from n. sicca, n. flava, n. subflava, n. mucosa, n. flavescens, n. cinerea and n. polysaccharea. as expected, tbpb genes from families tbpbc, d and e recombined often with most of this occurring from bases to discussion the aim of this study was to identify the origin of the isotype i tbpb gene. previous observations have determined that these were confined to meningococci belonging to the st- clonal complex. in contrast, isotype ii genes were widely distributed among n. meningitidis clonal complexes and n. lactamica isolates. the results presented here reveal the existence of isotype i tbpb genes among diverse neisseria spp. based on phylogenetic analysis these could be divided into two families: tbpba containing genes homologous to st- meningococci and tbpbb including more distantly related isotype i genes belonging to diverse non-pathogenic neisseria spp.. n. lactamica and n. polysaccharea isolates were found with both tbpb isotypes while, in agreement with previous studies, n. gonorrhoeae isolates solely contained isotype ii tbpb genes. phylogenetic analysis demonstrated the presence of three isotype ii families, named tbpbc through to tbpbe. family c contained genes belonging to n. lactamica, n. polysaccharea, n. meningitidis and n. gonorrhoeae isolates, family d included another subset of isotype ii tbpb genes belonging to n. lactamica and n. meningitidis isolates and finally, family e comprised n. gonorrhoeae genes. in light of the tbpb families now present a new nomenclature is proposed according to published guidelines in bacterial genetics. previously, studies in tbpb genetic diversity focussed on a specific neisseria spp. or meningococcal clonal complex and did not encompass all of the neisseria spp. included in the present work. this inclusion has provided a more detailed analysis of tbpb diversity with the proposed nomenclature allowing more flexibility for future tbpb genes. using this scheme genes can be grouped according to the family they belong to followed by an allele number. a number of features are shared between clonal complex st- n. meningitidis isolates and non-pathogenic neisseria. sequences upstream of the pile gene from the class ii pilin-producing n. meningitidis strain fam were identical to the short region characterised upstream from n. polysaccharea pile. the n. polysaccharea isolate analysed was included in the present study and harboured a tbpb gene similar to that of n. meningitidis isolate fam furthermore, opca genes are absent among meningococci belonging the st- clonal complex and were also undetectable among n. polysaccharea isolates and, which were found in this study with isotype i tbpb genes. the identification of isotype i family a and b genes among neisseria spp. is another characteristic shared with n. meningitidis isolates belonging to clonal complex st- and is indicative of the occurrence of several horizontal genetic transfer events between non-pathogenic neisseria, in particular n. polysaccharea and meningococci belonging to this clonal complex. the evolutionary reasons leading to the existence of two tbpb isotypes among neisseria are unknown. however, seclusion of isotype i tbpb to st- clonal complex meningococci may be due to clonal expansion or selection for this isotype. indeed, the isotype i tbpb protein has been shown to play an essential role in iron acquisition from human transferrin with isogenic mutants deficient in tbpb failing to grow on htf as a sole iron source. thus, both the tbpa and tbpb parts of the transferrin complex are critical. this was reflected in the lower diversity observed among tbpb genes belonging to families a and b, highlighting the importance of this gene in contributing to the fitness of the organism. there has been selection for isotype i tbpb among meningococci belonging to the st- clonal complex such that it has become restricted to this clonal complex. in contrast, isotype ii tbpb genes have been found to provide a purely facilitating role such that tbpb-deficient mutants were only incapacitated with slower growth. this has been confirmed in isogenic mutagenesis studies of both tbpa and tbpb in n. gonorrhoeae, h. influenzae and m. catarrhalis. the non-essential role the isotype ii tbpb gene has in iron acquisition may contribute to its hyper-variability. indeed, zhu et al found that the high rate of import among isotype ii tbpb genes, although providing a temporary advantage because of antigenic composition, resulted in reduced fitness of the isolates. the higher recombination patterns observed in the present study among isotype ii tbpb genes combined with the deeper phylogeny seen support this.  this work investigated the distribution of the two tbpb variants among neisseria spp. and aimed to discover the origin of the isotype i tbpb gene. results revealed this gene was found among diverse neisseria spp. indicating the occurrence of a horizontal genetic transfer event between n. meningitidis and non-pathogenic neisseria. three features shared between st- meningococci and non-pathogenic neisseria have now been described: the presence of isotype i tbpb genes the identical sequences upstream of the pile gene and the analogous genetic organisation of the opca negative locus. a revised nomenclature was proposed according to the published guidelines. the scheme now distinguishes isotype i tbpb genes into two new families: tbpba and tbpbb the former contained tbpb genes closely related to st- clonal complex meningococci, the latter included the more distantly related tbpb genes belonging to many non-pathogenic neisseria species. the scheme also separates isotype ii tbpb genes into three new families: tbpbc comprising tbpb genes from n. meningitidis, n. lactamica, n. polysaccharea and n. gonorrhoeae isolates, tbpbd consisting of tbpb genes from n. lactamica and n. meningitidis isolates and finally, tbpbe containing n. gonorrhoeae tbpb genes. 
two families of isotype i tbpb were identified: family a containing conserved genes belonging to st- meningococci, n. polysaccharea and n. lactamica isolates and family b including more diverse isotype i tbpb genes from n. sicca, n. mucosa, n. flava, n. subflava as well as n. cinerea, n. flavescens and n. polysaccharea isolates. three isotype ii tbpb families were identified with: family c containing diverse tbpb genes belonging to n. polysaccharea, n. lactamica, n. gonorrhoeae and n. meningitidis isolates, family d including another subset of isotype ii tbpb genes from n. lactamica isolates and family e solely composed of n. gonorrhoeae tbpb genes. two tbpb families have been documented in neisseria meningitidis: an isotype i tbpb gene of kb and an isotype ii tbpb gene of kb, the former expressed by meningococci in the disease-associated st- clonal complex and the latter found among meningococci belonging to the hyper-invasive clonal complexes including st- st- st- st-41/ as well as n. gonorrhoeae isolates. transferrin binding protein b, an outer membrane lipoprotein, is required for the acquisition of iron from human transferrin. as a result, the diversity of the tbpb gene was investigated in a defined collection of neisseria species. the origin of the isotype i tbpb gene is unknown, however several features in common with non-pathogenic neisseria and the st- clonal complex n. meningitidis isolate fam have been documented leading to the hypothesis that the isotype i tbpb gene may also be shared between non-pathogenic neisseria and st- meningococci. this study reveals another instance of similarity between meningococci of the st- clonal complex and non-pathogenic neisseria with the origin of the isotype i tbpb gene resulting from a horizontal genetic transfer event occurring between these two populations.
 transferrin binding protein b, an outer membrane lipoprotein, is required for the acquisition of iron from human transferrin. two tbpb families have been documented in neisseria meningitidis: an isotype i tbpb gene of kb and an isotype ii tbpb gene of kb, the former expressed by meningococci in the disease-associated st- clonal complex and the latter found among meningococci belonging to the hyper-invasive clonal complexes including st- st- st- st-41/ as well as n. gonorrhoeae isolates. the origin of the isotype i tbpb gene is unknown, however several features in common with non-pathogenic neisseria and the st- clonal complex n. meningitidis isolate fam have been documented leading to the hypothesis that the isotype i tbpb gene may also be shared between non-pathogenic neisseria and st- meningococci. as a result, the diversity of the tbpb gene was investigated in a defined collection of neisseria species.  two families of isotype i tbpb were identified: family a containing conserved genes belonging to st- meningococci, n. polysaccharea and n. lactamica isolates and family b including more diverse isotype i tbpb genes from n. sicca, n. mucosa, n. flava, n. subflava as well as n. cinerea, n. flavescens and n. polysaccharea isolates. three isotype ii tbpb families were identified with: family c containing diverse tbpb genes belonging to n. polysaccharea, n. lactamica, n. gonorrhoeae and n. meningitidis isolates, family d including another subset of isotype ii tbpb genes from n. lactamica isolates and family e solely composed of n. gonorrhoeae tbpb genes.  this study reveals another instance of similarity between meningococci of the st- clonal complex and non-pathogenic neisseria with the origin of the isotype i tbpb gene resulting from a horizontal genetic transfer event occurring between these two populations. 