BACKGROUND
spirochaetes represent a monophyletic lineage and a major branch in eubacterial evolution; brachyspira is the sole genus of the family brachyspiraceae within the order spirochaetales, which belongs to the spirochaete phylum  <cit> .
brachyspira are gram-negative, loosely coiled, aerotolerant anaerobes that colonise the lower gastrointestinal  tract of mammals and birds, but vary in pathogenicity.
there are seven brachyspira species that are currently officially recognised: b. aalborgi, a potential human pathogen  <cit> ; the porcine pathogen, b. hyodysenteriae <cit> ; the avian pathogens, b. alvinipulli <cit>  and b. intermedia <cit> ; the avian, porcine and human pathogen, b. pilosicoli <cit> ; non-pathogenic b. innocens <cit>  and b. murdochii, which is of uncertain pathogenic potential  <cit> .
in addition, there are a number of proposed species including “b.
canis”  <cit> , “b.
pulli”  <cit>  and “b.
suanatina”  <cit>  amongst others.
the classification of the genus is still immature and the often used descriptors of certain brachyspira as pathogenic, intermediate pathogenic or non-pathogenic is subject to debate.
b. pilosicoli is the only species considered to be a pathogen of birds, pigs and humans.
the species is quite diverse, and it seems unlikely that there are barriers to cross-species and zoonotic transmission  <cit> .
b. pilosicoli is an aetiological agent of colitis and occasional spirochaetaemia in humans  <cit> , and a cause of porcine intestinal spirochaetosis  and avian intestinal spirochaetosis   <cit> .
it may also cause disease in other species  <cit> .
b. pilosicoli is commonly found in humans living in densely populated areas with poor hygienic conditions  <cit> , and in homosexual males  <cit> .
b. pilosicoli infections are highly prevalent in intensively farmed swine and poultry, inducing inflammation in the colon and caeca, diarrhoea and reducing growth and productivity  <cit> .
chemotaxis and motility are deemed important virulence factors, and, as with b. hyodysenteriae, b. pilosicoli has a chemoattraction to mucin that facilitates penetration of the mucus and association with the underlying intestinal epithelial surface  <cit> .
the intimate contact with the epithelia induces a mucus outpouring and epithelial sloughing  <cit> .
an unusual feature of b. pilosicoli infection, and shared only by b. aalborgi, is the ability to insert one cell end into the luminal surface of enterocytes in the large intestine, forming a pit-like structure, with arrays of such attached spirochaetes giving the appearance of a “false brush-border”  <cit> .
this unusual form of attachment of b. pilosicoli also occurs in caco- <dig> cells in vitro, resulting in apoptosis, actin rearrangement and elevated interleukin expression  <cit> .
the paucity of genomic information and absence of tools for genetic manipulation are responsible, at least partly, for the lack of knowledge regarding the adaptations that brachyspira have undergone to colonise the lower gi tract, and for the pathogenic species to induce disease.
brachyspira whole genome sequences have only recently been made available for the following species: b. hyodysenteriae <cit> , b. intermedia <cit> , b. murdochii <cit>  and b. pilosicoli <cit> .
the four published sequences showed substantial genetic diversity, and their availability has facilitated research on the corresponding species.
however, the availability of only one genome sequence per species has limited the conclusions that can be drawn from the genome as a representation for the species as a whole, and does not allow analysis of intra-species genomic variation.
here, we report the whole genome sequence of b. pilosicoli b <dig>  isolated from a chicken exhibiting clinical symptoms of ais in the uk, and the partial genome sequence of b. pilosicoli wesb, isolated from an australian aboriginal child with diarrhoea.
experimentally, the latter strain has been shown to colonise and cause disease in pigs  <cit> .
although the strains were originally isolated from different host species, it is unlikely that the differences that were found between them were related to their host species of origin  <cit> .
the genomes are presented alongside the whole genome sequence of b. pilosicoli 95/ <dig>  isolated from a pig with pis in australia, and which has been confirmed to be virulent in experimental infection studies in pigs  <cit> .
we employed the biolog phenotype microarray™  technology  <cit>  to assess carbon utilisation in these strains.
these studies facilitated the validation of differences observed in genotype and permitted detailed correlation between genotype and phenotype.
methods
bacterial strains and growth conditions
b. pilosicoli b <dig> was isolated from a chicken displaying clinical symptoms of ais in the uk; wesb was isolated from an australian child with diarrhoea  <cit>  and 95/ <dig> from the diarrhoeic faeces of a pig with pis, in australia  <cit> .
the strains were cultured on fastidious anaerobe blood agar   <cit>  in an anaerobic atmosphere  at 37°c or 42°c for  <dig> days, for phenotypic studies.
for genomic dna extraction, strains were grown in pre-reduced anaerobic broth  <cit>  at 37°c and a cell pellet was prepared from mid-log phase broth growth.
genomic dna preparation, library construction and sequencing
cetyltrimethylammonium bromide  extraction was used to purify high molecular weight genomic dna  <cit> .
the b. pilosicoli b <dig> and wesb genomes were sequenced on a roche  <dig> flx platform, using a standard preparation for a  <dig> kb and  <dig> kb library, respectively.
for the b <dig> genome, a de novo assembly of the sequence reads into contiguous sequences was generated using newbler assembler software.
the reads were assembled into one scaffold of  <dig> contigs with an average coverage of ×  <dig>  remaining gaps were closed by pcr walking between unlinked, contiguous sequences  <cit> , followed by sanger sequencing.
in total,  <dig> sanger reads were incorporated into the assembly.
for the wesb genome, sequence data were initially assembled with short oligonucleotide alignment program   <cit>  and subsequently newbler assembler software was used to create a combined assembly with illumina reads.
iterative mapping and assembly for gap elimination   <cit>  improved genome assemblies by targeted re-assembly of illumina reads to span gaps within scaffolds.
to check for indels  and single nucleotide polymorphisms , iterative correction of reference nucleotides   <cit>  was applied to the genome and appropriate corrections were made.
all repeats over  <dig> bp were checked to ensure that they were confirmed by at least two spanning read pairs.
the incomplete wesb genome was sequenced within one scaffold, with an average coverage of ×  <dig>
sequence analysis and annotation
the complete nucleotide sequence and annotation of b. pilosicoli b <dig>  and partial nucleotide sequence and annotation of b. pilosicoli wesb b <dig>  have been deposited in genbank.
scaffold sequences for unpublished genomes b. alvinipulli c1t and b. intermedia hb <dig> can be accessed from the authors via e-mail request.
the draft genome scaffolds for b. aalborgii are available at the metahit website .
sequence and protein analysis and annotation  for the complete b. pilosicoli b <dig> and partial b. pilosicoli wesb genomes was as previously described for b. hyodysenteriae wa <dig>  <cit>  and b. pilosicoli 95/ <dig>  <cit>  unless otherwise stated.
the multi locus sequence typing  dendrogram of six brachyspira strains that have undergone genome sequencing, and three that are currently within unpublished genome sequencing projects being undertaken by the authors was calculated and constructed from the concatenation of  <dig> gene nucleotide sequences   <cit> .
these concatenated sequences were aligned by clustalw  <cit>  and the maximum likelihood dendrogram was generated via mega <dig>  <cit> .
the condensed bootstrap maximum likelihood dendrogram was constructed from the general time reversible  model with a gamma of  <dig>   and an assumption that a fraction of sites  are evolutionarily invariable .
the open source utility ‘freckle’ was used for sequence dot plotting .
gene prediction and gene and protein sequence extraction was achieved using prodigal  <dig>  .
initial coding region annotation was completed with an in-house updated compilation of the annotation pipeline autofact  <dig>   <cit> .
resulting annotations were manually checked and edited where appropriate to be consistent with previous brachyspira genome annotation methodologies for comparative purposes  <cit> .
final annotations were assessed with the ncbi microbial genome submission tool .
protein cluster analysis
protein reciprocal blast similarity searches with a threshold maximum expected value 1e- <dig> were conducted with blastlinemcl, which is an implementation of the markov clustering algorithm  for graphs .
the granularity of the output cluster was set with an inflation value of  <dig> .
biolog phenotype microarray™
b. pilosicoli 95/ <dig>  b <dig> and wesb were analysed using the biolog pm™ technology  <cit>  for high throughput substrate utilisation screening, which included  <dig> unique carbon sources .
pm panels and reagents were supplied by biolog and used according to the manufacturer’s instructions.
briefly, under anaerobic conditions, bacterial cells were aseptically picked from the faba agar surface with a sterile cotton swab and suspended in  <dig> ml of biolog inoculating fluid  until a cell density of 40% transmittance was reached on a biolog turbidimeter.
before the addition to pm microtitre plates, bacterial suspensions were further diluted into  <dig> ml of if- <dig>  in sterile water.
pm microtitre plates were pre-incubated with two ageless® oxygen absorbers   <dig> h prior to inoculation, at ambient temperature.
the resuspended bacterial cells were pipetted into the 96-well plates at a volume of  <dig> μl/well.
prior to removal from the anaerobic chamber, one ageless® oxygen absorber and one co2gen compact sachet  were attached per pm panel, which were then placed into  <dig> oz whirl-pak® long-term sample retention bags  with the open end heat-sealed.
substrate utilisation was measured via the reduction of a tetrazolium dye  to formazan , indicative of cellular respiration at 37°c.
experiments were also run at 42°c, using bacteria cultured at this temperature.
formazan formation was monitored at  <dig> min intervals for  <dig> h in omnilog apparatus.
kinetic data were analyzed with omnilog-pm software.
each experiment was performed at least twice per strain.
it was noted that although tetrazolium dye reduction is indicative of cellular respiration, it can occur independent of cell growth  <cit> .
blank pm <dig> and pm <dig> controls were run, whereby if- <dig> was added in place of the bacterial cell suspension, to assess for abiotic reactions that occur in the anaerobic atmosphere across the  <dig> h monitoring period.
the following compounds were omitted from analysis due to the nature of the abiotic reactions that occurred in wells containing these compounds, under the conditions of the study: d-arabinose and l-arabinose, dihydroxyacetone, d-glucosamine, 5-keto-d-gluconate, l-lyxose, palatinose, d-ribose, 2-deoxy-d-ribose, sorbate, d-tagatose and d-xylose.
RESULTS
comparison of general genome features
a dendrogram based on the mlst data for nine brachyspira strains highlighted the close relationship between the three b. pilosicoli strains, with b. aalborgi being distinct, but most closely related to b. pilosicoli and distantly related to b. hyodysenteriae , concordant with previous findings  <cit> .
the two b. intermedia strains appeared less closely related than might be expected, supporting reports of extensive diversity in this species based on results of pulse-field gel electrophoresis   <cit> , and a previous mlst study which indicated that these two strains belong to distinct groups  <cit> .
it has been suggested that not all isolates with the b. intermedia phenotype should be assigned to this species  <cit> .
the general genome features of the three sequenced b. pilosicoli genomes are compared in table  <dig>  the g + c content of the b. pilosicoli genomes were very similar to each other , and to that of other chromosomes in the genus, which range from  <dig> % to  <dig> %  <cit> .
the complete genome sequence of b. pilosicoli b <dig> consisted of a  <dig> , <dig> bp circular chromosome , whereas the incomplete wesb genome was larger, at  <dig> , <dig> bp.
the  <dig> , <dig> bp genome of strain 95/ <dig> was the smallest of the three genomes.
not only did the b. pilosicoli genomes show size variability, but also they were smaller than the genomes of the other sequenced species, apart from b. aalborgi 513t which our preliminary studies suggest is ~ <dig>  mb .
the relatively small size of the b. pilosicoli genomes is most likely due to them being members of a more specialised species that has undergone a high degree of reductive genome evolution.
if this is the case, then b. pilosicoli is likely to be an older pathogen than other brachyspira species such as b. hyodysenteriae <cit> .
such a reductive genome evolution may have allowed improved energy efficiency, and enhanced pathogenic potential.
reductive genome evolution is particularly evident in obligate, intracellular bacterial pathogens  <cit>  and consistent with this, of the brachyspira species, only b. pilosicoli and b. aalborgi show long-term intimate associations with the surface of enterocytes, into which they interdigitate one of their cell ends.
in addition to their small genomes, the sequenced b. pilosicoli strains lacked plasmids, whereas the genomes of the other fully sequenced brachyspira species have included plasmids  <cit> .
the comparison includes strains 95/ <dig> , b <dig>  and wesb .
a the incomplete wesb strain genome was within one scaffold.
b those genes with significant pid and/or query/target coverage hits; significance equals blastx/blastp pid of at least 25% and/or 75% query or target coverage.
c assigned to ko via kegg automatic annotation server
the disparity between the number of open reading frames  and genome size between the b <dig> and wesb strains and the high number of non-significant percentage identity  and coverage orfs in the wesb genome may be an artefact of the incomplete nature of this genome, which is the largest of the three strains.
in 95/ <dig>   <dig> % of orfs were assigned a kegg orthology , whereas only  <dig> % and  <dig> % of orfs were assigned in b <dig> and wesb, respectively.
a lower proportion of orfs were matched in cog database for b <dig> and wesb compared to 95/ <dig>
all three b. pilosicoli strains harboured the same number of transfer rna , rrna and transfer-messenger  genes .
the trna genes represented all  <dig> amino acids and there were single copies of the 5s, 16s and 23s rrna genes.
the rrf  and rrl  genes were co-located in all three b. pilosicoli genomes, with the rrs  gene located approximately  <dig> kb,  <dig> kb and  <dig> kb from the other rrna genes in the 95/ <dig>  b <dig> and wesb genomes, respectively.
this rrna gene organisation has been considered a distinguishing feature of brachyspira species  <cit> , since other spirochaetes typically have differing copy numbers and organisations  <cit> ; however, similar arrangements to brachyspira have been detected in the spirochaete borrelia burgdorferi <cit> .
situated between the rrs gene and rrfrrl cluster, which are either side of the oric, was the tmrna  gene and nine of the total  <dig> trnas that were otherwise dispersed throughout the genome .
the origin of replication of the b. pilosicoli genomes was set according to the position of the oric and gc-skew pattern, as previously suggested  <cit> ; this was supported by the ori-finder program  <cit> .
the origin of replication was originally considered to be adjacent to the dnaa gene  <cit> , however there was no association between the oric and dnaa genes in the b. pilosicoli b <dig> genome , as found in other brachyspira genomes  <cit> .
the arrangement of genes surrounding the dnaa gene was consistent between the b. pilosicoli strains, as with the other sequenced brachyspira genomes  <cit> .
the genes at the oric, although consistent between the b. pilosicoli strains, appear to vary extensively between the species.
b. pilosicoli genome architecture
on comparing b <dig> with 95/ <dig>  four major genome rearrangement events appeared to have occurred, whereas two rearrangements were evident when comparing wesb to 95/ <dig> .
mobile genetic elements  were found adjacent to or within close proximity of the sites where recombination events appear to have occurred in the b <dig> and wesb genomes.
sixty-one and  <dig> mges, including insertion sequence elements , integrases, recombinases and transposases were identified in the b <dig> and wesb genomes, respectively, compared to just four in the 95/ <dig> genome .
the proportion of these features therefore seems to correlate with the extent of rearrangement within the genome.
furthermore, multiple copies of an integrase gene that was absent from the 95/ <dig> genome were identified in the genomes of b <dig>  and wesb  .
the lower number of copies in wesb may be an artefact of the genes not assembling in the incomplete genome.
mges have been implicated in chromosomal rearrangements, gene disruptions resulting in pseudogenes, and eventual loss of genes, which may contribute to reductive genome evolution  <cit> .
species and strains that are undergoing or have recently undergone reductive genome evolution, and hence become more specialised pathogens, typically harbour large numbers of mges  <cit> .
there were fewest suspected pseudogenes  found in 95/ <dig> and most in b <dig> , a finding that correlates to the number of mges and degree of genome rearrangements in these strains.
of the total number of pseudogenes in each strain,  <dig> %,  <dig> % and  <dig> % were in a cluster with orthologs in the other two strains in 95/ <dig>  b <dig> and wesb, respectively.
most strikingly, all shared clusters included either multiple b <dig> and/or wesb pseudogenes with a complete 95/ <dig> gene.
differences in the number of mges in the three b. pilosicoli genomes may relate to their different stages of reductive genome evolution.
strain 95/ <dig>  which had the smallest genome, also had the fewest mges and this could be interpreted as indicating that the mges that induced the genome reduction in this strain have become lost.
alternatively, mge expansion may not have occurred in 95/ <dig> to the same degree as in b <dig> and wesb, as mges are generally lost in a fragmentary manner by pseudogenisation.
thus, it is unlikely that the 95/ <dig> genome, which has the fewest pseudogenes has been reduced in this way.
on the other hand, the greater number of pseudogenes in the larger b <dig> and wesb genomes does suggest that they may be undergoing genome reduction.
a possible explanation would be that these strains are in the initial stages of genome reduction, at the point at which mge expansion occurs  <cit> .
genome reduction and mge expansion is often associated with niche specialisation or host restriction  <cit> , however b. pilosicoli are not considered host-restricted, and wesb, of human origin, has been shown also to infect chickens and pigs  <cit> .
b. pilosicoli is a highly recombinant species  <cit> , and despite differences in genome arrangement and the number of pseudogenes, part of the difference in genome sizes simply reflects the carriage of different subsets of the pan-genome.
a dot plot comparison of the three b. pilosicoli genomes revealed that the chromosomal rearrangements were arranged symmetrically around the origin or terminus of replication, highlighted by the x-patterns in the alignments .
it has been postulated that symmetrical rearrangements occur because recombination events are determined by the replication forks that are approximately equal distance from the oric during bidirectional replication  <cit> .
it has also been argued that non-symmetrical rearrangements can be disadvantageous, and so genome rearrangements such as those found in the b. pilosicoli strains are a product of selection  <cit> .
despite the significant chromosomal rearrangements, genome alignments showed that the majority of genome sequence was shared between the three strains, with the larger b <dig> and wesb genomes possessing the greatest proportion of unique sequences .
furthermore, a  <dig> kb region, likely to have involvement in horizontal gene transfer , and that is partially conserved in all previously reported brachyspira genomes as well as enterococcus faecalis and escherichia coli <cit> , was identified in the b. pilosicoli b <dig>  and wesb  genomes.
functional genome comparisons
functional classifications assigned to each of the protein-coding genes of the three b. pilosicoli strains using the cog database showed that the general distribution of features into categories was similar for the three strains , and this highlighted their close relationship.
despite having the smallest genome, b. pilosicoli 95/ <dig> possessed the greatest number of features in six categories.
b <dig> contained the most features in eight categories, and wesb in one category.
a striking difference between the strains was in the carbohydrate , amino acid  and nucleotide  transport and metabolism categories, with the larger wesb genome containing considerably more features than the b <dig> and 95/ <dig> genomes.
in addition, compared to other brachyspira species the b. pilosicoli strains had a reduced number of features associated with inorganic ion transport and metabolism   <cit> .
athe number and percentage of the total genes within each of the genomes, assigned to each functional group are shownb.
bthose genes with significant pid and/or query/target coverage hits; significance equals blastx/blastp pid of at least 25% and/or 75% query or target coverage.
c the incomplete wesb strain genome was within one scaffold.
global genome feature comparisons
the three b. pilosicoli strains contained  <dig>  conserved genes, and these contribute to defining the b. pilosicoli pan-genome ; this related to  <dig> %,  <dig> % and  <dig> % of the total genes of the 95/ <dig>  b <dig> and wesb genomes, respectively.
as expected, there was a greater number of core genes between the strains of b. pilosicoli than between strains of different species; substantially fewer core genes  were identified for b. hyodysenteriae wa <dig>  b. pilosicoli 95/ <dig> and b. murdochii 56-150t <cit> .
b. pilosicoli wesb harboured the greatest number of unique genes, with  <dig> % of its genes being absent from the other genomes; b <dig> had a similar proportion , whereas 95/ <dig> had considerably fewer .
b. pilosicoli b <dig> and wesb shared the greatest proportion of genes  while b <dig> shared a greater percentage of its genes with 95/ <dig>  than with wesb .
global genome feature comparisons against other brachyspira species
complete genome sequences of b. hyodysenteriae wa <dig>  b. intermedia pws/at, b. pilosicoli 95/ <dig> and b. murdochii 56-150t have previously undergone comparative analysis  <cit> .
genome sequences of b. pilosicoli b <dig> and wesb can now be added to these comparisons, giving the first opportunity for a brachyspira intra-species genome comparison.
a protein blastmatrix comparison was performed on the four previously sequenced genomes, the two newly-sequenced b. pilosicoli genomes and the draft genome scaffolds of b. aalborgi 513t, b. alvinipulli c1t and b. intermedia hb <dig>  .
not unexpectedly, the analysis revealed that the b. pilosicoli strains shared the greatest proportion of proteins .
of b. pilosicoli strains, b <dig> had the greatest proportion of protein repeats relating to paralogs , despite not having the largest genome.
overall, the non-pathogenic b. murdochii had the greatest proportion of protein repeats , perhaps relating to its large genome.
high proportions of shared proteins highlighted the close relationships of b. hyodysenteriae with b. intermedia  and b. murdochii  .
b. aalborgi shared the lowest percentage of proteins with other brachyspira species, consistent with evidence that this is the most divergent species .
the percentage of the total cds that were identified in other genomes  and the proportion of protein repeats within the genome , is shown.
a cut-off e-value of 1e- <dig> was used.
a incomplete genome currently within a genome sequencing project.
b the incomplete wesb strain genome was within one scaffold.
a protein markov clustering analysis of the six published brachyspira genomes, identified  <dig>  protein clusters shared by all six strains , the encoding genes of which may be used to define a brachyspira species pan-genome.
this analysis revealed b. intermedia pws/at harboured the greatest number of clusters not found in the other sequenced brachyspira genomes  and it has the largest genome.
the greatest number of clusters shared only between two strains was with b. intermedia pws/at and b. hyodysenteriae wa <dig> , consistent with the close relationship of these species .
of the b. pilosicoli strains, b <dig> and wesb shared the most unique protein clusters , and wesb also shared the greatest number of clusters with a non-b.
pilosicoli strain, having  <dig> clusters in common with b. intermedia and  <dig> with b. murdochii.
the b. pilosicoli strains collectively shared the most clusters with b. murdochii 56-150t , and fewest with b. hyodysenteriae wa <dig> , as noted previously  <cit> .
non-b.
pilosicoli strains shared  <dig> clusters, whereas the b. pilosicoli strains shared  <dig> clusters, reflecting gene loss and genome reduction.
unique to the three strains of b. pilosicoli
of  <dig> protein clusters present only in the b. pilosicoli genomes ,  <dig> % were hypothetical or unclassified.
the majority of protein clusters were metabolic features, including an α-galactosidase , the activity of which is a distinguishing feature of the species  <cit> .
although it was suggested that b. pilosicoli had lost many transport-related genes during reductive evolution  <cit> ,  <dig> clusters were for transport proteins.
sialidase family-like protein genes unique to b. pilosicoli 95/ <dig>   <cit>  were also present in b <dig>  and wesb ; the products of such genes are produced by a variety of mucosal pathogens may play a role in colonisation or inducing tissue damage  <cit> .
clusters for an α- <dig> -fucosyl transferase , two membrane proteins   and two glycosyltransferases   were unique to b. pilosicoli and may contribute to host cell adherence.
other b. pilosicoli-specific clusters were for an ankyrin repeat protein , a β-lactamase , two peptidases   and phage proteins  .
unique and shared by two strains of b. pilosicoli
of the b. pilosicoli strains, b <dig> and wesb shared most unique clusters .
fewer clusters were shared with 95/ <dig>  but of twelve clusters unique to 95/ <dig> and b <dig>  all but n-acetyl mannosamine-6-phosphate 2-epimerase  were hypothetical.
six clusters were unique to 95/ <dig> and wesb, all lacking a specified function.
of  <dig> clusters unique to b <dig> and wesb,  <dig> % were hypothetical; notable clusters shared between these strains were for a further sialidase-like protein  and a peptidase .
the glycine reductase complex locus of 95/ <dig>  and b. murdochii 56-150t   <cit>  was identified in b <dig>  and wesb , but with an additional atp-binding cassette -type glycine betaine transport component in a separate locus .
moreover, a cluster for a transposase unique to b <dig>  and wesb  was detected.
genes that were shared only by the larger b <dig> and wesb genomes and were absent from 95/ <dig>  without apparent detriment, presumably have some specialised function that is not essential for survival.
these features may have been lost from 95/ <dig>  as they are not essential, or acquired in b <dig> and wesb, perhaps by hgt.
unique to one strain of b. pilosicoli
b. pilosicoli 95/ <dig> harboured the fewest and wesb the most unique features , correlating with their genome size.
as discussed above, the 95/ <dig> strain may have become more specialised, having lost non-essential features through reductive evolution  <cit> ; alternatively, the absence of orthologs in other strains or species may suggest that these features have been acquired via hgt.
of the strain-unique clusters,  <dig> %,  <dig> % and  <dig> % were for hypothetical proteins in 95/ <dig>  b <dig> and wesb, respectively.
in 95/ <dig>  unique clusters included a sodium/pantothenate symporter and an outer membrane lipoprotein  with a potential role in host cell adherence .
in b <dig>  unique clusters included putative phage proteins , additional glycine reductase complex proteins  and proteins involved in ascorbate metabolism  and mannitol metabolism .
in wesb, unique features included mannose/sorbose-specific phosphotransferase system  components , fructose-specific pts components  and a d-allose kinase .
six unique phage-related features and an integrase were identified at two loci in the wesb genome .
interestingly, each of the strains harboured unique genes for ankyrin proteins .
comparison of potential virulence features
virulence factor screening in brachyspira genomes was performed as described previously  <cit> , but with the analysis encompassing a greater array of genes, particularly in categories relating to adhesion and/or surface proteins and mges .
the greatest number of potential virulence features was in b <dig>  however additional features may be identified in the wesb genome once it is completed.
the analysis categorised the genes from the genomes of b. pilosicoli 95/ <dig>  b <dig> and wesb.
a the incomplete wesb strain genome was within one scaffold.
b core lipooligosaccharide  biosynthesis genes.
lipooligosaccharides
an rfbbadc cluster, encoding proteins for nucleotide sugar biosynthesis and with a suggested role in o-antigen assimilation in bacteria such as salmonella <cit> , was identified on the b. hyodysenteriae wa <dig> plasmid  <cit> .
although lacking this cluster, the three b. pilosicoli strains possessed rfba  and rfbb , but rfbc was noted only in b <dig>  and wesb  .
genes inferred to be involved in the biosynthesis of  <dig> -dideoxyhexose, an o-antigen component of lipopolysaccharide   <cit> , were found adjacent to the rfbc gene in b <dig> and wesb; both strains contained rfbf  and rfbg , but rfbh was present only in wesb .
the absence of such genes in the pathogenic strain 95/ <dig> suggests that they have a limited impact on virulence.
chemotaxis and motility
as with 95/ <dig>  the two other b. pilosicoli strains possessed fewer chemotaxis genes than b. hyodysenteriae and b. murdochii   <cit> .
no mcpc genes were found in the three b. pilosicoli strains, despite their detection in the genomes of the other fully sequenced brachyspira species.
the inter-species differences in the number and complement of chemotaxis-related genes may account for differences in their attraction to mucins and affinity to local host niches  <cit> .
no mcpa genes were identified in b <dig>  but two copies were found in the other b. pilosicoli strains.
the same complement of chemosensory transducer genes was identified in all three strains, as was the previously described cluster of seven such genes  <cit> .
differences in the number of chemotaxis-related genes between the three strains may translate from differences in genome size.
this may denote a redundancy of features that can be lost without apparent detriment to long-term survival.
the same flagella genes were shared by all three b. pilosicoli strains.
adhesion and membrane proteins
end-on attachment of the spirochaete to the luminal surface of the lower intestinal tract epithelia is characteristic of b. pilosicoli and b. aalborgi colonisation  <cit> , and hence surface-associated proteins or lipoproteins are potential candidates for virulence.
all lipoprotein genes in 95/ <dig> were found in b <dig> and wesb, but these strains also had a predicted secreted lipoprotein  and a lipoprotein carrier protein, lola , which anchors lipoproteins to the outer membrane  <cit> .
the same complement of genes encoding variable surface proteins found in 95/ <dig>  <cit>  and the putative integral membrane virulence factor, mvin  were noted in b <dig> and wesb.
genes for outer membrane proteins with a potential role in virulence were identified, including bspa antigens, which may bind fibronectin and initiate a serological response  <cit> , ompa proteins, similar to proteins implicated in leptospira virulence  <cit> , and tia invasion determinants.
genes encoding tolc were identified in all three b. pilosicoli strains, and this protein has been implicated in host invasion, virulence gene expression, and as an outer membrane component of efflux pumps  <cit> .
the periplasmic proteins identified were predicted to be primarily associated with other membrane proteins, and constitute abc transporters with putative roles in virulence  <cit> .
gene duplications were largely responsible for the greater number of inner membrane virulence factors in b <dig> and wesb, but since they were absent for 95/ <dig> they were unlikely to have significant impact on virulence.
wesb harboured two additional genes encoding oppa, which has suggested involvement in spirochaete-host interactions in treponema denticola <cit> .
genes encoding p-type atpase components, such as cada and znta, were noted in the three strains and these have been implicated in the ability of pathogens to sense and adapt to intracellular environments through heavy metal ion regulation  <cit> , in addition to trk potassium transport components, required for invasion and intracellular growth of salmonella <cit> .
genes encoding outer, periplasmic and inner membrane proteins that constitute transport systems implicated in bacterial virulence mechanisms were detected, such as polyamine abc-type transport, which is important for streptococcus pneumoniae pathogenesis  <cit> , tonb-dependant iron transport, which is related to shigella dysenteriae virulence  <cit> , and pts systems implicated in the virulence of mycobacterium tuberculosis and e. coli <cit> .
genes were found encoding components of the acrab-tolc complex, which confers antibiotic resistance and survival in the gi tract  <cit> , a ferrous iron transporter, feob, for iron acquisition, gut colonisation and intracellular survival of multiple enteropathogens  <cit> , and a glutamine transporter gene, glnq, which has been implicated in streptococcus adherence and virulence  <cit> .
in the b. pilosicoli strains, an mgl operon similar to one with a proposed role in virulence expression in treponema pallidum <cit>  was noted.
multidrug efflux features were found in all three strains, which aside from drug resistance, are attributed with a range of roles in pathogenesis  <cit> .
genes for the sec pathway described in 95/ <dig>  <cit> , with no needle-associated genes were also noted in b <dig> and wesb, with an additional seca-like gene in wesb .
host tissue degradation
the complement of haemolysis-related genes was identical between the three strains.
compared to previous analysis, other genes were detected including a haemolysin, previously undetected in 95/ <dig>  and three streptolysin genes, sagb , sagc  and sagd , involved in β-haemolysis and virulence in streptococci  <cit> .
a putative phospholipase/carboxylesterase  was found only in b <dig>  the three strains contained similar numbers of peptidases and proteases, which may participate in local degradation of host tissues, however 95/ <dig> lacked peptidase e, which had no effect on protein degradation in salmonella typhimurium  <cit> , and hence this non-essential enzyme may have been lost through reductive evolution.
oxidative stress
genes related to oxidative stress were shared by the three strains.
a partial bati  operon  <cit>  was noted in all strains, in close proximity to one of the nox genes and consisted of batb , batc , batd  and bate .
the bata gene was in a distinct locus in the three strains .
ankyrin-like protein
there was little difference in the number of genes encoding ankyrin-like proteins between the b. pilosicoli strains, which may be involved in host cell interactions through their ability to bind host chromatin as in orientia <cit> .
b. pilosicoli had consistently fewer of these genes than b. hyodysenteriae <cit> .
phage and other mobile genetic elements
outside of bacteriophage regions, four,  <dig> and  <dig> mges were identified in 95/ <dig>  b <dig> and wesb, respectively, correlating with the extent of genomic rearrangements.
the types and copy number of all mges in the b. pilosicoli genomes are detailed in additional file  <dig>  the region encoding genes related to the vsh- <dig> prophage-like gene transfer agent  in 95/ <dig>  <cit> , was identified in b <dig>  and wesb .
this region was ~ <dig> kb in 95/ <dig> compared to ~ <dig> kb in b <dig> and wesb due to an insertion between genes encoding orfe and hvp <dig>  containing genes for a monosaccharide-transporting atpase , an abc transporter-related protein , a rok family protein , an integrase in b <dig> only , and a periplasmic binding protein/laci transcriptional regulator .
generally, these features had high homology with those in clostridium carboxidivorans , consistent with the finding that brachyspira share a high degree of gene similarity with clostridium <cit> , and supporting the notion that the bacteriophages exchange genetic material between species  <cit> .
in wesb, an additional cluster of vsh-1-associated genes, flanked by a phage terminase, was detected ; the different genes in this region shared highest homology with c. carboxidivorans, b. hyodysenteriae, b. intermedia, b. pilosicoli and b. murdochii, suggesting that the gta had involvement in intra- and inter-species gene transfer.
the bacteriophage that was identified in b. pilosicoli 95/ <dig> , and in b. murdochii 56-150t   <cit> , was also found in b <dig>  and wesb  .
in b. pilosicoli, the bacteriophage size was proportional to genome size.
hypothetical proteins encoded in this region were shared between 95/ <dig> and b <dig>  however wesb contained four unique hypothetical genes.
the b <dig> pp <dig> bacteriophage possessed a unique ankyrin repeat protein .
an adenine-specific dna methyltransferase gene was present only in the wesb pp <dig> bacteriophage , adjacent to the dna methylase gene found in b. pilosicoli bacteriophages , but absent from those of b. murdochii 56-150t.
two separate novel bacteriophages regions were found in b <dig>  and wesb .
the ~ <dig> kb pp <dig> bacteriophage contained seven phage proteins , six predicted proteins with homology to sequences of other brachyspira species, and  <dig> unique hypothetical genes.
the ~ <dig> kb pp <dig> bacteriophage  shared all the components of the pi <dig> bacteriophage of b. intermedia pws/at, suggesting transfer of the bacteriophage in an inter-species hgt event.
interestingly, pp <dig> was flanked by vsh- <dig> components , and hence the vsh- <dig> gta may be responsible for mediating the hgt event.
two nuclease genes  and a number of unique hypothetical genes in pp <dig> were not identified in pi <dig>  clustered regularly interspaced short palindromic repeats , which provide bacteria with acquired resistance to bacteriophages  <cit> , were only identified in the non-pathogenic b. murdochii 56-150t, which suggests a role for bacteriophages in brachyspira pathogenicity.
b. pilosicoli b <dig> and b. intermedia pws/at did however possess a bacteriophage resistance protein  which has been implicated in protecting against bacteriophages  <cit> .
central metabolism and correlation with phenotype
analysis of the genomes of b. hyodysenteriae and b. pilosicoli has revealed that these species share many metabolic capabilities  <cit> .
in the current study the analysis of central metabolic pathway detection in brachyspira genomes was extended by application of biolog pm™ technology for phenotypic determination of carbon source utilisation of the b. pilosicoli strains.
the utilisation of  <dig> unique carbon compounds  by the strains was screened, and their metabolic capabilities were found to be highly conserved.
differences were found in the utilisation of just seven carbon sources, which were correlated with genotypic variations .
possible explanations for the differences in phenotype relate to differences in genomic features.
carbohydrate metabolism
high proportions of the b. pilosicoli genomes were associated with carbohydrate transport and metabolism , and from metabolic pathway reconstructions it is evident that glycolysis constitutes a major backbone of energy production  <cit> .
collectively the b. pilosicoli strains utilised  <dig> % of carbohydrate compounds tested, and more specifically  <dig> % of hexose sugars .
genes for enzymes involved in converting glucose-6-phosphate to ribulose-5-phosphate that were identified in b. hyodysenteriae wa <dig>  <cit> , were found in the b. pilosicoli genomes.
these features are likely to direct carbohydrate oxidation towards the non-oxidative pentose phosphate pathway, to generate reducing power required for biosynthetic pathways.
b. pilosicoli is characterised by an absence of β-glucosidase activity  <cit> , however a novel system for metabolising β-glucosides found in 95/ <dig>  <cit>  was also present in b <dig> and wesb, which, alongside specific pts systems, is likely to be involved in the utilisation of d-cellobiose and arbutin as carbon sources.
despite lacking β-glucosidase, metabolism of β-glucosides may be important to b. pilosicoli virulence as this phenotype is associated with growth, adhesion and colonisation in other bacteria  <cit> .
of the disaccharides tested,  <dig> % were utilised by the b. pilosicoli strains, whereas, of the oligosaccharides only dextrin was utilised, which is likely to be attributed to α-glucosidase activity .
amino acid metabolism
of the cog categories related to metabolism, the greatest proportion of the genome was related to amino acid transport and metabolism .
phenotypic studies revealed that despite the high number of genes for amino acid/ oligopeptide transporters found in the genomes, only five of the tested amino acids were able to support b. pilosicoli as a sole carbon source .
genes encoding enzymes to direct these amino acids towards pyruvate metabolism and hence energy production were identified, including alanine dehydrogenase , threonine aldolase , glycine hydroxymethyltransferase  and l-serine dehydratase .
moreover, a glycine reductase complex found in the b. pilosicoli strains, which catalyses the reductive deamination of glycine, forming atp, would be involved in the utilisation of glycine.
a high proportion of amino acid metabolic features in b. pilosicoli were related to biosynthesis and potentially maintaining intermediates of the partial tricarboxylic acid  cycle identified in this species  <cit> , rather than catabolism to produce energy.
l-glutamate and l-glutamine were insufficient to sustain b. pilosicoli as a sole carbon source; these amino acids are primary products of ammonia assimilation used in peptidoglycan, los and outer membrane protein biosynthesis  <cit> , hence their metabolism is redirected to energy yielding pathways.
the b. pilosicoli strains possessed genes for glutamate dehydrogenase , which catalyses the reversible synthesis of glutamate from α-ketoglutarate and ammonium.
since α-ketoglutarate was able to sustain b. pilosicoli, the presence of a transporter for α-ketoglutarate and not glutamate may explain this phenotype.
the ability to utilise certain amino acids as an energy source may have become redundant in brachyspira, which typically occupy the nutrient-rich lower gi tract, and thus associated features may have been lost through reductive evolution.
nucleotide metabolism
the b. pilosicoli strains were able to utilise three purine and two pyrimidine nucleosides tested as a sole carbon source .
the enzymes suggested to complete a metabolic link between nucleoside and central metabolism in b. hyodysenteriae wa <dig>  <cit>  were identified in the b. pilosicoli strains.
lipid metabolism
despite the presence of enzymes involved in the β-oxidation of fatty acids, including a long chain fatty acid-coa ligase , no long chain fatty acids tested were utilised by b. pilosicoli as a carbon source; however, the short chain fatty acids, butyric acid and propionic acid, were utilised .
glycerol was utilised as a carbon source, and genes for its metabolism were detected including those for a glycerol uptake facilitator , glycerol kinase  and glycerol-3-phosphate dehydrogenase .
the gene set required for fatty acid biosynthesis was incomplete in b <dig> and wesb, as it was in 95/ <dig>  <cit> .
CONCLUSIONS
in this study, we report the genome of b. pilosicoli strain b <dig> and the near complete genome of strain wesb.
together with the previously reported 95/ <dig> genome, this allowed the first intra-species genome comparison within the genus brachyspira.
our feature-based analysis revealed a high level of similarity between the three strains and identified genes that we suggest different strains of the spirochaete may have lost in a process of reductive genome evolution.
sequence-based comparisons showed the majority of sequence was shared between the strains, with few unique regions; however, genome rearrangements were observed around the oric.
mges were found associated to areas of rearrangements, and these features may be a factor that has driven or is driving reductive evolution.
novel bacteriophages were identified in the newly-sequenced genomes, which displayed evidence of intra- and inter-species hgt, and these may have key practical applications for use in genetic manipulation.
this is the first analysis of the spirochaete in a high-throughput phenotype screening tool, allowing correlation between genotype and phenotype.
future work will focus on the application of this technology to a wider range of brachyspira species to validate genome differences, potentially providing a means by which these phenotypes can be used for rapid screening to infer genotypes and improve current diagnostic methods.
with the increasing availability of brachyspira genome sequences, such technology should facilitate the validation of metabolic models based on genome sequence.
abbreviations
a: adenine; ais: avian intestinal spirochaetosis; abc: atp-binding cassette; act: artemis comparison tool; atp: adenosine triphosphate; bp: base pair; c: cytosine; cds: coding dna sequence; cog: cluster of orthologous genes; crispr: clustered regularly interspaced short palindromic repeats; ctab: cetyltrimethylammonium bromide; dna: deoxyribonucleic acid; faba: fastidious anaerobe blood agar; g: guanine; gi: gastrointestinal; gta: gene transfer agent; gtr: general time reversible; hgt: horizontal gene transfer; icorn: iterative correction of reference nucleotides; image: iterative mapping and assembly for gap elimination; indels: insertions/deletions; ise: insertion sequence element; kaas: kegg automatic annotation server; kb: kilobase; kegg: kyoto encyclopedia of genes and genomes; ko: kegg orthology; los: lipooligosaccharide; lps: lipopolysaccharide; mb: megabase; mcl: markov clustering algorithm; mge: mobile genetic element; mlst: multi locus sequence typing; ncbi: national center for biotechnology information; orf: open reading frame; pcr: polymerase chain reaction; pfge: pulse-field gel electrophoresis; pid: percentage identity; pis: porcine intestinal spirochaetosis; pm: phenotype microarraytm; pts: phosphotransferase system; rna: ribonucleic acid; rrna: ribosomal rna; snp: single nucleotide polymorphisms; soap: short oligonucleotide alignment program; t: thymine; tca: tricarboxylic acid; tmrna: transfer-messenger rna; trna: transfer rna.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
ljm, djh and rml conceived the study.
ljm and acd performed the genome sequencing and gap closure of b. pilosicoli b <dig>  jp and akt were involved in genome sequencing of the wesb.
djh, tl and ndp were involved in the genome sequencing of b. aalborgi 513t, b. alvinipulli c1t and b. intermedia hb <dig>  ljm, mlb, djh, mib and acd conceived and designed comparative genomic studies.
ljm and mlb performed comparative genomic analysis and analysed the data.
ljm, ma, mjw and rml conceived and designed phenotyping experiments.
ljm and ma conducted phenotyping experiments.
ljm wrote the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
type and copy number of mobile genetic elements  in the genomes ofb.
pilosicoli 95/ <dig>  b <dig> and wesb.
a combination of protein markov cluster analysis and reciprocal blast searches against the conserved domain database  was used to determine the copy number of each type of mge across the three b. pilosicoli genomes, using a cut-off e-value of 1e- <dig>  the orf number, position, and size of all mges identified in each of the b. pilosicoli genomes is displayed.
click here for file
additional file 2
conserved and shared protein clusters between the six genome-sequencedbrachyspira strains.
b. hyodysenteriae wa <dig> , b. intermedia pws/at , b. murdochii 56-150t  and b. pilosicoli 95/ <dig> , b <dig>  and wesb a strains we included in the protein cluster analysis.
a cut-off e-value of 1e- <dig> was used.
click here for file
additional file 3
comparison of the utilisation of unique carbon sources byb.
pilosicoli95/ <dig>  b <dig> and wesb.
biolog phenotype microarray™  technology was employed for these studies and omnilog apparatus was used to detect formazan formation and hence, respiration due to utilisation of the carbon source; +, able to utilise the compound; -, unable to utilise the compound.
click here for file
acknowledgments
the authors acknowledge receipt of funding for this study from the british egg marketing board research and education trust, the eu grant metahit , the royal college of veterinary surgeons , and murdoch university.
we are grateful to richard rance, david harris, ruth gilderthorp, nicola corton, and other members of the sequencing and genome improvement teams at the wellcome trust sanger institute for their assistance in the preparation of the incomplete genome sequence of b. pilosicoli wesb.

 spirochaetes represent a monophyletic lineage and a major branch in eubacterial evolution; brachyspira is the sole genus of the family brachyspiraceae within the order spirochaetales, which belongs to the spirochaete phylum. brachyspira are gram-negative, loosely coiled, aerotolerant anaerobes that colonise the lower gastrointestinal tract of mammals and birds, but vary in pathogenicity. there are seven brachyspira species that are currently officially recognised: b. aalborgi, a potential human pathogen; the porcine pathogen, b. hyodysenteriae; the avian pathogens, b. alvinipulli and b. intermedia; the avian, porcine and human pathogen, b. pilosicoli; non-pathogenic b. innocens and b. murdochii, which is of uncertain pathogenic potential. in addition, there are a number of proposed species including “b. canis”, “b. pulli” and “b. suanatina” amongst others. the classification of the genus is still immature and the often used descriptors of certain brachyspira as pathogenic, intermediate pathogenic or non-pathogenic is subject to debate. b. pilosicoli is the only species considered to be a pathogen of birds, pigs and humans. the species is quite diverse, and it seems unlikely that there are barriers to cross-species and zoonotic transmission. b. pilosicoli is an aetiological agent of colitis and occasional spirochaetaemia in humans, and a cause of porcine intestinal spirochaetosis and avian intestinal spirochaetosis. it may also cause disease in other species. b. pilosicoli is commonly found in humans living in densely populated areas with poor hygienic conditions, and in homosexual males. b. pilosicoli infections are highly prevalent in intensively farmed swine and poultry, inducing inflammation in the colon and caeca, diarrhoea and reducing growth and productivity. chemotaxis and motility are deemed important virulence factors, and, as with b. hyodysenteriae, b. pilosicoli has a chemoattraction to mucin that facilitates penetration of the mucus and association with the underlying intestinal epithelial surface. the intimate contact with the epithelia induces a mucus outpouring and epithelial sloughing. an unusual feature of b. pilosicoli infection, and shared only by b. aalborgi, is the ability to insert one cell end into the luminal surface of enterocytes in the large intestine, forming a pit-like structure, with arrays of such attached spirochaetes giving the appearance of a “false brush-border”. this unusual form of attachment of b. pilosicoli also occurs in caco- cells in vitro, resulting in apoptosis, actin rearrangement and elevated interleukin expression. the paucity of genomic information and absence of tools for genetic manipulation are responsible, at least partly, for the lack of knowledge regarding the adaptations that brachyspira have undergone to colonise the lower gi tract, and for the pathogenic species to induce disease. brachyspira whole genome sequences have only recently been made available for the following species: b. hyodysenteriae, b. intermedia, b. murdochii and b. pilosicoli. the four published sequences showed substantial genetic diversity, and their availability has facilitated research on the corresponding species. however, the availability of only one genome sequence per species has limited the conclusions that can be drawn from the genome as a representation for the species as a whole, and does not allow analysis of intra-species genomic variation. here, we report the whole genome sequence of b. pilosicoli b isolated from a chicken exhibiting clinical symptoms of ais in the uk, and the partial genome sequence of b. pilosicoli wesb, isolated from an australian aboriginal child with diarrhoea. experimentally, the latter strain has been shown to colonise and cause disease in pigs. although the strains were originally isolated from different host species, it is unlikely that the differences that were found between them were related to their host species of origin. the genomes are presented alongside the whole genome sequence of b. pilosicoli 95/ isolated from a pig with pis in australia, and which has been confirmed to be virulent in experimental infection studies in pigs. we employed the biolog phenotype microarray™ technology to assess carbon utilisation in these strains. these studies facilitated the validation of differences observed in genotype and permitted detailed correlation between genotype and phenotype. methods bacterial strains and growth conditions b. pilosicoli b was isolated from a chicken displaying clinical symptoms of ais in the uk; wesb was isolated from an australian child with diarrhoea and 95/ from the diarrhoeic faeces of a pig with pis, in australia. the strains were cultured on fastidious anaerobe blood agar in an anaerobic atmosphere at 37°c or 42°c for days, for phenotypic studies. for genomic dna extraction, strains were grown in pre-reduced anaerobic broth at 37°c and a cell pellet was prepared from mid-log phase broth growth. genomic dna preparation, library construction and sequencing cetyltrimethylammonium bromide extraction was used to purify high molecular weight genomic dna. the b. pilosicoli b and wesb genomes were sequenced on a roche flx platform, using a standard preparation for a kb and kb library, respectively. for the b genome, a de novo assembly of the sequence reads into contiguous sequences was generated using newbler assembler software. the reads were assembled into one scaffold of contigs with an average coverage of ×  remaining gaps were closed by pcr walking between unlinked, contiguous sequences, followed by sanger sequencing. in total, sanger reads were incorporated into the assembly. for the wesb genome, sequence data were initially assembled with short oligonucleotide alignment program and subsequently newbler assembler software was used to create a combined assembly with illumina reads. iterative mapping and assembly for gap elimination improved genome assemblies by targeted re-assembly of illumina reads to span gaps within scaffolds. to check for indels and single nucleotide polymorphisms, iterative correction of reference nucleotides was applied to the genome and appropriate corrections were made. all repeats over bp were checked to ensure that they were confirmed by at least two spanning read pairs. the incomplete wesb genome was sequenced within one scaffold, with an average coverage of ×  sequence analysis and annotation the complete nucleotide sequence and annotation of b. pilosicoli b and partial nucleotide sequence and annotation of b. pilosicoli wesb b have been deposited in genbank. scaffold sequences for unpublished genomes b. alvinipulli c1t and b. intermedia hb can be accessed from the authors via e-mail request. the draft genome scaffolds for b. aalborgii are available at the metahit website. sequence and protein analysis and annotation for the complete b. pilosicoli b and partial b. pilosicoli wesb genomes was as previously described for b. hyodysenteriae wa and b. pilosicoli 95/ unless otherwise stated. the multi locus sequence typing dendrogram of six brachyspira strains that have undergone genome sequencing, and three that are currently within unpublished genome sequencing projects being undertaken by the authors was calculated and constructed from the concatenation of gene nucleotide sequences. these concatenated sequences were aligned by clustalw and the maximum likelihood dendrogram was generated via mega. the condensed bootstrap maximum likelihood dendrogram was constructed from the general time reversible model with a gamma of and an assumption that a fraction of sites are evolutionarily invariable. the open source utility ‘freckle’ was used for sequence dot plotting. gene prediction and gene and protein sequence extraction was achieved using prodigal. initial coding region annotation was completed with an in-house updated compilation of the annotation pipeline autofact. resulting annotations were manually checked and edited where appropriate to be consistent with previous brachyspira genome annotation methodologies for comparative purposes. final annotations were assessed with the ncbi microbial genome submission tool. protein cluster analysis protein reciprocal blast similarity searches with a threshold maximum expected value 1e- were conducted with blastlinemcl, which is an implementation of the markov clustering algorithm for graphs. the granularity of the output cluster was set with an inflation value of. biolog phenotype microarray™ b. pilosicoli 95/ b and wesb were analysed using the biolog pm™ technology for high throughput substrate utilisation screening, which included unique carbon sources. pm panels and reagents were supplied by biolog and used according to the manufacturer’s instructions. briefly, under anaerobic conditions, bacterial cells were aseptically picked from the faba agar surface with a sterile cotton swab and suspended in ml of biolog inoculating fluid until a cell density of 40% transmittance was reached on a biolog turbidimeter. before the addition to pm microtitre plates, bacterial suspensions were further diluted into ml of if- in sterile water. pm microtitre plates were pre-incubated with two ageless® oxygen absorbers h prior to inoculation, at ambient temperature. the resuspended bacterial cells were pipetted into the 96-well plates at a volume of μl/well. prior to removal from the anaerobic chamber, one ageless® oxygen absorber and one co2gen compact sachet were attached per pm panel, which were then placed into oz whirl-pak® long-term sample retention bags with the open end heat-sealed. substrate utilisation was measured via the reduction of a tetrazolium dye to formazan, indicative of cellular respiration at 37°c. experiments were also run at 42°c, using bacteria cultured at this temperature. formazan formation was monitored at min intervals for h in omnilog apparatus. kinetic data were analyzed with omnilog-pm software. each experiment was performed at least twice per strain. it was noted that although tetrazolium dye reduction is indicative of cellular respiration, it can occur independent of cell growth. blank pm and pm controls were run, whereby if- was added in place of the bacterial cell suspension, to assess for abiotic reactions that occur in the anaerobic atmosphere across the h monitoring period. the following compounds were omitted from analysis due to the nature of the abiotic reactions that occurred in wells containing these compounds, under the conditions of the study: d-arabinose and l-arabinose, dihydroxyacetone, d-glucosamine, 5-keto-d-gluconate, l-lyxose, palatinose, d-ribose, 2-deoxy-d-ribose, sorbate, d-tagatose and d-xylose.  comparison of general genome features a dendrogram based on the mlst data for nine brachyspira strains highlighted the close relationship between the three b. pilosicoli strains, with b. aalborgi being distinct, but most closely related to b. pilosicoli and distantly related to b. hyodysenteriae, concordant with previous findings. the two b. intermedia strains appeared less closely related than might be expected, supporting reports of extensive diversity in this species based on results of pulse-field gel electrophoresis, and a previous mlst study which indicated that these two strains belong to distinct groups. it has been suggested that not all isolates with the b. intermedia phenotype should be assigned to this species. the general genome features of the three sequenced b. pilosicoli genomes are compared in table the g + c content of the b. pilosicoli genomes were very similar to each other, and to that of other chromosomes in the genus, which range from % to %. the complete genome sequence of b. pilosicoli b consisted of a, bp circular chromosome, whereas the incomplete wesb genome was larger, at, bp. the, bp genome of strain 95/ was the smallest of the three genomes. not only did the b. pilosicoli genomes show size variability, but also they were smaller than the genomes of the other sequenced species, apart from b. aalborgi 513t which our preliminary studies suggest is ~ mb. the relatively small size of the b. pilosicoli genomes is most likely due to them being members of a more specialised species that has undergone a high degree of reductive genome evolution. if this is the case, then b. pilosicoli is likely to be an older pathogen than other brachyspira species such as b. hyodysenteriae. such a reductive genome evolution may have allowed improved energy efficiency, and enhanced pathogenic potential. reductive genome evolution is particularly evident in obligate, intracellular bacterial pathogens and consistent with this, of the brachyspira species, only b. pilosicoli and b. aalborgi show long-term intimate associations with the surface of enterocytes, into which they interdigitate one of their cell ends. in addition to their small genomes, the sequenced b. pilosicoli strains lacked plasmids, whereas the genomes of the other fully sequenced brachyspira species have included plasmids. the comparison includes strains 95/, b and wesb. a the incomplete wesb strain genome was within one scaffold. b those genes with significant pid and/or query/target coverage hits; significance equals blastx/blastp pid of at least 25% and/or 75% query or target coverage. c assigned to ko via kegg automatic annotation server the disparity between the number of open reading frames and genome size between the b and wesb strains and the high number of non-significant percentage identity and coverage orfs in the wesb genome may be an artefact of the incomplete nature of this genome, which is the largest of the three strains. in 95/ % of orfs were assigned a kegg orthology, whereas only % and % of orfs were assigned in b and wesb, respectively. a lower proportion of orfs were matched in cog database for b and wesb compared to 95/ all three b. pilosicoli strains harboured the same number of transfer rna, rrna and transfer-messenger genes. the trna genes represented all amino acids and there were single copies of the 5s, 16s and 23s rrna genes. the rrf and rrl genes were co-located in all three b. pilosicoli genomes, with the rrs gene located approximately kb, kb and kb from the other rrna genes in the 95/ b and wesb genomes, respectively. this rrna gene organisation has been considered a distinguishing feature of brachyspira species, since other spirochaetes typically have differing copy numbers and organisations; however, similar arrangements to brachyspira have been detected in the spirochaete borrelia burgdorferi. situated between the rrs gene and rrfrrl cluster, which are either side of the oric, was the tmrna gene and nine of the total trnas that were otherwise dispersed throughout the genome. the origin of replication of the b. pilosicoli genomes was set according to the position of the oric and gc-skew pattern, as previously suggested; this was supported by the ori-finder program. the origin of replication was originally considered to be adjacent to the dnaa gene, however there was no association between the oric and dnaa genes in the b. pilosicoli b genome, as found in other brachyspira genomes. the arrangement of genes surrounding the dnaa gene was consistent between the b. pilosicoli strains, as with the other sequenced brachyspira genomes. the genes at the oric, although consistent between the b. pilosicoli strains, appear to vary extensively between the species. b. pilosicoli genome architecture on comparing b with 95/ four major genome rearrangement events appeared to have occurred, whereas two rearrangements were evident when comparing wesb to 95/. mobile genetic elements were found adjacent to or within close proximity of the sites where recombination events appear to have occurred in the b and wesb genomes. sixty-one and mges, including insertion sequence elements, integrases, recombinases and transposases were identified in the b and wesb genomes, respectively, compared to just four in the 95/ genome. the proportion of these features therefore seems to correlate with the extent of rearrangement within the genome. furthermore, multiple copies of an integrase gene that was absent from the 95/ genome were identified in the genomes of b and wesb. the lower number of copies in wesb may be an artefact of the genes not assembling in the incomplete genome. mges have been implicated in chromosomal rearrangements, gene disruptions resulting in pseudogenes, and eventual loss of genes, which may contribute to reductive genome evolution. species and strains that are undergoing or have recently undergone reductive genome evolution, and hence become more specialised pathogens, typically harbour large numbers of mges. there were fewest suspected pseudogenes found in 95/ and most in b, a finding that correlates to the number of mges and degree of genome rearrangements in these strains. of the total number of pseudogenes in each strain, %, % and % were in a cluster with orthologs in the other two strains in 95/ b and wesb, respectively. most strikingly, all shared clusters included either multiple b and/or wesb pseudogenes with a complete 95/ gene. differences in the number of mges in the three b. pilosicoli genomes may relate to their different stages of reductive genome evolution. strain 95/ which had the smallest genome, also had the fewest mges and this could be interpreted as indicating that the mges that induced the genome reduction in this strain have become lost. alternatively, mge expansion may not have occurred in 95/ to the same degree as in b and wesb, as mges are generally lost in a fragmentary manner by pseudogenisation. thus, it is unlikely that the 95/ genome, which has the fewest pseudogenes has been reduced in this way. on the other hand, the greater number of pseudogenes in the larger b and wesb genomes does suggest that they may be undergoing genome reduction. a possible explanation would be that these strains are in the initial stages of genome reduction, at the point at which mge expansion occurs. genome reduction and mge expansion is often associated with niche specialisation or host restriction, however b. pilosicoli are not considered host-restricted, and wesb, of human origin, has been shown also to infect chickens and pigs. b. pilosicoli is a highly recombinant species, and despite differences in genome arrangement and the number of pseudogenes, part of the difference in genome sizes simply reflects the carriage of different subsets of the pan-genome. a dot plot comparison of the three b. pilosicoli genomes revealed that the chromosomal rearrangements were arranged symmetrically around the origin or terminus of replication, highlighted by the x-patterns in the alignments. it has been postulated that symmetrical rearrangements occur because recombination events are determined by the replication forks that are approximately equal distance from the oric during bidirectional replication. it has also been argued that non-symmetrical rearrangements can be disadvantageous, and so genome rearrangements such as those found in the b. pilosicoli strains are a product of selection. despite the significant chromosomal rearrangements, genome alignments showed that the majority of genome sequence was shared between the three strains, with the larger b and wesb genomes possessing the greatest proportion of unique sequences. furthermore, a kb region, likely to have involvement in horizontal gene transfer, and that is partially conserved in all previously reported brachyspira genomes as well as enterococcus faecalis and escherichia coli, was identified in the b. pilosicoli b and wesb genomes. functional genome comparisons functional classifications assigned to each of the protein-coding genes of the three b. pilosicoli strains using the cog database showed that the general distribution of features into categories was similar for the three strains, and this highlighted their close relationship. despite having the smallest genome, b. pilosicoli 95/ possessed the greatest number of features in six categories. b contained the most features in eight categories, and wesb in one category. a striking difference between the strains was in the carbohydrate, amino acid and nucleotide transport and metabolism categories, with the larger wesb genome containing considerably more features than the b and 95/ genomes. in addition, compared to other brachyspira species the b. pilosicoli strains had a reduced number of features associated with inorganic ion transport and metabolism. athe number and percentage of the total genes within each of the genomes, assigned to each functional group are shownb. bthose genes with significant pid and/or query/target coverage hits; significance equals blastx/blastp pid of at least 25% and/or 75% query or target coverage. c the incomplete wesb strain genome was within one scaffold. global genome feature comparisons the three b. pilosicoli strains contained conserved genes, and these contribute to defining the b. pilosicoli pan-genome; this related to %, % and % of the total genes of the 95/ b and wesb genomes, respectively. as expected, there was a greater number of core genes between the strains of b. pilosicoli than between strains of different species; substantially fewer core genes were identified for b. hyodysenteriae wa b. pilosicoli 95/ and b. murdochii 56-150t. b. pilosicoli wesb harboured the greatest number of unique genes, with % of its genes being absent from the other genomes; b had a similar proportion, whereas 95/ had considerably fewer. b. pilosicoli b and wesb shared the greatest proportion of genes while b shared a greater percentage of its genes with 95/ than with wesb. global genome feature comparisons against other brachyspira species complete genome sequences of b. hyodysenteriae wa b. intermedia pws/at, b. pilosicoli 95/ and b. murdochii 56-150t have previously undergone comparative analysis. genome sequences of b. pilosicoli b and wesb can now be added to these comparisons, giving the first opportunity for a brachyspira intra-species genome comparison. a protein blastmatrix comparison was performed on the four previously sequenced genomes, the two newly-sequenced b. pilosicoli genomes and the draft genome scaffolds of b. aalborgi 513t, b. alvinipulli c1t and b. intermedia hb. not unexpectedly, the analysis revealed that the b. pilosicoli strains shared the greatest proportion of proteins. of b. pilosicoli strains, b had the greatest proportion of protein repeats relating to paralogs, despite not having the largest genome. overall, the non-pathogenic b. murdochii had the greatest proportion of protein repeats, perhaps relating to its large genome. high proportions of shared proteins highlighted the close relationships of b. hyodysenteriae with b. intermedia and b. murdochii. b. aalborgi shared the lowest percentage of proteins with other brachyspira species, consistent with evidence that this is the most divergent species. the percentage of the total cds that were identified in other genomes and the proportion of protein repeats within the genome, is shown. a cut-off e-value of 1e- was used. a incomplete genome currently within a genome sequencing project. b the incomplete wesb strain genome was within one scaffold. a protein markov clustering analysis of the six published brachyspira genomes, identified protein clusters shared by all six strains, the encoding genes of which may be used to define a brachyspira species pan-genome. this analysis revealed b. intermedia pws/at harboured the greatest number of clusters not found in the other sequenced brachyspira genomes and it has the largest genome. the greatest number of clusters shared only between two strains was with b. intermedia pws/at and b. hyodysenteriae wa, consistent with the close relationship of these species. of the b. pilosicoli strains, b and wesb shared the most unique protein clusters, and wesb also shared the greatest number of clusters with a non-b. pilosicoli strain, having clusters in common with b. intermedia and with b. murdochii. the b. pilosicoli strains collectively shared the most clusters with b. murdochii 56-150t, and fewest with b. hyodysenteriae wa, as noted previously. non-b. pilosicoli strains shared clusters, whereas the b. pilosicoli strains shared clusters, reflecting gene loss and genome reduction. unique to the three strains of b. pilosicoli of protein clusters present only in the b. pilosicoli genomes, % were hypothetical or unclassified. the majority of protein clusters were metabolic features, including an α-galactosidase, the activity of which is a distinguishing feature of the species. although it was suggested that b. pilosicoli had lost many transport-related genes during reductive evolution, clusters were for transport proteins. sialidase family-like protein genes unique to b. pilosicoli 95/ were also present in b and wesb; the products of such genes are produced by a variety of mucosal pathogens may play a role in colonisation or inducing tissue damage. clusters for an α- -fucosyl transferase, two membrane proteins and two glycosyltransferases were unique to b. pilosicoli and may contribute to host cell adherence. other b. pilosicoli-specific clusters were for an ankyrin repeat protein, a β-lactamase, two peptidases and phage proteins. unique and shared by two strains of b. pilosicoli of the b. pilosicoli strains, b and wesb shared most unique clusters. fewer clusters were shared with 95/ but of twelve clusters unique to 95/ and b all but n-acetyl mannosamine-6-phosphate 2-epimerase were hypothetical. six clusters were unique to 95/ and wesb, all lacking a specified function. of clusters unique to b and wesb, % were hypothetical; notable clusters shared between these strains were for a further sialidase-like protein and a peptidase. the glycine reductase complex locus of 95/ and b. murdochii 56-150t was identified in b and wesb, but with an additional atp-binding cassette -type glycine betaine transport component in a separate locus. moreover, a cluster for a transposase unique to b and wesb was detected. genes that were shared only by the larger b and wesb genomes and were absent from 95/ without apparent detriment, presumably have some specialised function that is not essential for survival. these features may have been lost from 95/ as they are not essential, or acquired in b and wesb, perhaps by hgt. unique to one strain of b. pilosicoli b. pilosicoli 95/ harboured the fewest and wesb the most unique features, correlating with their genome size. as discussed above, the 95/ strain may have become more specialised, having lost non-essential features through reductive evolution; alternatively, the absence of orthologs in other strains or species may suggest that these features have been acquired via hgt. of the strain-unique clusters, %, % and % were for hypothetical proteins in 95/ b and wesb, respectively. in 95/ unique clusters included a sodium/pantothenate symporter and an outer membrane lipoprotein with a potential role in host cell adherence. in b unique clusters included putative phage proteins, additional glycine reductase complex proteins and proteins involved in ascorbate metabolism and mannitol metabolism. in wesb, unique features included mannose/sorbose-specific phosphotransferase system components, fructose-specific pts components and a d-allose kinase. six unique phage-related features and an integrase were identified at two loci in the wesb genome. interestingly, each of the strains harboured unique genes for ankyrin proteins. comparison of potential virulence features virulence factor screening in brachyspira genomes was performed as described previously, but with the analysis encompassing a greater array of genes, particularly in categories relating to adhesion and/or surface proteins and mges. the greatest number of potential virulence features was in b however additional features may be identified in the wesb genome once it is completed. the analysis categorised the genes from the genomes of b. pilosicoli 95/ b and wesb. a the incomplete wesb strain genome was within one scaffold. b core lipooligosaccharide biosynthesis genes. lipooligosaccharides an rfbbadc cluster, encoding proteins for nucleotide sugar biosynthesis and with a suggested role in o-antigen assimilation in bacteria such as salmonella, was identified on the b. hyodysenteriae wa plasmid. although lacking this cluster, the three b. pilosicoli strains possessed rfba and rfbb, but rfbc was noted only in b and wesb. genes inferred to be involved in the biosynthesis of -dideoxyhexose, an o-antigen component of lipopolysaccharide, were found adjacent to the rfbc gene in b and wesb; both strains contained rfbf and rfbg, but rfbh was present only in wesb. the absence of such genes in the pathogenic strain 95/ suggests that they have a limited impact on virulence. chemotaxis and motility as with 95/ the two other b. pilosicoli strains possessed fewer chemotaxis genes than b. hyodysenteriae and b. murdochii. no mcpc genes were found in the three b. pilosicoli strains, despite their detection in the genomes of the other fully sequenced brachyspira species. the inter-species differences in the number and complement of chemotaxis-related genes may account for differences in their attraction to mucins and affinity to local host niches. no mcpa genes were identified in b but two copies were found in the other b. pilosicoli strains. the same complement of chemosensory transducer genes was identified in all three strains, as was the previously described cluster of seven such genes. differences in the number of chemotaxis-related genes between the three strains may translate from differences in genome size. this may denote a redundancy of features that can be lost without apparent detriment to long-term survival. the same flagella genes were shared by all three b. pilosicoli strains. adhesion and membrane proteins end-on attachment of the spirochaete to the luminal surface of the lower intestinal tract epithelia is characteristic of b. pilosicoli and b. aalborgi colonisation, and hence surface-associated proteins or lipoproteins are potential candidates for virulence. all lipoprotein genes in 95/ were found in b and wesb, but these strains also had a predicted secreted lipoprotein and a lipoprotein carrier protein, lola, which anchors lipoproteins to the outer membrane. the same complement of genes encoding variable surface proteins found in 95/ and the putative integral membrane virulence factor, mvin were noted in b and wesb. genes for outer membrane proteins with a potential role in virulence were identified, including bspa antigens, which may bind fibronectin and initiate a serological response, ompa proteins, similar to proteins implicated in leptospira virulence, and tia invasion determinants. genes encoding tolc were identified in all three b. pilosicoli strains, and this protein has been implicated in host invasion, virulence gene expression, and as an outer membrane component of efflux pumps. the periplasmic proteins identified were predicted to be primarily associated with other membrane proteins, and constitute abc transporters with putative roles in virulence. gene duplications were largely responsible for the greater number of inner membrane virulence factors in b and wesb, but since they were absent for 95/ they were unlikely to have significant impact on virulence. wesb harboured two additional genes encoding oppa, which has suggested involvement in spirochaete-host interactions in treponema denticola. genes encoding p-type atpase components, such as cada and znta, were noted in the three strains and these have been implicated in the ability of pathogens to sense and adapt to intracellular environments through heavy metal ion regulation, in addition to trk potassium transport components, required for invasion and intracellular growth of salmonella. genes encoding outer, periplasmic and inner membrane proteins that constitute transport systems implicated in bacterial virulence mechanisms were detected, such as polyamine abc-type transport, which is important for streptococcus pneumoniae pathogenesis, tonb-dependant iron transport, which is related to shigella dysenteriae virulence, and pts systems implicated in the virulence of mycobacterium tuberculosis and e. coli. genes were found encoding components of the acrab-tolc complex, which confers antibiotic resistance and survival in the gi tract, a ferrous iron transporter, feob, for iron acquisition, gut colonisation and intracellular survival of multiple enteropathogens, and a glutamine transporter gene, glnq, which has been implicated in streptococcus adherence and virulence. in the b. pilosicoli strains, an mgl operon similar to one with a proposed role in virulence expression in treponema pallidum was noted. multidrug efflux features were found in all three strains, which aside from drug resistance, are attributed with a range of roles in pathogenesis. genes for the sec pathway described in 95/, with no needle-associated genes were also noted in b and wesb, with an additional seca-like gene in wesb. host tissue degradation the complement of haemolysis-related genes was identical between the three strains. compared to previous analysis, other genes were detected including a haemolysin, previously undetected in 95/ and three streptolysin genes, sagb, sagc and sagd, involved in β-haemolysis and virulence in streptococci. a putative phospholipase/carboxylesterase was found only in b the three strains contained similar numbers of peptidases and proteases, which may participate in local degradation of host tissues, however 95/ lacked peptidase e, which had no effect on protein degradation in salmonella typhimurium, and hence this non-essential enzyme may have been lost through reductive evolution. oxidative stress genes related to oxidative stress were shared by the three strains. a partial bati operon was noted in all strains, in close proximity to one of the nox genes and consisted of batb, batc, batd and bate. the bata gene was in a distinct locus in the three strains. ankyrin-like protein there was little difference in the number of genes encoding ankyrin-like proteins between the b. pilosicoli strains, which may be involved in host cell interactions through their ability to bind host chromatin as in orientia. b. pilosicoli had consistently fewer of these genes than b. hyodysenteriae. phage and other mobile genetic elements outside of bacteriophage regions, four, and mges were identified in 95/ b and wesb, respectively, correlating with the extent of genomic rearrangements. the types and copy number of all mges in the b. pilosicoli genomes are detailed in additional file the region encoding genes related to the vsh- prophage-like gene transfer agent in 95/, was identified in b and wesb. this region was ~ kb in 95/ compared to ~ kb in b and wesb due to an insertion between genes encoding orfe and hvp containing genes for a monosaccharide-transporting atpase, an abc transporter-related protein, a rok family protein, an integrase in b only, and a periplasmic binding protein/laci transcriptional regulator. generally, these features had high homology with those in clostridium carboxidivorans, consistent with the finding that brachyspira share a high degree of gene similarity with clostridium, and supporting the notion that the bacteriophages exchange genetic material between species. in wesb, an additional cluster of vsh-1-associated genes, flanked by a phage terminase, was detected; the different genes in this region shared highest homology with c. carboxidivorans, b. hyodysenteriae, b. intermedia, b. pilosicoli and b. murdochii, suggesting that the gta had involvement in intra- and inter-species gene transfer. the bacteriophage that was identified in b. pilosicoli 95/, and in b. murdochii 56-150t, was also found in b and wesb. in b. pilosicoli, the bacteriophage size was proportional to genome size. hypothetical proteins encoded in this region were shared between 95/ and b however wesb contained four unique hypothetical genes. the b pp bacteriophage possessed a unique ankyrin repeat protein. an adenine-specific dna methyltransferase gene was present only in the wesb pp bacteriophage, adjacent to the dna methylase gene found in b. pilosicoli bacteriophages, but absent from those of b. murdochii 56-150t. two separate novel bacteriophages regions were found in b and wesb. the ~ kb pp bacteriophage contained seven phage proteins, six predicted proteins with homology to sequences of other brachyspira species, and unique hypothetical genes. the ~ kb pp bacteriophage shared all the components of the pi bacteriophage of b. intermedia pws/at, suggesting transfer of the bacteriophage in an inter-species hgt event. interestingly, pp was flanked by vsh- components, and hence the vsh- gta may be responsible for mediating the hgt event. two nuclease genes and a number of unique hypothetical genes in pp were not identified in pi clustered regularly interspaced short palindromic repeats, which provide bacteria with acquired resistance to bacteriophages, were only identified in the non-pathogenic b. murdochii 56-150t, which suggests a role for bacteriophages in brachyspira pathogenicity. b. pilosicoli b and b. intermedia pws/at did however possess a bacteriophage resistance protein which has been implicated in protecting against bacteriophages. central metabolism and correlation with phenotype analysis of the genomes of b. hyodysenteriae and b. pilosicoli has revealed that these species share many metabolic capabilities. in the current study the analysis of central metabolic pathway detection in brachyspira genomes was extended by application of biolog pm™ technology for phenotypic determination of carbon source utilisation of the b. pilosicoli strains. the utilisation of unique carbon compounds by the strains was screened, and their metabolic capabilities were found to be highly conserved. differences were found in the utilisation of just seven carbon sources, which were correlated with genotypic variations. possible explanations for the differences in phenotype relate to differences in genomic features. carbohydrate metabolism high proportions of the b. pilosicoli genomes were associated with carbohydrate transport and metabolism, and from metabolic pathway reconstructions it is evident that glycolysis constitutes a major backbone of energy production. collectively the b. pilosicoli strains utilised % of carbohydrate compounds tested, and more specifically % of hexose sugars. genes for enzymes involved in converting glucose-6-phosphate to ribulose-5-phosphate that were identified in b. hyodysenteriae wa, were found in the b. pilosicoli genomes. these features are likely to direct carbohydrate oxidation towards the non-oxidative pentose phosphate pathway, to generate reducing power required for biosynthetic pathways. b. pilosicoli is characterised by an absence of β-glucosidase activity, however a novel system for metabolising β-glucosides found in 95/ was also present in b and wesb, which, alongside specific pts systems, is likely to be involved in the utilisation of d-cellobiose and arbutin as carbon sources. despite lacking β-glucosidase, metabolism of β-glucosides may be important to b. pilosicoli virulence as this phenotype is associated with growth, adhesion and colonisation in other bacteria. of the disaccharides tested, % were utilised by the b. pilosicoli strains, whereas, of the oligosaccharides only dextrin was utilised, which is likely to be attributed to α-glucosidase activity. amino acid metabolism of the cog categories related to metabolism, the greatest proportion of the genome was related to amino acid transport and metabolism. phenotypic studies revealed that despite the high number of genes for amino acid/ oligopeptide transporters found in the genomes, only five of the tested amino acids were able to support b. pilosicoli as a sole carbon source. genes encoding enzymes to direct these amino acids towards pyruvate metabolism and hence energy production were identified, including alanine dehydrogenase, threonine aldolase, glycine hydroxymethyltransferase and l-serine dehydratase. moreover, a glycine reductase complex found in the b. pilosicoli strains, which catalyses the reductive deamination of glycine, forming atp, would be involved in the utilisation of glycine. a high proportion of amino acid metabolic features in b. pilosicoli were related to biosynthesis and potentially maintaining intermediates of the partial tricarboxylic acid cycle identified in this species, rather than catabolism to produce energy. l-glutamate and l-glutamine were insufficient to sustain b. pilosicoli as a sole carbon source; these amino acids are primary products of ammonia assimilation used in peptidoglycan, los and outer membrane protein biosynthesis, hence their metabolism is redirected to energy yielding pathways. the b. pilosicoli strains possessed genes for glutamate dehydrogenase, which catalyses the reversible synthesis of glutamate from α-ketoglutarate and ammonium. since α-ketoglutarate was able to sustain b. pilosicoli, the presence of a transporter for α-ketoglutarate and not glutamate may explain this phenotype. the ability to utilise certain amino acids as an energy source may have become redundant in brachyspira, which typically occupy the nutrient-rich lower gi tract, and thus associated features may have been lost through reductive evolution. nucleotide metabolism the b. pilosicoli strains were able to utilise three purine and two pyrimidine nucleosides tested as a sole carbon source. the enzymes suggested to complete a metabolic link between nucleoside and central metabolism in b. hyodysenteriae wa were identified in the b. pilosicoli strains. lipid metabolism despite the presence of enzymes involved in the β-oxidation of fatty acids, including a long chain fatty acid-coa ligase, no long chain fatty acids tested were utilised by b. pilosicoli as a carbon source; however, the short chain fatty acids, butyric acid and propionic acid, were utilised. glycerol was utilised as a carbon source, and genes for its metabolism were detected including those for a glycerol uptake facilitator, glycerol kinase and glycerol-3-phosphate dehydrogenase. the gene set required for fatty acid biosynthesis was incomplete in b and wesb, as it was in 95/.  in this study, we report the genome of b. pilosicoli strain b and the near complete genome of strain wesb. together with the previously reported 95/ genome, this allowed the first intra-species genome comparison within the genus brachyspira. our feature-based analysis revealed a high level of similarity between the three strains and identified genes that we suggest different strains of the spirochaete may have lost in a process of reductive genome evolution. sequence-based comparisons showed the majority of sequence was shared between the strains, with few unique regions; however, genome rearrangements were observed around the oric. mges were found associated to areas of rearrangements, and these features may be a factor that has driven or is driving reductive evolution. novel bacteriophages were identified in the newly-sequenced genomes, which displayed evidence of intra- and inter-species hgt, and these may have key practical applications for use in genetic manipulation. this is the first analysis of the spirochaete in a high-throughput phenotype screening tool, allowing correlation between genotype and phenotype. future work will focus on the application of this technology to a wider range of brachyspira species to validate genome differences, potentially providing a means by which these phenotypes can be used for rapid screening to infer genotypes and improve current diagnostic methods. with the increasing availability of brachyspira genome sequences, such technology should facilitate the validation of metabolic models based on genome sequence. abbreviations a: adenine; ais: avian intestinal spirochaetosis; abc: atp-binding cassette; act: artemis comparison tool; atp: adenosine triphosphate; bp: base pair; c: cytosine; cds: coding dna sequence; cog: cluster of orthologous genes; crispr: clustered regularly interspaced short palindromic repeats; ctab: cetyltrimethylammonium bromide; dna: deoxyribonucleic acid; faba: fastidious anaerobe blood agar; g: guanine; gi: gastrointestinal; gta: gene transfer agent; gtr: general time reversible; hgt: horizontal gene transfer; icorn: iterative correction of reference nucleotides; image: iterative mapping and assembly for gap elimination; indels: insertions/deletions; ise: insertion sequence element; kaas: kegg automatic annotation server; kb: kilobase; kegg: kyoto encyclopedia of genes and genomes; ko: kegg orthology; los: lipooligosaccharide; lps: lipopolysaccharide; mb: megabase; mcl: markov clustering algorithm; mge: mobile genetic element; mlst: multi locus sequence typing; ncbi: national center for biotechnology information; orf: open reading frame; pcr: polymerase chain reaction; pfge: pulse-field gel electrophoresis; pid: percentage identity; pis: porcine intestinal spirochaetosis; pm: phenotype microarraytm; pts: phosphotransferase system; rna: ribonucleic acid; rrna: ribosomal rna; snp: single nucleotide polymorphisms; soap: short oligonucleotide alignment program; t: thymine; tca: tricarboxylic acid; tmrna: transfer-messenger rna; trna: transfer rna. competing interests the authors declare that they have no competing interests. authors’ contributions ljm, djh and rml conceived the study. ljm and acd performed the genome sequencing and gap closure of b. pilosicoli b jp and akt were involved in genome sequencing of the wesb. djh, tl and ndp were involved in the genome sequencing of b. aalborgi 513t, b. alvinipulli c1t and b. intermedia hb ljm, mlb, djh, mib and acd conceived and designed comparative genomic studies. ljm and mlb performed comparative genomic analysis and analysed the data. ljm, ma, mjw and rml conceived and designed phenotyping experiments. ljm and ma conducted phenotyping experiments. ljm wrote the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 type and copy number of mobile genetic elements in the genomes ofb. pilosicoli 95/ b and wesb. a combination of protein markov cluster analysis and reciprocal blast searches against the conserved domain database was used to determine the copy number of each type of mge across the three b. pilosicoli genomes, using a cut-off e-value of 1e- the orf number, position, and size of all mges identified in each of the b. pilosicoli genomes is displayed. click here for file additional file 2 conserved and shared protein clusters between the six genome-sequencedbrachyspira strains. b. hyodysenteriae wa, b. intermedia pws/at, b. murdochii 56-150t and b. pilosicoli 95/, b and wesb a strains we included in the protein cluster analysis. a cut-off e-value of 1e- was used. click here for file additional file 3 comparison of the utilisation of unique carbon sources byb. pilosicoli95/ b and wesb. biolog phenotype microarray™ technology was employed for these studies and omnilog apparatus was used to detect formazan formation and hence, respiration due to utilisation of the carbon source; +, able to utilise the compound; -, unable to utilise the compound. click here for file acknowledgments the authors acknowledge receipt of funding for this study from the british egg marketing board research and education trust, the eu grant metahit, the royal college of veterinary surgeons, and murdoch university. we are grateful to richard rance, david harris, ruth gilderthorp, nicola corton, and other members of the sequencing and genome improvement teams at the wellcome trust sanger institute for their assistance in the preparation of the incomplete genome sequence of b. pilosicoli wesb.
in the first intra-species genome comparison within the brachyspira genus, we report the whole genome sequence of b. pilosicoli b an avian isolate, the incomplete genome sequence of b. pilosicoli wesb, a human isolate, and the comparisons with b. pilosicoli 95/ we also draw on incomplete genome sequences from three other brachyspira species. further application of such comparisons will improve understanding of the metabolic capabilities of brachyspira species. through comparison of the b and wesb genomes with the 95/ genome, features that we propose are non-essential due to their absence from 95/ include a peptidase, glycine reductase complex components and transposases. feature and sequence genome comparisons revealed a high degree of similarity between the three b. pilosicoli strains, although the genomes of b and wesb were larger than that of 95/. finally we report the first application of the high-throughput biolog phenotype screening tool on the b. pilosicoli strains for detailed comparisons between genotype and phenotype. genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. to date, the only available genome sequence of b. pilosicoli is that of strain 95/ a porcine isolate. the availability of multiple b. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer.
 the anaerobic spirochaete brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. to date, the only available genome sequence of b. pilosicoli is that of strain 95/ a porcine isolate. in the first intra-species genome comparison within the brachyspira genus, we report the whole genome sequence of b. pilosicoli b an avian isolate, the incomplete genome sequence of b. pilosicoli wesb, a human isolate, and the comparisons with b. pilosicoli 95/ we also draw on incomplete genome sequences from three other brachyspira species. finally we report the first application of the high-throughput biolog phenotype screening tool on the b. pilosicoli strains for detailed comparisons between genotype and phenotype.  feature and sequence genome comparisons revealed a high degree of similarity between the three b. pilosicoli strains, although the genomes of b and wesb were larger than that of 95/. genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. through comparison of the b and wesb genomes with the 95/ genome, features that we propose are non-essential due to their absence from 95/ include a peptidase, glycine reductase complex components and transposases. novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer. phenotypic differences predicted from genome analysis, such as the lack of genes for glucuronate catabolism in 95/ were confirmed by phenotyping.  the availability of multiple b. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. in addition, phenotype screening allowed determination of how genotypic differences translated to phenotype. further application of such comparisons will improve understanding of the metabolic capabilities of brachyspira species. brachyspira pilosicolispirochaetecolitiszoonosiswhole genome sequencinggenome comparisonhorizontal gene transferphenotype microarray™ 