BACKGROUND
chlamydia are widely distributed and highly successful bacterial pathogens that only replicate inside eukaryotic cells, which is a key factor in their ability to remain hidden from the host immune response and to cause persistent infections  <cit> .
seemingly contrary to this highly adapted intracellular lifestyle, the majority of the eleven currently described species in the genus chlamydia, can infect multiple host species.
the best example of this is chlamydia psittaci, primarily recognised as an avian pathogen, but is also known to infect and cause disease in cattle, sheep, pigs and horses, while posing a zoonotic risk for humans  <cit> .
other species such as c. pecorum, c. abortus and c. pneumoniae, can infect multiple hosts as well .
for each of these species, there is little understanding of the mechanisms involved in the adaptation to different niches, especially considering that different strains within a species share nearly identical genomes with a dna sequence similarity of > 98%  <cit> .
the adaptation of bacterial pathogens to specific niches is driven by the evolutionary “arms race” that takes place between the host and the bacterium  <cit> .
the host’s immune system can provide a selective pressure for the accumulation of mutations in the genes of the bacterium.
although the majority of polymorphisms within genes cause synonymous changes, which are indicative of purifying selection for the encoded protein to maintain its current function and structure, a small number of genes will accumulate non-synonymous substitutions that result in protein variation  <cit> .
recent data analysing the accumulation of these single nucleotide polymorphisms  in c. trachomatis has emphasized that this will be key to understanding the host adaptation of each the chlamydial species  <cit> .
an interesting example of chlamydial pathogenicity and intra-species host adaptation potentially lies in an analysis of the animal pathogen, c. pecorum.
c. pecorum is a widespread pathogen of economically important livestock species such as cattle, sheep, goats and pigs.
in livestock, infections of c. pecorum manifest as a range of diseases such as polyarthritis, pneumonia, conjunctivitis and encephalomyelitis, while also being linked to diseases of the gastrointestinal and urogenital tracts  <cit> .
beyond these reports, perhaps the most common outcome of c. pecorum infection is the absence of disease symptoms  <cit> .
however, even in animals that are asymptomatic, there is evidence for a subclinical pathological effect  <cit> .
while c. pecorum infections in livestock are of economic concern to primary producers globally, the best example of the pathogenic potential of this obligate intracellular bacterium is through the ongoing association between c. pecorum infection of the koala, a native australian marsupial, and debilitating ocular and urogenital tract diseases  <cit> .
in this capacity, c. pecorum is a key threatening process to the long-term survival of this native species  <cit> .
in the absence of genome sequence data for the representative strains infecting each host species, efforts to understand the genetic relationship and host adaptation of c. pecorum strains infecting livestock and koalas has centered on the use of molecular typing methods.
a previous study that investigated the molecular epidemiology of c. pecorum using a multi locus sequence analysis  typing scheme on both livestock and koala c. pecorum strains suggested that there is limited genetic diversity between strains and that they share a common ancestor  <cit> .
this finding was in contrast to previous reports utilising an alternative molecular target, ompa, encoding the chlamydia major outer membrane protein, a porin responsible for nutrient transfers, attachment and structural support  <cit> .
however, the ompa gene is likely to be evolving at a faster rate than the rest of the genome since it is located on the cell surface and, as a result, the gene is under high positive selection  <cit> .
more recently, three c. pecorum strains from ruminant animals were sequenced revealing a high level of sequence similarity and gene content  <cit> .
in this setting, it is vital to employ additional whole genome sequencing of non-ruminant c. pecorum strains in order to fully understand the genetic diversity between c. pecorum strains and to identify genes that could be involved in adaptation to different hosts.
in this study, the genome sequences of c. pecorum strains isolated from three koalas and a sheep were compared to identify genes that could play a potential role in adaptation to different hosts and to gain insight into genetic diversity and evolution.
in addition, the koala c. pecorum genomes were compared against each other in order to examine the genetic diversity between conjunctival and urogenital c. pecorum strains.
the survey of the broader diversity of selected regions of the c. pecorum genome in a range of clinical samples from australian livestock and koalas was also conducted.
RESULTS
phylogenetic relationship of c. pecorumas revealed by whole genome sequencing
the draft genomes of three c. pecorum strains isolated from koalas with clinical symptoms  each comprise a single scaffold of approximately  <dig>  mbp .
the chromosomes have a gc content of 41% and an average read coverage of 1500×.
the fourth draft genome was from the c. pecorum vr <dig>  strain that was isolated from the joint fluid of a sheep suffering from polyarthritis in the usa.
none of the strains sequenced possessed the cryptic chlamydial plasmid.
the three koala c. pecorum strains have been typed based on the ompa gene and each strain represents a different ompa serotype   <cit> .table  <dig>
c. pecorum
draft genomes sequenced in this study
genotype
source
year of isolation
country
isolation tissue
disease
total number of reads
number of contigs
n50
assembly length
number of cds
% gc content
to determine the relationship between the four sequenced c. pecorum strains, a phylogenetic tree was constructed based on the sequence of  <dig> conserved genes across eight chlamydia species.
the inferred topology of the phylogenetic tree  is consistent with other phylogenetic analysis of chlamydia species  <cit> .
the phylogenetic reconstruction indicates that the closest relative of c. pecorum is c. pneumoniae.
the phylogenetic analysis revealed that the three koala c. pecorum strains are part of a separate lineage from the ruminant c. pecorum strains sequenced in this study and those previously described  <cit> .
the two urogenital c. pecorum strains  grouped more closely together, away from the conjunctival c. pecorum iptale isolate.figure  <dig>
phylogeny of
chlamydia
species.
the maximum-likelihood tree was reconstructed using phyml with the gtr substitution model based on the nucleotide sequence of  <dig> orthologous genes.
bootstrap values are shown as percentages of  <dig> replicates.
the scale bars represent the number of substitutions per site.
the gray box shows the within-species phylogeny of the sequenced c. pecorum strains and is magnified.
whole genome comparison revealed a high level of gene conservation between c. pecorumstrains from koalas and livestock
whole genome comparisons confirmed that the four draft c. pecorum genomes included in this study are highly conserved and syntenic, with similar gene content.
comparisons against the genomes of other chlamydia species revealed that c. pecorum contains several regions that differ significantly to other chlamydia species .
the variable regions include the chlamydial plasticity zone , a genomic island located near the terminus of replication and a cluster of genes that encode polymorphic membrane proteins .
like the other c. pecorum genomes studied to date  <cit> , the four draft c. pecorum genomes sequenced encode a near intact tryptophan biosynthesis operon but they are missing the trpe/g genes.
the genomes of the c. pecorum strains are highly conserved with a dna sequence identity ranging from  <dig>  to  <dig> %.
the conservation of gene content and sequence similarity between c. pecorum strains is consistent with other chlamydia species  <cit> .
single nucleotide polymorphisms contribute to the genetic diversity between the c. pecorumgenomes
although the overall gene content is conserved between each c. pecorum genome, there are a significant number of snps that contribute to variation between the c. pecorum strains.
the number of predicted snps between livestock and koala c. pecorum strains ranges from  <dig> to  <dig> snps.
between the livestock c. pecorum strains the number of polymorphisms range from  <dig> to  <dig> snps with the exception of the phylogenetically distinct c. pecorum pv <dig> strain which differs from the other livestock strains by  <dig>  <dig> snps.
comparisons of the three koala c. pecorum genomes revealed that there are 1461- <dig> snps .
the distribution of snps was plotted across the whole genome using a custom r script to reveal regions that are accumulating mutations .
the region with the largest number of snps in the c. pecorum genomes is a 36 kb gene cluster encoding  <dig> pmps.
between c. pecorum e <dig> and c. pecorum vr <dig>  there are  <dig> synonymous and  <dig> non-synonymous snps within the pmp cluster, with the majority of snps found in the pmpg subfamily .
between the livestock c. pecorum e <dig> and koala c. pecorum mc/marsbar isolates, this pmp cluster contains  <dig> synonymous and  <dig> non-synonymous snps making this gene cluster the most variable region between c. pecorum strains from different hosts.
comparison between the genomes of koala c. pecorum strains indicates that most of the variation observed in this region occurs with the pmpg genes.
the adaptation to different hosts is likely the result of small and subtle changes that occurred in the genomes rather than differences in the gene content.table  <dig>
total snps in
c. pecorum
genomes using
c. pecorum
mc/marsbar as a reference
distribution of snps in
c. pecorum
genomes.
the histograms show the number of snps in relation to the genomic positions between different c. pecorum genomes with a window size of 10 kb.
the top left hand graph shows the snp distribution between the koala c. pecorum mc/marsbar genome and the livestock c. pecorum e <dig> genome while the bottom left hand graph shows the number of snps between two koala c. pecorum strains .
the right hand graphs display the distribution of snps observed between the two usa livestock c. pecorum strains  and two of the european livestock strains .
the red boxes mark the positions of the two snp hotspot regions identified in the koala c. pecorum strains.
the first polymorphic region starts at 300 kb and is approximately 77 kb in length and the second region includes the plasticity zone.
visual representation of the cluster of pmp genes in
c. pecorum
genome.
the blue arrows represent the pmps and the orange arrows represent hypothetical proteins.
the vertical lines mark the location and type of snps .
the gaps in the genome assembly are marked by the gray rectangles and sizes of these gaps were estimated using pair-end read data.
the base coordinates of this region in c. pecorum e <dig> are  <dig> .
<dig>  the image was generated using easyfig  <cit> .
snps within koala c. pecorumstrains highlight genes potentially associated with host adaptation to marsupials
the distribution of snps across the c. pecorum genomes revealed that there are two regions in the koala and european livestock c. pecorum strains that are hotspots for the accumulation of snps .
the corresponding highly polymorphic regions do not show a similar level of diversity between the sheep and cattle c. pecorum strains isolated from usa.
the first region, located near the origin of replication, is approximately  <dig> kbp in length.
a detailed analysis of this region revealed the presence of genes that encode for putative type iii effector proteins, chaperones and other potential virulence-related proteins.
also found here is a cluster of genes that encode the inner membrane components of the type iii secretion system .
in contrast to many other bacterial pathogens where the genes for the t3ss apparatus are clustered together on pathogenicity islands, chlamydia t3ss genes are located in four separate clusters disseminated throughout the genome  <cit> .
in addition, there is a sulfur transfer system encoded by the sufbcd operon and sufs similar to a system in e. coli; the rest of the genes located within this region are predicted to be involved in various metabolic processes  <cit> .
the  <dig> kbp region is a hotspot for snps in both the australian koala strains  and european livestock strains  but this region is conserved between the two usa livestock strains  .
between c. pecorum e <dig> and c. pecorum mc/marsbar, we observed  <dig> synonymous and  <dig> non-synonymous snps located within genes in the  <dig> kbp region.
the majority of the non-synonymous snps are associated with putative virulence factors, including the type iii effector proteins.
a selection analysis was carried out on each gene within this region between c. pecorum e <dig> and c. pecorum mc/marsbar to determine if any genes are under positive selection  <cit> .
between the livestock and koala strains, most of the genes in this region are under purifying selection with a dn/ds ratio of less than  <dig>  however, we observed three genes that are under positive selection, including one of the putative type iii effectors  and two conserved hypothetical proteins .
within the koala c. pecorum strains themselves, all the genes in this region are under purifying selection with mostly synonymous snp differences, with the exception of the srpa <dig> gene .
the srpa <dig> protein has 100% sequence identity between c. pecorum mc/marsbar and c. pecorum iptale, however it only shares 85% sequence identity to the srpa <dig> homolog from c. pecorum dbdeug .
the c-terminal domain of srpa <dig> was conserved across all the c. pecorum genomes and it is the n-terminal domain that is variable even between c. pecorum koala strains.figure  <dig>
visual representation of the  <dig> kbp snp hot-spot region.
the arrows represent genes and are coloured according to predicted functions inferred by blast searches.
the vertical lines mark the location of snps with the green lines indicating synonymous snps and the red lines indicating non-synonymous snps.
this region includes the sufbcd operon, which encodes an iron uptake system and a gene cluster that encodes the inner membrane components of the type iii secretion system.
also encoded in this region are several putative type iii effectors and chaperones.
the base coordinates of this region in c. pecorum e <dig> are  <dig> .
<dig>  the image was created with easyfig  <cit> .
the second genomic region with a high frequency of snps is the pz .
however, it should be noted that in the koala c. pecorum genomes, there are assembly gaps within the pz and that the number of predicted snps in the pz is likely an under estimation.
paired-end data is used to estimate the size of the gaps, which suggest that the gene content of the pz is identical in each of the genomes.
in c. pecorum, the pz is 42 kb with the acetyl-coa-carboxylases genes  located at the 5′ boundary and the guaab and add genes at the 3′ end  <cit> .
the pz in koala c. pecorum strains encodes two cytotoxin genes in tandem, in this study designated as toxa and toxb.
the pz also contains several other genes that encode proteins linked to pathogenesis, including the mac/perforin domain protein and five phospholipase d   <cit> .
the presence of two cytotoxic genes make the c. pecorum pz unique compared to the pz from other chlamydia species.
although of similar size, the toxa and toxb genes share only 43% sequence identity on the amino acid level with several conserved motifs.
the n-terminal regions of the toxa/b genes contain a catalytic glycosyltransferase domain.
interestingly, in the genome of c. pecorum mc/marsbar, a snp  in the toxb gene resulted in a premature stop codon at position  <dig>  truncating the gene.
the presence of the premature stop codon was confirmed with pcr amplification and sequencing of the toxb gene in the c. pecorum mc/marsbar strain, as well as the other two koala and a sheep strain.
the toxb gene is only truncated in c. pecorum mc/marsbar and full-length homologs are present in the other four c. pecorum genomes.figure  <dig>
visual representation of the
c. pecorum
plasticity zone.
the arrows represent genes and they are colored according to function, which is illustrated in the legend.
the vertical lines mark the location and type of snps .
the gaps in the genome assembly are marked by the gray rectangles and sizes of these gaps were estimated using pair-end read data.
the base coordinates of the plasticity zone in c. pecorum e <dig> are  <dig> .
<dig>  the image was generated using easyfig  <cit> .
the presence of a limited number of pseudogenes further highlights genetic variation between koala c. pecorumstrains
in addition to toxb, another five pseudogenes were identified in the three koala c. pecorum genomes, providing additional evidence of genetic diversity between c. pecorum strains isolated from koalas .
the pseudogenes in c. pecorum were caused by premature stop codons within the gene sequences as a result of snps.
c. pecorum iptale has three pseudogenes, one being the pyre gene  that codes for an orotate phosphoribosyltransferase.
the functions of the other two pseudogenes in c. pecorum iptale are unknown, although one of the genes  contains a signal peptide at the n-terminus suggesting that the encoded protein targets a secretory pathway.
the genomes of c. pecorum mc/marsbar and c. pecorum dbdeug each have a single pseudogene that contains transmembrane domains; however their actual functions are unknown.
all six pseudogenes, including toxb, identified in the koala c. pecorum genomes are intact in the all the livestock c. pecorum genomes.table  <dig>
pseudogenes caused by premature stop codons in
c. pecorum
strains isolated from koalas
some but not all predicted koala c. pecorumpseudogenes are widely distributed in c. pecorumstrains detected from across the koala’s host range
in a preliminary investigation into the broader genetic diversity of c. pecorum in australian animals, we pcr amplified and sequenced partial regions of the six pseudogenes identified in the koala c. pecorum genomes from  <dig> c. pecorum pcr positive samples collected from the  conjunctival and urogenital tract sinuses of koalas from populations in queensland, new south wales, victoria and south australia ; and  conjunctiva and rectums of australian sheep  .
the list of c. pecorum strains chosen was based on pcr-positive swab samples that have sufficient chlamydial dna for multiple pcr amplification and to ensure an even distribution of strains from each australian state.
out of the six pseudogenes identified from the genome sequences, four were also found to be pseudogenes in the clinical samples.
the toxb gene was found to be truncated in some  but not all of the koala c. pecorum positive samples from populations in south-east queensland, south australia and victoria.
indeed, we were able to identify  <dig> unique toxb gene fragment sequences amongst the collection examined, with sequences varying in similarity between  <dig>  –  <dig> % across the livestock and koala samples .
similarly, one of the pseudogenes  from c. pecorum iptale was also truncated in three urogenital c. pecorum positive swabs from three different koala hosts, however the truncation is caused by a different mutation than in c. pecorum iptale.
the pseudogene  from c. pecorum mc/marsbar was found to be truncated in another two c. pecorum positive samples collected from the cloaca and urethra of a koala in victoria and nsw, respectively.
the pseudogene  from c. pecorum dbdeug was also confirmed to be a pseudogene in seven c. pecorum positive samples from the conjunctiva and genital tracts of koalas in queensland only.
interestingly, the remaining two pseudogenes  from c. pecorum iptale were intact in all koala c. pecorum samples analysed.
although only a smaller sample set was analyzed, the evidence that all pseudogenes identified and confirmed in the koala c. pecorum genomes and clinical samples appeared to be intact in australian livestock was notable.
while this observation appeared to further distinguish koala from livestock c. pecorum strains, it is also worth noting that a number of the new partial gene sequences identified in this analysis for each of these marker genes were otherwise identical .
this was confirmed with a phylogenetic tree based on the concatenated sequences of five of the six pseudogenes – pyre gene sequences were difficult to amplify for a number of samples – not showing any clear distinction between koala and livestock strains from europe, the usa and australia .figure  <dig>
bayesian phylogenetic tree of concatenated sequence of five pseudogene fragments.
the tree was constructed using mrbayes hky <dig> model based on the concatenated sequences of the five pseudogenes from  <dig> c. pecorum detected in koalas and livestock.
posterior probabilities >  <dig>  are displayed on tree nodes.
the colouring of the strain name as per the legend indicates the location of each strain.
the following abbreviations denote anatomical site of the host where the swab sample was collected: rec , jnt , le , ure , pen , vag  and clo .
discussion
while the initial sequencing of chlamydial genomes has provided important insights into the general biology of the chlamydiae, reconciling the minor genetic differences between different strains of the same species with particular diseases, hosts and sites of infection has been more challenging.
c. pecorum is a useful model to investigate adaptation to different niches since it is a major pathogen with a broad host range and tissue tropisms .
like most chlamydia species, c. pecorum genomes are conserved in terms of gene content and order  <cit> .
this high degree of gene conservation is believed to be the result of chlamydia’s intracellular lifestyle, which reduces exposure to frequent lateral gene transfer events that often cause phenotypic changes in other bacterial genera  <cit> .
therefore, small variations in the c. pecorum genomes are the likely causes for different phenotypic traits that are observed in different strains.
when it comes to investigating the genetic diversity of c. pecorum, one of the limiting factors has been the lack of genome sequences for c. pecorum strains isolated from different animals, in particular strains isolated from koalas.
in this study we examined the draft genomes of three c. pecorum strains that were isolated from koalas in south-east queensland, australia.
each of the three koala c. pecorum genomes represents a different genotype, based on allelic differences in the ompa gene.
also sequenced, as part of this study, is the genome of c. pecorum vr <dig> , a strain that was isolated from the joint of a sheep suffering polyarthritis in the usa.
the c. pecorum vr <dig> genome along with the four publically available c. pecorum genomes serve as references for comparison to the koala c. pecorum genomes, to assess the level of genetic diversity between c. pecorum strains  <cit> .
the pz, the pmp gene cluster and the  <dig> kbp region near the origin of replication are hotspots for snps accumulation in the australian koala strains and the european livestock strains.
a similar high degree of snps is also observed in these same regions in c. trachomatis genomes .
the gene content of the pz and the  <dig> kbp region were also compared between c. trachomatis and c. pecorum using discontinuous megablast  <cit> .
while the gene content of the pz is significantly different between c. pecorum and c. trachomatis, the only difference in the  <dig> kbp hotspot region between both species is that the putative virulence genes in c. pecorum are absent in c. trachomatis.
the pz and the  <dig> kbp region are highly conserved between the two american c. pecorum strains but additional genome sequencing of isolates from the usa is needed to confirm if this level of conservation is unique to usa strains.
identification of snps between c. pecorum genomes from livestock and koalas has provided insight into which genes are likely to be involved in host adaptation.
the most rapidly evolving genes in c. pecorum are the genes coding for the pmps, a family of proteins that may be important for adhesion of chlamydia to host cells  <cit> .
in particular, the pmpg subfamily represents the most variable class of pmps in c. pecorum, which is also the most rapidly evolving pmp class in c. psittaci
<cit> .
despite the variation in nucleotide sequences, the members of the pmp family possess a conserved domain structure that includes the c-terminal autotransporter beta-domain, a central domain unique to this family of proteins and a n-terminal domain that is involved in adhesion  <cit> .
pmps share sequence similarity to the autotransporter class of proteins, which have a diverse array of functions and often play an essential role in pathogenesis, thus the observed variation could also contribute to immune evasion  <cit> .
this study has identified several novel putative virulence genes that could be specifically involved with adaptation to different hosts.
these virulence genes include a putative surface protein srpa <dig> and two putative type iii effectors.
type iii effectors are virulence factors often involved in interactions with host cellular proteins in order to enhance the survival of the bacteria  <cit> .
amino acid changes introduced by non-synonymous snps could alter the binding domains of the effectors, altering their affinity to cellular targets.
the other potential virulence factor of interest is the putative surface protein srpa1; the observed variation in the protein sequence could alter binding and/or recognition to surface receptors of host cells, however, experimental confirmation is obviously required for further investigations.
however, the accumulation of non-synonymous snps in srpa <dig> and the two putative effector genes could also be due to a selective pressure for avoiding antibody and cellular immune responses.
our comparison of the c. pecorum genomes revealed the presence of six pseudogenes in the strains isolated from koalas, one of these pseudogenes being the toxb gene located in the pz in c. pecorum mc/marsbar.
c. pecorum typically possesses two full-length cytotoxin genes in the pz, designated toxa and toxb.
the full-length chlamydia cytotoxin is similar to the large clostridial cytotoxin  from clostridium difficile, which also has the glycosyltransferase domain in the n-terminal region of the protein.
this domain has been shown to interfere with eukaryotic cells by glycosylating gtp binding proteins of the ras superfamily, inactivating them and leading to disassembly of the actin cytoskeleton  <cit> .
the lcts also include a domain for binding to the surface receptors of eukaryotic cells and a transmembrane segment that is involved in translocation into the cytoplasm  <cit> .
c. pecorum mc/marsbar has a full-length toxa gene and a shorter toxb gene that has been truncated by a premature stop codon.
the truncation of toxb was observed in  <dig> of the  <dig> c. pecorum positive swabs samples for which partial gene sequences could be amplified from wild koala, suggesting that it could be a recent mutation since the remaining sequences identified were otherwise intact.
it is plausible that the c-terminal domain of the toxb gene is being deleted to reduce the energetic cost of maintaining the full-length gene  <cit> .
out of the other five pseudogenes in the koala c. pecorum genomes, only the pyre gene has a predicted function.
the gene pyre encodes an orotate phosphoribosyltransferase, an enzyme that is part of the de novo pyrimidine biosynthesis pathway  <cit> .
chlamydia spp.
cannot synthesise pyrimidine nucleotides de novo as key genes involved in this pathway are missing  <cit> , but instead must rely on a salvaging pathway that involves transporting ribonucleotides directly from the host cell  <cit> .
the presence of the pyre gene could be a remnant from when chlamydia was able to synthesise pyrimidine de novo; this is supported by the observed fragmentation of pyre in c. pecorum iptale, indicating gene decay.
interestingly, despite this potentially important observation, the pyre gene was found to be intact in all koala c. pecorum strains that were screened, raising questions over whether this snp may have resolved during laboratory passage.
in addition to this pseudogene, there are two other pseudogenes that are unique to the c. pecorum iptale conjunctival isolate, compared to the sequenced urogenital dbdeug and mc/marsbar strains.
however, while one of these pseudogenes was found to be intact in all samples tested, the other  was confirmed to be a pseudogene in three koala urogenital samples from the same geographic region and therefore both of these genes are unlikely to be involved in tissue tropism.
sequence alignments of the pseudogene fragments revealed that some genotypes are specific to c. pecorum detected in australian livestock and koala hosts .
however, a phylogenetic analysis of the concatenated pseudogene sequences showed that, collectively, there was no host specific separation in contrast to our whole genome phylogenies .
the observation that  multiple c. pecorum genotypes can be circulating in a single population of animals; and  individual genotypes can be found across multiple populations of koalas or livestock is otherwise consistent with our previous fine-detailed molecular epidemiological investigations  <cit> .
CONCLUSIONS
while the primary focus of this study was to examine the genetic differences potentially associated with c. pecorum host adaptation, the observed accumulation of genetic changes in koala c. pecorum strains provides an opportunity to speculate on the origin of this pathogen in the koala.
the koala c. pecorum genomes contain a small number of pseudogenes and gene truncations, which are otherwise intact in the livestock genomes.
these mutations suggest that c. pecorum is evolving to adapt to the koala through the loss of genes that are no longer necessary.
although our genome phylogenies do not yet support this, the most parsimonious explanation for this observation is that koala c. pecorum strains have derived from livestock strains and are undergoing genetic changes to better adapt to the new host.
interestingly enough, if this is true, then it would be the reverse of what we have previously observed for koala chlamydia pneumoniae, whereby the genome of the koala strain was found to be largely intact compared to human strains, suggesting that the koala strains were ancestral  <cit> .
in order to validate that koala c. pecorum strains are indeed derived from livestock strains it is necessary to reconstruct the evolutionary history of c. pecorum using phylogenetic and molecular clock analyses.
however, additional c. pecorum strains will need to be sequenced in order to fill in the missing links from this comparison including analysis of c. pecorum isolates from australian livestock.
indeed, a limitation of the phylogenetic analysis performed in this study is the small number of available c. pecorum genomes.
c. pecorum pv <dig> was expected to cluster together with c. pecorum w <dig> and c. pecorum p <dig> based on the geographic location of these strains but the pv <dig> strain is phylogenetically distinct from the other c. pecorum strains.
therefore, it is important to sequence more c. pecorum strains in order to fully understand the phylogenetic structure of this species but also to learn more about the role of the observed limited gene variation in disease pathogenesis and tissue and host adaptation.

 chlamydia are widely distributed and highly successful bacterial pathogens that only replicate inside eukaryotic cells, which is a key factor in their ability to remain hidden from the host immune response and to cause persistent infections. seemingly contrary to this highly adapted intracellular lifestyle, the majority of the eleven currently described species in the genus chlamydia, can infect multiple host species. the best example of this is chlamydia psittaci, primarily recognised as an avian pathogen, but is also known to infect and cause disease in cattle, sheep, pigs and horses, while posing a zoonotic risk for humans. other species such as c. pecorum, c. abortus and c. pneumoniae, can infect multiple hosts as well. for each of these species, there is little understanding of the mechanisms involved in the adaptation to different niches, especially considering that different strains within a species share nearly identical genomes with a dna sequence similarity of > 98%. the adaptation of bacterial pathogens to specific niches is driven by the evolutionary “arms race” that takes place between the host and the bacterium. the host’s immune system can provide a selective pressure for the accumulation of mutations in the genes of the bacterium. although the majority of polymorphisms within genes cause synonymous changes, which are indicative of purifying selection for the encoded protein to maintain its current function and structure, a small number of genes will accumulate non-synonymous substitutions that result in protein variation. recent data analysing the accumulation of these single nucleotide polymorphisms in c. trachomatis has emphasized that this will be key to understanding the host adaptation of each the chlamydial species. an interesting example of chlamydial pathogenicity and intra-species host adaptation potentially lies in an analysis of the animal pathogen, c. pecorum. c. pecorum is a widespread pathogen of economically important livestock species such as cattle, sheep, goats and pigs. in livestock, infections of c. pecorum manifest as a range of diseases such as polyarthritis, pneumonia, conjunctivitis and encephalomyelitis, while also being linked to diseases of the gastrointestinal and urogenital tracts. beyond these reports, perhaps the most common outcome of c. pecorum infection is the absence of disease symptoms. however, even in animals that are asymptomatic, there is evidence for a subclinical pathological effect. while c. pecorum infections in livestock are of economic concern to primary producers globally, the best example of the pathogenic potential of this obligate intracellular bacterium is through the ongoing association between c. pecorum infection of the koala, a native australian marsupial, and debilitating ocular and urogenital tract diseases. in this capacity, c. pecorum is a key threatening process to the long-term survival of this native species. in the absence of genome sequence data for the representative strains infecting each host species, efforts to understand the genetic relationship and host adaptation of c. pecorum strains infecting livestock and koalas has centered on the use of molecular typing methods. a previous study that investigated the molecular epidemiology of c. pecorum using a multi locus sequence analysis typing scheme on both livestock and koala c. pecorum strains suggested that there is limited genetic diversity between strains and that they share a common ancestor. this finding was in contrast to previous reports utilising an alternative molecular target, ompa, encoding the chlamydia major outer membrane protein, a porin responsible for nutrient transfers, attachment and structural support. however, the ompa gene is likely to be evolving at a faster rate than the rest of the genome since it is located on the cell surface and, as a result, the gene is under high positive selection. more recently, three c. pecorum strains from ruminant animals were sequenced revealing a high level of sequence similarity and gene content. in this setting, it is vital to employ additional whole genome sequencing of non-ruminant c. pecorum strains in order to fully understand the genetic diversity between c. pecorum strains and to identify genes that could be involved in adaptation to different hosts. in this study, the genome sequences of c. pecorum strains isolated from three koalas and a sheep were compared to identify genes that could play a potential role in adaptation to different hosts and to gain insight into genetic diversity and evolution. in addition, the koala c. pecorum genomes were compared against each other in order to examine the genetic diversity between conjunctival and urogenital c. pecorum strains. the survey of the broader diversity of selected regions of the c. pecorum genome in a range of clinical samples from australian livestock and koalas was also conducted.  phylogenetic relationship of c. pecorumas revealed by whole genome sequencing the draft genomes of three c. pecorum strains isolated from koalas with clinical symptoms each comprise a single scaffold of approximately mbp. the chromosomes have a gc content of 41% and an average read coverage of 1500×. the fourth draft genome was from the c. pecorum vr strain that was isolated from the joint fluid of a sheep suffering from polyarthritis in the usa. none of the strains sequenced possessed the cryptic chlamydial plasmid. the three koala c. pecorum strains have been typed based on the ompa gene and each strain represents a different ompa serotype.table c. pecorum draft genomes sequenced in this study genotype source year of isolation country isolation tissue disease total number of reads number of contigs n50 assembly length number of cds % gc content to determine the relationship between the four sequenced c. pecorum strains, a phylogenetic tree was constructed based on the sequence of conserved genes across eight chlamydia species. the inferred topology of the phylogenetic tree is consistent with other phylogenetic analysis of chlamydia species. the phylogenetic reconstruction indicates that the closest relative of c. pecorum is c. pneumoniae. the phylogenetic analysis revealed that the three koala c. pecorum strains are part of a separate lineage from the ruminant c. pecorum strains sequenced in this study and those previously described. the two urogenital c. pecorum strains grouped more closely together, away from the conjunctival c. pecorum iptale isolate.figure phylogeny of chlamydia species. the maximum-likelihood tree was reconstructed using phyml with the gtr substitution model based on the nucleotide sequence of orthologous genes. bootstrap values are shown as percentages of replicates. the scale bars represent the number of substitutions per site. the gray box shows the within-species phylogeny of the sequenced c. pecorum strains and is magnified. whole genome comparison revealed a high level of gene conservation between c. pecorumstrains from koalas and livestock whole genome comparisons confirmed that the four draft c. pecorum genomes included in this study are highly conserved and syntenic, with similar gene content. comparisons against the genomes of other chlamydia species revealed that c. pecorum contains several regions that differ significantly to other chlamydia species. the variable regions include the chlamydial plasticity zone, a genomic island located near the terminus of replication and a cluster of genes that encode polymorphic membrane proteins. like the other c. pecorum genomes studied to date, the four draft c. pecorum genomes sequenced encode a near intact tryptophan biosynthesis operon but they are missing the trpe/g genes. the genomes of the c. pecorum strains are highly conserved with a dna sequence identity ranging from to %. the conservation of gene content and sequence similarity between c. pecorum strains is consistent with other chlamydia species. single nucleotide polymorphisms contribute to the genetic diversity between the c. pecorumgenomes although the overall gene content is conserved between each c. pecorum genome, there are a significant number of snps that contribute to variation between the c. pecorum strains. the number of predicted snps between livestock and koala c. pecorum strains ranges from to snps. between the livestock c. pecorum strains the number of polymorphisms range from to snps with the exception of the phylogenetically distinct c. pecorum pv strain which differs from the other livestock strains by snps. comparisons of the three koala c. pecorum genomes revealed that there are 1461- snps. the distribution of snps was plotted across the whole genome using a custom r script to reveal regions that are accumulating mutations. the region with the largest number of snps in the c. pecorum genomes is a 36 kb gene cluster encoding pmps. between c. pecorum e and c. pecorum vr there are synonymous and non-synonymous snps within the pmp cluster, with the majority of snps found in the pmpg subfamily. between the livestock c. pecorum e and koala c. pecorum mc/marsbar isolates, this pmp cluster contains synonymous and non-synonymous snps making this gene cluster the most variable region between c. pecorum strains from different hosts. comparison between the genomes of koala c. pecorum strains indicates that most of the variation observed in this region occurs with the pmpg genes. the adaptation to different hosts is likely the result of small and subtle changes that occurred in the genomes rather than differences in the gene content.table total snps in c. pecorum genomes using c. pecorum mc/marsbar as a reference distribution of snps in c. pecorum genomes. the histograms show the number of snps in relation to the genomic positions between different c. pecorum genomes with a window size of 10 kb. the top left hand graph shows the snp distribution between the koala c. pecorum mc/marsbar genome and the livestock c. pecorum e genome while the bottom left hand graph shows the number of snps between two koala c. pecorum strains. the right hand graphs display the distribution of snps observed between the two usa livestock c. pecorum strains and two of the european livestock strains. the red boxes mark the positions of the two snp hotspot regions identified in the koala c. pecorum strains. the first polymorphic region starts at 300 kb and is approximately 77 kb in length and the second region includes the plasticity zone. visual representation of the cluster of pmp genes in c. pecorum genome. the blue arrows represent the pmps and the orange arrows represent hypothetical proteins. the vertical lines mark the location and type of snps. the gaps in the genome assembly are marked by the gray rectangles and sizes of these gaps were estimated using pair-end read data. the base coordinates of this region in c. pecorum e are. the image was generated using easyfig. snps within koala c. pecorumstrains highlight genes potentially associated with host adaptation to marsupials the distribution of snps across the c. pecorum genomes revealed that there are two regions in the koala and european livestock c. pecorum strains that are hotspots for the accumulation of snps. the corresponding highly polymorphic regions do not show a similar level of diversity between the sheep and cattle c. pecorum strains isolated from usa. the first region, located near the origin of replication, is approximately kbp in length. a detailed analysis of this region revealed the presence of genes that encode for putative type iii effector proteins, chaperones and other potential virulence-related proteins. also found here is a cluster of genes that encode the inner membrane components of the type iii secretion system. in contrast to many other bacterial pathogens where the genes for the t3ss apparatus are clustered together on pathogenicity islands, chlamydia t3ss genes are located in four separate clusters disseminated throughout the genome. in addition, there is a sulfur transfer system encoded by the sufbcd operon and sufs similar to a system in e. coli; the rest of the genes located within this region are predicted to be involved in various metabolic processes. the kbp region is a hotspot for snps in both the australian koala strains and european livestock strains but this region is conserved between the two usa livestock strains. between c. pecorum e and c. pecorum mc/marsbar, we observed synonymous and non-synonymous snps located within genes in the kbp region. the majority of the non-synonymous snps are associated with putative virulence factors, including the type iii effector proteins. a selection analysis was carried out on each gene within this region between c. pecorum e and c. pecorum mc/marsbar to determine if any genes are under positive selection. between the livestock and koala strains, most of the genes in this region are under purifying selection with a dn/ds ratio of less than however, we observed three genes that are under positive selection, including one of the putative type iii effectors and two conserved hypothetical proteins. within the koala c. pecorum strains themselves, all the genes in this region are under purifying selection with mostly synonymous snp differences, with the exception of the srpa gene. the srpa protein has 100% sequence identity between c. pecorum mc/marsbar and c. pecorum iptale, however it only shares 85% sequence identity to the srpa homolog from c. pecorum dbdeug. the c-terminal domain of srpa was conserved across all the c. pecorum genomes and it is the n-terminal domain that is variable even between c. pecorum koala strains.figure visual representation of the kbp snp hot-spot region. the arrows represent genes and are coloured according to predicted functions inferred by blast searches. the vertical lines mark the location of snps with the green lines indicating synonymous snps and the red lines indicating non-synonymous snps. this region includes the sufbcd operon, which encodes an iron uptake system and a gene cluster that encodes the inner membrane components of the type iii secretion system. also encoded in this region are several putative type iii effectors and chaperones. the base coordinates of this region in c. pecorum e are. the image was created with easyfig. the second genomic region with a high frequency of snps is the pz. however, it should be noted that in the koala c. pecorum genomes, there are assembly gaps within the pz and that the number of predicted snps in the pz is likely an under estimation. paired-end data is used to estimate the size of the gaps, which suggest that the gene content of the pz is identical in each of the genomes. in c. pecorum, the pz is 42 kb with the acetyl-coa-carboxylases genes located at the 5′ boundary and the guaab and add genes at the 3′ end. the pz in koala c. pecorum strains encodes two cytotoxin genes in tandem, in this study designated as toxa and toxb. the pz also contains several other genes that encode proteins linked to pathogenesis, including the mac/perforin domain protein and five phospholipase d. the presence of two cytotoxic genes make the c. pecorum pz unique compared to the pz from other chlamydia species. although of similar size, the toxa and toxb genes share only 43% sequence identity on the amino acid level with several conserved motifs. the n-terminal regions of the toxa/b genes contain a catalytic glycosyltransferase domain. interestingly, in the genome of c. pecorum mc/marsbar, a snp in the toxb gene resulted in a premature stop codon at position truncating the gene. the presence of the premature stop codon was confirmed with pcr amplification and sequencing of the toxb gene in the c. pecorum mc/marsbar strain, as well as the other two koala and a sheep strain. the toxb gene is only truncated in c. pecorum mc/marsbar and full-length homologs are present in the other four c. pecorum genomes.figure visual representation of the c. pecorum plasticity zone. the arrows represent genes and they are colored according to function, which is illustrated in the legend. the vertical lines mark the location and type of snps. the gaps in the genome assembly are marked by the gray rectangles and sizes of these gaps were estimated using pair-end read data. the base coordinates of the plasticity zone in c. pecorum e are. the image was generated using easyfig. the presence of a limited number of pseudogenes further highlights genetic variation between koala c. pecorumstrains in addition to toxb, another five pseudogenes were identified in the three koala c. pecorum genomes, providing additional evidence of genetic diversity between c. pecorum strains isolated from koalas. the pseudogenes in c. pecorum were caused by premature stop codons within the gene sequences as a result of snps. c. pecorum iptale has three pseudogenes, one being the pyre gene that codes for an orotate phosphoribosyltransferase. the functions of the other two pseudogenes in c. pecorum iptale are unknown, although one of the genes contains a signal peptide at the n-terminus suggesting that the encoded protein targets a secretory pathway. the genomes of c. pecorum mc/marsbar and c. pecorum dbdeug each have a single pseudogene that contains transmembrane domains; however their actual functions are unknown. all six pseudogenes, including toxb, identified in the koala c. pecorum genomes are intact in the all the livestock c. pecorum genomes.table pseudogenes caused by premature stop codons in c. pecorum strains isolated from koalas some but not all predicted koala c. pecorumpseudogenes are widely distributed in c. pecorumstrains detected from across the koala’s host range in a preliminary investigation into the broader genetic diversity of c. pecorum in australian animals, we pcr amplified and sequenced partial regions of the six pseudogenes identified in the koala c. pecorum genomes from c. pecorum pcr positive samples collected from the conjunctival and urogenital tract sinuses of koalas from populations in queensland, new south wales, victoria and south australia; and conjunctiva and rectums of australian sheep. the list of c. pecorum strains chosen was based on pcr-positive swab samples that have sufficient chlamydial dna for multiple pcr amplification and to ensure an even distribution of strains from each australian state. out of the six pseudogenes identified from the genome sequences, four were also found to be pseudogenes in the clinical samples. the toxb gene was found to be truncated in some but not all of the koala c. pecorum positive samples from populations in south-east queensland, south australia and victoria. indeed, we were able to identify unique toxb gene fragment sequences amongst the collection examined, with sequences varying in similarity between – % across the livestock and koala samples. similarly, one of the pseudogenes from c. pecorum iptale was also truncated in three urogenital c. pecorum positive swabs from three different koala hosts, however the truncation is caused by a different mutation than in c. pecorum iptale. the pseudogene from c. pecorum mc/marsbar was found to be truncated in another two c. pecorum positive samples collected from the cloaca and urethra of a koala in victoria and nsw, respectively. the pseudogene from c. pecorum dbdeug was also confirmed to be a pseudogene in seven c. pecorum positive samples from the conjunctiva and genital tracts of koalas in queensland only. interestingly, the remaining two pseudogenes from c. pecorum iptale were intact in all koala c. pecorum samples analysed. although only a smaller sample set was analyzed, the evidence that all pseudogenes identified and confirmed in the koala c. pecorum genomes and clinical samples appeared to be intact in australian livestock was notable. while this observation appeared to further distinguish koala from livestock c. pecorum strains, it is also worth noting that a number of the new partial gene sequences identified in this analysis for each of these marker genes were otherwise identical. this was confirmed with a phylogenetic tree based on the concatenated sequences of five of the six pseudogenes – pyre gene sequences were difficult to amplify for a number of samples – not showing any clear distinction between koala and livestock strains from europe, the usa and australia.figure bayesian phylogenetic tree of concatenated sequence of five pseudogene fragments. the tree was constructed using mrbayes hky model based on the concatenated sequences of the five pseudogenes from c. pecorum detected in koalas and livestock. posterior probabilities >  are displayed on tree nodes. the colouring of the strain name as per the legend indicates the location of each strain. the following abbreviations denote anatomical site of the host where the swab sample was collected: rec, jnt, le, ure, pen, vag and clo. discussion while the initial sequencing of chlamydial genomes has provided important insights into the general biology of the chlamydiae, reconciling the minor genetic differences between different strains of the same species with particular diseases, hosts and sites of infection has been more challenging. c. pecorum is a useful model to investigate adaptation to different niches since it is a major pathogen with a broad host range and tissue tropisms. like most chlamydia species, c. pecorum genomes are conserved in terms of gene content and order. this high degree of gene conservation is believed to be the result of chlamydia’s intracellular lifestyle, which reduces exposure to frequent lateral gene transfer events that often cause phenotypic changes in other bacterial genera. therefore, small variations in the c. pecorum genomes are the likely causes for different phenotypic traits that are observed in different strains. when it comes to investigating the genetic diversity of c. pecorum, one of the limiting factors has been the lack of genome sequences for c. pecorum strains isolated from different animals, in particular strains isolated from koalas. in this study we examined the draft genomes of three c. pecorum strains that were isolated from koalas in south-east queensland, australia. each of the three koala c. pecorum genomes represents a different genotype, based on allelic differences in the ompa gene. also sequenced, as part of this study, is the genome of c. pecorum vr, a strain that was isolated from the joint of a sheep suffering polyarthritis in the usa. the c. pecorum vr genome along with the four publically available c. pecorum genomes serve as references for comparison to the koala c. pecorum genomes, to assess the level of genetic diversity between c. pecorum strains. the pz, the pmp gene cluster and the kbp region near the origin of replication are hotspots for snps accumulation in the australian koala strains and the european livestock strains. a similar high degree of snps is also observed in these same regions in c. trachomatis genomes. the gene content of the pz and the kbp region were also compared between c. trachomatis and c. pecorum using discontinuous megablast. while the gene content of the pz is significantly different between c. pecorum and c. trachomatis, the only difference in the kbp hotspot region between both species is that the putative virulence genes in c. pecorum are absent in c. trachomatis. the pz and the kbp region are highly conserved between the two american c. pecorum strains but additional genome sequencing of isolates from the usa is needed to confirm if this level of conservation is unique to usa strains. identification of snps between c. pecorum genomes from livestock and koalas has provided insight into which genes are likely to be involved in host adaptation. the most rapidly evolving genes in c. pecorum are the genes coding for the pmps, a family of proteins that may be important for adhesion of chlamydia to host cells. in particular, the pmpg subfamily represents the most variable class of pmps in c. pecorum, which is also the most rapidly evolving pmp class in c. psittaci. despite the variation in nucleotide sequences, the members of the pmp family possess a conserved domain structure that includes the c-terminal autotransporter beta-domain, a central domain unique to this family of proteins and a n-terminal domain that is involved in adhesion. pmps share sequence similarity to the autotransporter class of proteins, which have a diverse array of functions and often play an essential role in pathogenesis, thus the observed variation could also contribute to immune evasion. this study has identified several novel putative virulence genes that could be specifically involved with adaptation to different hosts. these virulence genes include a putative surface protein srpa and two putative type iii effectors. type iii effectors are virulence factors often involved in interactions with host cellular proteins in order to enhance the survival of the bacteria. amino acid changes introduced by non-synonymous snps could alter the binding domains of the effectors, altering their affinity to cellular targets. the other potential virulence factor of interest is the putative surface protein srpa1; the observed variation in the protein sequence could alter binding and/or recognition to surface receptors of host cells, however, experimental confirmation is obviously required for further investigations. however, the accumulation of non-synonymous snps in srpa and the two putative effector genes could also be due to a selective pressure for avoiding antibody and cellular immune responses. our comparison of the c. pecorum genomes revealed the presence of six pseudogenes in the strains isolated from koalas, one of these pseudogenes being the toxb gene located in the pz in c. pecorum mc/marsbar. c. pecorum typically possesses two full-length cytotoxin genes in the pz, designated toxa and toxb. the full-length chlamydia cytotoxin is similar to the large clostridial cytotoxin from clostridium difficile, which also has the glycosyltransferase domain in the n-terminal region of the protein. this domain has been shown to interfere with eukaryotic cells by glycosylating gtp binding proteins of the ras superfamily, inactivating them and leading to disassembly of the actin cytoskeleton. the lcts also include a domain for binding to the surface receptors of eukaryotic cells and a transmembrane segment that is involved in translocation into the cytoplasm. c. pecorum mc/marsbar has a full-length toxa gene and a shorter toxb gene that has been truncated by a premature stop codon. the truncation of toxb was observed in of the c. pecorum positive swabs samples for which partial gene sequences could be amplified from wild koala, suggesting that it could be a recent mutation since the remaining sequences identified were otherwise intact. it is plausible that the c-terminal domain of the toxb gene is being deleted to reduce the energetic cost of maintaining the full-length gene. out of the other five pseudogenes in the koala c. pecorum genomes, only the pyre gene has a predicted function. the gene pyre encodes an orotate phosphoribosyltransferase, an enzyme that is part of the de novo pyrimidine biosynthesis pathway. chlamydia spp. cannot synthesise pyrimidine nucleotides de novo as key genes involved in this pathway are missing, but instead must rely on a salvaging pathway that involves transporting ribonucleotides directly from the host cell. the presence of the pyre gene could be a remnant from when chlamydia was able to synthesise pyrimidine de novo; this is supported by the observed fragmentation of pyre in c. pecorum iptale, indicating gene decay. interestingly, despite this potentially important observation, the pyre gene was found to be intact in all koala c. pecorum strains that were screened, raising questions over whether this snp may have resolved during laboratory passage. in addition to this pseudogene, there are two other pseudogenes that are unique to the c. pecorum iptale conjunctival isolate, compared to the sequenced urogenital dbdeug and mc/marsbar strains. however, while one of these pseudogenes was found to be intact in all samples tested, the other was confirmed to be a pseudogene in three koala urogenital samples from the same geographic region and therefore both of these genes are unlikely to be involved in tissue tropism. sequence alignments of the pseudogene fragments revealed that some genotypes are specific to c. pecorum detected in australian livestock and koala hosts. however, a phylogenetic analysis of the concatenated pseudogene sequences showed that, collectively, there was no host specific separation in contrast to our whole genome phylogenies. the observation that multiple c. pecorum genotypes can be circulating in a single population of animals; and individual genotypes can be found across multiple populations of koalas or livestock is otherwise consistent with our previous fine-detailed molecular epidemiological investigations.  while the primary focus of this study was to examine the genetic differences potentially associated with c. pecorum host adaptation, the observed accumulation of genetic changes in koala c. pecorum strains provides an opportunity to speculate on the origin of this pathogen in the koala. the koala c. pecorum genomes contain a small number of pseudogenes and gene truncations, which are otherwise intact in the livestock genomes. these mutations suggest that c. pecorum is evolving to adapt to the koala through the loss of genes that are no longer necessary. although our genome phylogenies do not yet support this, the most parsimonious explanation for this observation is that koala c. pecorum strains have derived from livestock strains and are undergoing genetic changes to better adapt to the new host. interestingly enough, if this is true, then it would be the reverse of what we have previously observed for koala chlamydia pneumoniae, whereby the genome of the koala strain was found to be largely intact compared to human strains, suggesting that the koala strains were ancestral. in order to validate that koala c. pecorum strains are indeed derived from livestock strains it is necessary to reconstruct the evolutionary history of c. pecorum using phylogenetic and molecular clock analyses. however, additional c. pecorum strains will need to be sequenced in order to fill in the missing links from this comparison including analysis of c. pecorum isolates from australian livestock. indeed, a limitation of the phylogenetic analysis performed in this study is the small number of available c. pecorum genomes. c. pecorum pv was expected to cluster together with c. pecorum w and c. pecorum p based on the geographic location of these strains but the pv strain is phylogenetically distinct from the other c. pecorum strains. therefore, it is important to sequence more c. pecorum strains in order to fully understand the phylogenetic structure of this species but also to learn more about the role of the observed limited gene variation in disease pathogenesis and tissue and host adaptation. 
this pathogen is also a key factor in the decline of the koala in australia. single nucleotide polymorphisms appear to be key factors in understanding the adaptive process. in one koala strain, the toxb gene was truncated by a premature stop codon but is full-length in iptale and dbdeug. an examination of the distribution of these pseudogenes in c. pecorum strains from a variety of koala populations, alongside a number of sheep and cattle c. pecorum positive samples from australian livestock, confirmed the presence of four predicted pseudogenes in koala c. pecorum clinical samples. two regions of the chromosome were found to be accumulating a large number of snps within the koala strains. we sequenced the genomes of three koala c. pecorum strains, isolated from the urogenital tracts and conjunctiva of diseased koalas. the genomic data revealed that variation between c. pecorum strains were mainly due to the accumulation of snps, some of which cause gene inactivation. these regions include the chlamydia plasticity zone, which contains two cytotoxin genes, and a kbp region that codes for putative type iii effector proteins. another five pseudogenes were also identified, two unique to the urogenital strains c. pecorum mc/marsbar and c. pecorum dbdeug, respectively, while three were unique to the koala c. pecorum conjunctival isolate iptale. comparisons of the draft c. pecorum genomes against the complete genomes of livestock c. pecorum isolates revealed that these strains have a conserved gene content and order, sharing a nucleotide sequence similarity > 98%. the identification of these genetic differences will provide the basis for further studies to understand the biology and evolution of this important animal pathogen.
 chlamydia pecorum is an important pathogen of domesticated livestock including sheep, cattle and pigs. this pathogen is also a key factor in the decline of the koala in australia. we sequenced the genomes of three koala c. pecorum strains, isolated from the urogenital tracts and conjunctiva of diseased koalas. the genome of the c. pecorum vr strain, isolated from a sheep with polyarthritis, was also sequenced.  comparisons of the draft c. pecorum genomes against the complete genomes of livestock c. pecorum isolates revealed that these strains have a conserved gene content and order, sharing a nucleotide sequence similarity > 98%. single nucleotide polymorphisms appear to be key factors in understanding the adaptive process. two regions of the chromosome were found to be accumulating a large number of snps within the koala strains. these regions include the chlamydia plasticity zone, which contains two cytotoxin genes, and a kbp region that codes for putative type iii effector proteins. in one koala strain, the toxb gene was truncated by a premature stop codon but is full-length in iptale and dbdeug. another five pseudogenes were also identified, two unique to the urogenital strains c. pecorum mc/marsbar and c. pecorum dbdeug, respectively, while three were unique to the koala c. pecorum conjunctival isolate iptale. an examination of the distribution of these pseudogenes in c. pecorum strains from a variety of koala populations, alongside a number of sheep and cattle c. pecorum positive samples from australian livestock, confirmed the presence of four predicted pseudogenes in koala c. pecorum clinical samples. consistent with our genomics analyses, none of these pseudogenes were observed in the livestock c. pecorum samples examined. interestingly, three snps resulting in pseudogenes identified in the iptale isolate were not found in any other c. pecorum strain analysed, raising questions over the origin of these point mutations.  the genomic data revealed that variation between c. pecorum strains were mainly due to the accumulation of snps, some of which cause gene inactivation. the identification of these genetic differences will provide the basis for further studies to understand the biology and evolution of this important animal pathogen. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords chlamydia pecorumsingle nucleotide polymorphismpseudogenecytotoxinissue-copyright-statement© the author 2014 