Background The genome of  Protochlamydia amoebophila  UWE25, a  Parachlamydia -related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs).
Background
The genome of  Protochlamydia amoebophila  UWE25, a  Parachlamydia -related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs).Protochlamydia amoebophilaParachlamydiaResults On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical  gly-tRNA  genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer ( traF ,  traG ,  traH ,  traN ,  traU ,  traW , and  trbC ), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial  tra  unit is devoted to DNA transfer. A close relatedness of this  tra  unit to F-like  tra  operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of  gly-tRNA  distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial  tra  unit.
Results
On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical  gly-tRNA  genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer ( traF ,  traG ,  traH ,  traN ,  traU ,  traW , and  trbC ), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial  tra  unit is devoted to DNA transfer. A close relatedness of this  tra  unit to F-like  tra  operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of  gly-tRNA  distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial  tra  unit.gly-tRNAtraFtraGtraHtraNtraUtraWtrbCtratratragly-tRNAtraConclusions A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of  Parachlamydia  bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of  Chlamydiaceae , we hypothesize that it was acquired after the divergence between  Parachlamydiaceae  and  Chlamydiaceae , when the  Parachlamydia -related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since  Parachlamydiaceae  are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for  Chlamydiales .
Conclusions
A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of  Parachlamydia  bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of  Chlamydiaceae , we hypothesize that it was acquired after the divergence between  Parachlamydiaceae  and  Chlamydiaceae , when the  Parachlamydia -related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since  Parachlamydiaceae  are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for  Chlamydiales .ParachlamydiaChlamydiaceaeParachlamydiaceaeChlamydiaceaeParachlamydiaParachlamydiaceaeChlamydiales

Background First described in 1997,  Parachlamydia acanthamoebae  is an obligate intracellular bacterium naturally infecting free-living amoebae [ 1 , 2 ]. It was isolated from  Acanthamoeba  spp. recovered from the nasal mucosa of healthy volunteers [ 1 ]. Later, additional strains of  Parachlamydiaceae  have been found within about 5% of  Acanthamoeba  spp. and once within  Hartmanella vermiformis  [ 2 , 3 ]. The 16S rRNA sequences of these  Parachlamydiaceae  are about 14% different from those of both genera  Chlamydophila  and  Chlamydia  [ 2 , 3 ]. Since the 16S rRNA sequence difference between  Chlamydophila  sp. and  Chlamydia  sp. is 6% only, it clearly appears that the speciation between the two latter occurred after the divergence between  Parachlamydiaceae  and  Chlamydiaceae . Like other  Chlamydiales ,  Parachlamydiaceae  can present two developmental stages: the reticulate body, a metabolically active dividing form, and the elementary body, an infective stage; the crescent body is another infective form, not observed in  Chlamydiaceae  [ 4 ]. Differentiation of the infective stages in reticulate bodies and multiplication of the latter were recently shown to occur within amoebal vacuoles, that may contain hundreds of bacteria [ 4 ]. Depending on the symbiotic/pathogenic relationships prevailing between both organisms, the escape of the bacteria from the amoeba may occur either by the release of secreted vesicles or by the lysis of the host [ 4 ]. There is a growing evidence of the human pathogenicity of  Parachlamydiaceae  [ 2 ]. For instance, positive  Parachlamydia  serologies were shown to be associated with a febrile epidemic [ 5 ], community-acquired pneumonia [ 6 ], and inhalation pneumonia [ 7 ]. The role of  Parachlamydia -related bacteria as agents of inhalation pneumonia is further suggested by the temperature-dependent release of the bacteria from their amoebal reservoir [ 8 ]. PCR amplification of parachlamydial DNA from monocytes, sputa and bronchoalveolar lavages collected from patients suffering of bronchitis or pneumonia also supports the pathogenic potential of  Parachlamydia  [ 9 - 12 ]. The survival of these  Chlamydia -like organisms within human macrophages [ 13 ] is an additional hint of parachlamydial pathogenicity. Horn  et al . [ 14 ], by sequencing and annotating the whole genome of the  Parachlamydia -related UWE25 contributed much to the understanding of the evolution of chlamydiae. Indeed, they demonstrated that major virulence mechanisms of  Chlamydiaceae  such as the Type Three Secretion System (TTSS) and the Chlamydial Protease-like Activity Factor (CPAF) are also encoded by the chromosome of the evolutionary early-branching  Parachlamydiaceae  UWE25. Genome analysis of the parachlamydial endosymbiont also identified Open Reading Frames (ORFs) homologous to Type Four Secretion Systems (TFSS) and characterized by a high G+C content, suggesting that they result from an horizontal transfer. Based on their annotation revealing the apparent absence of genes necessary for DNA transfer, Horn  et al . [ 14 ] proposed that this TFSS was involved in protein export but not in DNA transfer. To date, numerous genomic islands (GIs) were already identified along whole chromosomal sequences of various bacterial species. For instance, 140 GIs are described in the Islander database, including GIs of proteobacteria, firmicutes, actinobacteria and cyanobacteria [ 15 ]. Thus, we wondered whether any GIs were located along the bacterial chromosome of the amoebal endosymbiont UWE25. GIs are genetic elements which length vary from 10 to 200 kb and are inserted in a chromosome after a lateral transfer occurring, in some instances, between phylogenetically-distant microorganisms. Their heterologous origins are generally evidenced by a G+C content different from that of the remaining bacterial chromosome and by the presence of various mobility genes (i.e. involved in transposition, transduction or conjugative transfer), that are occasionally source of GI instability [ 16 , 17 ]. They are often flanked by particular DNA sequences, such as direct repeats or insertion sequences. Moreover, tRNA  loci  are generally used as insertion sites by GIs for their chromosomal integration [ 16 - 18 ]. Since no genetic tools are available for the study of this obligate intracellular bacteria, a bioinformatic approach was chosen to locate putative GIs.
Background
First described in 1997,  Parachlamydia acanthamoebae  is an obligate intracellular bacterium naturally infecting free-living amoebae [ 1 , 2 ]. It was isolated from  Acanthamoeba  spp. recovered from the nasal mucosa of healthy volunteers [ 1 ]. Later, additional strains of  Parachlamydiaceae  have been found within about 5% of  Acanthamoeba  spp. and once within  Hartmanella vermiformis  [ 2 , 3 ]. The 16S rRNA sequences of these  Parachlamydiaceae  are about 14% different from those of both genera  Chlamydophila  and  Chlamydia  [ 2 , 3 ]. Since the 16S rRNA sequence difference between  Chlamydophila  sp. and  Chlamydia  sp. is 6% only, it clearly appears that the speciation between the two latter occurred after the divergence between  Parachlamydiaceae  and  Chlamydiaceae .Parachlamydia acanthamoebaeAcanthamoebaParachlamydiaceaeAcanthamoebaHartmanella vermiformisParachlamydiaceaeChlamydophilaChlamydiaChlamydophilaChlamydiaParachlamydiaceaeChlamydiaceaeLike other  Chlamydiales ,  Parachlamydiaceae  can present two developmental stages: the reticulate body, a metabolically active dividing form, and the elementary body, an infective stage; the crescent body is another infective form, not observed in  Chlamydiaceae  [ 4 ]. Differentiation of the infective stages in reticulate bodies and multiplication of the latter were recently shown to occur within amoebal vacuoles, that may contain hundreds of bacteria [ 4 ]. Depending on the symbiotic/pathogenic relationships prevailing between both organisms, the escape of the bacteria from the amoeba may occur either by the release of secreted vesicles or by the lysis of the host [ 4 ].ChlamydialesParachlamydiaceaeChlamydiaceaeThere is a growing evidence of the human pathogenicity of  Parachlamydiaceae  [ 2 ]. For instance, positive  Parachlamydia  serologies were shown to be associated with a febrile epidemic [ 5 ], community-acquired pneumonia [ 6 ], and inhalation pneumonia [ 7 ]. The role of  Parachlamydia -related bacteria as agents of inhalation pneumonia is further suggested by the temperature-dependent release of the bacteria from their amoebal reservoir [ 8 ]. PCR amplification of parachlamydial DNA from monocytes, sputa and bronchoalveolar lavages collected from patients suffering of bronchitis or pneumonia also supports the pathogenic potential of  Parachlamydia  [ 9 - 12 ]. The survival of these  Chlamydia -like organisms within human macrophages [ 13 ] is an additional hint of parachlamydial pathogenicity.ParachlamydiaceaeParachlamydiaParachlamydiaParachlamydiaChlamydiaHorn  et al . [ 14 ], by sequencing and annotating the whole genome of the  Parachlamydia -related UWE25 contributed much to the understanding of the evolution of chlamydiae. Indeed, they demonstrated that major virulence mechanisms of  Chlamydiaceae  such as the Type Three Secretion System (TTSS) and the Chlamydial Protease-like Activity Factor (CPAF) are also encoded by the chromosome of the evolutionary early-branching  Parachlamydiaceae  UWE25. Genome analysis of the parachlamydial endosymbiont also identified Open Reading Frames (ORFs) homologous to Type Four Secretion Systems (TFSS) and characterized by a high G+C content, suggesting that they result from an horizontal transfer. Based on their annotation revealing the apparent absence of genes necessary for DNA transfer, Horn  et al . [ 14 ] proposed that this TFSS was involved in protein export but not in DNA transfer.et alParachlamydiaChlamydiaceaeParachlamydiaceaeet alTo date, numerous genomic islands (GIs) were already identified along whole chromosomal sequences of various bacterial species. For instance, 140 GIs are described in the Islander database, including GIs of proteobacteria, firmicutes, actinobacteria and cyanobacteria [ 15 ]. Thus, we wondered whether any GIs were located along the bacterial chromosome of the amoebal endosymbiont UWE25.GIs are genetic elements which length vary from 10 to 200 kb and are inserted in a chromosome after a lateral transfer occurring, in some instances, between phylogenetically-distant microorganisms. Their heterologous origins are generally evidenced by a G+C content different from that of the remaining bacterial chromosome and by the presence of various mobility genes (i.e. involved in transposition, transduction or conjugative transfer), that are occasionally source of GI instability [ 16 , 17 ]. They are often flanked by particular DNA sequences, such as direct repeats or insertion sequences. Moreover, tRNA  loci  are generally used as insertion sites by GIs for their chromosomal integration [ 16 - 18 ]. Since no genetic tools are available for the study of this obligate intracellular bacteria, a bioinformatic approach was chosen to locate putative GIs.lociResults A genomic island is present in the genome of UWE25 Using standard G+C content analyses of  Parachlamydia -related UWE25 chromosome, we observed a G+C-rich region (Figure  1A  and  1B ), similar to that shown by Horn  et al . [ 14 ]. Using the residual cumulative G+C content analysis adapted from the GC profile of Zhang and Zhang [ 19 ], we were able to precisely define a 19-kb region (Figure  1C ). The presence of 17-bp direct repeats flanking a 100-kb chromosome region (1648 to 1748 kb, Table  1 ) that encompasses the 19-kb DNA sequence enabled us to define a new region composed of 100 ORFs (See  additional data file 1  for the description of these genes and their location on the chromosome of UWE25). Interestingly, this 100-kb region is characterized by a higher level of local gene coorientation (75/100) than that characterizing the remaining of the genome (1015/1931, 52.6%, p < 0.001) and by a particular signature in the cumulative GC skew analysis. Two identical  gly-tRNA  genes in tandem are located at the proximal end of this 100-kb genetic element (Figure  1A,1C  and Table  1 ). Several mobility genes (eight putative transposases, one recombinase and seven bacteriophage related-proteins) are encoded within the 100-kb region (Figure  1C , Table  1 ). Thus, this region largely fulfills the accepted criteria of GIs [ 16 - 18 ]. We termed this newly described GI "Pam100G" ( Protochlamydia amoebophila , 100-kb, Gly-tRNA) according to the nomenclature used in the Islander database [ 15 ]. Mosaicism of the 100-kb genomic island  Interestingly, this GI can be divided into clearly distinct regions, according to their G+C content (Figure  1B , Table  2 ). The residual cumulative G+C content analysis highlights a modular structure with different slopes, each   linear segment indicates that genes of this unit present a rather constant local G+C content (Figure  1C ). A positive or a negative slope would indicate that   each block of genes presents a G+C content higher or lower that of the UWE25 chromosome, respectively.  The first module begins with a direct repeat and two identical  gly-tRNAs  in tandem. Composed of 28 ORFs, this unit exhibits a G+C content (36.4%) similar to that of the remaining of the genome of UWE25 (1931 ORFs, 36.1%). Sixteen homologs to these 28 genes (57%) were found in databases, 12 of them (75%) exhibiting a best score in BLAST analyses with a  Chlamydiaceae  ORF (See  additional data file 1 ). Interestingly, no gene of the other modules of the 100-kb GI exhibited a best hit in similarity analyses with any  Chlamydiaceae  counterpart. Some of the genes present in the first module, such as  sctN  and  sctQ , are part of a TTSS also present in  Chlamydiaceae . The other TTSS genes are disseminated along the chromosome of UWE25. The presence of some TTSS genes in the first module and of a gene encoding a putative transposase at the distal end of the first module of this 100-kb GI suggests that this first unit was acquired by chromosomal rearrangements. (See  additional data file 1  for the results of BLAST analyses). Characterized by a low G+C content (34.1%), the 2 nd  module encodes 18 ORFs. Only five are similar to known protein sequences (28%), four of them being identified as mobility genes (three bacteriophage-related genes and one putative transposase encoding gene). The 3 rd  module (19 kb), exhibiting the second highest G+C content of the UWE25 genome (40.9%), comprises 21 ORFs. Some of these genes were identified as  tra  genes by Horn  et al . [ 14 ]. Using BLAST analyses and alignment tools, we re-annotated the whole module (see below) and, if we except two transposase genes and one ORF of unknown function, we unveiled that all ORFs of this module belong to a genetic unit similar to the  tra  operons encoding the TFSS previously described in proteobacterial genomes (Figure  2 , See  additional data file 1  for the re-annotations of this module). Presenting a low G+C content (33.4%), the 4 th  module (10 kb) is composed of 13 ORFs. All these ORFs were previously annotated by Horn et al. [ 14 ] as encoding hypothetical proteins or without homolog. Our BLAST analysis identified one ORF homologous to genes encoding bacteriophage-related proteins and two genes of proteins involved in DNA metabolism (Table  1  and  2 , see also the table in the  additional data file 1 ). Interestingly, a direct repeat is located between the 9th and 10th genes of the module. This 17-bp direct repeat, that presents 3 mismatches is similar to those present at the proximal and distal ends of the GI, exhibiting the same 14 conserved nucleotides. It may reflect a complex evolutionary history of the GI, possibly enabling it to be mobile as 25-kb, 75-kb or 100-kb DNA segments. A single large protein is encoded along the 5 th  module (6 kb). Its G+C content is one of the highest of the UWE25 chromosome (41.8%). By BLAST analysis, this protein exhibits the strongest similarity with the human Nod3 protein. The 6 th  module (12 kb) is characterized by a low G+C content (33.3%). This unit is composed of 13 ORFs, the first ORF encoding a product similar to the Death on cure (Doc) protein of P1 bacteriophage. Two ORFs code for proteins involved in DNA metabolism and an additional ORF encodes a putative transposase. The 7 th  module is short (2 kb) and present a G+C-rich unit (38.7%). Five of the six ORFs of this unit encode a probable resolvase, three putative transposases and a phage-related Doc protein. The final direct repeat is located at the end of this module. With the only exception of the phage-related protein, all other ORFs of the 7 th  module appear to be similar to gamma-proteobacterial proteins, possibly explaining the observed different signal in the G+C content analysis. Role of the type IV secretion system encoded by the 100-kb genomic island The functions of genes encoded by GIs may be related, among others, to pathogenicity such as the ability to exploit the host intracellular environment. Since no genetic system has been described for any obligate intracellular chlamydiae, we investigated the putative functions of this GI by bioinformatics. We focused our analyses on the TFSS, for which a previous annotation of the  tra  genes showed a genetic unit unable to transfer DNA [ 14 ]. Using different protein comparison methods described in the  additional data file 1 , we identified supplementary  tra  genes, and compared the general organization of this  tra  unit with other genetic elements encoding TFSS genes [ 20 ]. The UWE25  tra  unit displays a striking colinearity with  tra  operons encoding F-like conjugative DNA transfer system, especially to those of the F and pNL1 plasmids of  Escherichia coli  and  Novosphingobium aromaticivorans , respectively (Figure  2 ). All homologous genes essential for DNA transfer in plasmid F ( traF ,  traG ,  traH ,  traN ,  traU ,  traW , and  trbC ) and involved in sex pilus retraction and mating pair stabilization [ 20 ] are present, strongly suggesting that, similarly to the other F-like TFSSs, the gene products encoded by the UWE25  tra  unit are devoted to DNA transfer. With the only exception of  traG , these genes are not present on P-like and I-like plasmids, reinforcing the close relationship prevailing between the UWE25  tra  unit and their F-like plasmids counterparts. Figure  3  shows that the UWE25  tra  unit clusters within F-like TFSSs, confirming that it may function as a F-like conjugative system. Drawn as an UPGMA tree (Figure  3A ), the comparison of the genetic organization of all  tra  units was performed as a gene order breakpoint analysis developed for the study of the mitochondrial genome evolution [ 21 ]. This analysis clearly shows that the closest relatives of the UWE25  tra  units are the  tra  operons of the F-like conjugative plasmids. The Fitch-Margoliash- and the minimum evolution comparisons performed on the same dataset presented the same tree topologies, confirming the former UPGMA results (data not shown). An  omit  test performed on this tree confirms that the results are robust: with one exception (involving the deep branching of one cluster on one tree), all 11 trees were congruent in all their nodes. Figure  3B  shows an UPGMA tree comparing the Kimura corrected  p -distances (the proportion  p  of nucleotide sites at which two sequences are different, taking into account the proportion of transversion- and transition-substitution rates) of nucleotide sequences of the concatenated  traA ,  traK ,  traB ,  traV , and  traC  genes. A similar topology is observed with (i) neighbor-joining- and minimum evolution trees inferred using the Kimura-corrected  p -distances and (ii) UPGMA, neighbor-joining- and minimum evolution trees performed on  p -distance of the whole coding sequences of the concatenated  tra  genes (See  additional data file 2  for these trees). Neighbor-joining- and minimum evolution methods comparing Kimura-corrected  p -distances of the complete coding sequences confirmed that the  tra  unit of UWE25 is phylogenetically closely related to the  tra  operons of the F-like plasmids: the bootstrap values of 94% and 91% respectively, support the node separating the concatenated  tra  genes of the chromosomal UWE25 and the R27 plasmid, a gamma-proteobacterial F-like conjugative plasmid, from those of all other plasmids (See the  additional data file 2  for these trees). In neighbor-joining and minimum evolution analyses of  p -distances, the  tra  unit of UWE25 also clusters with the  tra  operons of gamma-proteobacterial F-like plasmids: the bootstrap of 96% and 92%, respectively, support the node separating the concatenated  tra  genes of UWE25 and RTS1, SXT, R391, three gamma-proteobacterial F-like conjugative plasmid, from their closest relative, R27 plasmid (See  additional data file 2  for these trees). Taken together, all these data strongly suggest that the UWE25  tra  unit is closely related to F-like conjugative  tra  operons. Origin of the genomic island and of its type four secretion system Our BLAST analyses [ 22 ] reveal that a majority (24/43) of genes not presenting a best hit for chlamydial genes but having homologs in other taxa are more related to proteobacterial genes (see Table  2  and the  additional data file 1  for similarity analyses indicating for each parachlamydial  tra  gene the most similar gene and its taxonomical background). Moreover, the BLAST analyses of the 21 ORFs of the third module, encoding the  tra  genes, show that most ORFs of this unit (15/21) are of proteobacterial origin. However, since six of them present the highest similarity to alpha-proteobacterial genes and six others to gamma-proteobacterial genes, a more precise origin of the parachlamydial  tra  unit could not be precisely defined by this first approach. The presence of  gly-tRNA  at the proximal end of the GI of UWE25 is consistent with a close relatedness between this GI and proteobacteria: out of 14 GIs described in the  Islander  database of Mantri  et al . [ 15 , 22 ] inserted along a chromosome by a  gly-tRNA  (14/140), 12 of them (86%) were sequenced in a proteobacterial genome. No GI of Gram-positives described in the Islander database are inserted in a chromosome within a  gly-tRNA  gene. Again, a precise proteobacterial origin could not be proposed, because the distribution of  gly-tRNA  genes in alpha- (4/22) and gamma-proteobacterial (8/72) GIs is not significantly different: by including only the non-redundant GIs, the distribution of  gly-tRNA  genes in alpha and gamma-proteobacterial GIs is 2/20 and 7/71, respectively. Comparison of gene order between all  tra  units also failed in assigning a precise origin to the UWE25  tra  unit since it branched near the alpha- and gamma-proteobacterial  tra  operons (Figure  3A ). The only first hint of a possible gamma-proteobacterial origin for the UWE25  tra  unit was brought by the phylogenetic analyses (Figure  3B  and additional files  1  & 2 ). Thus, bootstraps values of 94, 91, 96 and 92% supported the node separating the concatenated  tra  genes of UWE25 and several  tra  operons of gamma-proteobacterial F-like plasmids from the F-plasmids of an alpha-proteobacteria and of other gamma-proteobacteria. (See above, and  additional data file 2  for these trees).
Results
A genomic island is present in the genome of UWE25 Using standard G+C content analyses of  Parachlamydia -related UWE25 chromosome, we observed a G+C-rich region (Figure  1A  and  1B ), similar to that shown by Horn  et al . [ 14 ]. Using the residual cumulative G+C content analysis adapted from the GC profile of Zhang and Zhang [ 19 ], we were able to precisely define a 19-kb region (Figure  1C ). The presence of 17-bp direct repeats flanking a 100-kb chromosome region (1648 to 1748 kb, Table  1 ) that encompasses the 19-kb DNA sequence enabled us to define a new region composed of 100 ORFs (See  additional data file 1  for the description of these genes and their location on the chromosome of UWE25). Interestingly, this 100-kb region is characterized by a higher level of local gene coorientation (75/100) than that characterizing the remaining of the genome (1015/1931, 52.6%, p < 0.001) and by a particular signature in the cumulative GC skew analysis. Two identical  gly-tRNA  genes in tandem are located at the proximal end of this 100-kb genetic element (Figure  1A,1C  and Table  1 ). Several mobility genes (eight putative transposases, one recombinase and seven bacteriophage related-proteins) are encoded within the 100-kb region (Figure  1C , Table  1 ). Thus, this region largely fulfills the accepted criteria of GIs [ 16 - 18 ]. We termed this newly described GI "Pam100G" ( Protochlamydia amoebophila , 100-kb, Gly-tRNA) according to the nomenclature used in the Islander database [ 15 ].
A genomic island is present in the genome of UWE25
Using standard G+C content analyses of  Parachlamydia -related UWE25 chromosome, we observed a G+C-rich region (Figure  1A  and  1B ), similar to that shown by Horn  et al . [ 14 ]. Using the residual cumulative G+C content analysis adapted from the GC profile of Zhang and Zhang [ 19 ], we were able to precisely define a 19-kb region (Figure  1C ). The presence of 17-bp direct repeats flanking a 100-kb chromosome region (1648 to 1748 kb, Table  1 ) that encompasses the 19-kb DNA sequence enabled us to define a new region composed of 100 ORFs (See  additional data file 1  for the description of these genes and their location on the chromosome of UWE25). Interestingly, this 100-kb region is characterized by a higher level of local gene coorientation (75/100) than that characterizing the remaining of the genome (1015/1931, 52.6%, p < 0.001) and by a particular signature in the cumulative GC skew analysis. Two identical  gly-tRNA  genes in tandem are located at the proximal end of this 100-kb genetic element (Figure  1A,1C  and Table  1 ). Several mobility genes (eight putative transposases, one recombinase and seven bacteriophage related-proteins) are encoded within the 100-kb region (Figure  1C , Table  1 ). Thus, this region largely fulfills the accepted criteria of GIs [ 16 - 18 ]. We termed this newly described GI "Pam100G" ( Protochlamydia amoebophila , 100-kb, Gly-tRNA) according to the nomenclature used in the Islander database [ 15 ].Parachlamydiaet algly-tRNAProtochlamydia amoebophilaMosaicism of the 100-kb genomic island  Interestingly, this GI can be divided into clearly distinct regions, according to their G+C content (Figure  1B , Table  2 ). The residual cumulative G+C content analysis highlights a modular structure with different slopes, each   linear segment indicates that genes of this unit present a rather constant local G+C content (Figure  1C ). A positive or a negative slope would indicate that   each block of genes presents a G+C content higher or lower that of the UWE25 chromosome, respectively.  The first module begins with a direct repeat and two identical  gly-tRNAs  in tandem. Composed of 28 ORFs, this unit exhibits a G+C content (36.4%) similar to that of the remaining of the genome of UWE25 (1931 ORFs, 36.1%). Sixteen homologs to these 28 genes (57%) were found in databases, 12 of them (75%) exhibiting a best score in BLAST analyses with a  Chlamydiaceae  ORF (See  additional data file 1 ). Interestingly, no gene of the other modules of the 100-kb GI exhibited a best hit in similarity analyses with any  Chlamydiaceae  counterpart. Some of the genes present in the first module, such as  sctN  and  sctQ , are part of a TTSS also present in  Chlamydiaceae . The other TTSS genes are disseminated along the chromosome of UWE25. The presence of some TTSS genes in the first module and of a gene encoding a putative transposase at the distal end of the first module of this 100-kb GI suggests that this first unit was acquired by chromosomal rearrangements. (See  additional data file 1  for the results of BLAST analyses). Characterized by a low G+C content (34.1%), the 2 nd  module encodes 18 ORFs. Only five are similar to known protein sequences (28%), four of them being identified as mobility genes (three bacteriophage-related genes and one putative transposase encoding gene). The 3 rd  module (19 kb), exhibiting the second highest G+C content of the UWE25 genome (40.9%), comprises 21 ORFs. Some of these genes were identified as  tra  genes by Horn  et al . [ 14 ]. Using BLAST analyses and alignment tools, we re-annotated the whole module (see below) and, if we except two transposase genes and one ORF of unknown function, we unveiled that all ORFs of this module belong to a genetic unit similar to the  tra  operons encoding the TFSS previously described in proteobacterial genomes (Figure  2 , See  additional data file 1  for the re-annotations of this module). Presenting a low G+C content (33.4%), the 4 th  module (10 kb) is composed of 13 ORFs. All these ORFs were previously annotated by Horn et al. [ 14 ] as encoding hypothetical proteins or without homolog. Our BLAST analysis identified one ORF homologous to genes encoding bacteriophage-related proteins and two genes of proteins involved in DNA metabolism (Table  1  and  2 , see also the table in the  additional data file 1 ). Interestingly, a direct repeat is located between the 9th and 10th genes of the module. This 17-bp direct repeat, that presents 3 mismatches is similar to those present at the proximal and distal ends of the GI, exhibiting the same 14 conserved nucleotides. It may reflect a complex evolutionary history of the GI, possibly enabling it to be mobile as 25-kb, 75-kb or 100-kb DNA segments. A single large protein is encoded along the 5 th  module (6 kb). Its G+C content is one of the highest of the UWE25 chromosome (41.8%). By BLAST analysis, this protein exhibits the strongest similarity with the human Nod3 protein. The 6 th  module (12 kb) is characterized by a low G+C content (33.3%). This unit is composed of 13 ORFs, the first ORF encoding a product similar to the Death on cure (Doc) protein of P1 bacteriophage. Two ORFs code for proteins involved in DNA metabolism and an additional ORF encodes a putative transposase. The 7 th  module is short (2 kb) and present a G+C-rich unit (38.7%). Five of the six ORFs of this unit encode a probable resolvase, three putative transposases and a phage-related Doc protein. The final direct repeat is located at the end of this module. With the only exception of the phage-related protein, all other ORFs of the 7 th  module appear to be similar to gamma-proteobacterial proteins, possibly explaining the observed different signal in the G+C content analysis.
Mosaicism of the 100-kb genomic island
Interestingly, this GI can be divided into clearly distinct regions, according to their G+C content (Figure  1B , Table  2 ). The residual cumulative G+C content analysis highlights a modular structure with different slopes, each   linear segment indicates that genes of this unit present a rather constant local G+C content (Figure  1C ). A positive or a negative slope would indicate that   each block of genes presents a G+C content higher or lower that of the UWE25 chromosome, respectively.The first module begins with a direct repeat and two identical  gly-tRNAs  in tandem. Composed of 28 ORFs, this unit exhibits a G+C content (36.4%) similar to that of the remaining of the genome of UWE25 (1931 ORFs, 36.1%). Sixteen homologs to these 28 genes (57%) were found in databases, 12 of them (75%) exhibiting a best score in BLAST analyses with a  Chlamydiaceae  ORF (See  additional data file 1 ). Interestingly, no gene of the other modules of the 100-kb GI exhibited a best hit in similarity analyses with any  Chlamydiaceae  counterpart. Some of the genes present in the first module, such as  sctN  and  sctQ , are part of a TTSS also present in  Chlamydiaceae . The other TTSS genes are disseminated along the chromosome of UWE25. The presence of some TTSS genes in the first module and of a gene encoding a putative transposase at the distal end of the first module of this 100-kb GI suggests that this first unit was acquired by chromosomal rearrangements. (See  additional data file 1  for the results of BLAST analyses).gly-tRNAsChlamydiaceaeChlamydiaceaesctNsctQChlamydiaceaeCharacterized by a low G+C content (34.1%), the 2 nd  module encodes 18 ORFs. Only five are similar to known protein sequences (28%), four of them being identified as mobility genes (three bacteriophage-related genes and one putative transposase encoding gene).The 3 rd  module (19 kb), exhibiting the second highest G+C content of the UWE25 genome (40.9%), comprises 21 ORFs. Some of these genes were identified as  tra  genes by Horn  et al . [ 14 ]. Using BLAST analyses and alignment tools, we re-annotated the whole module (see below) and, if we except two transposase genes and one ORF of unknown function, we unveiled that all ORFs of this module belong to a genetic unit similar to the  tra  operons encoding the TFSS previously described in proteobacterial genomes (Figure  2 , See  additional data file 1  for the re-annotations of this module).traet altraPresenting a low G+C content (33.4%), the 4 th  module (10 kb) is composed of 13 ORFs. All these ORFs were previously annotated by Horn et al. [ 14 ] as encoding hypothetical proteins or without homolog. Our BLAST analysis identified one ORF homologous to genes encoding bacteriophage-related proteins and two genes of proteins involved in DNA metabolism (Table  1  and  2 , see also the table in the  additional data file 1 ). Interestingly, a direct repeat is located between the 9th and 10th genes of the module. This 17-bp direct repeat, that presents 3 mismatches is similar to those present at the proximal and distal ends of the GI, exhibiting the same 14 conserved nucleotides. It may reflect a complex evolutionary history of the GI, possibly enabling it to be mobile as 25-kb, 75-kb or 100-kb DNA segments.A single large protein is encoded along the 5 th  module (6 kb). Its G+C content is one of the highest of the UWE25 chromosome (41.8%). By BLAST analysis, this protein exhibits the strongest similarity with the human Nod3 protein.The 6 th  module (12 kb) is characterized by a low G+C content (33.3%). This unit is composed of 13 ORFs, the first ORF encoding a product similar to the Death on cure (Doc) protein of P1 bacteriophage. Two ORFs code for proteins involved in DNA metabolism and an additional ORF encodes a putative transposase.The 7 th  module is short (2 kb) and present a G+C-rich unit (38.7%). Five of the six ORFs of this unit encode a probable resolvase, three putative transposases and a phage-related Doc protein. The final direct repeat is located at the end of this module. With the only exception of the phage-related protein, all other ORFs of the 7 th  module appear to be similar to gamma-proteobacterial proteins, possibly explaining the observed different signal in the G+C content analysis.Role of the type IV secretion system encoded by the 100-kb genomic island The functions of genes encoded by GIs may be related, among others, to pathogenicity such as the ability to exploit the host intracellular environment. Since no genetic system has been described for any obligate intracellular chlamydiae, we investigated the putative functions of this GI by bioinformatics. We focused our analyses on the TFSS, for which a previous annotation of the  tra  genes showed a genetic unit unable to transfer DNA [ 14 ]. Using different protein comparison methods described in the  additional data file 1 , we identified supplementary  tra  genes, and compared the general organization of this  tra  unit with other genetic elements encoding TFSS genes [ 20 ]. The UWE25  tra  unit displays a striking colinearity with  tra  operons encoding F-like conjugative DNA transfer system, especially to those of the F and pNL1 plasmids of  Escherichia coli  and  Novosphingobium aromaticivorans , respectively (Figure  2 ). All homologous genes essential for DNA transfer in plasmid F ( traF ,  traG ,  traH ,  traN ,  traU ,  traW , and  trbC ) and involved in sex pilus retraction and mating pair stabilization [ 20 ] are present, strongly suggesting that, similarly to the other F-like TFSSs, the gene products encoded by the UWE25  tra  unit are devoted to DNA transfer. With the only exception of  traG , these genes are not present on P-like and I-like plasmids, reinforcing the close relationship prevailing between the UWE25  tra  unit and their F-like plasmids counterparts. Figure  3  shows that the UWE25  tra  unit clusters within F-like TFSSs, confirming that it may function as a F-like conjugative system. Drawn as an UPGMA tree (Figure  3A ), the comparison of the genetic organization of all  tra  units was performed as a gene order breakpoint analysis developed for the study of the mitochondrial genome evolution [ 21 ]. This analysis clearly shows that the closest relatives of the UWE25  tra  units are the  tra  operons of the F-like conjugative plasmids. The Fitch-Margoliash- and the minimum evolution comparisons performed on the same dataset presented the same tree topologies, confirming the former UPGMA results (data not shown). An  omit  test performed on this tree confirms that the results are robust: with one exception (involving the deep branching of one cluster on one tree), all 11 trees were congruent in all their nodes. Figure  3B  shows an UPGMA tree comparing the Kimura corrected  p -distances (the proportion  p  of nucleotide sites at which two sequences are different, taking into account the proportion of transversion- and transition-substitution rates) of nucleotide sequences of the concatenated  traA ,  traK ,  traB ,  traV , and  traC  genes. A similar topology is observed with (i) neighbor-joining- and minimum evolution trees inferred using the Kimura-corrected  p -distances and (ii) UPGMA, neighbor-joining- and minimum evolution trees performed on  p -distance of the whole coding sequences of the concatenated  tra  genes (See  additional data file 2  for these trees). Neighbor-joining- and minimum evolution methods comparing Kimura-corrected  p -distances of the complete coding sequences confirmed that the  tra  unit of UWE25 is phylogenetically closely related to the  tra  operons of the F-like plasmids: the bootstrap values of 94% and 91% respectively, support the node separating the concatenated  tra  genes of the chromosomal UWE25 and the R27 plasmid, a gamma-proteobacterial F-like conjugative plasmid, from those of all other plasmids (See the  additional data file 2  for these trees). In neighbor-joining and minimum evolution analyses of  p -distances, the  tra  unit of UWE25 also clusters with the  tra  operons of gamma-proteobacterial F-like plasmids: the bootstrap of 96% and 92%, respectively, support the node separating the concatenated  tra  genes of UWE25 and RTS1, SXT, R391, three gamma-proteobacterial F-like conjugative plasmid, from their closest relative, R27 plasmid (See  additional data file 2  for these trees). Taken together, all these data strongly suggest that the UWE25  tra  unit is closely related to F-like conjugative  tra  operons.
Role of the type IV secretion system encoded by the 100-kb genomic island
The functions of genes encoded by GIs may be related, among others, to pathogenicity such as the ability to exploit the host intracellular environment. Since no genetic system has been described for any obligate intracellular chlamydiae, we investigated the putative functions of this GI by bioinformatics. We focused our analyses on the TFSS, for which a previous annotation of the  tra  genes showed a genetic unit unable to transfer DNA [ 14 ]. Using different protein comparison methods described in the  additional data file 1 , we identified supplementary  tra  genes, and compared the general organization of this  tra  unit with other genetic elements encoding TFSS genes [ 20 ]. The UWE25  tra  unit displays a striking colinearity with  tra  operons encoding F-like conjugative DNA transfer system, especially to those of the F and pNL1 plasmids of  Escherichia coli  and  Novosphingobium aromaticivorans , respectively (Figure  2 ). All homologous genes essential for DNA transfer in plasmid F ( traF ,  traG ,  traH ,  traN ,  traU ,  traW , and  trbC ) and involved in sex pilus retraction and mating pair stabilization [ 20 ] are present, strongly suggesting that, similarly to the other F-like TFSSs, the gene products encoded by the UWE25  tra  unit are devoted to DNA transfer. With the only exception of  traG , these genes are not present on P-like and I-like plasmids, reinforcing the close relationship prevailing between the UWE25  tra  unit and their F-like plasmids counterparts.tratratratratraEscherichia coliNovosphingobium aromaticivoranstraFtraGtraHtraNtraUtraWtrbCtratraGtraFigure  3  shows that the UWE25  tra  unit clusters within F-like TFSSs, confirming that it may function as a F-like conjugative system. Drawn as an UPGMA tree (Figure  3A ), the comparison of the genetic organization of all  tra  units was performed as a gene order breakpoint analysis developed for the study of the mitochondrial genome evolution [ 21 ]. This analysis clearly shows that the closest relatives of the UWE25  tra  units are the  tra  operons of the F-like conjugative plasmids. The Fitch-Margoliash- and the minimum evolution comparisons performed on the same dataset presented the same tree topologies, confirming the former UPGMA results (data not shown). An  omit  test performed on this tree confirms that the results are robust: with one exception (involving the deep branching of one cluster on one tree), all 11 trees were congruent in all their nodes. Figure  3B  shows an UPGMA tree comparing the Kimura corrected  p -distances (the proportion  p  of nucleotide sites at which two sequences are different, taking into account the proportion of transversion- and transition-substitution rates) of nucleotide sequences of the concatenated  traA ,  traK ,  traB ,  traV , and  traC  genes. A similar topology is observed with (i) neighbor-joining- and minimum evolution trees inferred using the Kimura-corrected  p -distances and (ii) UPGMA, neighbor-joining- and minimum evolution trees performed on  p -distance of the whole coding sequences of the concatenated  tra  genes (See  additional data file 2  for these trees). Neighbor-joining- and minimum evolution methods comparing Kimura-corrected  p -distances of the complete coding sequences confirmed that the  tra  unit of UWE25 is phylogenetically closely related to the  tra  operons of the F-like plasmids: the bootstrap values of 94% and 91% respectively, support the node separating the concatenated  tra  genes of the chromosomal UWE25 and the R27 plasmid, a gamma-proteobacterial F-like conjugative plasmid, from those of all other plasmids (See the  additional data file 2  for these trees). In neighbor-joining and minimum evolution analyses of  p -distances, the  tra  unit of UWE25 also clusters with the  tra  operons of gamma-proteobacterial F-like plasmids: the bootstrap of 96% and 92%, respectively, support the node separating the concatenated  tra  genes of UWE25 and RTS1, SXT, R391, three gamma-proteobacterial F-like conjugative plasmid, from their closest relative, R27 plasmid (See  additional data file 2  for these trees). Taken together, all these data strongly suggest that the UWE25  tra  unit is closely related to F-like conjugative  tra  operons.tratratratraomitpptraAtraKtraBtraVtraCpptraptratratraptratratratratraOrigin of the genomic island and of its type four secretion system Our BLAST analyses [ 22 ] reveal that a majority (24/43) of genes not presenting a best hit for chlamydial genes but having homologs in other taxa are more related to proteobacterial genes (see Table  2  and the  additional data file 1  for similarity analyses indicating for each parachlamydial  tra  gene the most similar gene and its taxonomical background). Moreover, the BLAST analyses of the 21 ORFs of the third module, encoding the  tra  genes, show that most ORFs of this unit (15/21) are of proteobacterial origin. However, since six of them present the highest similarity to alpha-proteobacterial genes and six others to gamma-proteobacterial genes, a more precise origin of the parachlamydial  tra  unit could not be precisely defined by this first approach. The presence of  gly-tRNA  at the proximal end of the GI of UWE25 is consistent with a close relatedness between this GI and proteobacteria: out of 14 GIs described in the  Islander  database of Mantri  et al . [ 15 , 22 ] inserted along a chromosome by a  gly-tRNA  (14/140), 12 of them (86%) were sequenced in a proteobacterial genome. No GI of Gram-positives described in the Islander database are inserted in a chromosome within a  gly-tRNA  gene. Again, a precise proteobacterial origin could not be proposed, because the distribution of  gly-tRNA  genes in alpha- (4/22) and gamma-proteobacterial (8/72) GIs is not significantly different: by including only the non-redundant GIs, the distribution of  gly-tRNA  genes in alpha and gamma-proteobacterial GIs is 2/20 and 7/71, respectively. Comparison of gene order between all  tra  units also failed in assigning a precise origin to the UWE25  tra  unit since it branched near the alpha- and gamma-proteobacterial  tra  operons (Figure  3A ). The only first hint of a possible gamma-proteobacterial origin for the UWE25  tra  unit was brought by the phylogenetic analyses (Figure  3B  and additional files  1  & 2 ). Thus, bootstraps values of 94, 91, 96 and 92% supported the node separating the concatenated  tra  genes of UWE25 and several  tra  operons of gamma-proteobacterial F-like plasmids from the F-plasmids of an alpha-proteobacteria and of other gamma-proteobacteria. (See above, and  additional data file 2  for these trees).
Origin of the genomic island and of its type four secretion system
Our BLAST analyses [ 22 ] reveal that a majority (24/43) of genes not presenting a best hit for chlamydial genes but having homologs in other taxa are more related to proteobacterial genes (see Table  2  and the  additional data file 1  for similarity analyses indicating for each parachlamydial  tra  gene the most similar gene and its taxonomical background). Moreover, the BLAST analyses of the 21 ORFs of the third module, encoding the  tra  genes, show that most ORFs of this unit (15/21) are of proteobacterial origin. However, since six of them present the highest similarity to alpha-proteobacterial genes and six others to gamma-proteobacterial genes, a more precise origin of the parachlamydial  tra  unit could not be precisely defined by this first approach.tratratraThe presence of  gly-tRNA  at the proximal end of the GI of UWE25 is consistent with a close relatedness between this GI and proteobacteria: out of 14 GIs described in the  Islander  database of Mantri  et al . [ 15 , 22 ] inserted along a chromosome by a  gly-tRNA  (14/140), 12 of them (86%) were sequenced in a proteobacterial genome. No GI of Gram-positives described in the Islander database are inserted in a chromosome within a  gly-tRNA  gene. Again, a precise proteobacterial origin could not be proposed, because the distribution of  gly-tRNA  genes in alpha- (4/22) and gamma-proteobacterial (8/72) GIs is not significantly different: by including only the non-redundant GIs, the distribution of  gly-tRNA  genes in alpha and gamma-proteobacterial GIs is 2/20 and 7/71, respectively.gly-tRNAIslanderet algly-tRNAgly-tRNAgly-tRNAgly-tRNAComparison of gene order between all  tra  units also failed in assigning a precise origin to the UWE25  tra  unit since it branched near the alpha- and gamma-proteobacterial  tra  operons (Figure  3A ). The only first hint of a possible gamma-proteobacterial origin for the UWE25  tra  unit was brought by the phylogenetic analyses (Figure  3B  and additional files  1  & 2 ). Thus, bootstraps values of 94, 91, 96 and 92% supported the node separating the concatenated  tra  genes of UWE25 and several  tra  operons of gamma-proteobacterial F-like plasmids from the F-plasmids of an alpha-proteobacteria and of other gamma-proteobacteria. (See above, and  additional data file 2  for these trees).tratratratratratraDiscussion We showed that the  Parachlamydia -related endosymbiont UWE25 presents a 100-kb region largely fullfilling the criteria of GIs [ 16 - 18 ]. Indeed, this DNA region characterized by a high level of gene co-orientation presents a G+C content different from that of the remainder of the genome. The presence of direct repeats flanking this chromosome region enabled us to focus on 100 ORFs. Two identical  gly-tRNA  genes in tandem are present at the proximal end of this genetic element. Moreover, several mobility genes encoding transposases and bacteriophage related-proteins are located within this chromosome region. The cumulative residual G+C content analysis shows that this GI is composed of seven modules. Such a chimeric organization was already described in other GIs [ 23 , 24 ]. The first module contains chlamydiae genes probably brought by chromosome rearrangements. Some of these genes, homologous to TTSS genes of  Chlamydiaceae , might provide selective advantages to strains that retained the GI. The 2 nd , 4 th  and 6 th  modules are mainly composed of bacteriophage-related protein genes, that could reflect a putative phage implication in GI formation. The 3 rd  module codes for a TFSS similar to  tra  operons. We propose that this  tra  unit is devoted to DNA transfer, based (i) on similarity analyses demonstrating the presence of all genes encoding proteins used during a DNA transfer, (ii) on phylogenetic analyses of  tra  unit genes and, (iii) on comparison of gene order. These analyses clearly demonstrate that the UWE25  tra  unit is strongly more related to F-like conjugative system than to P-like and I-like secretion systems. The significant bootstraps of all trees obtained by standard gene phylogeny and their congruent topologies with others obtained by the gene order breakpoint analysis not biased by codon usage homing, strongly support the validity of these analyses confirming the F-like conjugative nature of the parachlamydial  tra  unit. Thus, our model significantly differs from the other proposed by Horn  et al . [ 14 ], who did not identify  traA ,  traL ,  traK ,  traV , and concluded that the UWE25  tra  unit is involved in protein export, and not in DNA transfer. The 5 th  module presents a nucleotide composition similar to the  tra  unit and is composed of a single high G+C 6-kb gene, whose product is similar to the human Nod3 protein. The Nod (Nucleotide-binding oligomerization domain) proteins are members of a family that also includes the apoptosis regulator Apaf1 (Apoptotic protease activating factor 1) and plant disease-resistance gene products [ 25 ]. The function of the human Nod3 is still unknown. Like Nod1 and Nod2, Nod3 might be involved in the recognition of conserved motifs present at the surface of bacteria, such as peptidoglycan. The nucleotide G+C composition of the 2 nd , 4 th , and 6 th  modules are similar, explaining the observed similar negative slope of the residual G+C curves. Moreover, these three modules encode phage-related proteins and proteins involved in DNA metabolism. These modules probably involved in mobility might have a common origin, the ancestral single phage module being currently separated in three pieces by the presence of the  tra  unit and of the Nod3-like protein encoding gene. The positive slope in the G+C analysis of the 7 th  module echoes those of the  tra  unit (3 rd  module) and of the Nod3-like protein (5 th  module). The 7 th  module encodes a transposition resolvase and three transposases similar to gamma-proteobacterial homologs. With the only exception of the phage-related Doc protein, that has an homolog at the beginning of the sixth module, and that might be located there after transposition, the 7 th  module appears thus to have a different origin than the 2 nd , 4 th  and 6 th  modules, though also encoding mobility genes. The presence of a F-like  tra  unit along the sequences of UWE25 is the first evidence of a putative conjugative system in chlamydiae. If conjugation occurs, it most probably takes place within free-living amoebae, that may contain several hundreds of  Parachlamydia  bacteria tightly packed in their vacuoles [ 4 ]. Such a conjugation system would be a mechanism to transfer DNA between internalized bacteria sharing an amoebal vacuole. Moreover, it may provide molecular genetic tools for obligate intracellular bacteria. The presence of  tra  units/operons in the parachlamydial UWE25 and in proteobacteria could be explained by an emergence of this unit in a common ancestor of both clades, and by its subsequent loss in  Chlamydiaceae . Another evolutionary scenario is that the  tra  unit was acquired from a proteobacteria by a  Parachlamydiaceae  in a common amoebal vacuole. Since the  tra  unit is absent from all sequenced  Chlamydiaceae  genomes, this transfer would have occurred after the divergence of  Parachlamydiaceae  and  Chlamydiaceae , at a time when  Parachlamydia  was already an intracellular bacteria. An intra-amoebal transfer of this GI is supported by the permissivity of free-living amoebae to proteobacteria [ 26 ], and by several hints suggesting its proteobacterial origin. Though phylogenetic analyses suggested a gamma-proteobacterial origin of the F-like parachlamydial  tra , further analyses have to confirm whether this GI module was acquired from an alpha-, beta-, or gamma-proteobacteria unit. We hypothesize that the F-like parachlamydial  tra  unit has been brought by a lateral transfer from a proteobacterial genome. This hypothesis is strongly supported by the cumulative GC skew analysis [ 27 - 30 ] producing a signal of the GI differing from that of the remaining of the genome (Figure  1A  and  1B ). The value of nucleotide skew analyses as good taxonomical markers is supported by (i) routine analyses on prokaryotic genome by cumulative TA-skews [ 30 ] and (ii) comparison of intragenic nucleotide skews of small subunit ribosomal RNA of the whole living world [ 31 ]. The genometric approach appeared to be able to identify GIs of  Chlamydiales . Sequencing additional genomes of environmental chlamydiae, that present a large biodiversity [ 3 ], will provide major insights on bacterial evolution and hopefully a better comprehension of the emergence of this parachlamydial GI.
Discussion
We showed that the  Parachlamydia -related endosymbiont UWE25 presents a 100-kb region largely fullfilling the criteria of GIs [ 16 - 18 ]. Indeed, this DNA region characterized by a high level of gene co-orientation presents a G+C content different from that of the remainder of the genome. The presence of direct repeats flanking this chromosome region enabled us to focus on 100 ORFs. Two identical  gly-tRNA  genes in tandem are present at the proximal end of this genetic element. Moreover, several mobility genes encoding transposases and bacteriophage related-proteins are located within this chromosome region.Parachlamydiagly-tRNAThe cumulative residual G+C content analysis shows that this GI is composed of seven modules. Such a chimeric organization was already described in other GIs [ 23 , 24 ]. The first module contains chlamydiae genes probably brought by chromosome rearrangements. Some of these genes, homologous to TTSS genes of  Chlamydiaceae , might provide selective advantages to strains that retained the GI. The 2 nd , 4 th  and 6 th  modules are mainly composed of bacteriophage-related protein genes, that could reflect a putative phage implication in GI formation.ChlamydiaceaeThe 3 rd  module codes for a TFSS similar to  tra  operons. We propose that this  tra  unit is devoted to DNA transfer, based (i) on similarity analyses demonstrating the presence of all genes encoding proteins used during a DNA transfer, (ii) on phylogenetic analyses of  tra  unit genes and, (iii) on comparison of gene order. These analyses clearly demonstrate that the UWE25  tra  unit is strongly more related to F-like conjugative system than to P-like and I-like secretion systems. The significant bootstraps of all trees obtained by standard gene phylogeny and their congruent topologies with others obtained by the gene order breakpoint analysis not biased by codon usage homing, strongly support the validity of these analyses confirming the F-like conjugative nature of the parachlamydial  tra  unit. Thus, our model significantly differs from the other proposed by Horn  et al . [ 14 ], who did not identify  traA ,  traL ,  traK ,  traV , and concluded that the UWE25  tra  unit is involved in protein export, and not in DNA transfer.tratratratratraet altraAtraLtraKtraVtraThe 5 th  module presents a nucleotide composition similar to the  tra  unit and is composed of a single high G+C 6-kb gene, whose product is similar to the human Nod3 protein. The Nod (Nucleotide-binding oligomerization domain) proteins are members of a family that also includes the apoptosis regulator Apaf1 (Apoptotic protease activating factor 1) and plant disease-resistance gene products [ 25 ]. The function of the human Nod3 is still unknown. Like Nod1 and Nod2, Nod3 might be involved in the recognition of conserved motifs present at the surface of bacteria, such as peptidoglycan.traThe nucleotide G+C composition of the 2 nd , 4 th , and 6 th  modules are similar, explaining the observed similar negative slope of the residual G+C curves. Moreover, these three modules encode phage-related proteins and proteins involved in DNA metabolism. These modules probably involved in mobility might have a common origin, the ancestral single phage module being currently separated in three pieces by the presence of the  tra  unit and of the Nod3-like protein encoding gene.traThe positive slope in the G+C analysis of the 7 th  module echoes those of the  tra  unit (3 rd  module) and of the Nod3-like protein (5 th  module). The 7 th  module encodes a transposition resolvase and three transposases similar to gamma-proteobacterial homologs. With the only exception of the phage-related Doc protein, that has an homolog at the beginning of the sixth module, and that might be located there after transposition, the 7 th  module appears thus to have a different origin than the 2 nd , 4 th  and 6 th  modules, though also encoding mobility genes.traThe presence of a F-like  tra  unit along the sequences of UWE25 is the first evidence of a putative conjugative system in chlamydiae. If conjugation occurs, it most probably takes place within free-living amoebae, that may contain several hundreds of  Parachlamydia  bacteria tightly packed in their vacuoles [ 4 ]. Such a conjugation system would be a mechanism to transfer DNA between internalized bacteria sharing an amoebal vacuole. Moreover, it may provide molecular genetic tools for obligate intracellular bacteria.traParachlamydiaThe presence of  tra  units/operons in the parachlamydial UWE25 and in proteobacteria could be explained by an emergence of this unit in a common ancestor of both clades, and by its subsequent loss in  Chlamydiaceae . Another evolutionary scenario is that the  tra  unit was acquired from a proteobacteria by a  Parachlamydiaceae  in a common amoebal vacuole. Since the  tra  unit is absent from all sequenced  Chlamydiaceae  genomes, this transfer would have occurred after the divergence of  Parachlamydiaceae  and  Chlamydiaceae , at a time when  Parachlamydia  was already an intracellular bacteria. An intra-amoebal transfer of this GI is supported by the permissivity of free-living amoebae to proteobacteria [ 26 ], and by several hints suggesting its proteobacterial origin. Though phylogenetic analyses suggested a gamma-proteobacterial origin of the F-like parachlamydial  tra , further analyses have to confirm whether this GI module was acquired from an alpha-, beta-, or gamma-proteobacteria unit. We hypothesize that the F-like parachlamydial  tra  unit has been brought by a lateral transfer from a proteobacterial genome. This hypothesis is strongly supported by the cumulative GC skew analysis [ 27 - 30 ] producing a signal of the GI differing from that of the remaining of the genome (Figure  1A  and  1B ). The value of nucleotide skew analyses as good taxonomical markers is supported by (i) routine analyses on prokaryotic genome by cumulative TA-skews [ 30 ] and (ii) comparison of intragenic nucleotide skews of small subunit ribosomal RNA of the whole living world [ 31 ]. The genometric approach appeared to be able to identify GIs of  Chlamydiales . Sequencing additional genomes of environmental chlamydiae, that present a large biodiversity [ 3 ], will provide major insights on bacterial evolution and hopefully a better comprehension of the emergence of this parachlamydial GI.traChlamydiaceaetraParachlamydiaceaetraChlamydiaceaeParachlamydiaceaeChlamydiaceaeParachlamydiatratraChlamydialesConclusions We showed that a GI present on the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of  Parachlamydiaceae  bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of  Chlamydiaceae , we hypothesize that it was acquired after the divergence between  Parachlamydiaceae  and  Chlamydiaceae , when the  Parachlamydia -related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired by a  Parachlamydiaceae  from phylogenetically-distant bacteria sharing an amoebal vacuole. Since  Parachlamydiaceae  are emerging agents of pneumonia [ 2 ] and since many GIs are also considered as pathogenicity islands [ 17 ], the Pam100G GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for studying  Chlamydiales .
Conclusions
We showed that a GI present on the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of  Parachlamydiaceae  bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of  Chlamydiaceae , we hypothesize that it was acquired after the divergence between  Parachlamydiaceae  and  Chlamydiaceae , when the  Parachlamydia -related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired by a  Parachlamydiaceae  from phylogenetically-distant bacteria sharing an amoebal vacuole. Since  Parachlamydiaceae  are emerging agents of pneumonia [ 2 ] and since many GIs are also considered as pathogenicity islands [ 17 ], the Pam100G GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for studying  Chlamydiales .ParachlamydiaceaeChlamydiaceaeParachlamydiaceaeChlamydiaceaeParachlamydiaParachlamydiaceaeParachlamydiaceaeChlamydialesMethods Sequence The genome sequence of UWE25 [ 14 ] (Accession number: NC_005861) is available at the NCBI website [ 32 , 33 ]. In this contribution, the acronym UWE25 refers only to the  Parachlamydia -related endosymbiont UWE25, and thus not to the  Acanthamoeba  sp. strain UWE25 from which the parachlamydial endosymbiont UWE25 was recovered [ 3 ]. Horn  et al . recently proposed UWE25 as the type strain of a new bacterial species:  Protochlamydia amoebophila  [ 34 ]. BLAST analyses BLAST analyses were performed with BLASTP 2.2.9 [ 35 ] available on the NCBI website [ 36 ] using the BLOSUM62 matrix, and gap penalties of 11 and 1. Each ORF was compared against all genes of non-redundant databases available at the NCBI website. An e-value of 0.001 was selected as a standard cut-off. To further identify possible homologous ORFs, we also BLASTed each  tra  gene of F plasmid versus all genes of the full genome of  Parachlamydia  and conversely, each ORF of the putative parachlamydial  tra  unit versus counterparts of the different F-like plasmids. CLUSTALW was used to detect the best relatedness of a given parachlamydial Tra protein with its possible homologs encoded by the F and pNL1 plasmids. Residual cumulative GC content The residual cumulative G+C content, a slightly modified version of the cumulative GC profile defined by Zhang and Zhang [ 19 ], was used to reveal local variations of G+C content of a genome, without using sliding windows of arbitrary size. First, a G+C content analysis was performed on 100-bp windows of the selected chromosome sequence, as for a cumulative GC skew analysis. The cumulative G+C content  GC n  of the  n th  window is obtained by cumulating the G+C contents from the first to the  n th  window: where, in the window  i ,  G i  and  C i  are the numbers of Gs and Cs, respectively, and  N i  is the total number of nucleotides. To visualize genomic regions differing from the average G+C content, a linear regression  y  defined by a slope  k  is performed on the cumulative curve using the least square methods: y ( n ) =  kn where  n  is the position of the center of the  n th  window. The residual cumulative G+C content curve  GC ' can then be drawn as a function of the position of each window center: GC ' n  =  GC n  -  kn Zhang and Zhang [ 19 ] recently demonstrated that, in some instances, abrupt changes in the residual cumulative G+C content curve correspond to genomic islands. Repeats identification The perfect tandem repeats identification was first performed using the EQUICKTANDEM software (Richard Durbin, Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK) [ 37 ] on a 200-kb DNA sequence (UWE25 genome position: 1.6 to 1.8 Mb) encompassing the  tra  genes previously identified by Horn  et al . [ 14 ]. The duplicated genes and the ORFs containing internal repeats were removed. For each pair of direct repeats, potential unperfect matches of flanking nucleotides were scanned using DNA strider 1.2.1 [ 38 ], with the following settings: a minimal size of 11 bp and 3 mismatches. Furthermore, sequences similar to direct repeats were searched along the whole chromosome, and sequences also found outside the selected 200-kb region were discarded from our analysis. Finally, the direct repeats positions were compared to the G+C content analysis, the cumulative GC skew curve, and to tRNA genes locations. Phylogenetic analyses Since Horn  et al . [ 14 ] did not identified  traA ,  traL ,  traK ,  traV , re-annotation of the UWE25  tra  unit was necessary for phylogenetic analyses. We used i) the genes of F-like plasmids encoding the following  tra  genes, i.e.  traA ,  traK ,  traB ,  traV ,  traC , and ii) the corresponding ORFs of P- and I-like plasmids [ 20 ], i.e.  trbC/VirB2 ,  trbG/VirB9 ,  trbI/VirB10 ,  trbH/VirB7 ,  trbE/VirB4  of P-plasmids and  traX ,  traN ,  traO ,  traI ,  traU  of I-plasmids, respectively. The genes were concatenated to obtain a single nucleotide sequence and aligned with CLUSTALW ([ 39 ] as it was already performed for genes of ribosomal proteins [ 40 ]. Using this alignment and the MEGA 2.1 software [ 41 ], we inferred phylogenetic relationships by drawing trees using  p -distances (the proportion  p  of nucleotide sites at which two sequences compared are different) and Kimura corrected  p -distance (correction for the rates of transition and transversion) with Unweighted Pair Group Method with Arithmetic Mean (UPGMA), neighbor-joining, and minimum evolution methods. To prevent alignment biases, trees were drawn using the complete deletion option implemented on MEGA 2.1. Gene order breakpoint analyses To quantify the inversion and transposition events leading to the current organization of  tra  operons, the gene order breakpoint analysis developed for small genomes (mitochondria) by Blanchette  et al . [ 21 ] was used to estimate the similarity of gene order existing between the  tra  unit of UWE25 and the  tra  operons reviewed by Lawley  et al . [ 20 ]. The distance calculated for two given operons  O i  and  O j  containing homologous genes proposed by Blanchette  et al . [ 21 ] was slightly modified to take into account the variation of gene numbers of  tra  operons: instead of counting the number of minimal breakpoints existing between two  tra  operons, a distance was estimated by measuring the proportion of conserved gene pairs between both genomic entities. Next, a comparison matrix is established by calculating the distance for each pairwise comparison. Finally, a dissimilarity matrix is obtained by subtracting each distance from 1. For instance, if the operon  O i  encodes sequentially four genes ( a ,  b ,  c , and  d ) and the operon  O j , six genes ( a ,  b ,  e ,  -d ,  -c , and  -f ; genes labeled by a minus sign are encoded by the complementary strand), the gene order breakpoint analysis reveals that two gene pairs are conserved:  ab  and  cd . The dissimilarity distances existing between the operons i)  O i  and  O j , and ii)  O j  and  O i  would be: 1-(2/3) = 1/3 and 1-(2/5) = 3/5, respectively. From the square dissimilarity matrix, phylogenetic trees were drawn. Three different distance-matrix analyses were used: the UPGMA, the Fitch-Margoliash- and the minimum evolution methods. To assess the robustness of the tree, an  omit  test [ 42 ] was performed on 11 UPGMA trees, in each one organism is missing.
Methods
Sequence The genome sequence of UWE25 [ 14 ] (Accession number: NC_005861) is available at the NCBI website [ 32 , 33 ]. In this contribution, the acronym UWE25 refers only to the  Parachlamydia -related endosymbiont UWE25, and thus not to the  Acanthamoeba  sp. strain UWE25 from which the parachlamydial endosymbiont UWE25 was recovered [ 3 ]. Horn  et al . recently proposed UWE25 as the type strain of a new bacterial species:  Protochlamydia amoebophila  [ 34 ].
Sequence
The genome sequence of UWE25 [ 14 ] (Accession number: NC_005861) is available at the NCBI website [ 32 , 33 ]. In this contribution, the acronym UWE25 refers only to the  Parachlamydia -related endosymbiont UWE25, and thus not to the  Acanthamoeba  sp. strain UWE25 from which the parachlamydial endosymbiont UWE25 was recovered [ 3 ]. Horn  et al . recently proposed UWE25 as the type strain of a new bacterial species:  Protochlamydia amoebophila  [ 34 ].ParachlamydiaAcanthamoebaet alProtochlamydia amoebophilaBLAST analyses BLAST analyses were performed with BLASTP 2.2.9 [ 35 ] available on the NCBI website [ 36 ] using the BLOSUM62 matrix, and gap penalties of 11 and 1. Each ORF was compared against all genes of non-redundant databases available at the NCBI website. An e-value of 0.001 was selected as a standard cut-off. To further identify possible homologous ORFs, we also BLASTed each  tra  gene of F plasmid versus all genes of the full genome of  Parachlamydia  and conversely, each ORF of the putative parachlamydial  tra  unit versus counterparts of the different F-like plasmids. CLUSTALW was used to detect the best relatedness of a given parachlamydial Tra protein with its possible homologs encoded by the F and pNL1 plasmids.
BLAST analyses
BLAST analyses were performed with BLASTP 2.2.9 [ 35 ] available on the NCBI website [ 36 ] using the BLOSUM62 matrix, and gap penalties of 11 and 1. Each ORF was compared against all genes of non-redundant databases available at the NCBI website. An e-value of 0.001 was selected as a standard cut-off. To further identify possible homologous ORFs, we also BLASTed each  tra  gene of F plasmid versus all genes of the full genome of  Parachlamydia  and conversely, each ORF of the putative parachlamydial  tra  unit versus counterparts of the different F-like plasmids. CLUSTALW was used to detect the best relatedness of a given parachlamydial Tra protein with its possible homologs encoded by the F and pNL1 plasmids.traParachlamydiatraResidual cumulative GC content The residual cumulative G+C content, a slightly modified version of the cumulative GC profile defined by Zhang and Zhang [ 19 ], was used to reveal local variations of G+C content of a genome, without using sliding windows of arbitrary size. First, a G+C content analysis was performed on 100-bp windows of the selected chromosome sequence, as for a cumulative GC skew analysis. The cumulative G+C content  GC n  of the  n th  window is obtained by cumulating the G+C contents from the first to the  n th  window: where, in the window  i ,  G i  and  C i  are the numbers of Gs and Cs, respectively, and  N i  is the total number of nucleotides. To visualize genomic regions differing from the average G+C content, a linear regression  y  defined by a slope  k  is performed on the cumulative curve using the least square methods: y ( n ) =  kn where  n  is the position of the center of the  n th  window. The residual cumulative G+C content curve  GC ' can then be drawn as a function of the position of each window center: GC ' n  =  GC n  -  kn Zhang and Zhang [ 19 ] recently demonstrated that, in some instances, abrupt changes in the residual cumulative G+C content curve correspond to genomic islands.
Residual cumulative GC content
The residual cumulative G+C content, a slightly modified version of the cumulative GC profile defined by Zhang and Zhang [ 19 ], was used to reveal local variations of G+C content of a genome, without using sliding windows of arbitrary size. First, a G+C content analysis was performed on 100-bp windows of the selected chromosome sequence, as for a cumulative GC skew analysis. The cumulative G+C content  GC n  of the  n th  window is obtained by cumulating the G+C contents from the first to the  n th  window:GCnnthnthwhere, in the window  i ,  G i  and  C i  are the numbers of Gs and Cs, respectively, and  N i  is the total number of nucleotides. To visualize genomic regions differing from the average G+C content, a linear regression  y  defined by a slope  k  is performed on the cumulative curve using the least square methods:iGiCiNiyky ( n ) =  knynknwhere  n  is the position of the center of the  n th  window. The residual cumulative G+C content curve  GC ' can then be drawn as a function of the position of each window center:nnGCGC ' n  =  GC n  -  knGCnGCnknZhang and Zhang [ 19 ] recently demonstrated that, in some instances, abrupt changes in the residual cumulative G+C content curve correspond to genomic islands.Repeats identification The perfect tandem repeats identification was first performed using the EQUICKTANDEM software (Richard Durbin, Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK) [ 37 ] on a 200-kb DNA sequence (UWE25 genome position: 1.6 to 1.8 Mb) encompassing the  tra  genes previously identified by Horn  et al . [ 14 ]. The duplicated genes and the ORFs containing internal repeats were removed. For each pair of direct repeats, potential unperfect matches of flanking nucleotides were scanned using DNA strider 1.2.1 [ 38 ], with the following settings: a minimal size of 11 bp and 3 mismatches. Furthermore, sequences similar to direct repeats were searched along the whole chromosome, and sequences also found outside the selected 200-kb region were discarded from our analysis. Finally, the direct repeats positions were compared to the G+C content analysis, the cumulative GC skew curve, and to tRNA genes locations.
Repeats identification
The perfect tandem repeats identification was first performed using the EQUICKTANDEM software (Richard Durbin, Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK) [ 37 ] on a 200-kb DNA sequence (UWE25 genome position: 1.6 to 1.8 Mb) encompassing the  tra  genes previously identified by Horn  et al . [ 14 ]. The duplicated genes and the ORFs containing internal repeats were removed. For each pair of direct repeats, potential unperfect matches of flanking nucleotides were scanned using DNA strider 1.2.1 [ 38 ], with the following settings: a minimal size of 11 bp and 3 mismatches. Furthermore, sequences similar to direct repeats were searched along the whole chromosome, and sequences also found outside the selected 200-kb region were discarded from our analysis. Finally, the direct repeats positions were compared to the G+C content analysis, the cumulative GC skew curve, and to tRNA genes locations.traet alPhylogenetic analyses Since Horn  et al . [ 14 ] did not identified  traA ,  traL ,  traK ,  traV , re-annotation of the UWE25  tra  unit was necessary for phylogenetic analyses. We used i) the genes of F-like plasmids encoding the following  tra  genes, i.e.  traA ,  traK ,  traB ,  traV ,  traC , and ii) the corresponding ORFs of P- and I-like plasmids [ 20 ], i.e.  trbC/VirB2 ,  trbG/VirB9 ,  trbI/VirB10 ,  trbH/VirB7 ,  trbE/VirB4  of P-plasmids and  traX ,  traN ,  traO ,  traI ,  traU  of I-plasmids, respectively. The genes were concatenated to obtain a single nucleotide sequence and aligned with CLUSTALW ([ 39 ] as it was already performed for genes of ribosomal proteins [ 40 ]. Using this alignment and the MEGA 2.1 software [ 41 ], we inferred phylogenetic relationships by drawing trees using  p -distances (the proportion  p  of nucleotide sites at which two sequences compared are different) and Kimura corrected  p -distance (correction for the rates of transition and transversion) with Unweighted Pair Group Method with Arithmetic Mean (UPGMA), neighbor-joining, and minimum evolution methods. To prevent alignment biases, trees were drawn using the complete deletion option implemented on MEGA 2.1.
Phylogenetic analyses
Since Horn  et al . [ 14 ] did not identified  traA ,  traL ,  traK ,  traV , re-annotation of the UWE25  tra  unit was necessary for phylogenetic analyses. We used i) the genes of F-like plasmids encoding the following  tra  genes, i.e.  traA ,  traK ,  traB ,  traV ,  traC , and ii) the corresponding ORFs of P- and I-like plasmids [ 20 ], i.e.  trbC/VirB2 ,  trbG/VirB9 ,  trbI/VirB10 ,  trbH/VirB7 ,  trbE/VirB4  of P-plasmids and  traX ,  traN ,  traO ,  traI ,  traU  of I-plasmids, respectively. The genes were concatenated to obtain a single nucleotide sequence and aligned with CLUSTALW ([ 39 ] as it was already performed for genes of ribosomal proteins [ 40 ]. Using this alignment and the MEGA 2.1 software [ 41 ], we inferred phylogenetic relationships by drawing trees using  p -distances (the proportion  p  of nucleotide sites at which two sequences compared are different) and Kimura corrected  p -distance (correction for the rates of transition and transversion) with Unweighted Pair Group Method with Arithmetic Mean (UPGMA), neighbor-joining, and minimum evolution methods. To prevent alignment biases, trees were drawn using the complete deletion option implemented on MEGA 2.1.et altraAtraLtraKtraVtratratraAtraKtraBtraVtraCtrbC/VirB2trbG/VirB9trbI/VirB10trbH/VirB7trbE/VirB4traXtraNtraOtraItraUpppGene order breakpoint analyses To quantify the inversion and transposition events leading to the current organization of  tra  operons, the gene order breakpoint analysis developed for small genomes (mitochondria) by Blanchette  et al . [ 21 ] was used to estimate the similarity of gene order existing between the  tra  unit of UWE25 and the  tra  operons reviewed by Lawley  et al . [ 20 ]. The distance calculated for two given operons  O i  and  O j  containing homologous genes proposed by Blanchette  et al . [ 21 ] was slightly modified to take into account the variation of gene numbers of  tra  operons: instead of counting the number of minimal breakpoints existing between two  tra  operons, a distance was estimated by measuring the proportion of conserved gene pairs between both genomic entities. Next, a comparison matrix is established by calculating the distance for each pairwise comparison. Finally, a dissimilarity matrix is obtained by subtracting each distance from 1. For instance, if the operon  O i  encodes sequentially four genes ( a ,  b ,  c , and  d ) and the operon  O j , six genes ( a ,  b ,  e ,  -d ,  -c , and  -f ; genes labeled by a minus sign are encoded by the complementary strand), the gene order breakpoint analysis reveals that two gene pairs are conserved:  ab  and  cd . The dissimilarity distances existing between the operons i)  O i  and  O j , and ii)  O j  and  O i  would be: 1-(2/3) = 1/3 and 1-(2/5) = 3/5, respectively. From the square dissimilarity matrix, phylogenetic trees were drawn. Three different distance-matrix analyses were used: the UPGMA, the Fitch-Margoliash- and the minimum evolution methods. To assess the robustness of the tree, an  omit  test [ 42 ] was performed on 11 UPGMA trees, in each one organism is missing.
Gene order breakpoint analyses
To quantify the inversion and transposition events leading to the current organization of  tra  operons, the gene order breakpoint analysis developed for small genomes (mitochondria) by Blanchette  et al . [ 21 ] was used to estimate the similarity of gene order existing between the  tra  unit of UWE25 and the  tra  operons reviewed by Lawley  et al . [ 20 ]. The distance calculated for two given operons  O i  and  O j  containing homologous genes proposed by Blanchette  et al . [ 21 ] was slightly modified to take into account the variation of gene numbers of  tra  operons: instead of counting the number of minimal breakpoints existing between two  tra  operons, a distance was estimated by measuring the proportion of conserved gene pairs between both genomic entities. Next, a comparison matrix is established by calculating the distance for each pairwise comparison. Finally, a dissimilarity matrix is obtained by subtracting each distance from 1.traet altratraet alOiOjet altratraFor instance, if the operon  O i  encodes sequentially four genes ( a ,  b ,  c , and  d ) and the operon  O j , six genes ( a ,  b ,  e ,  -d ,  -c , and  -f ; genes labeled by a minus sign are encoded by the complementary strand), the gene order breakpoint analysis reveals that two gene pairs are conserved:  ab  and  cd . The dissimilarity distances existing between the operons i)  O i  and  O j , and ii)  O j  and  O i  would be: 1-(2/3) = 1/3 and 1-(2/5) = 3/5, respectively.OiabcdOjabe-d-c-fabcdOiOjOjOiFrom the square dissimilarity matrix, phylogenetic trees were drawn. Three different distance-matrix analyses were used: the UPGMA, the Fitch-Margoliash- and the minimum evolution methods. To assess the robustness of the tree, an  omit  test [ 42 ] was performed on 11 UPGMA trees, in each one organism is missing.omitAuthors' contributions GG and CAR initiated the project. GG and FC reannotated the parachlamydial  tra  unit and performed all BLAST analyses. FC and LG delimited the GI by direct repeat analyses. GG drew the phylogenetic trees of the concatenated  tra  genes. After developing the residual cumulative G+C content analysis used for a software development, LG performed the G+C content analyses and the gene order comparison of  tra  units by the gene order breakpoint analysis. CAR established the correlation existing between the tRNA genes and the GIs according to taxonomy and coordinated the team work. GG wrote the first draft of the paper. All authors improved the manuscript and approved its final version.
Authors' contributions
GG and CAR initiated the project. GG and FC reannotated the parachlamydial  tra  unit and performed all BLAST analyses. FC and LG delimited the GI by direct repeat analyses. GG drew the phylogenetic trees of the concatenated  tra  genes. After developing the residual cumulative G+C content analysis used for a software development, LG performed the G+C content analyses and the gene order comparison of  tra  units by the gene order breakpoint analysis. CAR established the correlation existing between the tRNA genes and the GIs according to taxonomy and coordinated the team work. GG wrote the first draft of the paper. All authors improved the manuscript and approved its final version.tratratraSupplementary Material Additional File 1 Supplementary table. Results of BLAST [ 35 , 36 ] analyses of 100 ORFs present in the 100-kb region. BLAST analyses were performed using BLOSUM62 matrix and gap penalties of 11 and 1. Chromosome location of each ORF, its G+C content, coding strand, and the presence of at least one homolog in  Chlamydiaceae  are presented. Direct repeats (DR),  gly-tRNA  genes and limits of each modules of the GI are highlighted. Click here for file Additional File 2 Supplementary figure. Phylogenetic analyses suggest that the UWE25  tra  unit is phylogenetically closely related to F-like DNA conjugative  tra  operons: (A) UPGMA-, (B) Neighbor-joining-, and (C) minimum evolution-trees comparing p-distances and Kimura corrected  p -distances of nucleotide sequences of the concatenated  traA ,  traK ,  traB ,  traV , and  traC  genes (the UPGMA tree comparing the Kimura corrected  p -distances of  tra  genes, shown in Figure  3B , is presented here to facilitate comparison with the other trees). Interestingly, in neighbor-joining and minimum evolution analyses of the  p -distances, the  tra  unit of UWE25 is clustered with  tra  operons of gamma-proteobacterial F-like plasmids: bootstrap values of 96% and 92%, respectively, support the node separating the concatenated  tra  genes of UWE25 and RTS1, SXT, R391, three gamma-proteobacterial F-like conjugative plasmids, from their closest relative R27 plasmid. Similarly, in neighbor-joining and minimum evolution analyses of the Kimura corrected  p -distances, bootstrap values of 94% and 91%, respectively, support the node separating the concatenated  tra  genes of the chromosomal UWE25 and the R27 plasmid, another gamma-proteobacterial F-like conjugative plasmid, from those of all other plasmids. Click here for file
Supplementary Material
Additional File 1
Supplementary table. Results of BLAST [ 35 , 36 ] analyses of 100 ORFs present in the 100-kb region. BLAST analyses were performed using BLOSUM62 matrix and gap penalties of 11 and 1. Chromosome location of each ORF, its G+C content, coding strand, and the presence of at least one homolog in  Chlamydiaceae  are presented. Direct repeats (DR),  gly-tRNA  genes and limits of each modules of the GI are highlighted.Chlamydiaceaegly-tRNAClick here for fileAdditional File 2
Supplementary figure. Phylogenetic analyses suggest that the UWE25  tra  unit is phylogenetically closely related to F-like DNA conjugative  tra  operons: (A) UPGMA-, (B) Neighbor-joining-, and (C) minimum evolution-trees comparing p-distances and Kimura corrected  p -distances of nucleotide sequences of the concatenated  traA ,  traK ,  traB ,  traV , and  traC  genes (the UPGMA tree comparing the Kimura corrected  p -distances of  tra  genes, shown in Figure  3B , is presented here to facilitate comparison with the other trees). Interestingly, in neighbor-joining and minimum evolution analyses of the  p -distances, the  tra  unit of UWE25 is clustered with  tra  operons of gamma-proteobacterial F-like plasmids: bootstrap values of 96% and 92%, respectively, support the node separating the concatenated  tra  genes of UWE25 and RTS1, SXT, R391, three gamma-proteobacterial F-like conjugative plasmids, from their closest relative R27 plasmid. Similarly, in neighbor-joining and minimum evolution analyses of the Kimura corrected  p -distances, bootstrap values of 94% and 91%, respectively, support the node separating the concatenated  tra  genes of the chromosomal UWE25 and the R27 plasmid, another gamma-proteobacterial F-like conjugative plasmid, from those of all other plasmids.tratraptraAtraKtraBtraVtraCptraptratratraptraClick here for file