Extrachromosomal elements of the Chlamydiaceae
Extrachromosomal plasmid sequences pCpnKo from C. pneumoniae koala LPCoLN [24], pCpnE1 from C. pneumoniae horse N16 [34], pCpA1 from C. psittaci avian N352 (Lusher ME, Gregory J, Storey CC, Richmond SJ: Analysis of the complete nucleotide sequence of the plasmid pCpA1 isolated from an avian strain of Chlamydia psittaci, Submitted), pCfe1 from C. felis feline Fe/C-56 [35], pCpGP1 from C. caviae guinea pig GPIC [36], pMoPn from C. muridarum mouse Nigg [25], and pCTA, pJALI, pSW2 and pLVG440 from C. trachomatis human serovars A [37], B [38], E [38] and L1 [39], respectively, were compared for their overall synteny and relationship (Additional file 9). These plasmids each contain eight major open reading frames (ORFs – potentially encode a protein) designated ORF1-8. Although the plasmid sequences vary in size (7169-7966 bp), alignment of their amino acid sequences revealed a high degree of similarity and large conserved regions (Additional files 9). The pCpnKo and pCpnE1 plasmid sequences share 96.2% identity and are more closely related to pCpA1 (81.3 and 78.9% identity), pCpGP1 (81.6 and 79.1% identity) and pCfe1 (77.5 and 75.1% identity) than with pMoPn (69.5 and 67.7% identity), pCTA, pJALI, pSW2 and pLVG440 (63.9-69.5% identity) (Additional file 10). Overall, there were approximately 30 indels among the species; three of the longest indels were identified in ORF1 of pCpnE1 (deletion), pCfe1 (insertion) and pSW2 (deletion). A phylogenetic tree was inferred from multiple sequence alignment of the amino acid sequence (Figure 2). Three main branches (supported with a high bootstrap value) were evident: (1) C. pneumoniae LPCoLN and N16; (2) C. psittaci N352, C. felis Fe/C-56 and C. caviae GPIC; (3) C. muridarum and C. trachomatis isolates.
Figure 2  Phylogeny of the chlamydial plasmid. Phylogenetic relationships of C. pneumoniae koala LPCoLN (pCpnKo), C. pneumoniae horse N16 (pCpnE1), C. psittaci avian N352 (pCpA1), C. felis feline Fe/C-56 (pCfe1), C. caviae guinea pig GPIC (pCpGP1), C. muridarum mouse Nigg (pMoPn) and C. trachomatis human serovars A (pCTA), B (pJALI), E (pSW2) and L1 (pLVG440) were inferred from predicted amino acid sequences, and were constructed by Neighbor-Joining analysis and 1,000 bootstrap replicates. The bacteriophage is another strain-specific extrachromosomal element reported in chlamydial species. In C. pneumoniae, AR39 is the only human genome to have an extrachromosomal bacteriophage (4524 nt single-stranded DNA) [25], whereas the koala LPCoLN genome showed remnants of a phage. The first remnant of a phage in the koala LPCoLN genome was evident in CPK_ORF00729, a 366 nt incomplete ORF that is presumably defective, sharing approximately 79% similarity (nt 301/382) to the partial-length of the human AR39 phage. CPK_ORF00729 also shares 97% similarity (nt 324/333) to the Chp1 remnant (C. psittaci phage), which is present in the four C. pneumoniae human genomes. This suggests earlier integration of the phage genome in the C. pneumoniae genome (Geng MM, Schuhmacher A, Muehldorfer I, Bensch KW, Schaefer KP, Schneider S, Pohl T, Essig A, Marre R, Melchers K: The genome sequence of Chlamydia pneumoniae TW183 and comparison with other Chlamydia strains based on whole genome sequence analysis, submitted). The second koala LPCoLN phage remnant was a 445 nt ORF, termed CPK_ORF00730, which appears to be 'intact', sharing approximately 77% similarity (nt 342/445) to the human AR39 phage. The koala LPCoLN phage remnants are positioned between hypothetical genes in the genome and there appear to be no further remnants of the phage within this region of the genome. A comparison of the C. pneumoniae koala LPCoLN phage with other chlamydial species revealed approximately 77-79% sequence identity to partial-length sequences of the C. psittaci Chp2 phage, C. pecorum phage 3, C. caviae phiCPG1 phage and C. abortus Chp4 phage.