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Chlamydial plasmids

 [Lay reader: The genetic material, DNA, consists of various sequences of just four nucleotide bases forming the genetic code. Proteins similarly consist of sequences of amino acids which are specified by the nucleotides on DNA. A unique combination of three nucleotide bases is required to specify a particular amino acid, the so-called triplet code. Open reading frames are nucleotide sequences lying between recognisable start and stop signals, which are potentially (not necessarily) translatable into protein].

All plasmids from human C. trachomatis isolates are extremely similar, with less than 1% nucleotide sequence variation  All are about 7,500 nucleotides in size, with eight open reading frames computer-predicted to code for proteins of more than 100 amino acids, with short non-coding sequences between some of them only [Thomas and Clarke, 1997]. These open reading frames are shown in the diagram below for the cryptic plasmid of C. trachomatis L1/ 440/LN.

All chlamydial plasmids have four 22 base pair tandem repeats in the intergenic region between ORFs 1 and 8, plus AT rich clusters upstream of this region and an inverted repeat. This degree of conservation suggests that this region is extremely important. It is known to be analogous to the origin of replication (ORI in Fig 1 above) of some E. coli plasmids. In the figure below adapted from Thomas and Clarke, 1997 the black box at the left of each sequence [which has been linearized for convenience] represents the start of the 22 base pair repeats at nucleotide 1. The computer predicted initiation codon for each ORF is also conserved for each plasmid, ATG for ORFs 1, 3, 4, 5, & 6 and GTG for ORFs 7/8. The shaded ORFs are those with a predicted GTG initiation codon. Codon usage appears to be similar to that in the C. trachomatis chromosome. All ORFs are transcribed from the same strand, with the exception of ORF 2, which is transcribed from the complementary strand in the direction indicated.  Overall, the organization of the ORFs in these 4 Chlamydiaceae species is essentially the same, the only major difference being the deletion of part of ORF 1 in the equine Chlamydophila pneumoniae strain N16. In vitro all of these ORFs are transcribed [Pearce 1990] but the suggestion that multiple sigma factors might regulate the transcription of plasmid genes [Ricci et al., 1995] is not supported by the results of chlamydial genomic sequencing. Plasmid specified proteins have been identified in proteomic maps of C. trachomatis [Shaw et al., 2002].

In their nucleotide sequence, chlamydial plasmids are more closely related than is the corresponding chromosomal DNA. The two Chlamydia species shown below share 80% identity, while the two Chlamydophila species share 69% identity. There is 60% identity between the Chlamydia trachomatis L1 and Chlamydophila psittaci A1 explaining why cross hybridization between C. trachomatis and C. psittaci plasmids has been observed only under conditions of low stringency  [see Thomas and Clarke, 1997 for further references].

Some of the possible functions of these open reading frames are summarized in the table below, together with the relevant literature sources.

The chlamydial plasmid has great practical importance. It is a favoured target for DNA-based diagnosis of C. trachomatis infection for two reasons. Firstly, there are approximately 7-10 copies of the plasmid present per chlamydial particle [Tam et al., 1992]. Use of a multi-copy gene [the gene encoding 16S chlamydial rRNA is another example] is an in built amplification factor enhancing the possibility of detecting an individual particle. Thus the plasmid has been used to develop new diagnostic methods based on DNA microarray technology [Westin et al., 2001]. Secondly, chlamydial plasmids and bacteriophages are also of great interest for the development of shuttle vectors for the genetic manipulation of chlamydiae. In the case of the plasmid The lack of such methodology is undoubtedly a major impediment to chlamydial research. Progress has been made [Tam et al., 1994] but the lack of major non-coding regions in the plasmid makes a plasmid shuttle vector tricky to design. Thus this crucially important goal remains as one of the Mount Everests of chlamydial research. 

 The plasmid is also of theoretical interest. Its sequence is highly conserved among different isolates of C. trachomatis. There are authenticated examples of C. trachomatis strains lacking the plasmid [Farencena et al., 1997; Miyashita et al., 2001] and it does have effects. Plaque purified C. trachomatis free of the plasmid has unusual inclusion morphology, is glycogen free, and shows no alteration in antibiotic sensitivity [Miyashita et al., 2001]. However, the fact that such strains exist shows that the plasmid is not essential for C. trachomatis survival.  The question then, is why has the plasmid has been so effectively maintained through chlamydial evolution? Is this simply an example of the selfish gene, or does the plasmid confer some unknown advantage to chlamydiae bearing in mind that not all the functions of the ORFs have yet been identified.  Fortunately for chlamydial diagnosis and diagnostics company shareholders, plasmid-free C. trachomatis strains are rare. 

[Comment elicited from [JWM]: "If you promise to remember that I have not followed the chlamydial plasmid literature closely, here are a few thoughts on their evolution. The high degree of similarity among the plasmids from both Chlamydia and Chlamydophila species suggests that an ancestral plasmid was acquired by the chlamydial lineage a very long time ago, perhaps before divergence of the two Chlamydiaceae genera.  It would be of great interest to know if the other families also have plasmids and what their relation of the Chlamydiaceae plasmids might be. That plasmid-less chlamydiae are rare suggests that plasmid loss is selected against--that the plasmids do have a function.  The near-identity of the C. trachomatis plasmids can be taken as evidence for strong selection.  The plasmids of other bacteria are seldom required for in vitro growth and any of them contribute to the virulence of their bacterial hosts.  It may be that the chlamydial plasmids are needed for continued existence under conditions more demanding than cell culture--multiplication and indefinite serial transmission in natural hosts living under natural conditions.  This is another example of a general idea I presented several times in the Evolution section"].

[INC & MEW] May 2002

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References

Buissan, J. P. & Roy, P. H. (1991). The 7.5kb plasmid of Chlamydia trachomatis codes for a site-specific recombinase. In: Abstracts of the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, abstract 81, p112. American Society of Microbiology, Washington DC.

Comanducci, M., Ricci, S. & Ratti, G. (1988). The structure of a plasmid of Chlamydia trachomatis believed to be required for growth within mammalian cells. Molecular Microbiology 2, 531 - 538.

Comanducci, M., Ricci, S., Cevenini, R. & Ratti, G. (1990). Diversity of the Chlamydia trachomatis common plasmid in biovars with different pathogenicity. Plasmid 23, 149 - 154.

Comanducci M, Cevenini R, Moroni A, Giuliani MM, Ricci S, Scarlato V, Ratti G. (1993). Expression of a plasmid gene of Chlamydia trachomatis encoding a novel 28 kDa antigen. Journal of General Microbiology 139, 1083 - 1092.

Comanducci, M., Manetti, R., Bini, L., Santucci, A., Pallini, V., Cevenini, R., Sueur, J. M., Orfila, J. & Ratti, G. (1994). Humoral immune response to plasmid protein pgp3 in patients with Chlamydia trachomatis infection. Infection and Immunity 62, 5491 - 5497.

Fahr, M. J., Sriprakash, K. S. & Hatch, T. P. (1992). Convergent and overlapping transcripts of the Chlamydia trachomatis 7.5-kb plasmid. Plasmid 28, 247 - 257.

Farencena, A., Comanducci, M., Donati, M., Ratti, G. &  Cevenini, R. (1997). Characterization of a new isolate of Chlamydia trachomatis which lacks the common plasmid and has properties of biovar trachoma. Infection and Immunity 65, 2965 - 2969. Full article

Hatt, C., Ward, M. E., Clarke, I. N. (1988). Analysis of the entire nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar L1. Evidence for involvement in DNA replication. Nucleic Acids Res. 16, 4053- 4067.

Lovett, M., Kuo, K-K., Holmes, K & Falkow, S. (1980). Plasmids of the genus Chlamydia. In: Current chemotherapy and infectious diseases, vol 2, pp 1250-1252 (Eds Nelson, J. & Grassi, C.) published by American Society of Microbiology, Washington DC.

Miyashita, N., Matsumoto, A., Fukano, H., Niki, Y. & Matsushima, T. (2001). The 7.5-kb common plasmid is unrelated to the drug susceptibility of Chlamydia trachomatis. Journal of Infection and Chemotherapy 7, 113 - 116.

Pearce, B. J., Fahr, M. J., Hatch, T. P. & Sriprakash, K. S. (1991). A chlamydial plasmid is differentially transcribed during the life cycle of Chlamydia trachomatis. Plasmid 26, 116 - 122.

Ratti, G., Comanducci, M., Orfila, J., Sueur, J. M. & Gommeaux, A. (1995). New chlamydial antigen as a serological marker in HIV infection. Lancet. 346, 912. [Letter] 

Ricci, S., Ratti, G. & Scarlato, V. (1995). Transcriptional regulation in the Chlamydia trachomatis pCT plasmid. Gene 154, 93 - 98. Full article

Shaw, A. C., Gevaert, K., Demol, H. et al., (2002). Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2. Proteomics 2, 164 - 186.

Sriprakash, K. S. & Macavoy, E. S. (1987). Characterization and sequence of a plasmid from the trachoma biovar of Chlamydia trachomatis. Plasmid 18, 205 - 214.

Sriprakash KS, Pearce BJ. (1990). Mapping of transcripts encoded by the plasmid in Chlamydia trachomatis. FEMS Microbiology Letters 59, 299 - 303.

Stothard, D.R., Williams, J. A., Van Der Pol, B. & Jones, R. B. (1998). Identification of a Chlamydia trachomatis serovar E urogenital isolate which lacks the cryptic plasmid. Infection and Immunity 66, 6010 - 6013. Full article

Tam, J. E., Davis, C. H., Thresher, R. J. & Wyrick, P. B. (1992). Location of the origin of replication for the 7.5-kb Chlamydia trachomatis plasmid. Plasmid 27, 231- 236.

Tam, J. E., Davis, C. H. & Wyrick, P. B. (1994). Expression of recombinant DNA introduced into Chlamydia trachomatis by electroporation. Canadian Journal of Microbiology 40, 583 - 591.

Thomas, N. S & Clarke I. N. (1992). Revised map of the Chlamydia trachomatis L1/440/LN plasmid. In: Proceedings of the 2nd meeting of the European Society for chlamydial research, p42. [Eds P-A Mardh et al.,] published by Societa Editrice Esculapio Bologna Italy.

Thomas, N. S. & Clarke, I. N. (1994). Molecular characterization of the plasmid from the Chlamydia trachomatis mouse pneumonitis biovar. In : Chlamydial Infections. Proceedings of the 8th international symposium on human chlamydial infections, pp251-254 [Eds Orfila, J et al.,] published by Societa Editrice Esculapio, Bologna Italy.

Thomas, N. S., Lusher, M., Storey, C. C., Clarke, I. N. (1997). Plasmid diversity in Chlamydia. Microbiology (UK) 143, 1847 - 1854. Full article

Westin, L., Miller, C., Vollmer, D., Canter, D., Radtkey, R., Nerenberg, M. & O'Connell, J. P. (2001). Antimicrobial resistance and bacterial identification utilizing a microelectronic chip array. Journal of Clinical Microbiology 39, 1097 - 1104. Full article

Wills, J. M., Watson, G., Lusher, M., Mair, T. S., Wood, D., Richmond, S. J. (1990). Characterisation of Chlamydia psittaci isolated from a horse. Veterinary Microbiology 1990 Jul;24(1):11 - 19. [N16 is an unusual isolate currently regarded as being an equine Chlamydophila pneumoniae. See: Infections in horses]. 

NEXT: Chlamydial phages