Chlamydial genes and genomics

Chlamydial plasmids

Introduction

Chlamydial plasmids were first described by Lovett et al., (1980) in C. trachomatis and the former C. psittaci. The plasmid was first sequenced unidirectionally by Sriprakash & McAvoy, 1987 [_C. trachomatis serovar B_] and bidirectionally by Hatt et al., 1988 [C. trachomatis serovar L1]. Plasmid sequences of C. trachomatis were subsequently published for serovar L2 [Comanducci et al., 1988; Black et al., 1989]; serovar L1 [Thomas & Clarke 1992] and serovar D [Comanducci et al., 1990]. The studies by Comanducci et al., 1988 and Thomas & Clarke 1992 were particularly important in identifying minor errors in earlier sequencing which lead to differences in open reading frames.

Comparison of C. trachomatis plasmids

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.

pLGV440.gif (15955 bytes)

Fig 1. Double click on the diagram to open. Figure shows the cryptic plasmid of C. trachomatis L1/LGV440, kindly provided by Prof I Clarke, Southampton. The position of the 8 open reading frames is shown, together with the position of 'marker' restriction endonuclease cleavage sites. The nucleotide bases are numbered from the start of the 22 bp tandem repeats. he computer-generated ORFs are numbered so that ORF 1 follows directly after the origin of replication. For an explanation of the function of these open reading frames, refer to the text below.

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].


C. trachomatis L1/440/LN





C. muridarum (Nigg strain)





Equine Chlamydophila pneumoniae (Wills et al.,_1990)





Avian Chlamydophila psittaci
A1
Fig 2. Comparative organisation of the open reading frames on the cryptic plasmid of 4 Chlamydiaceae spp.

(Adapted from Thomas & Clarke 1997, Microbiology 143, 1847 - 1854).

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

ORF number

Function / Remarks

References

1

305 and amino acids in C. trachomatis L1 and C. muridarum Nigg strain. 307 amino acids in avian C. psittaci N352. Major deletion, split ORF comprising only 185 amino acids in equine C. pneumoniae pCpnE1. Plasmid encoded replication protein thought to bind to 1 or more sets of tandem repeats. Possible replication forks identified by electron microscopy. Shares 32-35% amino acid identity with ORF 2. Tam et al., 1992
Thomas & Clarke 1997

2

330 - 335 amino acids in above strains. Similarity in properties to ORF1 suggests probably also involved in plasmid replication. ORF1 but not ORF2 has a deletion in the equine C. pneumoniae strain, suggesting that ORF2 may be more important than ORF1 for plasmid replication. Unlike other ORFs, transcribed from the complementary strand. Contains conserved domains belonging to a family of recombinase proteins. Activity probably regulated by short antisense transcripts, which have been identified in vitro in C. muridarum and the LGV biovar of C. trachomatis. Buissan & Roy, 1991
Fahr et al., 1992
Pearce 1990
Ricci et al., 1993
Thomas & Clarke, 1994
Thomas & Clarke, 1997

3

Homologous with DnaB genes of E. coli and S. typhimurium & gene 12 of phage P22. DnaB is a helicase involved in unwinding double stranded DNA during replication. An E. coli promoter-like sequence has been found to occur upstream from the plasmid-encoded dnaB gene. Sriprakash & Pearce (1990) suggest such sequences may be evolutionary relics. Hatt et al., 1988
Sriprakash & Pearce, 1990
Thomas & Clarke, 1997

4

345 - 354 amino acids in the above strains. Function not known. Thomas & Clarke, 1997

5

Encodes a 28 KDalton protein, pgp 3, which is known to be produced in the chlamydial inclusion. Minor differences between strains. Antibody to this protein can be detected in patient sera and also, for reasons that are unclear, in the sera of HIV-infected patients. Comanducci et al., 1993; 1994
Ratti _et al.
, 1995

6

Predicted translation product is 101 - 102 amino acids in above strains Thomas & Clarke, 1997

7

243 - 261 amino acids in above strains. Shares partial homology to several E. coli plasmid and phage encoded proteins including SopA and ParA involved in regulation of partitioning and copy number. Thomas & Clarke, 1997

8

Conserved. May function together with ORF 7 in similar manner to Sop A/B and ParA/B operons. Ricci et al., 1995
Comanducci et al., 1990

Practical importance of the chlamydial plasmid in chlamydial diagnostics

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 as an amplification target for nucleic acid-based diagnostic tests and also to develop DNA microarray procedures [Westin et al., 2001]. Secondly, chlamydial plasmids and phages are 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 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. This crucially important goal remains one of the 'Mount Everests' of chlamydial research.

Functional role of the plasmid

The plasmid 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. Why, then, 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?

Comment by Jim Moulder (2002).

"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

NEXT: Chlamydial phages

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 [Acrobat]

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 [Acrobat]

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 [Acrobat]

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: Equine infections].

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Topic revision: r5 - 2011-04-02 - MeWard
 
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