The plasmids of Chlamydia trachomatis and Chlamydophila pneumoniae (N16): accurate determination of copy number and the paradoxical effect of plasmid-curing agents

Mark A. Pickett, J. Sylvia Everson, Patrick J. Pead and Ian N. Clarke

Molecular Microbiology Group, University of Southampton Medical School, MP814, Southampton General Hospital, Hampshire SO16 6YD, UK

Correspondence
Mark A. Pickett
map{at}soton.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A 7·5 kbp cryptic plasmid is found in almost all isolates of Chlamydia trachomatis. Real-time PCR assays, using TaqMan chemistry, were set up to quantify accurately both the chlamydial plasmid and the single copy, chromosomal omcB gene in the infectious, elementary bodies (EBs) of C. trachomatis L1 440. Plasmid copy number was also determined in the EBs of six other lymphogranuloma venereum (LGV) isolates (serovars L1–L3), ten trachoma isolates (serovars A–C) and nine urogenital isolates (serovars D–J). The results indicated an average plasmid copy number of 4·0±0·8 (mean±95 % confidence interval) plasmids per chromosome. During the chlamydial developmental cycle, up to 7·6 plasmids per chromosome were detected, indicating an increased plasmid copy number in the actively replicating reticulate bodies. Attempts to eliminate the plasmid from strain L1 440 using the plasmid-curing agents ethidium bromide, acridine orange or imipramine/novobiocin led to a paradoxical increase in plasmid copy number. It is speculated that the stress induced by chemical curing agents may stimulate the activity of plasmid-encoded replication (Rep) proteins. In contrast to C. trachomatis, only a single isolate of Chlamydophila pneumoniae bears a plasmid. C. pneumoniae strain N16 supports a 7·4 kbp plasmid in which ORF1, encoding one of the putative Rep proteins, is disrupted by a deletion and split into two smaller ORFs. Similar assay techniques revealed 1·3±0·2 plasmids per chromosome (mean±95 % confidence interval) in EBs of this strain. These findings are in agreement with the hypothesis that the ORF1-encoded protein is involved in, but not essential for, plasmid replication and control of copy number.


Abbreviations: AcOr, acridine orange; EB, elementary body; EtBr, ethidium bromide; MCC, minimum cytotoxic concentration; QPCR, quantitative PCR; RB, reticulate body


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Members of the Chlamydiaceae are obligately intracellular, Gram-negative bacteria of significant importance in both human and animal pathogenesis. These organisms exhibit a unique developmental cycle, in which a metabolically active reticulate body (RB) gives rise to a dense, infectious elementary body (EB) (Rockey & Matsumoto, 2000). The family comprises two genera, Chlamydophila and Chlamydia (Everett et al., 1999), although this classification remains controversial (Schachter et al., 2001).

Chlamydophila pneumoniae is an important human pathogen implicated in arterial disease (Saikku et al., 1992); infection of the respiratory tract causes an atypical pneumonia (Hahn et al., 2002). Although predominantly a human pathogen, C. pneumoniae strains have been isolated from animal species, e.g. equines (Everett et al., 1999; Storey et al., 1993; Wills et al., 1990) and koala (Wardrop et al., 1999).

Chlamydia trachomatis is not only the major infectious agent of preventable blindness in the developing world (Thylefors et al., 1995), but also the commonest cause of non-specific urethritis in developed countries (Burstein & Zenilman, 1999). Sexually transmitted disease caused by C. trachomatis is frequently undiagnosed in women, whereupon an ascending infection may lead to pelvic inflammatory disease, salpingitis and consequent infertility (Paavonen, 1998).

Extrachromosomal elements have been detected in several chlamydial species: both a cryptic plasmid (Lovett et al., 1980; Thomas et al., 1997) and a chlamydiaphage of the microvirus family (Everson et al., 2003; Richmond et al., 1982) have been described. Almost all strains of C. trachomatis harbour the plasmid, but some plasmid-free isolates have been described (An et al., 1992; Farencena et al., 1997; Matsumoto et al., 1998; Peterson et al., 1990; Stothard et al., 1998, 1999). There is a marked conservation (<1 % variation) of plasmid DNA sequences within this species (Black et al., 1989; Comanducci et al., 1988, 1990; Hatt et al., 1988; Sriprakash & MacAvoy, 1987b; Thomas & Clarke, 1992). The copy number of the plasmid has not been determined accurately, but isotopic methods suggest the presence of between seven and ten plasmid copies per bacterial chromosome (Palmer & Falkow, 1986; Tam et al., 1992).

Eight major ORFs (>100 bases) have been assigned to the chlamydial plasmid (Black et al., 1989; Comanducci et al., 1988, 1990; Hatt et al., 1988; Sriprakash & MacAvoy, 1987b; Thomas & Clarke, 1992). The four 22-bp tandem repeats located in the intergenic region between ORF8 and ORF1, together with an upstream AT-rich region, are indicative of an origin of replication (Chattoraj, 2000). This location of the origin has been confirmed by electron microscopy (Tam et al., 1992). The position of ORF1 and ORF2 immediately downstream of the iteron-like, tandem repeats, together with the size and net positive charge of their hypothetical products (which share 32–35 % amino acid identity), indicates that they may function as replication proteins (Thomas et al., 1997). In other systems, plasmid copy number is controlled by the cross-linking, or ‘handcuffing’, of iteron regions by such plasmid-encoded replication proteins (Park et al., 2001).

Investigation of the molecular biology of the Chlamydiaceae has been hampered by the lack of a gene transfer system. Consequently, there is no method available to introduce foreign DNA stably into these organisms. The chlamydiaphage and plasmid are obvious candidates as vectors in such a system. Previous attempts to transform chlamydiae with a recombinant plasmid containing the chloramphenicol acetyltransferase gene inserted into ORF1 yielded only transient expression (Tam et al., 1994). Such a shuttle vector system must comprise a replication-competent vector with a selectable marker into which foreign DNA sequences may be inserted. The limited understanding of the function of plasmid ORFs and the lack of intergenic regions may restrict the placement of foreign DNA sequences. In addition, a gene transfer system must overcome potential restriction barriers and incorporate a host able to support replication of the vector. As plasmid-free strains of chlamydiae may not be able to support plasmid replication, the most suitable host would be a plasmid-containing strain cured of its plasmid.

In this work, quantitative PCR (QPCR) assays were used to determine accurately chlamydial plasmid copy number in both C. trachomatis and C. pneumoniae. Changes in plasmid copy number during the developmental cycle were also investigated. Finally, attempts were made to eliminate the chlamydial plasmid from the organism by using chemical curing agents.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Culture of chlamydiae and EB purification.
Strains used in this study are listed in Table 1. Chlamydiae were grown as previously described (Garner et al., 2004). Host buffalo green monkey kidney (BGMK) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal calf serum (FCS; 10 % v/v). Cells were infected with chlamydial EBs in medium containing cycloheximide (1 µg ml–1) and gentamicin (25 µg ml–1). All strains except those belonging to the C. trachomatis lymphogranuloma venereum (LGV) biovar required centrifugation onto host cells for efficient infection. Infected cells were detached from the flask with PBS containing trypsin/EDTA and pelleted by centrifugation in DMEM/FCS. Intracellular chlamydiae were released using a Dounce homogenizer and EBs were purified by centrifugation through Urografin 370 (32 % v/v; Schering) or Triosil 440 (25 % w/v; Nycomed).


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Table 1. Bacterial strains

 
Use of plasmid-curing agents.
BGMK cells were cultured in the presence of increasing amounts of the plasmid-curing agents ethidium bromide (EtBr), acridine orange (AcOr), imipramine or novobiocin to determine their minimum cytotoxic concentrations (MCCs), assessed by cell rounding and detachment from the flask. In subsequent experiments, each agent was used at levels below its MCC. Initially, the agents were used at concentrations known to exhibit antiplasmid effects in free-living bacteria (Courtright et al., 1988; Molnar & Foldeak, 1989; Trevors, 1986); these were 25 µg EtBr ml–1, 2·5 µg AcOr ml–1 or a mixture of 20 µg novobiocin and 20 µg imipramine ml–1. Subsequently, a range of EtBr concentrations up to 80 % of the MCC was used. C. trachomatis L1 440 was grown in BGMK cells for seven passages with plasmid-curing agent and infected cells were then harvested at 48 h post-infection (p.i.) for PCR analysis.

Extraction of DNA for QPCR analysis.
Lysis of infected cells and/or chlamydiae was achieved by heating in the presence of ammonia using a modified method of Lanham et al. (2001). Infected cells or purified EBs were centrifuged at 12 000 g for 10 min. The supernatant was discarded and the pellet resuspended in PBS. The sample was centrifuged under the same conditions, the supernatant discarded and the pellet resuspended in 135 µl ammonia solution (2 M). Each sample was heated at 96 °C in an open microfuge tube or microplate well until dryness (approx. 50 min). The residue was then resuspended in 50 µl high-quality water. Samples were diluted 1 : 1000 prior to QPCR analysis.

Preparation of DNA standards.
Laboratory strains of Escherichia coli harbouring the recombinant plasmids listed in Table 2 were grown in liquid culture at 37 °C to late exponential phase in the presence of ampicillin (25 µg ml–1). Plasmids were prepared by alkaline lysis using a Plasmid Midi kit (Qiagen) according to the manufacturer's instructions. Estimates of plasmid purity were obtained by agarose gel electrophoresis and measurement of A260/A280 ratios. Plasmid quantification was carried out by the measurement of A260 and also using a fluorometric PicoGreen (Molecular Probes) assay in an SLT Fluostar (BMG Labtech), with a pBR322 (Promega) standard. To minimize errors during QPCR analysis, the C. trachomatis standard DNAs (pCTL12A and pSRP1A) were mixed prior to serial dilution in high-quality water containing pBR322 (100 µg ml–1); the latter was used to block absorption of standard DNA to the polypropylene tubes. The C. pneumoniae standards (pH17 and pMWU1A) were co-diluted by the same method. Standard dilutions used in the QPCR ranged between 100 and 107 DNA molecules per reaction.


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Table 2. Recombinant plasmid standards

 
Real-time QPCR.
The absolute number of chlamydial plasmids and omcB genes in each sample was determined by performing 5'-exonuclease (TaqMan) assays using unlabelled primers and carboxyfluorescein/carboxytetramethylrhodamine (FAM/TAMRA) dual-labelled probes. Due to the lack of sequence similarity between the genera, separate primer and probe sets were designed for C. trachomatis and C. pneumoniae (Table 3). For the C. trachomatis assay, primers and probes were selected from DNA sequence regions conserved between the serovars. A region within ORF2 of the chlamydial plasmid displays appropriate sequence conservation. A single copy of the omcB gene is located on the chlamydial chromosome, hence the ratio of chlamydial plasmid : omcB is equivalent to the number of plasmid copies per bacterium. Five microlitres of sample was added to 20 µl reaction mixture containing forward primer (300 nM), reverse primer (300 nM), probe (100 nM) and TaqMan Universal PCR Master Mix (Applied Biosystems). Real-time PCR cycles were performed in an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. Amplification plots showing the relative change in fluorescence ({Delta}Rn) during the PCR were assessed and an arbitrary threshold level of {Delta}Rn set close to, but above, baseline levels. The threshold cycle (Ct) for each sample is the PCR cycle number at which the threshold level of {Delta}Rn is achieved. For each sample, the Ct approximates to the cycle at which fluorescence change is first detectable. Standard curves of Ct against the logarithm of the number of DNA molecules per reaction were plotted. The Ct value obtained for an unknown may then be used to interpolate the number of DNA molecules present.


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Table 3. Real-time PCR primer and probe sequences

 

   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Real-time PCR assay for C. trachomatis
Assays were set up using recombinant plasmids as DNA standards (Fig. 1). The specificity of oligonucleotide primers and probes for C. trachomatis was confirmed by performing real-time PCRs using total DNA derived from cultured human cells and C. pneumoniae. Results were negative in both cases (data not shown). Amplification plots (Fig. 1a, c) show typical sigmoid shape, with the fluorescence change becoming detectable during the exponential phase of the reaction, followed by saturation due to depletion of reactants. As expected, an increase in the starting amount of DNA allows an earlier detectable fluorescence change. The total change in fluorescence throughout the reactions is greater for the plasmid (approx. 1·0 relative unit) than for omcB (approx. 0·6 relative units). The standard curves of Ct against log (DNA molecules per reaction) are linear, with correlation coefficients r2=0·91 for both plasmid and omcB assays. The slope of the standard curve is proportional to the efficiency of the PCR and indicates an efficiency of 0·84 for the plasmid assay and 0·81 for omcB, where an efficiency of 1·0 would represent a doubling of total DNA amount every cycle. Both assays consistently detected as few as ten molecules of target DNA, but replicates were unreliable when there were fewer than ten target molecules present in the reaction.



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Fig. 1. Real-time PCR analysis of C. trachomatis DNA standards. (a) Amplification plot of the C. trachomatis plasmid ORF2 assay using the pCTL12A standard. (b) Standard curve derived from (a). (c) Amplification plot of the C. trachomatis omcB assay using the pSRP1A standard. (d) Standard curve derived from (c). Serial, tenfold dilutions of purified plasmid DNA were used as template in the reactions: 100 ({blacklozenge}),10–1 ({lozenge}),10–2 ({blacksquare}),10–3 ({square}),10–4 ({blacktriangleup}),10–5 ({triangleup}). 100 pCTL12A is equivalent to 9·56x105 DNA molecules per reaction, and 100 pSRP1A is equivalent to 2·10x106 DNA molecules per reaction. The amplification plots are representative and illustrate, for each template concentration, the change in fluorescence as the PCR cycle number increases. The threshold cycle (Ct) for each reaction is the cycle number at which a fixed threshold value of relative fluorescence is attained. Standard curves of Ct against number of DNA molecules per reaction were used to calculate unknowns. Standard reactions were performed in triplicate.

 
Both circular and linearized plasmid standards were subjected to the ammonia/heat treatment used for cell lysis. Linearization did not affect the efficiency of detection by real-time PCR (data not shown).

Determination of C. trachomatis plasmid copy number
Purified C. trachomatis EBs were lysed as described and QPCR analyses carried out for the plasmid and omcB gene. The plasmid copy number per EB was calculated as 4·0±0·8 (mean±95 % confidence interval).

Plasmid copy number variation during the developmental cycle
BGMK cells infected with C. trachomatis L1 440 were removed for ammonia lysis and QPCR analysis at 0, 16, 24, 41 and 48 h p.i. The level of omcB detected in each sample (Fig. 2a) reflects the amount of chlamydial genome (and hence chlamydiae) in each aliquot. An increase in omcB is first detected at 24 h p.i. Copies of chlamydial genome then increase exponentially – a doubling time of approximately 3 h is indicated. Lysis of host cells was observed approximately 40 h p.i. Fig. 2(b) indicates that, during the infectious cycle, there is an initial increase in plasmid copy number, which reaches a maximum at 16 h p.i. Thereafter, a decrease in plasmid copy number is observed.



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Fig. 2. Variation of C. trachomatis plasmid copy number during the developmental cycle. Infected cells were removed for quantitative PCR analysis at 0, 16, 24, 41 and 48 h p.i. (a) Relative number of chlamydial genomes during the developmental cycle. omcB assay results equate to relative genome copies, which were normalized to one at 0 h p.i. (b) Plasmid copy number (plasmid : omcB ratio) during the chlamydial developmental cycle.

 
Plasmid copy number in other C. trachomatis serovars
The plasmid copy number in 25 other strains from 12 serovars of C. trachomatis was determined by QPCR. All strains had a similar plasmid content to L1 440; copy number variation was statistically not significant (ANOVA).

Effect of plasmid-curing agents on BGMK cells and C. trachomatis
The MCCs of the curing agents for BGMK cells were 250 µg ml–1 (EtBr), 25 µg ml–1 (AcOr), 25 µg ml–1 (novobiocin) and 25 µg ml–1 (imipramine). When an imipramine/novobiocin mixture was used for chlamydial plasmid curing, these agents were present in the cell culture medium at all times. However, if EtBr or AcOr was present at the time of infection, no chlamydial inclusions were produced, suggesting chlamydicidal properties associated with these two agents. Therefore, in subsequent experiments, EtBr or AcOr was added 16 h p.i. When C. trachomatis was cultured in the presence of AcOr or EtBr alone, or a mixture of novobiocin and imipramine, the ratio of plasmid : omcB did not decrease (Fig. 3a). This indicated that these agents did not have a curative effect on the chlamydial plasmid. Paradoxically, the ratio of plasmid : omcB increased. To investigate this effect further, we used EtBr at increasing concentrations and again determined the plasmid : omcB ratio. The results (Fig. 3b) indicate that, as the concentration of EtBr increases, the plasmid copy number rises. TaqMan assay sensitivity was not affected by the addition of EtBr (200 ng ml–1) or AcOr (20 ng ml–1) to the reaction (data not shown).



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Fig. 3. Effect of curing agents on chlamydial plasmid copy number. C. trachomatis L1 440 was passaged seven times through BGMK cells in the presence of selected curing agents. When an imipramine/novobiocin mixture was used, these agents were present in the medium throughout the passages. When either EtBr or AcOr was used, the agent was added 16 h p.i. in each passage. Infected cells were subsequently lysed for quantitative PCR analysis. Plasmid copy number is calculated from the ratio chlamydial plasmid : omcB. (a) Effect of curing agents. Cell culture medium contained EtBr (25 µg ml–1) or AcOr (2·5 µg ml–1) or a mixture of novobiocin (20 µg ml–1) and imipramine (20 µg ml–1). No curing agent was added to the control culture. (b) Effect of increasing EtBr concentration. EtBr was added to the culture medium at different concentrations, up to 80 % of the MCC for BGMK cells.

 
Real-time PCR assay for C. pneumoniae
Due to the lack of sequence similarity between the C. trachomatis and C. pneumoniae plasmids, discrete primers and probes were designed for the QPCR assay of plasmid and omcB of the latter organism. Recombinant plasmids containing the C. pneumoniae N16 plasmid or omcB gene were used as assay standards. Serial dilutions of these plasmids were used as templates to produce amplification plots and standard curves as described above (results not shown). The efficiency of the C. pneumoniae PCR, calculated from the slope of the standard curves, was 0·90 for the plasmid and 0·92 for omcB. The sensitivity of the two assays differs slightly. The plasmid assay reproducibly detects as few as ten DNA molecules, a similar sensitivity to the C. trachomatis assays. However, the threshold sensitivity for the omcB assay is between ten and 100 DNA molecules, with only the latter being detected reproducibly.

Determination of C. pneumoniae plasmid copy number
Purified EBs of C. pneumoniae N16 EBs were lysed and quantitative plasmid and omcB PCRs were performed. The ratio of plasmid to omcB is equivalent to the plasmid copy number and was calculated as 1·3±0·2 plasmids per EB (mean±95 % confidence interval).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We describe the use of real-time QPCR assays to determine plasmid copy number accurately in both genera of the Chlamydiaceae. Of the chemistries available (Wittwer et al., 1997), the 5'-exonuclease (TaqMan) assay was selected. Unlike SYBR Green I chemistry, TaqMan utilizes a complementary oligonucleotide probe internal to the PCR primers, affording the assay an additional level of specificity. This was considered important when assaying total DNA extracted from infected cells rather than from purified EBs. Separate oligonucleotide primer and probe sets were selected for C. trachomatis and C. pneumoniae, as neither their plasmids (Thomas et al., 1997) nor their omcB genes (Watson et al., 1991) display sufficient DNA sequence similarity to enable the design of pan-Chlamydiaceae assays. Plasmid and omcB assays were performed in separate tubes, as multiplexing requires an optimization process to limit primer concentrations (Livak, 2001). Such limitation reduces assay sensitivity. Absolute quantification using standard curves was chosen for the following reasons: (i) only this method permits the determination and comparison of analytical sensitivities, (ii) recombinant plasmids were available that could be used as assay standards and (iii) the assay could be used in future infectious dose and/or diagnostic studies. The source of standard DNA for QPCR may be the whole organism (Apfalter et al., 2003; van Doornum et al., 2003) or recombinant plasmid. The use of both closed, circular (Coker et al., 2003; Yates et al., 2001) and linearized (Pusterla et al., 1999; Weidmann et al., 2003) plasmid standards has been described in the literature. There may be different degrees of supercoiling in the chlamydial plasmid, chlamydial genome and plasmid standards, and the effect of this on PCR efficiency was considered. Although linearization of a plasmid has been found to affect significantly its functioning as a PCR target (Pogozelski et al., 2003), Tasker et al. (2003) reported that chromosomal and plasmid templates were amplified by PCR with similar efficiencies. This result was confirmed in the present study by submitting identical amounts of (i) supercoiled, closed circular and (ii) linearized, standard plasmid template for TaqMan PCR assay. Presumably, any topological constraints (due to supercoiling) affecting polymerase access to its template would be significant only in the first few PCR cycles; subsequently, linear product would represent the major template species. Efficiencies of the four TaqMan assays described in this work compare favourably with those reported for other real-time PCR assays (Tichopad et al., 2003). Analytical sensitivities of 10–100 molecules were observed; these are similar to those observed in other TaqMan assays (Apfalter et al., 2003; Weidmann et al., 2003). Poor reproducibility below ten molecules per reaction is probably due to sampling errors, as previously predicted for low concentrations of PCR target (Coupland, 1994).

Bacterial DNA was released from infected cells and chlamydiae by heating in the presence of ammonia, followed by the removal of alkali by evaporation for a fixed length of time (Lanham et al., 2001). Using the procedure as described produced PCR results with unacceptable replicate variance (results not shown). Residual ammonia was detected in some of the extracted DNA, and it was postulated that this was affecting the QPCR to varying extents. Although the spectral properties of the TaqMan probe fluors, FAM and TAMRA, should be unaffected by pH values from 7 to 10, the efficiency of the PCR will be altered (Payne et al., 1995). Therefore, in this study, the procedure of Lanham et al. (2001) was modified by heating the samples to dryness to ensure the complete removal of ammonia.

The QPCR methodology described above was used to determine the plasmid copy number in EBs of C. trachomatis L1 440. The result was 4·0±0·8 plasmid copies per chromosome in the EB. Previously, C. trachomatis LGV2 EBs have been reported to contain 10 plasmid copies per chromosome, by a method using the incorporation of 3H-adenosine and liquid scintillation counting (Palmer & Falkow, 1986). This higher figure may be due to the preferential loss of chromosomal DNA during harvesting after its separation from plasmid DNA by CsCl gradient centrifugation or agarose gel electrophoresis. In addition, supercoiled plasmid DNA may be less prone to degradation by contaminating nuclease during the separation of plasmid and chromosome. The QPCR procedure does not require separation of these DNA species. There is less than 1 % nucleotide sequence variation in the plasmids of human isolates of C. trachomatis (Black et al., 1989; Comanducci et al., 1988; Hatt et al., 1988; Sriprakash & MacAvoy, 1987b; Thomas & Clarke, 1992) – a significant factor in the laboratory diagnosis of C. trachomatis infections. Diagnostic methods targeting a multicopy plasmid display an increase in sensitivity compared with those that target a single copy gene. Kits that target the chlamydial plasmid are commercially available and use either PCR (Amplicor CT/NG; Roche Diagnostics) or strand displacement amplification (ProbeTec ET; Becton Dickinson). Significant plasmid DNA sequence similarity also suggests that the same plasmid replication and partition control mechanisms are in operation in different strains of C. trachomatis. The analysis of 26 strains of this species indicated no significant differences in plasmid copy number.

Bacterial plasmid copy number has been demonstrated to vary with both growth rate (Kapralek et al., 1998; Lin-Chao & Bremer, 1986; Sayadi et al., 1989) and growth phase (Coker et al., 2003). The plasmid copy number in chlamydial RBs has been previously estimated as between 7 and 10 copies per chromosome (Tam et al., 1992), using filter lysis and radioactive probes to plasmid and chromosome as described previously (Shields et al., 1986). However, the method used was insufficiently sensitive to detect small changes in copy number during the developmental cycle. QPCR revealed that there is an increase in plasmid to chromosome ratio during the first 15 h of the developmental cycle. At 15 h p.i., light microscopy indicated that all infecting EBs had differentiated to RBs within inclusions. Thereafter, when RBs are observed to differentiate into EBs, the ratio of plasmid to chromosome decreases. These results suggest that the plasmid copy number in chlamydial RBs is almost twice that found in EBs.

The plasmid copy number determined for C. trachomatis indicates that the plasmid is a low-copy-number replicon and that its replication is tightly controlled. As plasmid-free isolates of C. trachomatis are rare, low plasmid copy number also suggests the presence of an efficient partitioning system. Interestingly, plasmid ORF7 and ORF8 share several features with SopA/B and ParA/B operons (Ricci et al., 1995; Thomas et al., 1997). DNA sequence analysis has shown that iteron binding and/or inhibitor targeting mechanisms may operate to control chlamydial plasmid copy number (Thomas et al., 1997). There is significant similarity between ORF1 and ORF2 of the C. trachomatis plasmid, and it was suggested that both ORFs encode products that function as replication proteins. Control by twin replication proteins has been described in other plasmid systems (Basu et al., 2002). Although there is only 60 % nucleotide sequence identity between the C. pneumoniae and C. trachomatis plasmids, their genomic organization is remarkably similar (Thomas et al., 1997). It is interesting to note that the C. pneumoniae N16 plasmid ORF1 contains a 150 bp deletion and is split into two separate ORFs, 1A and 1B. Therefore, the ORF2 product may be the sole replication protein in the C. pneumoniae plasmid. As it is possible that plasmid replication control might be damaged in C. pneumoniae N16, we also set up assays to determine plasmid copy number in this strain. Results showed that the C. pneumoniae N16 plasmid copy number is significantly lower than that of C. trachomatis. It is possible that the ORF1 product is not essential for plasmid replication, but stimulates the ORF2 product in its origin-binding activity. Such a mechanism has been postulated for the mycobacterial plasmid pAL5000 (Basu et al., 2002). This is an encouraging observation for vector development as it suggests that ORF1 is not essential for plasmid maintenance.

All human C. pneumoniae isolates (Campbell et al., 1987; Lusher et al., 1989) and several isolates of Chlamydophila psittaci (Lusher et al., 1991) lack the cryptic plasmid. Although it has been suggested that the C. trachomatis plasmid encodes an essential protein (Sriprakash & MacAvoy, 1987a, b), plasmid-free isolates have been reported. These data suggest that the cryptic plasmid is not essential for chlamydial viability and prompted our investigation into the effect of chemical curing agents on C. trachomatis. The intercalating agents EtBr (Crameri et al., 1986) and AcOr (Mesas et al., 2004) have been used successfully as plasmid-curing agents in other organisms. The DNA gyrase inhibitor novobiocin (McHugh & Swartz, 1977) and the antidepressant tricyclic drug imipramine (Molnar et al., 1978) have also been used for this purpose, both alone and in combination (Molnar & Foldeak, 1989). However, the successful curing of plasmids in obligately intracellular organisms has not been reported, and no reduction in plasmid copy number was observed when these agents were added to chlamydial cell culture medium. Even in free-living bacteria, the effective concentration of curing agent varies widely depending on the replicon, the host species and the conditions employed, and many plasmids cannot be cured (reviewed by Trevors, 1986). Chlamydiae replicate entirely within the inclusion, a membrane-limited vacuole in the host-cell cytoplasm (Hackstadt et al., 1997). Therefore, to be effective, a curing agent must first pass through the host-cell plasma membrane and inclusion membrane in addition to the bacterial membranes. It may be impossible to achieve effective intrabacterial concentrations of the chemicals without cytotoxicity. The MCCs for uninfected BGMK cells were determined and the curing agents were then used at up to 80 % of these concentrations in the chlamydial infection experiments. It was found that the presence of EtBr or AcOr at the time of infection totally prevented the formation of chlamydial inclusions. As it has been suggested that EtBr is chlamydicidal for EBs and not RBs (Sriprakash & Woodroffe, 1988), it was decided to add EtBr or the related agent AcOr after most infecting EBs had differentiated to RBs, i.e. 16 h p.i.

All the curing agents used in this study caused a paradoxical increase in plasmid copy number. Initially, there was concern that contamination of the PCRs with curing agent may cause this effect, as both EtBr and AcOr have been shown to inhibit PCR by intercalation (Nath et al., 2000). As plasmid topology limits such intercalation, it was postulated that preferential inhibition of the omcB assay (during the first PCR cycle) may occur. This is unlikely for the following reasons. (i) The concentrations of EtBr and AcOr present in the PCRs in this work are no greater than 200 ng ml–1 and 20 ng ml–1, respectively, and did not affect the TaqMan assays. Nath et al. (2000) found that 10 µg EtBr ml–1 was required to inhibit the reaction. (ii) The DNA gyrase inhibitor novobiocin does not intercalate and, furthermore, its induction of positive supercoiling inhibits duplex melting (Sioud et al., 1988) and would be expected to decrease the chlamydial plasmid's availability as a template. (iii) The non-planar molecule imipramine also does not intercalate and has not been reported to inhibit PCR or affect the supercoiling of plasmids (Molnar et al., 1978).

The paradoxical increase in plasmid copy number may be explained by the induction of chlamydial stress responses. In other bacteria, both the SOS response and the heat-shock response are induced by nutrient starvation (Janion et al., 2002; Zhang & Griffiths, 2003). As homologous genes for both these regulons have been identified in the chlamydial genome (Stephens et al., 1998), and chlamydiae are auxotrophic for most nucleotides (Tipples & McClarty, 1993), it is feasible that the detrimental effect of curing agent on host-cell metabolism induces stress responses. The chlamydial recA gene homologue encodes an active protein (Hintz et al., 1995; Zhang et al., 1995), which may regulate an SOS response within the organism. It is known that the SOS response affects plasmid copy number in other systems (Bertrand-Burggraf et al., 1989); indeed, in E. coli, it has been implicated as an additional control mechanism of plasmid copy number (Ingmer et al., 2001). In chlamydiae, heat-shock protein expression is regulated by the HrcA repressor/CIRCE operator (Wilson & Tan, 2004). Chlamydial heat-shock proteins may induce plasmid replication by promoting the binding of Rep protein to the plasmid origin, as described for plasmid P1 in E. coli (Sozhamannan & Chattoraj, 1993).

The assays described in this work have allowed the true plasmid copy number, i.e. plasmids per chromosome, to be determined accurately in chlamydiae. They may be used to investigate further the molecular biology of the plasmid in strains of C. trachomatis and C. pneumoniae grown in mutant host-cell lines deficient in, for example, specific enzymes of nucleotide or energy metabolism. The ability to detect small quantities of plasmid will aid future chlamydial transformation and shuttle vector studies. The high sensitivity of the method described will allow the estimation of plasmid copy number directly in clinical specimens, without cell culture of the organism. Plasmid-free isolates will be readily detected, and plasmid copy number correlated with bacterial virulence and other pathogenic traits.


   ACKNOWLEDGEMENTS
 
This work was supported by Wellcome Trust grant number 074062.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Alexander, E. R., Wang, S. P. & Grayston, J. T. (1967). Further classification of TRIC agents from ocular trachoma and other sources by the mouse toxicity prevention test. Am J Ophthalmol 63 (Suppl.), 1469–1478.[Medline]

An, Q., Radcliffe, G., Vassallo, R., Buxton, D., O'Brien, W. J., Pelletier, D. A., Weisburg, W. G., Klinger, J. D. & Olive, D. M. (1992). Infection with a plasmid-free variant chlamydia related to Chlamydia trachomatis identified by using multiple assays for nucleic acid detection. J Clin Microbiol 30, 2814–2821.[Abstract]

Apfalter, P., Barousch, W., Nehr, M., Makristathis, A., Willinger, B., Rotter, M. & Hirschl, A. M. (2003). Comparison of a new quantitative ompA-based real-time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays. J Clin Microbiol 41, 592–600.[Abstract/Free Full Text]

Bailey, R. L., Hayes, L., Pickett, M., Whittle, H. C., Ward, M. E. & Mabey, D. C. W. (1994). Molecular epidemiology of trachoma in a Gambian village. Br J Ophthalmol 78, 813–817.[Abstract]

Basu, A., Chawla-Sarkar, M., Chakrabarti, S. & Das Gupta, S. K. (2002). Origin binding activity of the mycobacterial plasmid pAL5000 replication protein RepB is stimulated through interactions with host factors and coupled expression of repA. J Bacteriol 184, 2204–2214.[Abstract/Free Full Text]

Bertrand-Burggraf, E., Oertel, P., Schnarr, M., Daune, M. & Granger-Schnarr, M. (1989). Effect of induction of SOS response on expression of pBR322 genes and on plasmid copy number. Plasmid 22, 163–168.[CrossRef][Medline]

Black, C. M., Barnes, R. C., Birkness, K. A., Holloway, B. P. & Mayer, L. W. (1989). Nucleotide sequence of the common plasmid of Chlamydia trachomatis serovar L2 – use of compatible deletions to generate overlapping fragments. Curr Microbiol 19, 67–74.

Burstein, G. R. & Zenilman, J. M. (1999). Nongonococcal urethritis – a new paradigm. Clin Infect Dis 28 (Suppl. 1), S66–S73.[Medline]

Campbell, L. A., Kuo, C.-C. & Grayston, J. T. (1987). Characterization of the new Chlamydia agent, TWAR, as a unique organism by restriction endonuclease analysis and DNA-DNA hybridization. J Clin Microbiol 25, 1911–1916.[Medline]

Chattoraj, D. K. (2000). Control of plasmid DNA replication by iterons: no longer paradoxical. Mol Microbiol 37, 467–476.[CrossRef][Medline]

Clarke, I. N. & Lambden, P. R. (1988). Stable cloning of the amino terminus of the 60 kDa outer membrane protein of Chlamydia trachomatis serovar L1. FEMS Microbiol Lett 51, 81–86.[CrossRef]

Coker, P. R., Smith, K. L., Fellows, P. F., Rybachuck, G., Kousoulas, K. G. & Hugh-Jones, M. E. (2003). Bacillus anthracis virulence in guinea pigs vaccinated with anthrax vaccine adsorbed is linked to plasmid quantities and clonality. J Clin Microbiol 41, 1212–1218.[Abstract/Free Full Text]

Comanducci, M., Ricci, S. & Ratti, G. (1988). The structure of a plasmid of Chlamydia trachomatis believed to be required for growth within mammalian cells. Mol Microbiol 2, 531–538.[Medline]

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.[Medline]

Coupland, R. W. (1994). Quality-control, analytical sensitivity, and the polymerase chain-reaction. J Clin Immunoass 17, 237–242.

Courtright, J. B., Turowski, D. A. & Sonstein, S. A. (1988). Alteration of bacterial DNA structure, gene expression, and plasmid encoded antibiotic resistance following exposure to enoxacin. J Antimicrob Chemother 21 (Suppl. B), 1–18.[Medline]

Crameri, R., Davies, J. E. & Hutter, R. (1986). Plasmid curing and generation of mutations induced with ethidium bromide in streptomycetes. J Gen Microbiol 132, 819–824.[Medline]

Everett, K. D. E., Bush, R. M. & Andersen, A. A. (1999). Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol 49, 415–440.[Abstract]

Everson, J. S., Garner, S. A., Lambden, P. R., Fane, B. A. & Clarke, I. N. (2003). Host range of chlamydiaphages {pi}CPAR39 and Chp3. J Bacteriol 185, 6490–6492.[Abstract/Free Full Text]

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. Infect Immun 65, 2965–2969.[Abstract]

Garner, S., Everson, J. S., Lambden, P. R., Fane, B. & Clarke, I. N. (2004). Isolation, molecular characterisation and genome sequence of a bacteriophage (Chp3) from Chlamydophila pecorum. Virus Genes 28, 207–214.[CrossRef][Medline]

Hackstadt, T., Fischer, E. R., Scidmore, M. A., Rockey, D. D. & Heinzen, R. A. (1997). Origins and functions of the chlamydial inclusion. Trends Microbiol 5, 288–293.[CrossRef][Medline]

Hahn, D. L., Azenabor, A. A., Beatty, W. L. & Byrne, G. I. (2002). Chlamydia pneumoniae as a respiratory pathogen. Front Biosci 7, e66–e76.[Medline]

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.[Abstract]

Hayes, L. J., Pickett, M. A., Conlan, J. W., Ferris, S., Everson, J. S., Ward, M. E. & Clarke, I. N. (1990). The major outer-membrane proteins of Chlamydia trachomatis serovars A and B: intra-serovar amino acid changes do not alter specificities of serovar- and C subspecies-reactive antibody-binding domains. J Gen Microbiol 136, 1559–1566.[Medline]

Hayes, L. J., Bailey, R. L., Mabey, D. C. W., Clarke, I. N., Pickett, M. A., Watt, P. J. & Ward, M. E. (1992). Genotyping of Chlamydia trachomatis from a trachoma-endemic village in The Gambia by a nested polymerase chain reaction: identification of strain variants. J Infect Dis 166, 1173–1177.[Medline]

Hayes, L. J., Yearsley, P., Treharne, J. D., Ballard, R. A., Fehler, G. H. & Ward, M. E. (1994). Evidence for naturally occurring recombination in the gene encoding the major outer membrane protein of lymphogranuloma venereum isolates of Chlamydia trachomatis. Infect Immun 62, 5659–5663.[Abstract]

Hintz, N. J., Ennis, D. G., Liu, W. F. & Larsen, S. H. (1995). The recA gene of Chlamydia trachomatis: cloning, sequence, and characterization in Escherichia coli. FEMS Microbiol Lett 127, 175–180.[CrossRef][Medline]

Ingmer, H., Miller, C. & Cohen, S. N. (2001). The RepA protein of plasmid pSC101 controls Escherichia coli cell division through the SOS response. Mol Microbiol 42, 519–526.[CrossRef][Medline]

Janion, C., Sikora, A., Nowosielska, A. & Grzesiuk, E. (2002). Induction of the SOS response in starved Escherichia coli. Environ Mol Mutagen 40, 129–133.[CrossRef][Medline]

Kapralek, F., Tichy, P. J., Fabry, M. & Sedlacek, J. (1998). Effects of temperature and novobiocin on the expression of calf prochymosin gene and on plasmid copy number in recombinant Escherichia coli. Folia Microbiol (Praha) 43, 63–67.[Medline]

Lanham, S., Herbert, A., Basarab, A. & Watt, P. (2001). Detection of cervical infections in colposcopy clinic patients. J Clin Microbiol 39, 2946–2950.[Abstract/Free Full Text]

Lin-Chao, S. & Bremer, H. (1986). Effect of the bacterial growth rate on replication control of plasmid pBR322 in Escherichia coli. Mol Gen Genet 203, 143–149.[Medline]

Livak, K. J. (2001). Relative quantification of gene expression. ABI Prism 7700 Sequence Detection System user bulletin #2. Foster City, CA: Applied Biosystems. http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf

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. Edited by J. Nelson & C. Grassi. Washington, DC: American Society for Microbiology.

Lusher, M., Storey, C. C. & Richmond, S. J. (1989). Plasmid diversity within the genus Chlamydia. J Gen Microbiol 135, 1145–1151.[Medline]

Lusher, M., Storey, C. C. & Richmond, S. J. (1991). Extrachromosomal elements of the genus Chlamydia. In Advances in Gene Technology, vol. 2, pp. 261–285. Edited by P. J. Greenaway. New York: Elsevier Science.

Matsumoto, A., Izutsu, H., Miyashita, N. & Ohuchi, M. (1998). Plaque formation by and plaque cloning of Chlamydia trachomatis biovar trachoma. J Clin Microbiol 36, 3013–3019.[Abstract/Free Full Text]

McHugh, G. L. & Swartz, M. N. (1977). Elimination of plasmids from several bacterial species by novobiocin. Antimicrob Agents Chemother 12, 423–426.[Medline]

Mesas, J. M., Rodriguez, M. C. & Alegre, M. T. (2004). Plasmid curing of Oenococcus oeni. Plasmid 51, 37–40.[CrossRef][Medline]

Molnar, J. & Foldeak, S. (1989). Antiplasmid action of phenothiazines and related compounds. In Recent Advances in Chemotherapy. Proceedings of the 16th International Congress of Chemotherapy. Edited by E. Rubinstein & A. Dieter. Washington, DC: American Society for Microbiology.

Molnar, J., Beladi, I. & Holland, I. B. (1978). The plasmid curing action of imipramine in Escherichia coli K12. Genet Res 31, 197–201.[Medline]

Nath, K., Sarosy, J. W., Hahn, J. & Di Como, C. J. (2000). Effects of ethidium bromide and SYBR Green I on different polymerase chain reaction systems. J Biochem Biophys Methods 42, 15–29.[CrossRef][Medline]

Paavonen, J. (1998). Pelvic inflammatory disease. From diagnosis to prevention. Dermatol Clin 16, 747–756.[Medline]

Palmer, L. & Falkow, S. (1986). A common plasmid of Chlamydia trachomatis. Plasmid 16, 52–62.[Medline]

Park, K., Han, E., Paulsson, J. & Chattoraj, D. K. (2001). Origin pairing (‘handcuffing’) as a mode of negative control of P1 plasmid copy number. EMBO J 20, 7323–7332.[Abstract/Free Full Text]

Payne, D., Hoskins, S., Schouten, H., van Vleuten, H. & Tyring, S. (1995). Increased buffer pH enhances sensitivity and specificity of human papillomavirus detection using consensus primer based PCR. J Virol Methods 52, 105–110.[CrossRef][Medline]

Peterson, E. M., Markoff, B. A., Schachter, J. & de la Maza, L. M. (1990). The 7·5-kb plasmid present in Chlamydia trachomatis is not essential for the growth of this microorganism. Plasmid 23, 144–148.[Medline]

Pogozelski, W. K., Hamel, C. J. C., Woeller, C. F., Jackson, W. E., Zullo, S. J., Fischel-Ghodsian, N. & Blakely, W. F. (2003). Quantification of total mitochondrial DNA and the 4977-bp common deletion in Pearson's syndrome lymphoblasts using a fluorogenic 5'-nuclease (TaqMan) real-time polymerase chain reaction assay and plasmid external calibration standards. Mitochondrion 2, 415–427.[CrossRef]

Pusterla, N., Huder, J. B., Leutenegger, C. M., Braun, U., Madigan, J. E. & Lutz, H. (1999). Quantitative real-time PCR for detection of members of the Ehrlichia phagocytophila genogroup in host animals and Ixodes ricinus ticks. J Clin Microbiol 37, 1329–1331.[Abstract/Free Full Text]

Ricci, S., Ratti, G. & Scarlato, V. (1995). Transcriptional regulation in the Chlamydia trachomatis pCT plasmid. Gene 154, 93–98.[CrossRef][Medline]

Richmond, S. J., Stirling, P. & Ashley, C. R. (1982). Virus infecting the reticulate bodies of an avian strain of Chlamydia psittaci. FEMS Microbiol Lett 14, 31–36.[CrossRef]

Rockey, D. D. & Matsumoto, A. (2000). The chlamydial developmental cycle. In Prokaryotic Development, pp. 403–425. Edited by Y. V. Brun & L. J. Shimkets. Washington, DC: American Society for Microbiology.

Saikku, P., Leinonen, M., Tenkanen, L., Linnanmaki, E., Ekman, M.-R., Manninen, V., Mänttäri, M., Frick, M. H. & Huttunen, J. K. (1992). Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki heart study. Ann Intern Med 116, 273–278.[Medline]

Sayadi, S., Nasri, M., Barbotin, J. N. & Thomas, D. (1989). Effect of environmental growth conditions on plasmid stability, plasmid copy number, and catechol 2,3-dioxygenase activity in free and immobilized Escherichia coli cells. Biotechnol Bioeng 33, 801–808.

Schachter, J. & Meyer, K. F. (1969). Lymphogranuloma venereum. II. Characterization of some recently isolated strains. J Bacteriol 99, 636–638.[Medline]

Schachter, J., Stephens, R. S., Timms, P. & 28 other authors (2001). Radical changes to chlamydial taxonomy are not necessary just yet. Int J Syst Evol Microbiol 51, 249.[Free Full Text]

Shields, M. S., Kline, B. C. & Tam, J. E. (1986). A rapid method for the quantitative measurement of gene dosage: mini-F plasmid concentration as a function of cell growth rate. J Microbiol Methods 6, 33–46.[CrossRef]

Sioud, M., Baldacci, G., de Recondo, A. M. & Forterre, P. (1988). Novobiocin induces positive supercoiling of small plasmids from halophilic archaebacteria in vivo. Nucleic Acids Res 16, 1379–1391.[Abstract]

Sozhamannan, S. & Chattoraj, D. K. (1993). Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin. J Bacteriol 175, 3546–3555.[Abstract]

Sriprakash, K. S. & MacAvoy, E. S. (1987a). A gene for DnaB like protein in chlamydial plasmid. Nucleic Acids Res 15, 10596.[Medline]

Sriprakash, K. S. & MacAvoy, E. S. (1987b). Characterization and sequence of a plasmid from the trachoma biovar of Chlamydia trachomatis. Plasmid 18, 205–214.[Medline]

Sriprakash, K. S. & Woodroffe, S. (1988). Early stages of chlamydial growth cycle are sensitive to ethidium bromide. In Proceedings of the European Society for Chlamydia Research (1st meeting), p. 97. Edited by P. A. Mardh & M. La Placa. Bologna: Societa Editrice Esculapio.

Stephens, R. S., Kalman, S., Lammel, C. & 9 other authors (1998). Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282, 754–759.[Abstract/Free Full Text]

Storey, C., Lusher, M., Yates, P. & Richmond, S. (1993). Evidence for Chlamydia pneumoniae of non-human origin. J Gen Microbiol 139, 2621–2626.[Medline]

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. Infect Immun 66, 6010–6013.[Abstract/Free Full Text]

Stothard, D. R., Williams, J. A., Van der Pol, B. & Jones, R. B. (1999). Identification of a Chlamydia trachomatis serovar E urogenital isolate which lacks the cryptic plasmid (author's correction). Infect Immun 67, 2051.[Free Full Text]

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.[Medline]

Tam, J. E., Davis, C. H. & Wyrick, P. B. (1994). Expression of recombinant DNA introduced into Chlamydia trachomatis by electroporation. Can J Microbiol 40, 583–591.[Medline]

Thomas, N. S. & Clarke, I. N. (1992). Revised map of the Chlamydia trachomatis (L1/440/LN) plasmid. In Proceedings of the European Society for Chlamydial Research (2nd meeting), p. 42. Edited by P. A. Mardh, M. la Placa & M. E. Ward. Bologna: Societa Editrice Esculapio.

Thomas, N. S., Lusher, M., Storey, C. C. & Clarke, I. N. (1997). Plasmid diversity in Chlamydia. Microbiology 143, 1847–1854.[Medline]

Thylefors, B., Negrel, A. D., Pararajasegaram, R. & Dadzie, K. Y. (1995). Global data on blindness. Bull World Health Organ 73, 115–121.[Medline]

Tichopad, A., Dilger, M., Schwarz, G. & Pfaffl, M. W. (2003). Standardized determination of real-time PCR efficiency from a single reaction set-up. Nucleic Acids Res 31, e122.[Abstract/Free Full Text]

Tipples, G. & McClarty, G. (1993). The obligate intracellular bacterium Chlamydia trachomatis is auxotrophic for three of the four ribonucleoside triphosphates. Mol Microbiol 8, 1105–1114.[Medline]

Trevors, J. T. (1986). Plasmid curing in bacteria. FEMS Microbiol Rev 32, 149–157.[CrossRef]

Tuffrey, M., Falder, P., Gale, J. & Taylor-Robinson, D. (1986). Salpingitis in mice induced by human strains of Chlamydia trachomatis. Br J Exp Pathol 67, 605–616.[Medline]

van Doornum, G. J. J., Guldemeester, J., Osterhaus, A. D. M. E. & Niesters, H. G. M. (2003). Diagnosing herpesvirus infections by real-time amplification and rapid culture. J Clin Microbiol 41, 576–580.[Abstract/Free Full Text]

Wang, S. P. & Grayston, J. T. (1963). Classification of trachoma virus strains by protection of mice from toxic death. J Immunol 90, 849–856.[Medline]

Wang, S. & Grayston, J. T. (1975). Chlamydia trachomatis immunotype J. J Immunol 115, 1711–1716.[Abstract]

Wardrop, S., Fowler, A., O'Callaghan, P., Giffard, P. & Timms, P. (1999). Characterization of the koala biovar of Chlamydia pneumoniae at four gene loci – ompAVD4, ompB, 16S rRNA, groESL spacer region. Syst Appl Microbiol 22, 22–27.[Medline]

Watson, M. W., Lambden, P. R., Ward, M. E. & Clarke, I. N. (1989). Chlamydia trachomatis 60 kDa cysteine rich outer-membrane protein: sequence homology between trachoma and LGV biovars. FEMS Microbiol Lett 65, 293–298.[CrossRef]

Watson, M. W., al-Mahdawi, S., Lambden, P. R. & Clarke, I. N. (1990). The nucleotide sequence of the 60 kDa cysteine rich outer membrane protein of Chlamydia pneumoniae strain IOL-207. Nucleic Acids Res 18, 5299.[Medline]

Watson, M. W., Lambden, P. R. & Clarke, I. N. (1991). Genetic diversity and identification of human infection by amplification of the chlamydial 60-kilodalton cysteine-rich outer-membrane protein gene. J Clin Microbiol 29, 1188–1193.[Medline]

Weidmann, M., Meyer-Konig, U. & Hufert, F. T. (2003). Rapid detection of herpes simplex virus and varicella-zoster virus infections by real-time PCR. J Clin Microbiol 41, 1565–1568.[Abstract/Free Full Text]

Wills, J. M., Watson, G., Lusher, M., Mair, T. S., Wood, D. & Richmond, S. J. (1990). Characterisation of Chlamydia psittaci isolated from a horse. Vet Microbiol 24, 11–19.[CrossRef][Medline]

Wilson, A. C. & Tan, M. (2004). Stress response gene regulation in Chlamydia is dependent on HrcA-CIRCE interactions. J Bacteriol 186, 3384–3391.[Abstract/Free Full Text]

Wittwer, C. T., Herrmann, M. G., Moss, A. A. & Rasmussen, R. P. (1997). Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22, 130–138.[Medline]

Yates, S., Penning, M., Goudsmit, J., Frantzen, I., van de Weijer, B., van Strijp, D. & van Gemen, B. (2001). Quantitative detection of hepatitis B virus DNA by real-time nucleic acid sequence-based amplification with molecular beacon detection. J Clin Microbiol 39, 3656–3665.[Abstract/Free Full Text]

Zhang, Y. & Griffiths, M. W. (2003). Induced expression of the heat shock protein genes uspA and grpE during starvation at low temperatures and their influence on thermal resistance of Escherichia coli O157 : H7. J Food Prot 66, 2045–2050.[Medline]

Zhang, D. J., Fan, H., McClarty, G. & Brunham, R. C. (1995). Identification of the Chlamydia trachomatis RecA-encoding gene. Infect Immun 63, 676–680.[Abstract]

Received 14 September 2004; revised 23 November 2004; accepted 23 November 2004.



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