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
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ABSTRACT |
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INTRODUCTION |
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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 3235 % 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.
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METHODS |
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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 ml1). 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 ml1); 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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RESULTS |
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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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Effect of plasmid-curing agents on BGMK cells and C. trachomatis
The MCCs of the curing agents for BGMK cells were 250 µg ml1 (EtBr), 25 µg ml1 (AcOr), 25 µg ml1 (novobiocin) and 25 µg ml1 (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 ml1) or AcOr (20 ng ml1) to the reaction (data not shown).
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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).
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DISCUSSION |
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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 ml1 and 20 ng ml1, respectively, and did not affect the TaqMan assays. Nath et al. (2000)
found that 10 µg EtBr ml1 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.
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ACKNOWLEDGEMENTS |
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Received 14 September 2004;
revised 23 November 2004;
accepted 23 November 2004.
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