Identification of Plasmid-Free Chlamydia muridarum Organisms Using a Pgp3 Detection-Based Immunofluorescence Assay

Abstract

Chlamydia possesses a conserved 7.5 kb plasmid that is known to play an important role in chlamydial pathogenesis, since some chlamydial organisms lacking the plasmid are attenuated. The chlamydial transformation system developed recently required the use of plasmid-free organisms. Thus, the generation and identification of plasmid-free organisms represent a key step in understanding chlamydial pathogenic mechanisms. A tricolor immunofluorescence assay for simultaneously detecting the plasmid-encoded Pgp3 and whole organisms plus DNA staining was used to screen C. muridarum organisms selected with novobiocin. PCR was used to detect the plasmid genes. Next-generation sequencing was then used to sequence the genomes of plasmid-free C. muridarum candidates and the parental C. muridarum Nigg strain. We generated five independent clones of plasmid-free C. muridarum organisms by using a combination of novobiocin treatment and screening plaque-purified clones with anti-Pgp3 antibody. The clones were confirmed to lack plasmid genes by PCR analysis. No GlgA protein or glycogen accumulation was detected in cells infected with the plasmid-free clones. More importantly, whole-genome sequencing characterization of the plasmid-free C. muridarum organism and the parental C. muridarum Nigg strain revealed no additional mutations other than loss of the plasmid in the plasmid-free C. muridarum organism. Thus, the Pgp3-based immunofluorescence assay has allowed us to identify authentic plasmid-free organisms that are useful for further investigating chlamydial pathogenic mechanisms.

Introduction

Chlamydia trachomatis, consisting of 19 different serovars, can cause a broad spectrum of diseases, including trachoma and tubal factor infertility, and the even disseminated sexually transmitted infection. Recently, several studies have associated chlamydial infection with cervical and ovarian cancer development [4] . However, the pathogenic mechanisms of C. trachomatis-induced diseases remain unknown and there is still no licensed anti-C. trachomatis vaccine. The mouse chlamydial pathogen Chlamydia muridarum (also known as mouse pneumonitis, MoPn) is closely related to C. trachomatis. Although C. muridarum organisms cause no known human diseases, they have been and are still extensively used to study the mechanisms of C. trachomatis pathogenesis and immunity in mouse models [11, 19].

Most C. trachomatis clinical isolates carry a highly conserved plasmid comprising eight predicted coding sequences (CDSs; the gene products are designated as Pgp1-8) [8, 9], and naturally occurring plasmid-deficient clinical isolates are very rare. The plasmid may play a significant role in C. trachomatis pathogenesis since plasmidfree trachoma serovar A organisms no longer caused pathology in primate ocular tissues [7].This finding was consistent with an earlier observation that a C. muridarum strain after depletion of the plasmid failed to induce hydrosalpinx in mice [12]. It is now known that the chlamydial plasmid not only encodes eight Pgps but also regulates expression of more than 20 chlamydial genomic genes [2]. Plasmid-free chlamydial organisms generally displayed a reduced expression of glycogen synthesis genes and lacked glycogen accumulation in the inclusions. Recently, researchers developed a chlamydial plasmid shuttle vector-based transformation system that allowed the full complementation of the plasmid-free organisms with an engineered plasmid [21].This technology has offered an opportunity for chlamydiologists to genetically manipulate chlamydial organisms and define the molecular basis of plasmid-dependent pathogenesis [6, 17]. However, this genetic manipulation depends on the availability of plasmid-free organisms. Thus, the generation and identification of plasmid-free chlamydial organisms from different serovars/clinic isolates have become important. Herein, we report a convenient immunofluorescence assay for identifying plasmid-free chlamydial organisms using C. muridarum selected with novobiocin as a model system.

 

Materials and Methods

Chlamydial Organisms and Infection

Chlamydia muridarum Nigg strain was propagated in HeLa cells (human cervical carcinoma epithelial cells) or McCoy cells (mouse fibroblast cells). Briefly, cells were grown in 24-well plates with or without coverslips or 6-well plates containing DMEM supplemented with 10% FBS overnight at 37℃ in an incubator supplied with 5% CO2. The following day, the cell monolayers were pretreated with DEAE-dextran and chlamydial stocks were diluted in serum-free DMEM for inoculation. The chlamydial organisms were allowed to attach to the monolayers for 30 min at 37℃ before being centrifuged at 483 RCF (relative centrifugal force) for 1 h at 37℃. Following centrifugation, the cell culture supernatant was removed and DMEM with 10% FBS and 2 µg/ml of cycloheximide was added to the infected cells, which were incubated in 5% CO2 for 24-30 h. Elementary bodies (EBs) were purified by density gradient centrifugation using MD-76 (MD-76%: 0.1 g diatrizoate sodium and 0.66 g diatrizoate meglumine; Mallinckrodt Inc.).

In some experiments, novobiocin was added to the culture medium at final concentrations of 50, 62.5, 100, 125, and 150 µg/ml at 4 h post-infection. After incubation for another 40 h, the novobiocin-treated cultures were harvested by trypsinization and the cell pellets were lysed by vortexing with glass beads in a sucrose-phosphate-glutamate (SPG) buffer (218 mM sucrose, 3.76 mM KH2PO4, 7.1 mM K2HPO4, and 4.9 mM glutamate (pH 7.2)). The lysates were serially diluted for titrating live organisms on HeLa cell monolayers in 24-well plates or for plaque forming on McCoy cell monolayers in 6-well plates.

Reagents, Antibodies, and Constructs

Chemicals were obtained from Sigma-Aldrich. Secondary Cy2-conjugated antibodies against rabbit IgG, and Cy3-conjugated against mouse IgG were obtained from Jackson ImmunoResearch Laboratories, Inc. The AccuPrime Pfx SuperMix and 1 kb plus DNA ladder were from Invitrogen; mAb MC22 against chlamydial major outer membrane protein (MOMP) were from Zhong’s Lab [22]; and HRP-conjugated goat anti-mouse IgG were from Santa Cruz Biotech.

Antiserum against Pgp3 was raised by immunization of a mouse with GST-Pgp3 fusion protein. The pgp3 gene (TCA04) from the C. muridarum plasmid was used to build the fusion construct. Purification of GST-Pgp3 was performed as described previously [3].The mouse anti-GlgA (TC0181) polyclonal antiserum was raised with a GST-GlgA fusion protein.

Chlamydial Plaque-Forming Assay

A plaque-forming assay was adapted from a previously described protocol [13, 14].Briefly, the survived chlamydial organisms harvested from novobiocin-treated cultures were serially diluted and inoculated onto confluent monolayers of McCoy cells in 6-well tissue culture plates. The infection was facilitated by centrifugation at 483 RCF for 1 h at 37℃. The inoculum was then removed and replaced with DMEM containing 1 µg/ml of cycloheximide and 10% FBS. Four hours later, the medium was replaced with 2 ml of agarose overlay medium (1× EMEM, 3.5 g/l glucose, 2 mM L -glutamine, 1 mM sodium pyruvate, 10% FBS, 10 µg/ml gentamicin, 2 µg/ml cycloheximide, and a final concentration of 0.55% of agarose) followed by dispensing 4 ml of pre-warmed DMEM supplemented with 10% FBS on top of the agarose layer. The cultures were then incubated for 3-5 days to allow plaques to form. Individual plaques (well separated) were picked up into 100 µl of SPG buffer for making lysates by vortexing with glass beads and stored at -80℃. A portion of each plaque-derived lysate was used to infect fresh monolayers of HeLa cells grown in 96-well microplates. The 96-well plate cultures were processed for tricolor immunofluorescence screening for lack of the plasmid-encoded Pgp3 antigen.

Indirect Immunofluorescence Assay

HeLa cells grown in 96- or 24-well plates with or without chlamydial infection were washed once with PBS, and fixed with 4% paraformaldehyde dissolved in PBS for 30 min at room temperature (RT), followed by permeabilization with 2% saponin for an additional 30 min. After being washed three times with PBS and blocked with 4% BSA in PBS for 1 h, the cell samples were stained with primary antibodies diluted in 2% BSA in PBS overnight at 4℃. A mouse anti-Pgp3 or anti-GlgA antibody was used to detect Pgp3 or GlgA, whereas a rabbit anti-chlamydial organism antibody, raised by immunization with purified C. muridarum EBs, was used to monitor chlamydial organisms. After intensive washing, the binding of primary antibodies was visualized by a Cy3-conjugated goat anti-mouse (red) or a Cy2-conjugated goat anti-rabbit (green) IgG, and Hoechst 33258 (blue) was used to visualize nuclear DNA. The immunofluorescence images were acquired using an Olympus IX-80 fluorescence microscope equipped with multiple filter sets and Simple PCI imaging software as described previously [3].

For titrating inclusion forming units (IFUs), the inclusions were counted under a fluorescence microscope, where five random fields were counted per coverslip. The total number of IFUs was calculated based on the number of IFUs per field, number of fields per coverslip, and dilution factors. An average was taken from the serially diluted samples.

Iodine Staining

HeLa cells cultured overnight on glass coverslips in 24-well plates were infected with plasmid-free or wild-type C. muridarum as described above. After 28 h incubation, the culture medium was removed and the monolayer was rinsed several times with PBS and fixed with ice-cold methanol for 10 min at RT. The cell samples were stained with 5% iodine stain solution (5% potassium iodide and 5% iodine in 50% ethanol) for 40 min at RT. The coverslips were mounted in 50% glycerol containing 5% potassium iodide and 5% iodine. Stained cells were immediately photographed and the images were acquired by using an Olympus CH-30 microscope equipped with a Canon EOS Rebel T3i Digital SLR Camera.

PCR

The chlamydial organisms were lysed with 0.1% SDS and the lysates were used as PCR templates after dilution at 1:1,000. The PCR conditions were as follows: initial denaturation at 94℃ for 5 min, followed by 30 cycles of denaturation (94℃) for 30 sec, annealing (50℃) for 45 sec, and elongation (72℃) for 2 min. We separated PCR products in a 1% (w/v) agarose gel and visualized them by staining with ethidium bromide. The gene-specific primers used are listed in Table 1.

Table 1.TC0199: 648 bp TC0602N: M1~S245, 735 bp TCA01: 918 bp; TCA02: 1,023 bp; TCA03: 1,356 bp; TCA04: 723 bp, TCA05: 309 bp; TCA06: 807 bp; TCA07: 741 bp;

Western Blot Assay

HeLa cells with or without C. muridarum infection were solubilized in 2% SDS sample buffer and loaded onto 15% SDS-polyacrylamide gel wells. After electrophoresis, the proteins were wet transferred onto nitrocellulose membrane. Following transfer, the membrane was incubated overnight at 4℃ with mouse polyclonal antiserum against Pgp3 diluted 1:2,000 in 5% skim milk, prior to being incubated with the appropriate HRPconjugated goat anti-mouse IgG secondary antibody for 1 h at RT before detection using an ECL kit (Santa Cruz Biotech). After that, the membrane was incubated and agitated in stripping buffer (25 mM glycine, 1% SDS, pH 2.0) for 30 min at RT, and then washed with 0.05% PBST for 3× 10 min. It was re-blocked with milk and subjected to the western blot procedure. The primary antibody was mAb MC22.

Preparation of Chlamydial DNA for Sequencing

Genomic DNA was prepared as previously described [1] with some modifications. A plasmid-free clone, CMUT3, and the parent C. muridarum Nigg strain wild type were selected for experimentation. Briefly, plasmid-free or wild-type C. muridarum was large-scale propagated in HeLa cells. At 30 h post-infection, infected monolayers were detached with PBS containing 0.05% trypsin/0.02% EDTA, suspended in DMEM with 10% FBS and centrifuged at 500 RCF for 10 min at 4℃. The pellet was combined and resuspended in ice-cold PBS and ruptured by sonication to release the chlamydial organisms from the host cells. Low-speed spun supernatant (10 min at 500 RCF and 4℃) was subjected to high-speed centrifugation (30 min at 14,000 rpm and 4℃) to pellet the chlamydial organisms. The final pellet was resuspended in PBS, homogenized by sonication, and incubated with DNaseI (0.03 IU/ml) (Invitrogen) and RNase A (100 µg/ml) (Sigma) in a water bath at 37℃ for 1 h. This suspension was layered onto 35% MD-76 in Hepes/NaCl and centrifuged at 16,000 rpm for 1 h at 4℃ in a Beckman SW32Ti rotor. The resulting pellet was homogenized and resuspended in cold PBS. The suspension was overlaid onto a discontinuous-density gradients consisting of 40%, 44%, and 52% MD-76 and centrifuged at 17,000 rpm for 1.5 h at 4℃. After centrifugation, the EB fraction (located at the 44-52% interface) was collected, diluted in PBS, and then centrifuged at 14,000 rpm for 30 min at 4℃. The resulting pellets were washed with PBS to remove residual MD-76 and resuspended in PBS. Purified EBs were lysed with proteinase K (100 µg/ml) (Life Technologies) at 50℃ overnight. The DNA was extracted twice with 25:24:1 phenol:chloroform:isoamyl alcohol and once with chloroform and precipitated with alcohol. The genomic DNA was then purified using a QIAamp DNA mini-kit according to the manufacturer’s protocol. Prior to sequencing, several assays were performed to assess the purity and quality of DNA recovered by this method. The quality of DNA was verified by agarose gel electrophoresis, and the DNA was quantified by nanodrop method, and 2 µg of highly pure DNA for use was finally stored at 4℃.

Genome Sequencing and Sequence Analysis

Next-generation sequencing of purified genomic DNA was completed by the UTHSCSA Greehey Children’s Cancer Research Institute Genome Sequencing Facility TX, USA. Briefly, purified DNA was fragmented by a Covaris S220 Ultra Sonicator and a DNA sequencing (DNA-Seq) library was prepared by using the TruSeq DNA Sample Preparation kit (Illumina, Inc.) according to the manufacturer’s protocol. DNA-Seq libraries were sequenced with a 50 bp single-end sequencing module on the Illumina HiSeq2000 platform. After demultiplexing with CASAVA, sequence reads and associated qualities were exported in FASTQ format. Reads in FASTQ format were mapped to the C. muridarum strain Nigg reference chromosome (NC_002620.2) and pMoPn plasmid (NC_02182.1)[15]. Sequencing reads and associated coverage were visualized using the IGV browser [18].

 

Results

Generation and Identification of Plasmid-Free C. muridarum Organisms

To generate plasmid-free C. muridarum organisms, we first titrated novobiocin for its ability to inhibit C. muridarum replication. We found that novobiocin at 62.5 µg/ml achieved 99.5% killing of C. muridarum organisms. This was the dose chosen for the subsequent selection experiment. As described in Fig. 1A, after the novobiocin selection, the remaining live organisms were harvested from novobiocin-treated cultures and serially diluted for plating on McCoy cell monolayers grown on 6-well plates supplied with soft agar medium to allow plaque formation. The well-separated visible plaques were each individually picked up for making lysates with glass beads. A portion of each lysate was used to infect fresh monolayers of HeLa cells grown in 96-well cell culture microplates, while the remaining lysates were stored at -80℃. After culturing for 30 h, the 96-well microplate cultures were processed for tricolor immunofluorescence screening for lack of the plasmid-encoded Pgp3 antigen (Fig. 1B). Among the 153 plaques screened, five plaques were found to contain 100% (CMUT2, 3, & 15, panels c, d, & f) or >50% (CMUT1 & 9, panels b & e) of C. muridarum organisms lacking the plasmid-encoded Pgp3. The organisms from these five plaques were re-plaqued and the second-round plaques that contained 100% plasmid-free organisms were plaqued for the third time to ensure monoclonality of the plasmidfree organisms (data not shown).

Fig. 1.Plaque-forming assay and screening for C. muridarum organisms lacking plasmid. (A) Live organisms freshly harvested from novobiocin-treated cultures were serially diluted and plated on McCoy cell monolayers grown on 6-well plates. After the 6-well plate cultures were incubated for 3 to 5 days in soft agar medium, visible plaques (well separated from each other) were picked up and a portion of each plaque-derived lysate (made with glass beads) was used to infect fresh monolayers of HeLa cells grown in 96-well cell plates. The 96-well plate cultures were processed for tricolor immunofluorescence screening for lack of the plasmid-encoded Pgp3 antigen. (B) Pgp3 was detected with a mouse anti-Pgp3 antibody that was visualized with a goat anti-mouse IgG conjugated with Cy3 (red), whereas the chlamydial organisms were visualized with a rabbit anti-C. muridarum organism antibody plus a Cy2-conjugated goat anti-rabbit IgG (green). The DNA was visualized with Hoechst dye (blue). Among the 153 plaques screened, five plaques were found to contain 100% (CMUT2, 3, & 15, panels c, d, & f) or >50% (CMUT1 & 9, panels b & e) of C. muridarum organisms lacking the plasmid-encoded Pgp3. The organisms from these five plaques were re-plaqued, and the second-round plaques that contained 100% plasmid-free organisms were identified as shown in the bottom row images (panels h-l). The organisms harvested from the secondround of plaques were re-plaqued for a third time to ensure monoclonality of the plasmid-free organisms (data not shown).

In Vitro Characterization of Plasmid-Free C. muridarum Organisms

As shown in Fig. 2, the five plasmid-free clones along with the wild-type C. muridarum organisms were detected with PCR for DNA sequences of both genomic open reading frames (ORFs) TC0199 (coding for a hypothetical protein homologous to CT813) and TC0602N (N-terminal portion of C. muridarum -specific helicase) and plasmid CDSs TCA01 (coding for Pgp7), TCA02 (Pgp8), TCA03 (Pgp1), TCA04 (Pgp3), TCA05 (Pgp4), TCA06 (Pgp5), and TCA07 (Pgp6). The PCR primers for amplifying the corresponding DNA sequences are listed in Table 1. Although all expected PCR products were detected in the wild-type C. muridarum organisms, only the genomic ORF PCR products were detected in the plasmid-free C. muridarum organisms. These observations demonstrated that the plasmid-free C. muridarum organisms indeed lacked all plasmid CDSs. We further characterized plasmid-free C. muridarum in cell cultures by detecting Pgp3, GlgA, or glycogen (Fig. 3). The wild-type but not plasmid-free C. muridarum organisms expressed both Pgp3 and GlgA. Glycogen accumulation was only detected in the wild-type but not plasmid-free C. muridarum -infected cultures. On a western blot (Fig. 4), Pgp3 was detected only in C. muridarum wild-type-infected HeLa cell lysates, not in plasmid-free C. muridarum -infected HeLa cell lysates. As a control, the anti-MOMP antibody detected MOMP in the wild-type and plasmid-free C. muridarum -infected HeLa cell samples and ensured that all lanes with C. muridarum organism-containing samples had equivalent amounts of the organisms loaded. These results together have independently confirmed that plasmid-free C. muridarum candidates lacked the plasmid.

Fig. 2.PCR detection of plasmid genes. The five plasmid-free (panels b-f) and wild-type C. muridarum (a) organisms were subjected to PCR detection of DNA sequences of genomic open reading frames (ORFs) TC0199 (coding for a hypothetical protein homologous to CT813) and TC0602N (N-terminal portion of C. muridarum-specific helicase) and plasmid CDSs TCA01 (coding for Pgp7), TCA02 (Pgp8), TCA03 (Pgp1), TCA04 (Pgp3), TCA05 (Pgp4), TCA06 (Pgp5), and TCA07 (Pgp6) as shown on top of the gel images. All expected PCR products were detected in the wild-type C. muridarum organisms whereas only the genomic ORF PCR products were detected in the plasmid-free C. muridarum organisms (data are representative of two independent experiments). All PCR primers are listed in Table 1.

Fig. 3.In vitro characterization of plasmid-free C. muridarum. HeLa cells infected with C. muridarum wild type (panels a, g, and m) or deficient in plasmid (b-f, h-l, n-r) were subjected to immunofluorescence detection of Pgp3 (a-f) and GlgA (g-l) or iodine detection of glycogen (m-r). Mouse antibodies plus a Cy3-conjugated goat anti-mouse IgG were used to detect either Pgp3 or GlgA (red for both), and a rabbit antibody plus a Cy2-conjugated goat anti-rabbit IgG for detecting C. muridarum organisms (green). Hoechst dye was used to detect DNA (blue). The wild-type but not plasmid-free C. muridarum organisms expressed both Pgp3 and GlgA. Representative inclusions stained positive (arrows, panel a) or negative (arrowheads, n-r) with iodine are marked. Glycogen accumulation was detected in wild-type but not plasmid-free C. muridarum cultures. Data are representative of three independent experiments.

Fig. 4.Western blot detection of Pgp3 in fractions of plasmidfree or wild-type C. muridarum-infected HeLa cells. Normal HeLa cell lysates, or plasmid-free or wild-type C. muridarum (Nigg)-infected HeLa cell lysates were resolved on a SDS polyacrylamide gel and the resolved protein bands were transferred onto nitrocellulose membrane for western blot detection with anti-Pgp3 antibody. Pgp3 was detected only in wild-type C. muridarum infected-HeLa cell lysates. The same membrane was stripped and reprobed with anti-MOMP. Data are representative of two independent experiments.

Genomic Characterization of Plasmid-Free C. muridarum Clone CMUT3

Compared with the genomic information of the parental C. muridarum Nigg strain (GenBank Accession No. CP009608.1.), CMUT3 (GenBank Accession No. CP006974.1) lacked the plasmid. Interestingly, a single mutation was detected in CMUT3 within TC0412: an additional T at 472839 that led to a premature stop at amino acid 47. Significantly, we determined that this mutation was also present in the parental C. muridarum Nigg strain, indicating that the mutation in CMUT3 was present prior to novobiocin treatment. The gene TC0412 is predicted to encode a protein of unknown function that is 365 amino acids in length.

 

Discussion

Chlamydial plasmids are small, highly conserved, nonconjugative, and nonintegrative DNA molecules that are nearly ubiquitous in many chlamydial species [16]. Studies in both C. trachomatis [7] and C. muridarum [12] have revealed an important role for the chlamydial plasmid in the expression of key virulence properties. Meanwhile, the need to identify plasmid-free chlamydial organisms has become even greater owing to the recent advancement in transforming chlamydial organisms [6, 20].This is because the current transformation method is dependent on delivering a chlamydial plasmid-based shuttle vector back into plasmid-free chlamydial organisms. The traditional approach for identification of plasmid-free chlamydial organisms was based on glycogen staining [10],which is inherently insensitive and inconsistent. Various groups have successfully used PCR-based screening for lack of plasmid genes to identify plasmid-free chlamydial plaques selected with novobiocin [7, 13]. In the current study, we have demonstrated that a convenient immunofluorescence assay can be used for identifying plasmid-free chlamydial organisms. This tricolor immunofluorescence assay for simultaneously detecting the plasmid-encoded Pgp3 (red) and whole organisms (green) plus DNA staining (blue) was used to screen cells infected with survived chlamydial clones from novobiocin-treated cultures. In a single screening assay carried out in a 96-well microplate, five clones with the organisms labeled green but without Pgp3-labeling were identified. These clones lacked plasmid genes with undetectable levels of GlgA protein and glycogen. These observations have both revealed a rapid/ reliable immunofluorescence assay for identifying plasmidfree chlamydial organisms and validated the phenotype of plasmid-free chlamydial organisms.

Pgp3 is a highly conserved plasmid protein that is both abundant and immunogenic [8, 9].It has been shown that Pgp3 is both associated with the chlamydial outer membrane and secreted into the host cell cytosol. Thus, the Pgp3 detection signal is both strong and widely distributed across the entire infected cells. The roles of plasmid in pathogenesis of C. trachomatis ocular infection and C. muridarum urogenital tract infection have been demonstrated using plasmid-free organisms [7, 12].However, not all chlamydial organisms can be attenuated by depleting the plasmid. For example, the C. caviae GPIC organisms retained virulence even after losing the plasmid [5].Clearly, chlamydial pathogenic determinants can be coded by both the plasmid and the genome. The successful transformation of plasmidfree chlamydial organisms with engineered chlamydial plasmids has made it possible for identifying the plasmidencoded and/or regulated virulence factors [6, 17, 21]. Since only C. muridarum but not all C. trachomatis serovars can induce hydrosalpinx in mice, it is necessary to use the C. muridarum infection of mouse urogenital tract model to define plasmid-dependent virulence factors. Thus, the generation and identification of plasmid-free C. muridarum organisms represent our first step towards understanding the molecular mechanisms of chlamydial pathogenesis. The availability of plasmid-free CMUT series of C. muridarum organisms will provide chlamydial researchers with additional experimental tools in using the murine model system for investigating chlamydial pathogenesis. In vitro characterization of the newly generated CMUT organisms revealed that all five plasmid-free C. muridarum clones lacked plasmid genes and failed to accumulate GlgA protein or synthesize glycogen. These in vitro characteristics are typical features of plasmid-free organisms [13].More importantly, whole-genome sequencing characterization of the plasmid-free C. muridarum organism and the parental Nigg strain revealed no additional mutations other than loss of the plasmid in plasmid-free C. muridarum clone CMUT3. Efforts are under way to use the plasmidless CMUT organisms for dissecting the mechanisms of the chlamydial plasmid-dependent pathogenicity.