Figure 1.
Map of the shuttle vector pBR325::L2.
The inner circle represents the plasmid pL2 from C. trachomatis L2/434/Bu (blue) and the vector sequences of pBR325 (red). The coding sequences and their direction of transcription are represented by the boxes of the outer circle. The β-lactamase (bla), chloramphenicol acetyl transferase (cat) and inactivated tetracycline resistance gene (Δtet) are as indicated, as are key restriction endonuclease cleavage sites.
Figure 2.
Southern blot of C. trachomatis L2/434/Bu transformed by plasmid pBR325::L2.
Equal amounts of C. trachomatis DNA were loaded in each gel track, the DNA is uncut (track 1) or has been digested to completion with Bam HI (track 2) or Nde I (track 3). (A) DNA from wild type (untransformed C. trachomatis L2/434/Bu) was probed with the complete plasmid sequence pL2. (B) DNA from the transformed strain also probed with pL2. (C) DNA from the transformed strain probed with the complete β-lactamase gene sequence (generated by PCR). The wild type pL2 plasmid (7.5 kb) carries a unique Bam HI restriction site (which is used as the cloning site for insertion into the vector pBR325) but no Nde I site. The bands in tracks 1 and 3 in panel A represent various forms of the plasmid pL2. The recombinant plasmid pBR325::L2 is ∼13.5 kb, thus digestion with Bam HI releases the pBR325 vector (6 Kb – hybridizing to the bla probe in (C) and the chlamydial plasmid pL2 (7.5 kb – hybridizing to pL2 probe in panel B). The recombinant plasmid (pBR325::L2) carries a unique Nde I site (located in pBR325) and digestion with this enzyme linearises the vector at 13.5 kb; this band is detected with both hybridization probes (track 3 panels B and C).
Figure 3.
Growth characteristics of C. trachomatis L2 and C. trachomatis L2 transformed by pBR325::L2.
(A) C. trachomatis L2/434/Bu, (B) C. trachomatis L2/434/Bu transformed by shuttle vector pBR325::L2. McCoy cells in a 24 well tissue culture tray grown to confluence were infected with C. trachomatis at MOI = 1 and were cultured without (-PEN) and with (+PEN) penicillin G at 10 units/ml. The yield of C. trachomatis (IFU) per culture or single well is shown on the y – axis and time of sampling post infection is shown on the x –axis. The experiments were repeated in quadruplicate and standard error bars are shown for each sample point.
Figure 4.
Purified, transformed RBs have β – lactamase activity.
In Panel A equal amounts of purified RBs (measured by qPCR of the genome) from transformed C. trachomatis L2/434/Bu and the wild–type parental C. trachomatis L2/434/Bu were assayed for β – lactamase activity. In Panel B equal amounts of E. coli and E. coli transformed with pBR325::L2 were used as controls to verify the assay. Hydrolysis of Nitrocefin is measured in pH 7.0 buffer by absorbance at 486 nm over 60 min. Nitrocefin is a chromogenic β – lactamase substrate.
Figure 5.
Map of the plasmid vector pGFP::SW2.
The inner circle represents the plasmid pSW2 from C. trachomatis SW2 (blue) and the vector sequences (red). The coding sequences and their direction of transcription are represented by the boxes of the outer circle. Some key restriction endonuclease cleavage sites used in the construction of this vector are indicated on the inner circle. The cat gene is fused with RSGFP (shaded green) and expressed by a promoter derived from Neisseria meningitidis (nmP); the direction of transcription of this promoter is designated by the arrow.
Figure 6.
Green fluorescent inclusions in McCoy cells infected with C. trachomatis L2/434/Bu transformed with pGFP::SW2.
Untransformed C. trachomatis L2/434/Bu (control) and C. trachomatis L2/434/Bu transformed by pGFP::SW2 were grown on coverslips for two days before fixing in 4% formaldehyde (diluted in DPBS). Panel A shows untransformed C. trachomatis L2/434/Bu under white light (arrows indicate inclusions). Panel B is the same image under blue light. Panel C shows C. trachomatis L2/434/Bu transformed with plasmid pGFP::SW2 under white light, and panel D is the same field under blue light. The scale bar represents 20 µm.
Figure 7.
Southern blot of C. trachomatis L2 (25667R) transformed by plasmid pGFP::SW2.
Southern hybridization analyses of plasmid-free C. trachomatis L2 (25667R) (abbreviated as L2P-) and C. trachomatis L2 (25667R) transformed with plasmid pGFP::SW2 (in short L2P-/pSW2). Six chlamydial genomic DNA samples were loaded on the gel in pairs together with HyperLadder I (5 µl) from BIOLINE (Cat No. BIO-33025). The agarose gel image was taken before DNA transfer (A). The DNA blot was first hybridized with the pSW2 probe (B), and then stripped and re-probed with the GFP probe (C) or an ompA probe (D). Bam HI digestion of pGFP::SW2 created 3 fragments: 7169 bp (containing pSW2 probe sequence), 2925 bp and 1445 bp (containing GFP probe sequence). Bgl II digestion of pGFP::SW2 created 4 fragments: 5555 bp (containing the pSW2 probe sequence), 3625 bp (containing the GFP probe sequence), 1693 bp and 666 bp. When the C. trachomatis L2 genomic DNA (similar to L2P-) was digested with Bam HI or Bgl II, the fragment containing ompA sequence was 8837 bp (Bam HI digestion) or 5518 bp (Bgl II digestion). Southern hybridization using the pSW2 probe or the GFP probe showed hybridization signals at expected positions in all L2P-/pGFP::SW2 samples (uncut or digested); whilst no hybridization signal, as expected, was detected in all L2P- samples (uncut or digested) (B) and (C). Southern hybridization using the ompA probe showed that the hybridization signals were similar in L2P- and L2P-/pGFP::SW2 samples (uncut or digested) (D).
Figure 8.
Acquisition of the plasmid pGFP::SW2 restores the ability of plasmid-free C. trachomatis L2 (25667R) to synthesize glycogen.
The presence of glycogen within inclusions was detected by iodine staining of cells on coverslips. (A) McCoy cells control, (B) C. trachomatis L2 (25667R) in McCoy cells, (C) C. trachomatis L2/434/Bu in McCoy cells, (D) pGFP::SW2- transformed C. trachomatis L2 (25667R) in McCoy cells. The scale bar represents 20 µm.