Figure 1.
Shuttle vector with GFP expression under Tet control.
The E. coli cloning vector pASK-IBA33plus, with GFP inserted under Tet control, was ligated to the wild-type plasmid from C. trachomatis strain L2 using PCR-generated restriction endonuclease sites (see Materials and Methods for details). A variation of this shuttle vector was later constructed with a gene encoding mKate2 inserted at the indicated KasI site.
Figure 2.
Effect of anhydrotetracycline on C. trachomatis.
L929 cells were infected with wild-type C. trachomatis L2 and then incubated in the presence of various concentrations of ATc throughout the infection (0 to 24 hpi). Cultures were fixed at 24 hpi and stained for MOMP (green). Representative images from three different concentrations are shown (A). Additionally, the same samples (and additional samples) were analyzed using Cell Profiler to calculate the average inclusion size (B). Over 700 inclusions were analyzed at each concentration. Separately, infected L929 cell cultures were treated with different concentrations of ATc and lysed at 24 hpi. Lysates were used to infect a fresh monolayer (in the absence of ATc) and infection levels were enumerated at 24 hpi using immunofluorescence microscopy. Percent inhibition of progeny was determined relative to a lysate from an untreated control (C).
Figure 3.
GFP expression after induction at 24 hpi.
L929 cell cultures infected with pASK-GFP-L2 transformed C. trachomatis were induced with various concentrations of ATc at 24 hpi. Images were captured every 10 minutes for a total of 6 hours. Representative images from the culture induced with 10 ng/mL and an uninduced control are shown (A). The images were also analyzed using the Otsu segmentation algorithm to determine the mean fluorescence intensities in arbitrary units within the inclusions (B). Three microscope fields were averaged for each sample with approximately 40 infected cells per field. The mean fluorescence intensity of an uninduced sample was also calculated at each time point and the resulting values were subtracted from the induced samples at the corresponding time points.
Figure 4.
Detection of constitutive mKate2 and inducible GFP.
L929 cell cultures infected with pASK-GFP/mKate2-L2 transformed C. trachomatis were induced at 24 hpi and images were then taken at indicated time points. Representative images from an uninduced sample and a sample induced with 10 ng/mL ATc at 24 hpi are shown in (A). Representative images of a sample induced with 10 ng/mL ATc at 16 hpi (from a separate experiment) are shown in (B). White arrows highlight the relatively few C. trachomatis cells within the inclusion that did not express detectable GFP at 19 hpi and 20 hpi.
Figure 5.
Induction of GFP with lower ATc concentrations.
L929 cells were infected with pASK-GFP/mKate2-L2 transformed C. trachomatis. At 24 hpi, samples were induced for 4 hours with the indicated concentrations of ATc. After induction, soluble GFP and mKate2 in each sample was quantified using a microplate fluorometer. To normalize samples, GFP expression was divided by mKate2 expression and the resulting ratio was graphed (A). Increases observed in GFP fluorescence of samples induced with 0.25 ng/mL or higher were statistically significant compared to the 0 ng/mL sample (P < 0.01; Student’s t test). Induction from 16 to 20 hpi was also tested and the resulting GFP and mKate2 expression quantified as described above (B). All ATc concentrations tested for this induction period resulted in statistically significant increases in GFP expression. GFP fluorescence from the experiments shown in panels A and B are also graphed with mKate2 normalization (C). Error bars indicate standard deviation of three separate samples.
Figure 6.
Detection of GFP induction early in the developmental cycle.
L929 cell cultures infected with transformed C. trachomatis were induced at the start of infection (0 hpi). Samples were taken and lysates prepared at the indicated time points after the start of infection. GFP fluorescence was measured using a microplate fluorometer with the results quantified in arbitrary units. Error bars indicate standard deviation from two separate experiments.
Figure 7.
Detection of GFP gene transcription by qPCR.
L929 cell cultures infected with pASK-GFP-L2 transformed C. trachomatis were induced with 10 ng/mL ATc from 20-24 hpi. The culture medium was then replaced with ATc-free medium and samples were collected at the indicated time points for processing and analysis of GFP transcription by qPCR. Relative expression levels are in comparison to the uninduced sample. Error bars indicate standard deviation from two separate experiments.