A platform supporting generation and isolation of random transposon mutants in Chlamydia trachomatis

Chlamydia species represent a paradigm for understanding successful obligate intracellular parasitism. Despite limited genetic malleability, development of genetic tools has facilitated the elucidation of molecular mechanisms governing infectivity. Random mutagenesis approaches provide one of the most powerful strategies available to accomplish untargeted elucidation of gene function. Unfortunately, initial progress in transposon-mediated mutagenesis of Chlamydia has been challenging. To increase efficiency, we developed a plasmid-based system that couples conditional plasmid maintenance with a previously described strategy leveraging inducible expression of the Himar1-derived C9 transposase. Our pOri-Tn(Q) construct was maintained in C. trachomatis cultivated with antibiotics but was rapidly cured in the absence of antibiotic selection. pOri-Tn(Q) supported transposition events when transposase expression was induced during infection. Induction was accompanied by loss of the plasmid backbone when Penicillin G was used to select for only the transposable element. C9 induction during iterative passaging was used to increase overall insertion frequency and accumulate an expanded pool of transposon mutants. The approach supported isolation of individual mutant strains from the mixed pool, and whole-genome sequencing confirmed that the recovered strains harbored single insertions.

Methods
These data represent source data from the manuscript entitiled: A platform supporting generation and isolation of random transposon mutants in Chlamydia trachomatis

The raw data are qPCR, immunoblots, immunofluorescent images,and  DNA agarose gels. 

C. trachomatis serovar L2 (LGV 434) was used as the parent strain in these studies. Chlamydiae was maintained in HeLa 229 epithelial cells (CCL-1.2; ATCC) for routine culture and for genetic manipulations. HeLa were grown in RPMI 1640 medium containing 2 mM L-glutamine (Life Technologies) supplemented with 10 % (vol/vol) heat-inactivated fetal bovine serum (FBS; Sigma).  All cultures were maintained at 37 °C in an environment with 5 % CO2 and 95 % humidified air. Infections were accomplished by centrifugation of EBs onto cell monolayers at 20 °C for 1h at 900 x g. Where appropriate, cultures were supplemented with 2 µg/ml cycloheximide (Cyclo), 50 ng/ml anhydrotetracycline (aTc), and/or 2 mM Theophylline (Theo; Sigma).nd chlamydiae.

Plasmid abundance was assessed during serial passage of cultures under various induction and selection conditions. DNA was extracted from wells at 24 hrs during respective passages of C. trachomatis-infected monolayers. Relative counts of chlamydial 16S or plasmid-encoded c9 were determined by quantitative real-time PCR using the Bio-Rad CFX96 Real-Time System (Bio-Rad), iTaq Universal SYBR Green Supermix (Bio-Rad). Primers for c9 were those used for qRT-PCR and 16S-s (5’-CCTGGTAGTCCTTGCCGTAAAC-3’) and 16S-as (5’-TACTCCTCAGGCGGCATACTTA-3’) were used to amplify 16S DNA. Detection of mCherry-derived fluorescence was used as an indicator for the presence of pOri-Tn(Q) in live cultures. Quantitative enumeration of chlamydial infectious forming units (IFU) was performed via indirect immunofluorescence.

Immunodetection and microscopy. For immunoblot analyses, proteins were separated on 4 to 15 % SDS-PAGE gels (Bio-Rad) and transferred to 0.45 µm PVDF membranes (Millipore). Primary antibodies were specific for C9 transposase provided by Dr. David Lampe (Duquesne University) or Hsp60 (A57-B9, Santa Cruz). Peroxidase-conjugated secondary antibodies (Sigma) and Amersham ECL Plus (GE Healthcare UK Limited) detection reagents were used to visualize proteins. For indirect immunofluorescence, respective cultures were fixed with paraformaldehyde, permeabilized with 0.1% Triton X100, and probed with primary antibodies specific to MOMP or Hsp60 to detect chlamydiae. Visualization was accomplished using secondary antibodies conjugated to AlexaFluor-594 or -488 (Invitrogen). Cells were examined via epifluorescence microscopy using a Nikon E800 Eclipse with 100X oil immersion objective or via confocal using a Nikon A1R inverted confocal microscope with 63X oil immersion objective. All images were processed equivalently using auto contrast and unsharp-mask filter functions in Adobe® Photoshop® 6.0 (Adobe Systems).