DOI: 10.5061/dryad.f1vhhmh6d
title: Data from: A platform supporting generation and isolation of random transposon mutants in Chlamydia trachomatis

description: 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.
description: These data represent source data from the manuscript entitiled:
 <strong>A platform supporting generation and isolation of random
 transposon mutants in <em>Chlamydia
 trachomatis</em></strong> The raw data are
 qPCR, immunoblots, immunofluorescent images,and  DNA agarose
 gels.  <em>C. trachomatis</em> 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 <em>C. trachomatis</em>-infected monolayers.
 Relative counts of chlamydial <em>16S</em> or plasmid-encoded
 <em>c9</em> 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 <em>c9</em> 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. <strong>Immunodetection and
 microscopy. </strong>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).
description: # A platform supporting generation and isolation of random transposon
 mutants in Chlamydia trachomatis
 [https://doi.org/10.5061/dryad.f1vhhmh6d](https://doi.org/10.5061/dryad.f1vhhmh6d) #### Description of the data and file structure These data were collected as part of a study to develop an efficient transposon mutagenesis system for C. trachomatis. The data were collected from HeLa cultures infected with C. trachomatis expressing pOri-Tn(Q). For qRT-PCR and qPCR analyses DNA was extracted from triplicate infected cultures and processed for respective amplification in triplicate. Raw CT values are shown for all reactions.  For immunoblot analyses, whole culture samples were concentrated by TCA precipitation and resolved in SDS-PAGE gels. Immunoblots were probed with Chlamydia-specific or transposase-specific (C9). antibodies. For immunofluorescence, infected cultures were fixed and probed with Chlamydia-specific antibodies (indirect) or visualized for mCherry fluorescence (direct). For all infections, induction of transposition was accomplished by supplementing with anhydrotetracycline (aTC) and Theophyline (Theo). Antibiotic selection included the addition of spectinomycin (Spec) or Penicillin G (PenG). PCR amplification of specific genes was accomplished using specific primers. PCR products were resolved in 1% agarose gels.   #### Files and variables #### File: Raw\_Data\_JB00500-24.xlsx **Description:** Each tab corresponds to a manuscript-derived figure and contains the raw, unprocessed source data. ##### Variables * Tab 1(Figure 1C):  The dataset comprises CT reads from qRT-PCR corresponding to samples obtained from cultures that were untreated or treated with aTc, Theophylline, or both aTC and Theophylline. Each sample was probed with primer sets specific for rpoD and C9 transposase.  * Tab 2(Figure 1D):  The dataset comprises immunoblot data derived from whole-culture material probed with Chlamydia-specific (Hsp60) or transposase-specific (C9) antibodies. Arrow indicated position of the C9 transposase.  * Tab 3(Figure 2A):  The dataset comprises CT reads from qPCR corresponding to samples obtained from cultures that were infected with C. trachomatis expressing pSW2-RiboA-C9Q and serially passaged (P0-P5) in the absence of antibiotic selection.  Amplification of 16s and C9 was used to assess normalized abundance of the plasmid. * Tab4(Figure 2B):  The dataset comprises CT reads from qPCR corresponding to samples obtained from cultures that were infected with C. trachomatis expressing pSW2-RiboA-C9Q and serially passaged (P0-P5) in the absence of antibiotic selection, but presence of aTc and Theo.  Amplification of 16s and C9 was used to assess normalized abundance of the plasmid. * Tab 5(Figure 2C):  The dataset comprises CT reads from qPCR corresponding to samples obtained from cultures that were infected with C. trachomatis expressing pOri-Tn(Q) and serially passaged (P0-P5) in the absence of antibiotic selection.  Amplification of 16s and C9 was used to assess normalized abundance of the plasmid. * Tab 6(Figure 2D):  The dataset comprises CT reads from qPCR corresponding to samples obtained from cultures that were infected with C. trachomatis expressing pOri-Tn(Q) and serially passaged (P0-P5) in the absence of antibiotic selection, but presence of aTc and Theo.  Amplification of 16s and C9 was used to assess normalized abundance of the plasmid. * Tab 7(Figure 3A). Direct (mCherry) and indirect (total inclusions) fluorescence images obtained from 24 hr C. trachomatis cultures cultivated in the absence of induction (uninduced) or presence of aTc and Theophylline (Induced). Cultures were supplemented with antibiotics (PenG or Spec) where indicated.  * Tab 8(Figure 3B).  The dataset is comprised of inclusion forming unit counts from C. trachomatis pOri-Tn(Q) primary cultures that were maintained for 24 hrs without treatment (mock) or with induction (aTc+Theo) in the presence of spec or PenG. Numbers of total bacteria (green) and mCherry positive (red) inclusion counts are presented for 10 fields of view in triplicate samples of each treatment. * Tab 9(Figure 3C). This dataset is comprised of progeny inclusion forming counts from C. trachomatis pOri-Tn(Q) primary cultures that were maintained for 24 hrs without treatment (mock) or with induction (aTc+Theo) in the presence of spec or PenG. Average whole-well counts are provided for triplicate samples for each of the triplicate replicates for each treatment. * Tab 10(Figure 5B). This dataset is comprised of PCR products resolved in agarose gels. Each panel represents the amplification of a specific gene from individual DNAs obtained from group mutagenesis isolates. 10 Isolates (C1-10) were assayed. PCR products were resolved with 1kb size standards and specific amplicons are indicated by an arrow.