Chlamydia trachomatis arrives at a physiologically similar ‘persistent’ state in response to starvation of either nutrient remains unclear. Here, we report on the iron-dependent regulation of the trpRBA tryptophan salvage pathway in C. trachomatis. Iron starvation specifically induces trpBA expression from a novel promoter element within an intergenic region flanked by trpR and trpB. YtgR, the only known iron-dependent regulator in Chlamydia, can bind to the trpRBA intergenic region upstream of the alternative trpBA promoter to repress transcription. Simultaneously, YtgR binding promotes the termination of transcripts from the primary promoter upstream of trpR. This is the first description of an iron-dependent mechanism regulating prokaryotic tryptophan biosynthesis that may indicate the existence of novel approaches to gene regulation and stress response in Chlamydia.During infection, pathogens are starved of essential nutrients such as iron and tryptophan by host immune effectors. Without conserved global stress response regulators, how the obligate intracellular bacterium  Chlamydia trachomatis, a bacterium that causes many sexually-transmitted infections, is no exception. These bacteria do not normally make one of the building blocks of proteins, the amino acid tryptophan, but instead scavenge it from their human host.All forms of life must take up nutrients from their environment to survive. Chlamydia bacteria to take up a molecule found in the female genital tract that they can use to make their own tryptophan. Yet, how do the bacteria know when to activate these genes?One way that the immune system tries to fight a chlamydia infection is by cutting off the supply of tryptophan in an attempt to starve the bacteria. But the microbes have evolved to respond to these hardships and keep themselves alive. The ‘tryptophan salvage pathway’ is a set of genes that, when switched on, allows the Chlamydia bacteria, Pokorzynski et al. show that iron starvation switches on the tryptophan salvage pathway. Chlamydia most likely senses changes in iron levels via a protein called YtgR, and a closer look at the bacterial DNA revealed that YtgR interacts with the genes of the tryptophan salvage pathway. When iron levels were high, YtgR locked on to the DNA in the middle of this set of genes. This effectively switched off the genes on either side of the binding site. When iron levels dropped, YtgR came away from the DNA, releasing the genes and allowing the cell to use them to start making its own tryptophan. Together these findings indicate that, when the bacteria sense that iron levels have dropped, they prepare for a shortage of tryptophan too.Tryptophan starvation is not the only strategy that the immune system uses to fight chlamydia. It also restricts the supply of the essential metal iron to these bacteria. Now, using human cells grown in the laboratory and infected with Chlamydia is the most common bacterial sexually transmitted infection worldwide. Left untreated, it can cause infertility and blindness. This and future studies aimed at understanding how these bacteria respond to immune attack may reveal new ways to prevent or treat these infections. Chlamydia trachomatis are relatively poorly characterized, leaving unanswered many fundamental questions about the biology of this pathogen.Nutrient acquisition is critical for the success of pathogenic bacteria. Many pathogenic bacteria must siphon nutrients from their hosts, such as nucleotides, amino acids and biometals . This coC. trachomatis is the leading cause of bacterial sexually transmitted infections (STIs) and infection-derived preventable blindness worldwide , suggesting that this pathogen may utilize novel mechanisms to respond to nutrient stress  have not been shown to respond to Trp limitation (The best described inducer of persistence is the pro-inflammatory cytokine interferon-gamma (IFN-γ). The bacteriostatic effect of IFN-γ has been primarily attributed to host cell tryptophan (Trp) catabolism, an amino acid for which otrophic . Followiole ring . C. trac species , and thucription . This memitation .Escherichia coli, trpR is monocistronic and distal to the Trp biosynthetic operon. In C. trachomatis, TrpR is encoded in an operon, trpRBA, which also contains the Trp synthase α- and β- subunits , and possesses a 348 base-pair (bp) intergenic region (IGR) that separates trpR from trpBA. TrpBA catalyzes the final steps of Trp biosynthesis in bacteria; TrpA converts indoleglycerol-3-phosphate (IGP) to indole which is then condensed with serine by TrpB to form Trp. In C. trachomatis, TrpA cannot bind IGP and thus C. trachomatis requires indole as a substrate to synthesize Trp -containing endosomes  stress, we utilized the membrane-permeable iron chelator 2,2-bipyridyl (Bpdl), which has the advantage of rapidly and homogeneously starving C. trachomatis of iron (C. trachomatis serovar L2 of iron starting at 12 hr post-infection (hpi), or roughly at the beginning of mid-cycle growth. At this point the chlamydial organisms represent a uniform population of replicative RBs that are fully competent, both transcriptionally and translationally, to respond to stress. We treated infected HeLa cell cultures with 100 μM Bpdl or mock for either 6 or 12 hr (hrs) to determine a condition sufficient to limit iron to C. trachomatis without inducing hallmark persistent phenotypes. We stained infected cells seeded on glass coverslips with convalescent human sera and analyzed chlamydial inclusion morphology under both Bpdl- and mock-treated conditions by laser point-scanning confocal microscopy (=0.00151). We then assayed the transcript expression of two markers for persistence by reverse transcription quantitative PCR (RT-qPCR): the early gene euo, encoding a transcriptional repressor of late-cycle genes . This was unexpected, but we note that omcB expression has been shown to vary between chlamydial serovars and species when starved for iron , consistent with a more characteristic persistent phenotype. These data therefore also indicated that 6 hr of treatment would be ideal to monitor non-persistent responses to Trp limitation.We additionally assayed these same metrics following 6 or 12 hr of Trp starvation by culturing cells in either Trp-replete or Trp-depleted DMEM-F12 media supplemented with fetal bovine serum (FBS) pre-dialyzed to remove amino acids. We observed no discernable change in inclusion morphology out to 12 hr of Trp starvation , but geneuo  and omcBuo . However, we did not observe induction of ytgA transcript expression relative to the 12 hpi time-point. Here, we considered that apparent increases in transcription could be due to two factors: developmental regulation and transcriptional response to stress. Therefore, expression of genes of interest were monitored over time, for example 18 versus 12 hpi, in addition to single-timepoint comparisons, for example 18 hpi only. While we did not observe induction of ytgA over time, which would be more consistent with an iron-starved phenotype (i.e. ‘turning on’ gene expression), we reason that this is a consequence of the brief treatment period. This is in agreement with the need to prolong iron chelation to observe the transcriptional induction of ytgA  but was not induced relative to the 12 hpi time-point. The modestly elevated expression of these genes likely represents bona fide transcriptional responses to iron starvation given that the treatment condition was optimized to avoid gross changes in chlamydial development. The transcript expression of devB, encoding a 6-phosphogluconolactonase involved in the pentose phosphate pathway, was not observed to significantly respond to our brief iron limitation condition, suggesting that it is not a component of the primary iron starvation stress response in C. trachomatis. As expected, the transcript expression of dnaB, a replicative DNA helicase, was not altered by our iron starvation condition, consistent with its presumably iron-independent regulation . The marginal elevation in trpR expression at the 18 hpi time-point was surprising given that this gene was not identified as iron-responsive in a recent genome-wide RNA-sequencing study  downstream of the trpR ORF. Indeed, a previous study reported the presence of an alt. TSS in the trpRBA IGR, located 214 nucleotides upstream of the trpB translation start position (trpRBA IGR (trpBA. We therefore performed Rapid Amplification of 5’-cDNA Ends (5’-RACE) on RNA isolated from C. trachomatis L2-infected HeLa cells using the SMARTer 5’/3’ RACE Kit workflow (Takara Bio). Given the low expression of the trpRBA operon during normal development, we utilized two sequential gene-specific amplification steps (nested 5’-RACE) to identify 5’ cDNA ends in the trpRBA operon. These nested RACE conditions resulted in amplification that was specific to infected-cells . Interestingly, the 1.0 kb product displayed a very similar apparent enrichment following Bpdl treatment, suggesting that this RACE product represented a specifically iron-regulated TSS. Both the 1.5 and 1.0 kb RACE products were detectable in the Trp-depleted and iron-depleted conditions, respectively, during the primary RACE amplification, consistent with their induction under these conditions . Additionally, we observed that at 12 and 18 hpi in iron-replete conditions, the ratio of trpB to trpR was approximately 1.0, suggesting non-preferential basal expression across the three putative TSSs. Another factor contributing to this ratio is the synthesis of the full-length trpRBA polycistron. In support of this, the trpB to trpR ratio remained near 1.0 under the Trp-starved condition, which would be expected during transcription read-through of the whole operon. The apparent lack of preferential promoter utilization as described above could be attributed to the relatively low basal expression of the operon at 12 and 18 hpi under Trp- and iron-replete conditions, thus precluding quantitative detection of differential promoter utilization in this assay.If iron depletion was inducing  samples . In agretrpRBA operon, we isolated the 5’-RACE products across all conditions by gel extraction and cloned the products into the pRACE vector supplied by the manufacturer. We then sequenced the ligated inserts and BLASTed the sequences against the C. trachomatis L2 434/Bu genome to identify the location of the 5’-most nucleotides  covering 67% of the putative operator sequence  E. coli along with an IPTG-inducible pET151 expression vector with (pET151-YtgR) or without (pET151-EV) the C-terminal 139 amino acid residues of CTL0325 (YtgC). Note that we have previously demonstrated that this region is a functional iron-dependent repressor domain (C. trachomatis dnaB (pCCT-dnaB), and the promoter region of the ytgABCD operon  consistent with its previously reported auto-regulation of this promoter (To investigate the ability of YtgR to bind and repress transcription from the putative pression . In brier domain . To veripromoter .trpR promoter element (pCCT-trpR), 2) the putative trpBA promoter element represented by the IGR (pCCT-trpBA), and 3) the same putative trpBA promoter element with a mutated YtgR operator sequence that was diminished for both palindromicity and A-T richness, two typical features of prokaryotic promoter elements . This suggested that YtgR was capable of repressing transcription from the trpBA promoter element specifically. Interestingly, this repression phenotype was abrogated in the pCCT-trpBAΔOp background, where we observed no statistically meaningful difference in β-galactosidase activity, demonstrating that YtgR transcriptional repression of trpBA is operator-dependent. We subsequently addressed whether the region of the trpRBA IGR containing the YtgR operator site was sufficient to confer YtgR repression in this assay (trpRBA IGR into the pCCT reporter plasmid: the first fragment represented the 5’-end of the IGR containing the operator site at the 3’-end (pCCT-IGR1), the second fragment represented a central region of the IGR containing the operator site at the 5’-end (pCCT-IGR2), and the third fragment represented the 3’-end of the IGR and did not contain the operator site (pCCT-IGR3). Surprisingly, we observed that none of these fragments alone were capable of producing a significant repression phenotype in our reporter system.Using this same assay, we then inserted into the pCCT reporter plasmid 1) the dentical . When Ytis assay . We clontrpBA promoter element was dependent upon iron availability, we assessed repressor activity in the presence or absence of 500 µM Bpdl, mimicking previously utilized approaches for assessing iron-dependent repressor activity of the homologous DtxR  qPCR method to detect the abundance of co-immunoprecipitated promoter fragments with the recombinant YtgR domain. As before, we co-transformed the pET151-YtgR expression vector with pCCT-trpRBA YtgR operator site may disadvantage the processivity of RNAP reading-through the IGR from the upstream trpR promoter, possibly leading to transcript termination. Similar systems of RNAP read-through blockage have been reported; the transcription factor Reb1p ‘roadblocks’ RNAPII transcription read-through in yeast by promoting RNAP pausing and subsequent degradation  which we modified to include annotations for non-operonic IGRs. Using this coverage map, we sought to semi-quantitatively assess the mapping of reads across the trpRBA IGR to gain insight regarding possible transcription readthrough. Our analysis revealed that under Bpdl-treated conditions, there was a 2.21-fold increase in reads mapping to the trpRBA IGR (IGR_trpB) relative to mock treatment  using the genomewide RNA-seq analysis toolkit in the CLC Genomics Workbench. Regardless, a comparable increase in reads mapping to the upstream trpR CDS was not observed , suggesting that under iron replete conditions, transcripts originating from the primary trpR promoter may be terminated before reading through the IGR, thereby accounting for the increase in reads mapping to IGR_trpB following Bpdl treatment.We hypothesized that YtgR binding at the radation . To invechomatis . Using dreatment . This obeuo  and lpdA . For both IGR_euo  and IGR_lpdA , we did not observe a similar increase in read coverage following Bpdl treatment indicating that the increased coverage at IGR_trpB is likely specific  while CTL0819, upstream of IGR_lpdA, was 1.13-fold increased (p=0.56). We extracted individual RPKM values for each of these regions from this dataset and observed the same trend in mean fold-change differences reported from the genomewide analysis: only IGR_trpB was substantially increased relative to its upstream ORF  in the trpRBA operon in C. trachomatis, we utilized 3’-RACE to map the 3’-ends of transcripts using gene-specific primers within the trpR CDS . Notably, Trp-depletion did not increase the readthrough value, suggesting that Trp-depletion does not relieve YtgR repression at the alt. TSS and the levels of trpA remain relatively constant to those of trpR. It is therefore possible that under Trp-depleted, but iron-replete conditions, YtgR repression of the alternative trpBA promoter acts as a rheostat for full-length trpRBA transcription, helping to maintain a constant ratio of trpR to trpBA. Together, these analyses indicated that iron limitation resulted in transcription readthrough specifically at the YtgR operator site.Consistent with our model, readthrough 5’ of the YtgR operator site was not affected by Bpdl treatment, whereas Trp-depletion resulted in significantly more readthrough than all other conditions  E. coli co-transformed with pCCT-RT and either pET151-EV or pET151-YtgR. We observed that ectopic expression of YtgR significantly reduced readthrough into the lacZ gene by RT-qPCR  that harbored the entire ene lacZ . Note thtrpBA expression mediated by the repressor YtgR, representing a novel instance of integrated stress adaptation in Chlamydia. Utilizing an infected-epithelial cell culture model, we identified a previously undescribed iron-regulated promoter element independent of trpR within the trpRBA IGR that is responsible for the iron-limited induction of trpBA expression. Using in silico and biochemical methods, we demonstrate that YtgR binds the trpRBA IGR to regulate iron-dependent trpBA expression. Importantly, transcriptional repression in our heterologous system was shown to be dependent on an unaltered operator sequence that bears significant homology to the previously defined operator element in the ytgA promoter. Furthermore, our infected-cell culture studies revealed that transcripts originating from the primary trpR promoter terminate within the IGR, notably at the putative YtgR operator site, and that transcription read-through at this locus is iron- and YtgR-dependent. Thus, we propose that YtgR regulates trpBA expression at two levels: repression of the trpBA promoter and premature termination of the major transcript generated from the trpR promoter Ss across the trpRBA operon in our studies. However, the contribution of such a Trp-dependent alt. TSS as identified by Carlson et al. to the general stress response of C. trachomatis remains unclear given its association with presumably abnormal organisms. Does utilization of this alt. TSS indicate abnormal growth or a bona fide stress adaptation? Moreover, Akers and Tan were unable to verify TrpR binding to the trpRBA IGR by EMSA, suggesting that some other Trp-dependent mechanism may control transcription from this site , where most significant pathology is identified following infection with C. trachomatis, the pathogen must first navigate the lower genital tract (LGT). Chlamydia infections of the female LGT are associated with bacterial vaginosis (BV), which is characterized by obligate and facultative anaerobe colonization, some of which produce indole , an iron-binding glycoprotein, which can starve pathogens for iron . Ideally, genetic approaches could be employed to distinguish the regulatory effects of YtgR independent of TrpR. However, the genetic manipulation of trans-acting factors (e.g. YtgR) will presumably have unpredictable off-target effects. Genetically altering cis-acting factors – such as operator sequences – is more feasible, but at present we lack the information necessary to rationally mutate these sequences in C. trachomatis to interrogate these questions. The tight regulatory coordination at both the transcription initiation and termination steps would likely mean any mutation in the cis-acting sequences would affect both processes indiscriminately. Furthermore, in vivo infection models present challenges: attempting to answer these questions will likely require the use of non-human primate studies, as mouse models of Chlamydia infection do not recapitulate immune-mediated Trp starvation  cells were cultured at 37° C with 5% atmospheric CO2 in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 μg/mL gentamicin, 2 mM L-glutamine, and 10% (v/v) filter sterilized fetal bovine serum (FBS). For all experiments, HeLa cells were cultured between passage numbers 4 and 16. Chlamydia trachomatis serovar L2 (434/Bu) was originally obtained from Dr. Ted Hackstadt . Chlamydial EBs were isolated from infected HeLa cells at 36–40 hr post-infection (hpi) and purified by density gradient centrifugation essentially as described  prior to the monolayer being overlaid with inoculum (un-supplemented DMEM) at the indicated multiplicity of infection (MOI). Tissue culture plates were then centrifuged at 4° C with a speed of 1000 RPM  for 5 min to synchronize the infection. Inoculum was aspirated and cells were washed again with pre-warmed HBSS prior to the media being replaced with pre-warmed complete DMEM. Infected cultures were then returned to the tissue culture incubator until the indicated times post-infection. This procedure was replicated exactly for the infection of 24-well tissue culture plates.Chlamydia trachomatis L2-infected HeLa cell cultures were starved for iron by supplementation of the media with the iron chelator 2,2-bipyridyl  essentially as described  powder media was prepared following manufacture instructions and supplemented with 10% (v/v) filter-sterilized FBS which had been previously dialyzed 16–20 hr at 4° C in PBS in a 10 kDa MWCO dialysis cassette. Media was then further supplemented with 10 μg/mL gentamicin. At the indicated times post-infection, complete DMEM was aspirated and wells were washed with pre-warmed HBSS prior to the addition of tryptophan-replete or –deplete medium. Infected cell cultures were returned to the incubator for the indicated treatment periods.e.g. restriction enzyme, homology-directed, etc. All primers and plasmids used in this study can be found in ytgA vector (to excise the ytgA promoter fragment). The vector was then treated with antarctic phosphatase (New England Biolabs) prior to having the promoter of interest ligated into the pCCT backbone. Inserted promoters were verified to be in the correct orientation and free of sequence errors by PCR and sequencing. All pET vectors were cloned following manufacturer instructions .All constructs were cloned using standard molecular cloning techniques, C. trachomatis L2-infected HeLa cell cultures seeded on glass coverslips in 24-well tissue cultures plates were first washed with pre-warmed HBSS prior to fixation with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 20 min at RT ° C. Fixation solution was aspirated and wells were washed with PBS prior to permeabilization with 0.2% Triton X-100 in PBS for 5 min at RT° C. Permeabilization solution was then decanted and cells were washed with PBS. The coverslips were blocked for 30 min with 1% bovine serum albumin (BSA) in PBS at RT° C. To stain for Chlamydia, coverslips were washed with PBS and PBS supplemented with 1% BSA and 1:500 convalescent human sera was added to wells and incubated at RT° C for 1 hr with rocking. Primary antibody solution was decanted and coverslips were again washed with PBS. Goat anti-human Alexa-647  diluted 1:1000 in PBS with 1% BSA was then added to the wells and incubated in the dark for another hour at RT° C with rocking. Secondary antibody solution was then decanted, coverslips were washed again with PBS and coverslips were either immediately mounted on microscopy slides using ImmuMount (ThermoFisher Scientific) or VectaShield H-1000  or stored in the dark at 4° C until mounting. All images were acquired on a Leica TCS SP8 laser scanning confocal microscope, using identical settings, in the Integrative Physiology and Neuroscience Advanced Imaging Center at Washington State University. All images are Z-projections and were processed in Fiji . Samples were then pooled and split into two technical replicates (RT-qPCR) or kept as one biological replicate (RACE). Trizol-extracted samples were then thoroughly vortexed with a 100 μL volume of Zirconia beads prior to chloroform extraction. 100% ethanol was added to the aqueous phase and RNA was isolated using the Ambion RiboPure RNA Purification kit for bacteria following manufacturer instructions (ThermoFisher Scientific). DNA was removed from RNA samples using the Invitrogen DNA-free DNA Removal Kit following manufacturer instructions (ThermoFisher Scientific). RNA was stored at −20° C until further use. For E. coli, RNA was harvested using the Ambion RiboPure RNA Purification kit for bacteria following manufacturer instructions (ThermoFisher Scientific) from 9 mL of bacterial culture prepared as described below for the Two-Plasmid Reporter Assay. RNA was subsequently DNased as described above. cDNA was generated using either SuperScript IV Reverse Transcriptase  or SMARTScribe Reverse Transcriptase (RACE and RACE-specific qRT-PCR); Takara Bio, Kusatsu, Shiga Prefecture, Japan) essentially as described by the respective manufacturers. For cDNA generated for RT-qPCR, 650 ng of total RNA was used as a template in a 20 μL total reaction volume. For every RT reaction, a ‘no-RT’ control, generated from 350 ng of total RNA template in a 10 μL total volume, was included. For 5’-RACE, cDNA was generated from 250 ng of total RNA using random primers in a 10 μL total volume and further processed in the RACE workflow. cDNA was stored at −20° C.RNA was harvested from C. trachomatis-infected HeLa cell monolayers by scraping 3 wells of a 6-well plate in ice-cold PBS + 10% Proteinase K (ThermoFisher Scientific). Samples were then pooled and split into two technical replicates for analysis of genome copy number by qPCR. gDNA was isolated using the DNeasy Blood and Tissue Kit following manufacturer protocols . gDNA was stored at −20° C until further use.gDNA was harvested from 2O depending on the experimental condition being assayed . On ice, 3.3 μL of diluted sample was added to 79 μL of PowerUp SYBR Green Master Mix (ThermoFisher Scientific) with specific qPCR primers diluted to 500 nM. From this master mix, each experimental sample was assayed in triplicate 25 μL reactions. Assays were run on an Applied Biosystems 7300 Real Time PCR System with cycling conditions as follows: Stage 1: 50.0° C for 2 min, one rep. Stage 2: 95.0° C for 10 min, one rep. Stage 3: 95.0° C for 15 s, 40 reps. Stage 4: 60.0° C for 1 min, one rep. Primers were subjected to dissociation curve analysis to ensure that a single product was generated. For each primer set, a standard curve was generated using purified C. trachomatis L2 gDNA from EB preparations diluted from 2 × 10−3 to 2 × 100 ng per reaction. Ct values generated from each experimental reaction were then fit to standard curves (satisfying an efficiency of 95 ± 5%) for the respective primer pair and from the calculated ng quantities, transcript or genome copy number was calculated as follows:Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) cDNA (or gDNA in qPCR), prepared as described above, was diluted 1:10 or 1:100 in nuclease-free Hdf = dilution factor and the number of copies/ng DNA is calculated based on the size of the C. trachomatis L2 genome assuming that the molar mass per base pair is 650 (g/mol)/bp . All quantifications of genome copy number were determined using the ahpC qPCR primer set. Values from replicate assays were averaged, and values from replicate RNA/gDNA isolations were averaged to obtain the mean and standard deviation for one biological replicate. For some experiments, to account for batch effects across biological replicates, data was transformed such that the mean of all samples in each replicate was identical. In some instances, batch correcting generated negative values, and in this case data sets were scaled such that the lowest value equaled 1.0. HeLa cells were infected at an MOI of 2 for all RT-qPCR studies.For analysis of transcriptional readthrough, RT-qPCR was performed as described above and the readthrough value was computed as:Where Readthrough values were then batch corrected such that the mean of each replicate was identical.trpRBA operon, a ‘nested’ RACE protocol was used as outlined in the SMARTer RACE 5’/3’ Kit user manual. Briefly, 1.25–2.5 μL of cDNA generated for RACE was added to a 25 μL reaction volume and run in a thermal cycler for 40 cycles using the touch-down PCR conditions described by the manufacturer. In brief, five cycles were run at an annealing temperature of both 72° C and 70° C prior to 30 cycles run with an annealing temperature of 68° C. Following this primary amplification, the RACE products were diluted 1:50 in Tricine-EDTA Buffer supplied by the manufacturer, and 2.5 μL of diluted primary RACE product was added to a 25 μL reaction volume and subjected to another 20 cycles of nested PCR, as described by the manufacturer, using primers designed within the amplicon of the primary RACE products. Samples were electrophoresed on a 2% agarose gel for visualization and analysis. HeLa cells were infected at a MOI of 5 for all RACE studies.All RACE studies were performed using the SMARTer RACE 5’/3’ Kit (Takara Bio). To observe 5’-RACE products from the trpRBA polycistronic message.3’-RACE studies were performed essentially identical to 5’-RACE with the exception that total RNA was subjected to poly(A) tailing with a Poly(A) Polymerase following manufacturer instructions (New England Biolabs). In brief, at least 3.5 μg of total RNA was incubated at 37° C with Poly(A) Polymerase in reaction buffer supplemented with ATP and murine RNase Inhibitor (New England Biolabs) for 30 min prior to heat-inactivation at 65° C for 20 min. RNA was re-isolated through an RNA clean-up filter cartridge . A total of 125 ng of poly(A)-tailed total RNA was then used to generate 3’-RACE ready cDNA in a 10 μL reaction volume following manufacturer instructions. Primary and nested RACE was performed using 3’-RACE gene-specific primers following the same protocol for amplification described for 5’-RACE, with the exception that the extension time was adjusted to accommodate amplification of the full ~3 kb E. coli cells by heat shock. Transformed bacteria were plated on LB agar containing 50 μg/mL carbenicillin and incubated overnight at 37° C. Colonies were selected and screened for relevant inserts by PCR. Positive colonies were cultured overnight at 37° C in LB liquid broth containing 50 μg/mL carbenicillin and plasmids were isolated using the QIAprep Spin Miniprep kit (QIAGEN). Inserts were then sequenced by Eurofins Genomics using the default M13 Reverse sequencing primer. Returned sequencing data was aligned to the C. trachomatis L2 (434/Bu) genome (NCBI Accession: NC_010287) by BLAST and the most 5’ aligned nucleotide was considered the 5’ end of the insert. In the case of 3’-RACE data, the reverse complement sequence was first generated prior to alignment. Grouping of individual products was determined 1.) by clusters being greater than 30 nucleotides apart and 2.) by the specific RACE band that the alignment was derived from. These two criteria were not both satisfied in all cases and in those cases criteria 1.) was favored.5’-RACE products generated from either primary or nested RACE reactions were excised from the agarose gel and DNA was isolated using the NucleoSpin Gel and PCR Clean-up kit . The isolated RACE products were then cloned into the pRACE vector supplied in the SMARTer RACE 5’/3’ Kit using the In-Fusion HD cloning kit (Takara Bio). Ligated vectors were transformed into chemically competent Stellar C. trachomatis L2 434/Bu genome sequences were obtained from NCBI Accession NC_010287. Global pairwise sequence alignments were made using the EMBOSS Needle algorithm. Alignment parameters were set as follows: Matrix: DNAfull, Gap Open: 20, Gap Extend: 0.8, Output Format: pair, End Gap Penalty: True, End Gap Open: 10, End Gap Extend: 0.5. These conditions were sufficient to replicate the previously published alignment between the putative YtgR operator sequence and the TroR operator . The alignment parameters were set as follows: Matrix: DNAfull, Gap Open:10, Gap Extend: 0.5, Output Format: pair. These are the default conditions and were chosen to remove bias from the alignment results.All operator . Local pC. trachomatis L2 (434/Bu) genome by PCR using the indicated primer sets, which included KpnI restriction endonuclease sites at the 5’ and 3’ ends of the promoter amplicon. The amplified fragments and the pCCT-EV plasmid were then KpnI-digested and the promoters ligated into the vector using T4 or Quick Ligase (New England BioLabs). Insert directionality was confirmed by directional colony PCR and positive clones were sequence verified. pCCT-trpBAΔOperator was cloned by amplifying two fragments of the pCCT-trpBA vector with one ~ 60 mer primer containing the bases to be substituted for each fragment. Thus, the whole vector was split into two half-fragments containing the substituted bases. The two fragments were then cloned back together using In-Fusion Homology-Directed cloning (Takara Bio) to yield the final vector. Electrocompetent BL21(DE3) E. coli (Sigma Aldrich) were co-transformed by electroporation with the pCCT reporter plasmid and the pET151 expression vector (-EV or –YtgR) and plated on double selective LB agar containing 50 μg/mL carbenicillin and 15 μg/mL tetracycline. Prior to plating of transformed cells, 50 μL of 40 mg/mL X-Gal in DMSO  was applied to the plate for colorimetric determination of β-galactosidase expression. Transformants were incubated overnight at 37° C. The following evening, blue colonies from each experimental condition were selected and cultured overnight in LB liquid broth containing 0.2% (w/v) D-glucose (for catabolite repression of expression vectors), 50 μg/mL carbenicillin and 15 μg/mL tetracycline. Cultures were incubated overnight at 37° C. The following morning, overnight cultures were spun down to remove glucose-containing media and sub-cultured in LB liquid broth medium containing 50 μM FeSO4, 50 μg/mL carbenicillin and 15 μg/mL tetracycline to an OD600 of 0.45. Cultures were incubated for 1 hr at 37° C and sub-cultured a second time in the same media to an OD600 of 0.1. Cultures were returned to the incubator for another hour prior to the addition of 500 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce pET151 expression from the lac promoter. Cultures were incubated another hour prior to the addition of 0.2% L-arabinose to induce lacZ expression from the araBAD promoter. Cultures were incubated a final 2 hr prior to the collection of a 0.1 mL volume of cells for assaying β-galactosidase activity by the Miller Assay . 50 μL of 0.1% SDS and 100 μL of chloroform were then added to each sample prior to thorough vortexing. Samples were equilibrated for 5 min at 30° C and 200 μL of 4 mg/mL ortho-nitrophenyl-β-galactoside (ONPG) prepared in Phosphate Buffer  were added to the samples to initiate the reaction. Reactions were stopped by the addition of 500 μL 1 M Na2CO3. Absorbance was measured on a FLUOStar Optima plate reader  at 420 nm and Miller Units were calculated as:The YtgR-binding reporter assay was performed essentially as described, with minor modifications . Promoteer Assay . Cell pet = reaction time, v = volume of cells and finalOD = OD600 at the time of sample collection. It was empirically determined that the subtraction of absorbance at 550 nm had a negligible effect on the calculated value. A blank sample lacking cells was included in each experimental batch and used as a reference for absorbance. For each experimental condition, three independent co-transformed colonies were assayed in technical triplicate. In some instances, significantly high Miller Unit outliers were excluded by Grubb’s Test (p<0.05) under the assumption that extreme lacZ expression may reflect plasmid copy number or reporter gene expression issues.Where E. coli were cultured exactly as described for the Two-Plasmid assay with the exception that volumes were scaled up proportionately to increase sample size. For bipyridyl-treated experiments, 50 µM FeSO4 was excluded and 500 µM 2,2-bipyridyl in DMSO was added for the final three hours of culturing alongside IPTG. At the end of culturing, samples were fixed by diluting fresh 37% methanol-stabilized formaldehyde solution (Sigma Aldrich) to a final concentration of 1% and incubating the sample for 10 mins with periodic vortexing. Fixation was then quenched by the addition of 250 mM glycine to each sample and incubation proceeded for another five mins with periodic vortexing. Samples were then centrifuged for 15 mins at 3000xg, 4 ˚C to pellet fixed cells. The supernatant was discarded and samples were stored at −80˚ C. Fixed samples were thawed on ice and resuspended in 1 mL of ice-cold Pierce RIPA Lysis Buffer (ThermoFisher Scientific) supplemented with cOmplete EDTA-free Protease Inhibitor cocktail . Samples were then divided into three 330 µL aliquots  and immediately processed for sonication. Sonication was performed using the Biorupter Plus sonication device  with an attached water cooler. Samples were sonicated on high power for a total of 20 thirty-second on/off cycles at 4° C. These sonication conditions were not optimized for a particular DNA fragment size given that the target DNA promoter element was located on a plasmid and therefore isolated from possible off-target genomic binding sites. Sonicated lysates were then centrifuged to clear cell debris at 14,800 RPM for 5 mins at 4° C in a Sorvall Legend Mirco 21R centrifuge (ThermoFisher Scientific). Lysates were pre-cleared by incubating with 10 μL of Pierce Protein A/G Magnetic beads (ThermoFisher Scientific) at 4° C with rotation for 1 hr. Cleared lysates were then immediately processed for immunoprecipitation.Co-transformed BL21(DE3) For experimental IPs, Pierce Protein A/G Magnetic beads were loaded with His-Tag (D3I1O) XP Rabbit monoclonal antibody . Control IPs were performed with MNormal Rabbit IgG, . Antibody-loaded beads were prepared by incubating 25 μL of beads with 10 μL of antibody in 500 μL TBS-T (ThermoFisher Scientific) at 4° C for 2.5 hr with rotation. Antibody beads were separated on a magnetic rack and then blocked in 500 μL 5% BSA +200 mg/mL sheared salmon sperm DNA (Invitrogen) by incubating for 1 hr at 4° C with rotation. Blocked antibody beads were separated on a magnetic rack and then resuspended in TBS-T to their original volume. These conditions were scaled proportionately when doing multiple IPs. 25 μL of blocked antibody beads were then added to 330 μL of cleared sonicated lysate and incubated overnight at 4° C with rotation. The following morning, beads were separated on a magnetic rack and washed first with 300 μL ice-cold Low Salt Immune Complex Wash Buffer (EMD Millipore) incubated for five mins at RT° C with rotation. Beads were separated on a magnetic rack and washed a second time with 300 μL ice-cold High Salt Immune Complex Wash Buffer (EMD Millipore) incubated for five mins at RT° C with rotation. A final wash was carried out using 300 μL ice-cold LiCl Immune Complex Wash Buffer (EMD Millipore) incubated for five mins at RT° C with rotation. Protein-DNA complexes were then eluted off of the washed beads by incubating the beads in 420 μL Pierce IgG Elution Buffer, pH 2.0 (ThermoFisher Scientific) for five mins at RT° C with rotation. The beads were then immediately separated on a magnetic rack and the eluate was added to 80 μL 1 M Tris, pH 8.5 to neutralize pH.Cross-links were reversed by incubating eluted Protein-DNA complexes and input lysates at 95° C for 1 hr, with 750 RPM shaking. De-crosslinked samples were then centrifuged at 14,800 RPM for 5 mins at 4° C to clear debris. DNA was isolated using the Macherey-Nagel NucleoSpin Gel and PCR Clean-up Kit as described by the manufacturer (TakaraBio) with the stipulation that the input lysates were bound to the column in Buffer NTB and the immunoprecipitated DNA was bound to the column in Buffer NTI. DNA was eluted in a total of 30–60 μL Buffer NE and then processed for qPCR using the indicated primer pairs. ChIP-Input was diluted 1:50 or 1:100 in nuclease-free water prior to qPCR.t values obtained from triplicate qPCR assays were averaged and dilution correction was calculated as:The Ce.g. 50).Where dilution factor equals the fold-dilution reported above  represents the Ct value of the dilution-corrected ChIP-Input sample and Ct(ChIP) represents the dilution-corrected Ct value of the experimental or control antibody IPs.Where CThe fold enrichment relative to the control antibody IP was calculated as:These calculations assume 100% qPCR efficiency.The Fold Enrichment value was then normalized to the copy number of pET151-YtgR to account for variation in YtgR expression and thus ChIP efficacy. Copy number for pET151-YtgR was determined using primer pairs specific for the ampicillin resistance cassette and copy number was calculated as described above based on the size of the pET151-YtgR vector. Experimental values obtained from these assays were then batch corrected such that the mean of all samples in each replicate was identical. For aesthetic reasons, the values were then scaled such that the lowest value equaled 1.0.C. trachomatis L2 434/Bu genome (Accession: NC_010287) was modified to contain annotations for intergenic regions that fell between two genes in the same coding orientation, and this genome was used as the reference for read mapping. Read mapping and differential expression analysis was performed using default settings in CLC Genomics Workbench. Data aggregation in the Reads track was set to aggregate above 1 bp.RNA-Sequencing experiments were performed as described in their original publication . Raw andst and 3rd quartile range within the box, while the whiskers represent data within 1.5 interquartile ranges of the 1st or 3rd quartile. Extreme values outside this range are plotted as open circles. The 2nd quartile (median) is plotted as a black line within the box. Histogram plots were generated with a bin width of 20 and are plotted on a density scale. The overlaid density plots represent a statistical approximation of the data over a continuous scale. All statistical analyses were carried out in R Studio. All statistical computations were performed on the mean values of independent biological replicates calculated from the indicated number of respective technical replicates. For single pairwise comparisons, a two-sided unpaired Student’s t-test with Welch’s correction for unequal variance was used to determine statistical significance. For multiple pairwise comparisons, a One-Way Analysis of Variance (ANOVA) was conducted to identify significant differences within groups. If a significant difference was detected, then the indicated post-hoc pairwise test was used to identify the location of specific statistical differences. A p-value less than 0.05 was considered statistically significant. For all figures, *=p < 0.05, **=p < 0.01, and ***=p < 0.005.All graphs were generated using the ggplot2 package  in R Stu In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Chlamydia trachomatis" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Gisela Storz as the Reviewing and Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Derek Fisher (Reviewer #2).Thank you for submitting your article "A bipartite iron-dependent transcriptional regulation of the tryptophan salvage pathway in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:C. trachomatis tryptophan biosynthetic genes, which have been linked to surviving IFN-Ɣ stimulation of host cells and thus virulence. The authors established experimental conditions to decouple the effects of iron and trp starvation, both of which can be induced by IFN-Ɣ stimulation, and they showed discoordinate regulation of the trpRBA operon by iron. Iron regulation appears to be mediated through binding of the iron-dependent regulator, YtgR, to the trpR-trpB intergenic region (IGR) upstream of a newly-identified, iron-regulated promoter. The authors also explored the possibility that YtgR binding to the IGR causes transcriptional termination to reduce expression of trpBA, although the data for this model are less convincing.This study investigates iron-regulated expression of Essential revisions:1) The βgal results reported in Figure 5 and Figure 5—figure supplement 1 indicate that binding of YtgR to the IGS may require more than the putative operator sequence. Demonstration of iron-dependent, direct interactions between YtgR and the IGS/operator site would provide clarity on this point. EMSA or YtgR/operator DNA dot blot assays previously reported by the Carabeo group (PMID: 22689982) measuring iron-dependent, direct binding to the IGS region would provide strong support for the trp IGS YtgR operator site designation and iron-regulation, and could allow for rapid screening of sequence elements needed for YtgR binding. The latter approach might allow for the in silico identification of other YtgR operator sites.2) The section on iron limitation and transcription read-through was not well written. It was hard to understand and not fully explained. The three graphs should be shown at the same scale so that they can be fairly compared. What is the significance of decreased read-through at 18 hpi compared to 12 hpi, and which should BpdI treatment be compared to? This is not a trivial consideration because the difference between 12 and 18 hpi could potentially be due to increased attenuation by tryptophan and/or iron levels. What does it mean that there was less read-through with depletion or iron than tryptophan? The presence of residual read-through despite iron depletion should be acknowledged and discussed. The result with tryptophan depletion was described as being expected but the reasoning was not given. How do you know if tryptophan and iron affect read-through through the same or different mechanisms?3) It's critical for the study to establish iron-starvation conditions that don't induce persistence, but 1 of the 2 persistence markers did not respond as expected. Additional transcriptional markers of persistence  should be tested to confirm that 6 hours of BpdI treatment did not induce persistence. Ideally these persistence marker should include iron-dependent and iron-independent genes.C. trachomatis serovar D isolate  which showed decreased omcB transcript levels. This is touched on by the authors, who point out that omcB levels and iron stress responses seem to vary across chlamydial species and C. trachomatis serovars. This creates a potential concern that trpBA iron-mediated regulation might be LGV specific and may not occur in serovar D-K isolates which are more prevalent causes of infection in females . This becomes problematic in relation to the hypothesis that YtgR-mediated regulation allows for differential responses to environments in the lower versus upper genital tract if serovars D-K do not show the LGV-type response. Alignment of the IGS regions across serovars might address this point. Experimentally, the βgal assay  using non-LGV IGS regions and confirmation of iron regulated control of trpBA  in non-LGV serovars would support that the results are not LGV-specific and occur in serovars more frequently isolated from infected women. This would increase the significance of the study.4) The RT-qPCR results in Figure 1D showing an increase in transcript levels of the late gene omcB following iron depletion are at odds with prior work from the group using a 5) While it is clear that trpR transcription terminates in the IGR, the data for iron-regulated termination within the IGR are fairly weak. For one, why were the comparisons in Figure 7C-E made to the 12hr instead of the 18hr time point, the latter of which appears to indicate a decrease in the ratio of both trpR and the IGR transcripts compared to read-through transcripts. Secondly, Figure 3C shows increased trpR levels in iron depleted condition when compared to mock treatment at the same infection time point, similar to the iron-regulated genes shown in Figure 2, indicating this transcript is regulated by iron. Without more in-depth analysis of trpR promoter activity and trpR transcript stability, I'm not certain these results can be interpreted as iron-regulation of transcriptional read through.6) The impact of the study would be increased by data supporting the model that YtgR binding to its operator decreases RNA polymerase processivity. While these studies would be challenging in Chlamydia especially in a 2-3 month time frame expected for the revision, they could be done with the heterologous transcription assay. This approach would allow the effect of YtgR and other transcription factors on read-through transcription to be compared. It could also be used to show if this effect is dependent on iron or tryptophan. One concern regarding iron-regulated termination is that the data are not wholly consistent with this idea. Specifically, the data show some modest iron regulation of trpR, depending on what time points are used to evaluate the data . Moreover, there are two TTS in the IGR downstream of the YtgR operator, suggesting termination can occur independently of YtgR binding. Thus it is possible that multiple factors, including RNA stability, RNA processing, and termination, are contributing to their observations. In the absence of additional data, the authors should temper their conclusions.C. trachomatis virulence trait? This should be put into broader context of bacterial pathogenesis, where iron regulation of virulence is a recurrent theme for many pathogens, and highlighted as a new twist on this theme.7) Is this the first demonstration of iron regulating a  Essential revisions:1) The βgal results reported in Figure 5 and Figure 5—figure supplement 1 indicate that binding of YtgR to the IGS may require more than the putative operator sequence. Demonstration of iron-dependent, direct interactions between YtgR and the IGS/operator site would provide clarity on this point. EMSA or YtgR/operator DNA dot blot assays previously reported by the Carabeo group (PMID: 22689982) measuring iron-dependent, direct binding to the IGS region would provide strong support for the trp IGS YtgR operator site designation and iron-regulation, and could allow for rapid screening of sequence elements needed for YtgR binding. The latter approach might allow for the in silico identification of other YtgR operator sites.trpBA reporter plasmid in the presence of 500 µM bipyridyl. This approach has been used in previous publications to determine the iron-dependency of DtxR repression in similar reporter systems . Using this approach, we observed a moderate but significant increase in Β-galactosidase activity, which would be expected if the deprivation of iron inactivated YtgR DNA-binding and alleviated repression of lacZ.To demonstrate iron-dependency of YtgR repression, we performed an additional two-plasmid experiment with the pET151-YtgR expression vector and the pCCT-trpBA promoter element in the two-plasmid reporter system. This experiment revealed that YtgR specifically interacts with the trpBA promoter element, but not the promoter of trpR or dnaB, and that this interaction depends on the native operator sequence within the trpBA promoter. These studies were attempted in Chlamydia-infected cells with a polyclonal antibody raised against YtgC, but we were unable to efficiently immunoprecipitate cross-linked YtgR-DNA complexes with the raised antibody.We have optimized a targeted chromatin immunoprecipitation qPCR assay to demonstrate direct and operator-dependent interactions between YtgR and the E. coli, but its presence affected isolation of YtgR-DNA fragment interaction.We were unable to conduct the biolayer interferometry (BLI) assay that we used in our previous publication because those experiments were performed by a collaborator (Dr. Scott Grieshaber) using the core facilities at the University of Florida at Gainesville. We do not have access to the BLI equipment anymore because Dr. Grieshaber has left Florida. Instead we decided to take advantage of our two-plasmid assay to demonstrate transcriptional repression of the lacZ reporter, and binding of YtgR to the IGR operator sequence via chromatin immunoprecipitation. While we were successful in establishing iron-dependent transcriptional repression by YtgR, we could not achieve consistent results with the controls and “unknown” samples in our ChIP assay in the presence of bipyridyl. We were able to pinpoint the issue with the addition of bipyridyl because other experiments we conducted, including the effects of the mutated operator sequence on DNA binding of YtgR, gave the expected outcomes with the control samples. However, we were unable to resolve the issue. The addition of bipyridyl relieved transcriptional repression in the doubly transformed Despite these technical issues, we believe that our data collectively point to YtgR being an iron-dependent transcriptional repressor that targets the operator sequence within the conserved intergenic region, in conjunction with our previously published data that demonstrated YtgR binding to a different operator sequence in an iron-dependent manner.Note that we have replaced the previous pCCT-lpdA negative control with that of pCCT-dnaB for consistency, i.e. other figures used the dnaB as negative control .2) The section on iron limitation and transcription read-through was not well written. It was hard to understand and not fully explained.We have rewritten the section on transcription readthrough to enhance clarity.The three graphs should be shown at the same scale so that they can be fairly compared.We have revised the figure for clarity .What is the significance of decreased read-through at 18 hpi compared to 12 hpi, and which should BpdI treatment be compared to? This is not a trivial consideration because the difference between 12 and 18 hpi could potentially be due to increased attenuation by tryptophan and/or iron levels.trpBA promoter. We have also included a control amplicon as before at the 3’ end of the trpA sequence to indicate transcripts originating from the major trp promoter, AND those originating from the trpBA promoter, which when combined results in a ratio of >1.We have re-analyzed our RT-qPCR data on transcription readthrough. To simplify the analysis, we are now only comparing two amplicons: one constant upstream “normalization” and various downstream “readthrough” amplicons. They include amplicons located upstream of the putative YtgR-dependent termination site, one that is located downstream of this site, and one at the predicted termination immediately after the TrpA CDS. RT-qPCR was performed for each amplicon separately for each mRNA sample. To further account for possible variation, the normalization amplicon was also included. Because this amplicon was common to all transcripts driven from the major trp promoter, it was used for normalization. This analysis is comparable to the analysis we performed in Figure 4B. In this instance, however, the collection of amplicons used to determine readthrough at the YtgR operator site excluded any that would detect transcripts originating from the alternative transcriptional start site, i.e. downstream of the The previous figure was not as intuitive as we would have liked, but to answer the reviewer’s question in reference to the previous figure, we can only speculate that the decreased readthrough at 18 h relative to 12 h could have been due to differences in iron availability that Chlamydia experienced during its development. Having said this, we reanalyzed the data using a different normalization amplicon. By doing so, there was no longer a difference between 12 and 18 h. This lack of difference was consistent across all amplicons monitored. We hope that this reanalysis addressed the reviewer’s additional concerns.What does it mean that there was less read-through with depletion or iron than tryptophan? The presence of residual read-through despite iron depletion should be acknowledged and discussed.This could be due to differences in promoter strength or robustness of transcription from the major trp promoter and the alternative promoter within the IGR that would translate differences in levels of transcripts. Assuming different promoter strengths, under Trp-starved condition, transcription initiates robustly from the major trp promoter, while under iron-limiting condition, transcription starts from the relatively weaker alternative promoter. The former would be expected to yield a higher ratio relative to the latter. This would be consistent with the data shown in Figure 7B where the readthrough ratio approaches 1 for the Trp-starved samples, indicating readthrough to complete transcription.The result with tryptophan depletion was described as being expected but the reasoning was not given.The results  were expected based on previous reports by Carlson et al., Akers and Tan, Woods et al. that have already demonstrated the transcriptional induction of the trp operon under Trp depletion.How do you know if tryptophan and iron affect read-through through the same or different mechanisms?Regarding the mechanism of readthrough under iron- or Trp-depleted conditions, our argument would be that the mechanism is different. YtgR DNA-binding mediates readthrough under iron-limited conditions as a function of transcript termination, and inactivation of TrpR repression in the absence of the co-repressor Trp mediates readthrough under Trp-depleted conditions by inducing transcription initiation. However, the mechanism of Trp-dependent readthrough is only a side-note, with YtgR modulation of transcription termination being one of the major findings. Trp-depletion was simply used as a control because of its established regulation of trp operon transcription.3) It's critical for the study to establish iron-starvation conditions that don't induce persistence, but 1 of the 2 persistence markers did not respond as expected. Additional transcriptional markers of persistence  should be tested to confirm that 6 hours of BpdI treatment did not induce persistence. Ideally these persistence marker should include iron-dependent and iron-independent genes.euo and omcB). We essentially discounted the time point where one or more of these criteria indicated persistence, and focused on the 6-h treatment for the rest of the manuscript. Rather than focus on the unexpected behavior of omcB at the “12+12” timepoint, we highlight the identification of a treatment protocol (e.g. “12+6”) that maintained normal developmental expression of omcB and euo. As further support for the “12+6” treatment condition, we  published a genomewide analysis of the C. trachomatis transcriptome, and observed only a small collection of genes differentially expressed during iron starvation. These genes included trpB and trpA.We assume that the reviewer is referring to Figure 1. The purpose of this experiment was to identify a treatment duration that did not result in reduced replication (e.g. genome copy number), and aberrant expression of developmentally regulated genes (e.g. 4) The RT-qPCR results in Figure 1D showing an increase in transcript levels of the late gene omcB following iron depletion are at odds with prior work from the group using a C. trachomatis serovar D isolate  which showed decreased omcB transcript levels. This is touched on by the authors, who point out that omcB levels and iron stress responses seem to vary across chlamydial species and C. trachomatis serovars. This creates a potential concern that trpBA iron-mediated regulation might be LGV specific and may not occur in serovar D-K isolates which are more prevalent causes of infection in females . This becomes problematic in relation to the hypothesis that YtgR-mediated regulation allows for differential responses to environments in the lower versus upper genital tract if serovars D-K do not show the LGV-type response.omcB expression between this study and previous findings , the latter typically involved treatments at the start of infection. Not only has omcB expression been observed to vary under different stress conditions, but our decision to begin treatment at midcycle of chlamydial development necessarily means that previous reports that began stress treatment at the time of infection do not apply to this work and are not predictive for our studies. To discount the potential confounding effects of variations in omcB expression, we constrained our experiments to the 6 h treatment starting at 12 h post-infection.We have edited the manuscript to discuss the inconsistency of Alignment of the IGS regions across serovars might address this point. Experimentally, the βgal assay  using non-LGV IGS regions and confirmation of iron regulated control of trpBA  in non-LGV serovars would support that the results are not LGV-specific and occur in serovars more frequently isolated from infected women. This would increase the significance of the study.trpRBA IGR between serovars L2, D, A and B which demonstrates a >99% sequence identity, with 100% conservation at the putative YtgR operator site . We had also noted this in the text in the initial submission.To address the concern about LGV vs. urogenital serovars, we have provided a sequence alignment of the 5) While it is clear that trpR transcription terminates in the IGR, the data for iron-regulated termination within the IGR are fairly weak. For one, why were the comparisons in Figure 7C-E made to the 12hr instead of the 18hr time point, the latter of which appears to indicate a decrease in the ratio of both trpR and the IGR transcripts compared to read-through transcripts.trpRBA operator site”). We refer the reviewer to our third response to point 2 for details.As noted above in our third response to point 2, we have re-analyzed the transcription readthrough data, which we believe addresses several of the concerns mentioned  in our 6) The impact of the study would be increased by data supporting the model that YtgR binding to its operator decreases RNA polymerase processivity. While these studies would be challenging in Chlamydia especially in a 2-3 month time frame expected for the revision, they could be done with the heterologous transcription assay. This approach would allow the effect of YtgR and other transcription factors on read-through transcription to be compared. It could also be used to show if this effect is dependent on iron or tryptophan. One concern regarding iron-regulated termination is that the data are not wholly consistent with this idea. Specifically, the data show some modest iron regulation of trpR, depending on what time points are used to evaluate the data . Moreover, there are two TTS in the IGR downstream of the YtgR operator, suggesting termination can occur independently of YtgR binding. Thus it is possible that multiple factors, including RNA stability, RNA processing, and termination, are contributing to their observations. In the absence of additional data, the authors should temper their conclusions.trpR ORF and trpRBA IGR upstream of the lacZ reporter gene . Thus, RNAP initiated at the pBAD element would be required to readthrough the whole endogenous trpR-IGR sequence prior to reaching lacZ. We then monitored readthrough by RT-qPCR (as explained above) in the presence or absence of YtgR . Using this system, we observed a significant decrease in the amount of readthrough to the lacZ gene when YtgR was expressed, consistent with the idea that YtgR binding to the IGR inhibits RNAP processivity and induces transcript termination. Moreover, we were able to use this system to demonstrate a significant reduction in β-galactosidase activity when YtgR is ectopically expressed , confirming that lacZ is indeed not being transcribed similarly under these conditions. We have commented on the fact that we cannot discount a possible role for the other TTSs in this iron-dependent regulation , but we argue that any iron-dependent regulation of the other TTSs would produce the same result of limiting trpBA expression. Nevertheless, we have demonstrated that there is iron-dependent termination at the YtgR operator and that YtgR regulation of the IGR inhibits readthrough. The most parsimonious model is therefore that YtgR binding to its operator site in the IGR terminates transcription from the upstream trpR promoter, limiting downstream trpBA expression.To address the above concern about RNA polymerase processivity, we have adapted our two-plasmid system such that the reporter plasmid contained the entire 7) Is this the first demonstration of iron regulating a C. trachomatis virulence trait?With obligate intracellular pathogens like Chlamydia, it is difficult to make distinctions between virulence and survival. However, factors that are considered virulence factors in other pathogens have been demonstrated in Chlamydia to respond to iron starvation. For example, a report from Slepenkin et al., 2007 described the induction of transcription of various components of the type III secretion system upon treatment with the iron chelator Desferal. We found a similar transcriptional induction of components of the type III secretion system in shorter iron starvation by bipyridyl .This should be put into broader context of bacterial pathogenesis, where iron regulation of virulence is a recurrent theme for many pathogens, and highlighted as a new twist on this theme.trpBA: the possibility of deploying a unified response to counter the pleiotropic insults of IFN-γ stimulation , as well as the possibility that iron-dependent regulation of trpBA may have a specific and unique role in activating tryptophan biosynthesis in the lower genital tract, where the substrate for salvage, indole, is available, allowing C. trachomatis to prepare for oncoming IFN-γ stress . We have added an additional statement that this may play an important role in virulence in genital serovars of C. trachomatis.We have commented on two aspects of chlamydial virulence (or pathogenesis) in the female genital tract that are likely impacted by iron-dependent YtgR regulation of


Due to lower farrowing rate and reduced litter size with frozen-thawed semen, over 90% of artificial insemination (AI) is conducted using liquid stored boar semen. Although substantial progress has been made towards optimizing the cryopreservation protocols for boar sperm, the influencing factors and underlying mechanisms related to cryoinjury and freeze tolerance of boar sperm remain largely unknown. In this study, we report the differential expression of mRNAs and miRNAs between fresh and frozen-thawed boar sperm using high-throughput RNA sequencing. Our results showed that 567 mRNAs and 135 miRNAs were differentially expressed (DE) in fresh and frozen-thawed boar sperm. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the majority of DE mRNAs were enriched in environmental information processing such as cytokine-cytokine receptor interactions, PI3K-Akt signaling, cell adhesion, MAPK, and calcium signaling pathways. Moreover, the targets of DE miRNAs were enriched in significant GO terms such as cell process, protein binding, and response to stimuli. In conclusion, we speculate that DE mRNAs and miRNAs are heavily involved in boar sperm response to environment stimuli, apoptosis, and metabolic activities. The differences in expression also reflect the various structural and functional changes in sperm during cryopreservation. Artificial insemination (AI) has arguably been the most vital management tool employed for improving herd productivity in modern animal farming. Long-term semen storage has brought forth additional advantages to farmers of agriculturally important animals and to the AI industry . In pork2O2 synthesis could alter the DNA integrity in frozen bovine sperm [During cryopreservation, the structural and functional damages in sperm may result in reduced fertilization rates and could presumably lead to induction of apoptosis ,8. Mitocne sperm . The advne sperm , while ine sperm ,23. Simine sperm ,25, partne sperm . In factne sperm . Alteratne sperm .Sperm discards the majority of its cytoplasm during the final stages of differentiation. The mature sperm, therefore, lacks the significant cytoplasmic elements that harbor the antioxidants that are necessary for counterbalancing the detrimental effects of ROS and lipid peroxidation (LPO) . The maig-force (2400g) and short centrifugation time (3 min) are thought to be beneficial for reducing freezing injury and improving quality of sperm after thawing [Previous studies have demonstrated that low cooling rate provides more time for sperm dehydration, thereby reduce ice crystals-induced damage to the sperm and preserve sperm motility compared to fast cooling rate ,29. Simi thawing . Moreove thawing .ADD1, MYC, CYP19, ADAM2, PEG1/MEST, PRM1, and PRM2 are downregulated after cryopreservation [DNMT3A, DNMT3B, JHDM2A, KAT8, and PRM1, are also affected by the cryopreservation process [Mounting evidence suggests that cryodamage is not only limited to sperm membrane but also has considerable bearing on the integrity of sperm chromatin (nucleoprotein structure and DNA). Cryodamage is also involved in the degradation of certain mRNAs, therefore, it could impair the function of some relevant proteins ,4. Varioervation ,34,35. F process .MicroRNAs (miRNAs) are a class of evolutionarily conserved small non-coding RNAs  that are implicated in wide variety of complex biological mechanisms such as cell differentiation, proliferation, and metabolism. They are reported to act as guide molecules and play important roles in post-transcriptional gene regulatory mechanisms by complementary base pairing with specific target mRNAs ,38,39.miR-17-5p, miR-26a-5p, miR-486-5p, miR-122-5p, miR-184, and miR-20a-5p, are involved in regulating PTEN, PI3K/AKT, and STAT signaling and therefore can influence the motility, viability, and sperm apoptosis of frozen-thawed sperm [In a recent study, 86 miRNAs were found to be differentially expressed in bovine sperm of high and low viability after freezing-thawing. Forty of the known miRNAs were involved in the regulation of sperm cell function and sperm apoptosis. It is worth noting that certain miRNAs, such as ed sperm . Recentled sperm .In this study, in order to achieve a better understanding of the transcriptomic events and transcriptional regulatory mechanisms, we used the integrated approaches of high-throughput transcriptome and small RNA sequencing to explore the differential expression profiles of mRNAs and miRNAs in fresh and post-thaw boar sperm. We speculate that our findings may serve as a helpful tool for further elucidation of the underlying molecular mechanism relevant to boar sperm cryopreservation.A total of 18,956,444 and 16,507,275 raw reads and 12,561,033 and 11,100,601 high-quality (clean) reads (18–30 nt) were obtained from small RNA libraries of fresh and frozen-thawed sperm, respectively . HoweverAfter stringent filtering, we obtained 26,843,452 and 24,611,191 high-quality (clean) reads in fresh and frozen-thawed sperm, respectively; these were subsequently used for assembly . After aUpon further analysis, a total of 567 (482 known and 85 novel) genes were found to be differentially expressed in fresh and frozen-thawed sperm . CompareGene Ontology (GO) enrichment analysis was performed to classify DEGs in fresh and frozen-thawed sperm . A totalThe detailed results of enrichment analysis of KEGG pathway are shown in n = 16) was correlated with chemokine and cytokine signaling pathway, followed by interleukin signaling pathway and integrin signaling pathway. Other enriched pathways were apoptosis signaling pathway, cell cycle, Fas signaling pathway, and various other metabolic pathways  miRNAs and 95 new (novel) miRNAs. The miRNAs such as lization . Meanwhish sperm . Moreovein sperm ,53. Meanng ADAR1 . In thisPLCZ1 and CRISP2, have also been implicated in the activation of calcium ion pump, sperm–egg interaction, regulation of sperm intake, and fertilization process [VEGFA, vascular endothelial growth factor A, is involved in the regulation of PI3K/AKT signaling pathway, and is associated with AKT phosphorylation [VEGFA positively affects male reproductive stem cells by promoting self-renewal and maintenance of male spermatogonial stem cells. VEGF can activate the AKT signaling pathway and thereby maintain the stability of mitochondrial membrane potential and improve sperm motility. However, abnormal concentration of VEGF significantly inhibits sperm motility, acrosome reaction, and sperm–egg interaction, which ultimately lead to infertility [VEGFA in frozen-thawed sperm was significantly down-regulated, which may be related to changes in the mitochondrial membrane potential and sperm motility after cryopreservation. CDKN1B, cyclin-dependent kinase 2 and 4  inhibitor, is involved in G1 arrest, cell responses, and inhibition of cell proliferation. Akt/PKB is a central component of the PI3K signaling pathway. Akt/PKB can increase the expression of CDKN1B or inhibit its expression by phosphorylation of the forkhead transcription factor mediated by Akt/PKB, which may lead to CDKN1B proteolysis, cell cycle arrest, and apoptosis [CDKN1B expression is significantly reduced in frozen-thawed sperm, which may be related to DNA damage and sperm apoptosis caused by the rigorous freezing process.In light of the sequencing results of bovine frozen sperm transcripts, it has been inferred that mRNAs are mainly involved in biological processes such as protein translation, transport, and hydrolysis. Highly expressed mRNAs such as  process . In this process . VEGFA, rylation ,57. Receertility ,59,60. Epoptosis ,62. The SC5D, encodes a cholesterol biosynthesis enzyme, which catalyzes the production of 7-dehydrocholesterol (7DHC) in the cholesterol synthesis pathway, where cholesterol is metabolized by complete decomposition into carbon dioxide, water, and energy [SC5D was significantly increased in frozen-thawed sperm. This may be attributed to cholesterol efflux and metabolic changes occurring after cryopreservation of sperm. However, further studies are warranted to fully elucidate the putative regulatory mechanisms. Additionally, in this study, ACADS and SCD were also significantly down-regulated in frozen-thawed sperm, which may be attributed to the effects of diminished metabolic activity following cryopreservation of sperm. ACADS, acyl-CoA dehydrogenase, short chain (C2-C3) is involved in mitochondrial fatty acid beta oxidation. It has been shown that misfolded short-chain acyl-CoA dehydrogenase results in mitochondrial fission and oxidative stress [SCD, stearyl-CoA desaturase, plays a vital role in lipid biosynthesis, regulation of genes involved in adipogenesis, biosynthesis of triglycerides, and regulation of mitochondrial fatty acid oxidation, membrane phospholipids and cholesterol esters [In mammalian hibernation, reversible protein phosphorylation catalyzes the inhibition of pyruvate dehydrogenase activity, leading to inhibition of carbohydrate metabolism, which leads to lipid metabolism replacing carbohydrate metabolism as a major source of energy. d energy ,64. In te stress . SCD, stl esters ,67.PRKD2 or PKD2) in human and drosophila encodes a Ca2+-activated nonselective cation channel. Previously it has been reported that sperm motility is regulated by intra-flagellar calcium concentrations; in particular, the PKD2 calcium channel has been shown to affect sperm storage. In drosophila model, PKD2 cation channel on sperm flagellum has been implicated in modulating the directional sperm movement inside the female reproductive tract [PKD2 has no role in sperm hyperactivation [PKD2 calcium channel is also reportedly involved in adopting the correct flagellar waveform and wave propagation direction of drosophila sperm [PRKD2 was up-regulated in frozen-thawed boar sperm. However, it should be noted that we were unable to find any previous study reporting potential functional association between boar sperm and PKD2 gene. Therefore, we envisage that further studies would be of great value in elucidating the putative mechanism by PKD2 might affect post-thawed boar sperm motility, hyper-activation and fertility.Protein kinase D2  and glutamate-cysteine ligase catalytic subunit (GCLC) [COX7C) was abundantly expressed in cryopreserved sperm in low-fertility bulls [Nevertheless, at present, one of the most intriguing questions that require thorough elucidation is up-regulation of mRNA expression such as ne sperm . The up-t (GCLC) . Similarty bulls . These aty bulls . Moreovety bulls . Neverth2+ and Na+ concentrations, which induces membrane depolarization [2+ channel (CatSper), can activate signal transduction factors that are generally needed for initiating the cAMP-PKA signaling pathway and the subsequent steps in sperm capacitation [Interestingly, in a very recent report from our lab, we reported 226 DE mRNAs in fresh and frozen-thawed giant panda sperm. From these, 126 DE mRNAs were annotated to the olfactory transduction pathway . Additiorization , mediaterization . Moreovecitation . It has citation . Comparecitation . Therefon = 11) [8 mL−1. Finally, 11 ejaculates were equally divided into two aliquots. One aliquot was used directly for RNA extraction , and another aliquot (frozen-thawed sperm) was cryopreserved till further use in subsequent experiments.Procedures for ejaculates collection and sampling have been reported in our recent published paper on the same boars (n = 11)  using thn = 11) . Collectv:v) with Beltsville thawing solution , and cooled slowly to 17 °C in a constant temperature chamber. After centrifugation , sperm precipitates were diluted with 2 mL lactose-egg yolk extender  and cooled slowly to 4 °C. Then, the lactose-egg yolk extender supplemented with 6% glycerol was added to adjust the final concentration of glycerol to 3%. The mixtures were packaged into 0.25 mL polyvinyl chloride straws  and equilibrated in liquid nitrogen vapor for 10 min, then plunged into liquid nitrogen till further use.Immediately after collection, cryopreservation of boar sperm was performed according to our laboratory’s protocol . BrieflyPrior to total RNA extraction, ejaculates were washed three times to remove seminal plasma. Then, precipitated sperm were treated with 0.5% of Triton X-100 to avoid somatic cells contamination according to procedures in our previous study . The totp values were adjusted using the Benjamini and Hochberg’s approach for controlling false discovery rate (FDR). The miRNAs with an adjusted p < 0.05, |log2 (Fold Change)| ≥1, q value ≤ 0.01 found by DESeq were referred to as differentially expressed. Then, using BLAST software, we compared the predicted target gene sequences to the NR, Swiss-Prot, GO, COG, KEGG, KOG, Pfam database, and subsequently, the target gene annotation information was obtained. Finally, GO and KEGG analyses of predicted target genes were conducted.Raw data (raw reads) of fasta format were first processed through in-house Perl scripts. During this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads. The raw reads were trimmed and cleaned by removing sequences shorter than 18 nt or longer than 30 nt. Simultaneously, Q20, Q30, GC-content, and sequence duplication level of clean data was calculated. All the downstream analyses were based on clean data with high quality. Differential miRNA expression analyses between fresh and cryopreserved (frozen-thawed) sperm samples were performed using the DESeq R package . The resulting Sus scrofa 10.2, ftp://ftp.ensembl.org/pub/release-75/fasta/sus_scrofa/) using HISAT2  [q-value <0.01 and |log2 (fold change)| >1 were set as the threshold for significant differential expression. GO enrichment analysis of DE mRNAs was performed using the GOseqR package. Enrichment of DE mRNAs in KEGG pathways were analyzed by the KOBAS software .After total RNA extraction, the mRNA was isolated by NEBNext Poly (A) mRNA Magnetic Isolation Module . The cDNA library was constructed using NEBNext Ultra RNA Library Prep Kit for Illumina  and NEBNext Multiplex Oligos for Illumina  following the manufacturer’s instructions. The constructed cDNA libraries of the two groups of boar sperm were sequenced using the Illumina HiSeq2500 platform. The clean reads filtered from raw reads were mapped to pig genome  . Gene exn = 5) was extracted using TRIzol RNA isolation reagent  according to manufacturer’s protocol. Relative differences for miRNA or mRNA in fresh and frozen-thawed sperm were determined using the 2CT−△△ method. In order to verify the accuracy of high throughput sequencing data, we randomly selected and validated the differentially expressed miRNAs (n = 6) and mRNAs (n = 6) using qRT-PCR according to our laboratory’s protocol [Total RNA from sperm samples (protocol . Primersprotocol ) are shot-test. All data are shown as the means ± SEM. p values < 0.05 are regarded as statistically significant.The statistical differences were determined using the SPSS  by independent-sample Our study is the first to report the differential expression of mRNAs and miRNAs between fresh and post-thawed boar sperm obtained by high-throughput transcriptome and small RNA sequencing. Based on previous studies and the novel findings of this study, we speculate that the differentially expressed miRNAs in fresh and post-thaw boar sperm play considerable roles in regulation of important processes such as sperm cell processes, protein binding and response to stimuli. Similarly, GO and KEGG analyses revealed that these differentially expressed target mRNAs may be involved in important sperm specific biological processes such as sperm freezing, membrane integrity and function, carbohydrate transport, and metabolism. Our findings represent an important contribution to the thorough understanding of the mRNA and miRNA profiles in boar sperm and may serve as a helpful tool for further elucidation of the underlying molecular mechanism relevant to the mammalian sperm cryopreservation, particularly in porcine species.