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• W2983571208 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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 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. https://doi.org/10.7554/eLife.42295.001 eLife digest All forms of life must take up nutrients from their environment to survive. 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. 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 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? 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 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. 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. https://doi.org/10.7554/eLife.42295.002 Introduction 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 (Brown et al., 2008; Eisenreich et al., 2010; Ray et al., 2009; Skaar, 2010). This common feature among pathogens renders them susceptible to nutrient limitation strategies associated with the host immune response (Hood and Skaar, 2012). Counteractively, bacterial pathogens have evolved sophisticated molecular mechanisms to respond to nutrient deprivation, involving increasingly complex and sophisticated nutrient-sensing regulatory networks. These stress response mechanisms are essential for pathogens to avoid clearance by the immune system. By delineating their function at the molecular level, we can better target aspects of the host-pathogen interface suitable for therapeutic manipulation. However, stress responses in the obligate intracellular bacterium Chlamydia trachomatis are relatively poorly characterized, leaving unanswered many fundamental questions about the biology of this pathogen. C. trachomatis is the leading cause of bacterial sexually transmitted infections (STIs) and infection-derived preventable blindness worldwide (CDC, 2017; Newman et al., 2015; Taylor et al., 2014). Genital infections of chlamydia disproportionately affect women and are associated with serious sequelae in the female reproductive tract such as tubal factor infertility (Hafner, 2015). Chlamydiae are Gram-negative bacterial parasites that develop within a pathogen-specified membrane-bound organelle termed the inclusion (Moore and Ouellette, 2014). Chlamydial development is uniquely characterized by a biphasic interconversion of an infectious elementary body (EB) with a non-infectious, but replicative reticulate body (RB) (Abdelrahman and Belland, 2005). An obligate intracellular lifestyle has led to reductive genome evolution across chlamydial species; Chlamydiae have retained genes uniquely required for their survival, but have become nutritionally dependent on their hosts by discarding many metabolism-related genes (Clarke, 2011). Of note, C. trachomatis does not possess genes necessary for eliciting a stringent response to nutrient starvation (e.g. relA, spoT), suggesting that this pathogen may utilize novel mechanisms to respond to nutrient stress (Stephens et al., 1998). It is well established that in response to various stressors, Chlamydiae deviate from their normal developmental program to initiate an aberrant developmental state, termed ‘persistence’ (Wyrick, 2010). This persistent state is distinguished by the presence of viable, but non-cultivable, abnormally enlarged chlamydial organisms that display dysregulated gene expression. Importantly, Chlamydia can be reactivated from persistence by abatement of the stress condition. As such, chlamydial persistence at least superficially resembles a global stress response mechanism. Yet the mechanistic underpinnings of this phenotype are poorly understood, with most published studies focusing on the molecular and metabolic character of the aberrant, persistent form. It is therefore unclear to what extent primary stress responses contribute to the global persistent phenotype in Chlamydia. 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 C. trachomatis is auxotrophic (Byrne et al., 1986; Fehlner-Gardiner et al., 2002; Taylor and Feng, 1991). Following IFN-γ stimulation, infected host cells up-regulate expression of indoleamine-2,3-dioxygenase (IDO1), which catabolizes Trp to N-formylkynurenine via cleavage of the indole ring (Macchiarulo et al., 2009). C. trachomatis cannot recycle kynurenines, unlike some other chlamydial species (Wood et al., 2004), and thus IFN-γ stimulation effectively results in Trp starvation to C. trachomatis. The primary regulatory response to Trp starvation in C. trachomatis is mediated by a TrpR ortholog, whose Trp-dependent binding to cognate promoter elements represses transcription (Akers and Tan, 2006; Carlson et al., 2006). This mechanism of regulatory control is presumably limited in C. trachomatis, as homologs of genes regulated by TrpR in other bacteria (e.g. trpF, aroH, aroL) have not been shown to respond to Trp limitation (Wood et al., 2003). In many Gram-negative bacteria, such as 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 (TrpA and TrpB, respectively), 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 (Fehlner-Gardiner et al., 2002). Despite significant research on the chlamydial trpRBA operon, the functional significance of the trpRBA IGR is poorly characterized. While a putative TrpR operator sequence was identified in the IGR overlapping an alternative transcriptional origin for trpBA (Carlson et al., 2006), TrpR binding was not observed (Akers and Tan, 2006). Based on in silico predictions, an attenuator sequence has been annotated within the trpRBA IGR (Merino and Yanofsky, 2005), but this has not been thoroughly validated experimentally. Regardless, the IGR is >99% conserved at the nucleotide sequence level across ocular, genital and lymphogranuloma venereum (LGV) serovars of C. trachomatis, indicating functional importance (Carlson et al., 2005; Seth-Smith et al., 2009; Stephens et al., 1998; Thomson et al., 2008). Therefore, outside of TrpR-mediated repression, the complete detail of trpRBA regulation remains poorly elucidated and previous reports have indicated the possibility of more complex mechanisms of regulation (Brinkworth et al., 2018). The requirement of TrpBA for C. trachomatis to survive IFN-γ-mediated Trp starvation is well documented. However, IFN-γ is also known to limit iron and other essential biometals to intracellular pathogens as a component of host nutritional immunity (Cassat and Skaar, 2013; Hood and Skaar, 2012). Whether C. trachomatis has adapted to respond to these various IFN-γ-mediated insults remains unclear. Chlamydia have a strict iron dependence for normal development, evidenced by the onset of persistence following prolonged iron limitation (Raulston, 1997). Importantly, Chlamydia presumably acquire iron via vesicular interactions between the chlamydial inclusion and slow-recycling transferrin (Tf)-containing endosomes (Ouellette and Carabeo, 2010). IFN-γ is known to down-regulate transferrin receptor (TfR) expression in both monocytes and epithelial cells with replicative consequences for resident intracellular bacteria (Byrd and Horwitz, 1993; Byrd and Horwitz, 1989; Igietseme et al., 1998; Nairz et al., 2008). However, iron homeostasis in Chlamydia is poorly understood due to the lack of functionally characterized homologs to iron acquisition machinery that are highly conserved in other bacteria (Pokorzynski et al., 2017). Only the ytgABCD operon, encoding a metal permease, has been clearly linked to iron acquisition (Miller et al., 2009). Intriguingly, the YtgC (CTL0325) open reading frame (ORF) encodes a N-terminal permease domain fused to a C-terminal DtxR-like repressor domain, annotated YtgR (Akers et al., 2011; Thompson et al., 2012). YtgR is cleaved from the permease domain during infection and functions as an iron-dependent transcriptional repressor to autoregulate the expression of its own operon (Thompson et al., 2012). YtgR represents the only identified iron-dependent transcriptional regulator in Chlamydia. Whether YtgR maintains a more diverse transcriptional regulon beyond the ytgABCD operon has not yet been addressed and remains an intriguing question in the context of immune-mediated iron limitation to C. trachomatis. Consistent with the highly reduced capacity of the chlamydial genome, it is likely that C. trachomatis has a limited ability to tailor a specific response to each individual stress. In the absence of identifiable homologs for most global stress response regulators in C. trachomatis, we hypothesized that primary stress responses to pleiotropic insults may involve mechanisms of co-regulation by stress-responsive transcription factors. Here, we report on the unique iron-dependent regulation of the trpRBA operon in Chlamydia trachomatis. We propose a model of iron-dependent transcriptional regulation of trpRBA mediated by the repressor YtgR binding specifically to the IGR, which would enable C. trachomatis to respond similarly to the antimicrobial deprivation of Trp or iron mediated by IFN-γ. Such a mechanism of iron-dependent regulation of Trp biosynthesis has not been previously described in any other prokaryote and adds to the catalog of regulatory models for Trp biosynthetic operons in bacteria. Further, it reveals a highly dynamic mode of regulatory integration within the trpRBA operon, employing bipartite control at the transcription initiation and termination steps. Results Brief iron limitation via 2,2-bipyridyl treatment yields iron-starved, but non-persistent Chlamydia trachomatis To identify possible instances of regulatory integration between iron and Trp starvation in C. trachomatis, we optimized a stress response condition that preceded the development of a characteristically persistent phenotype. We reasoned that in order to effectively identify regulatory integration, we would need to investigate the bacterium under stressed, but not aberrant, growth conditions such that we could distinguish primary stress responses from abnormal growth. To specifically investigate the possible contribution of iron limitation to a broader immunological (e.g. IFN-γ-mediated) 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 (Thompson and Carabeo, 2011). We chose to starve 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 (Figure 1A). Following 6 hr of Bpdl treatment, chlamydial inclusions were largely indistinguishable from mock-treated inclusions, containing a homogeneous population of larger organisms, consistent with RBs in mid-cycle growth. However, by 12 hr of Bpdl treatment, the inclusions began to display signs of aberrant growth: they were perceptibly smaller, more comparable in size to 18 hpi, and contained noticeably fewer organisms, perhaps indicating a defect in bacterial replication or RB-to-EB differentiation. These observations were consistent with our subsequent analysis of genome replication by quantitative PCR (qPCR; Figure 1B). At 6 hr of Bpdl treatment, there was no statistically distinguishable difference in genome copy number when compared to the equivalent mock-treated time-point. However, by 12 hr of treatment, genome copy number was significantly reduced 4.47-fold in the Bpdl-treated group relative to mock-treatment (p=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 (Figure 1C), and the adhesin omcB, which is expressed late in the developmental cycle (Figure 1D). Characteristic persistence would display elevated euo expression late into infection and suppressed omcB expression throughout development. We observed that at 6 hr of Bpdl treatment, there was no statistically distinguishable difference in either euo or omcB expression when compared to the mock-treatment. Still at 12 hr of Bpdl treatment, euo expression was unchanged. However, omcB expression was significantly up-regulated following 12 hr of Bpdl-treatment (p=0.0024). This was unexpected, but we note that omcB expression has been shown to vary between chlamydial serovars and species when starved for iron (Pokorzynski et al., 2017). The decision to begin our brief iron starvation at 12 hpi may produce notable transcriptional differences from previous studies in which iron starvation was induced at the beginning of the chlamydial developmental cycle, and thereby prevented the establishment of a normal transcriptional program by Chlamydia. Collectively, these data indicated that 6 hr of Bpdl treatment was a more suitable time-point at which to monitor iron-limited stress responses. Figure 1 with 1 supplement see all Download asset Open asset Brief iron limitation via 2,2-bipyridyl treatment precedes the onset of characteristic chlamydial persistence. (A) C. trachomatis L2-infected HeLa cells were fixed and stained with convalescent human sera to image inclusion morphology by confocal microscopy following Bpdl treatment at the indicated times post-infection. Arrowheads indicate inclusions with visibly fewer organisms in the 12 hr Bpdl-treated condition. Figure shows representative experiment of three biological replicates. Scale bar = 25 μm, Inset scale bar = 5 μm. (B) Genomic DNA (gDNA) was harvested from infected HeLa cells at the indicated times post-infection under iron-replete (blue) and -depleted (red) conditions. Chlamydial genome copy number was quantified by qPCR. Chlamydial genome replication is stalled following 12 hr of Bpdl treatment, but not 6. N = 3. (C) Total RNA was harvested from infected HeLa cells at the indicated times post-infection under iron-replete (teal) and -depleted (orange) conditions. The transcript abundance of hallmark persistence genes euo and (D) omcB were quantified by RT-qPCR and normalized against genome copy number. Only at 12 hr of Bpdl treatment is omcB expression significantly affected. N = 3. Statistical significance was determined by One-Way ANOVA followed by post-hoc pairwise t-tests with Bonferroni’s correction for multiple comparisons. *=p < 0.05, **=p < 0.01, ***=p < 0.005. https://doi.org/10.7554/eLife.42295.003 Figure 1—source data 1 Source data for Figure 1A–D, Figure 1—figure supplement 1. https://doi.org/10.7554/eLife.42295.005 Download elife-42295-fig1-data1-v2.xlsx 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 (Figure 1—figure supplement 1A), but genome copy numbers were significantly reduced 2.7-fold at this time-point (p=0.00612; Figure 1—figure supplement 1B). The transcript expression of euo (Figure 1—figure supplement 1C) and omcB (Figure 1—figure supplement 1D) did not significantly change at either treatment duration, but Trp-depletion did result in a 1.88-fold reduction in omcB expression (p=0.0544), 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 next sought to determine whether our brief 6 hr Bpdl treatment was sufficient to elicit a transcriptional iron starvation phenotype. We chose to analyze the expression of three previously identified iron-regulated transcripts, ytgA (Figure 2A), ahpC (Figure 2B) and devB (Figure 2C), by RT-qPCR under Bpdl- and mock-treated conditions (Dill et al., 2009; Thompson and Carabeo, 2011). In addition, we analyzed the expression of one non-iron-regulated transcript, dnaB (Figure 2D), as a negative control (Brinkworth et al., 2018). Following 6 hr of Bpdl treatment, we observed that the transcript expression of the periplasmic iron-binding protein ytgA was significantly elevated 1.75-fold relative to the equivalent mock-treated time-point (p=0.0052). 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 (Miller et al., 2009; Raulston et al., 2007; Thompson and Carabeo, 2011). Similarly, we observed that the transcript expression of the thioredoxin ahpC was significantly elevated 2.15-fold relative to the equivalent mock-treated time-point (p=0.038) 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 (Brinkworth et al., 2018). Overall, these data confirmed that our 6 hr Bpdl treatment condition was suitable to produce a mild iron starvation phenotype at the transcriptional level, facilitating our investigation of iron-dependent regulatory integration. Figure 2 Download asset Open asset Brief iron limitation condition produces mild iron-starved transcriptional phenotype. (A) Total RNA and gDNA was harvested from infected HeLa cells at the indicated times post-infection under iron-replete (teal) and -depleted (orange) conditions. The transcript abundance of iron-regulated ytgA, (B) ahpC, (C) devB and (D) non-iron regulated dnaB were quantified by RT-qPCR and normalized against genome copy number. The transcript expression of ytgA and ahpC were significantly elevated following 6 hr Bpdl treatment, indicative of iron starvation to C. trachomatis. N = 3. Statistical significance was determined by One-Way ANOVA followed by post-hoc pairwise t-tests with Bonferroni’s correction for multiple comparisons. *=p < 0.05, **=p < 0.01, ***=p < 0.005. https://doi.org/10.7554/eLife.42295.006 Figure 2—source data 1 Source data for Figure 2A–D. https://doi.org/10.7554/eLife.42295.007 Download elife-42295-fig2-data1-v2.xlsx Transcript expression of the trpRBA operon is differentially regulated by iron in Chlamydia trachomatis Upon identifying an iron limitation condition that produced a relevant transcriptional phenotype while avoiding the onset of persistent development, we aimed to investigate whether the immediate response to iron starvation in C. trachomatis would result in the consistent induction of pathways unrelated to iron utilization/acquisition, but nevertheless important for surviving immunological stress. The truncated Trp biosynthetic operon, trpRBA (Figure 3A), has been repeatedly linked to the ability of genital and LGV serovars (D-K and L1-3, respectively) of C. trachomatis to counter IFN-γ-mediated stress. This is due to the capacity of the chlamydial Trp synthase in these serovars to catalyze the β synthase reaction, that is the condensation of indole to the amino acid serine to form Trp (Fehlner-Gardiner et al., 2002). In the presence of exogenous indole, C. trachomatis is therefore able to biosynthesize Trp such that it can prevent the development of IFN-γ-mediated persistence. Correspondingly, the expression of trpRBA is highly induced following IFN-γ stimulation of infected cells (Belland et al., 2003; Østergaard et al., 2016). These data have historically implicated Trp starvation as the primary mechanism by which persistence develops in C. trachomatis following exposure to IFN-γ. However, these studies have routinely depended on prolonged treatment conditions that monitor the terminal effect of persistent development, as opposed to the immediate molecular events which may have important roles in the developmental fate of Chlamydia. As such, these studies may have missed the contribution of other IFN-γ-stimulated insults such as iron limitation. Figure 3 Download asset Open asset Expression of the trpRBA operon in C. trachomatis is differentially regulated by brief iron limitation. (A) Cartoon depiction of the trpRBA operon (drawn to scale) with the primary transcriptional start site upstream of trpR annotated. (B) Total RNA and gDNA were harvested from infected HeLa cells at the indicated times post-infection under Trp-replete (black) and -depleted (red) conditions. The transcript expression of trpRBA operon was quantified by RT-qPCR and normalized against genome copy number. All three ORFs are significantly induced relative to 12 hpi following Trp starvation. N = 3. (C) Total RNA and gDNA were harvested from infected HeLa cells at the indicated times post-infection under iron-replete (blue) and -depleted (red) conditions. The transcript expression of trpRBA operon was quantified by RT-qPCR and normalized against genome copy number. Only trpB and trpA expression was significantly induced relative to 12 hpi. N = 3. Statistical significance was determined by One-Way ANOVA followed by post-hoc pairwise t-tests with Bonferroni’s correction for multiple comparisons. *=p < 0.05, **=p < 0.01, ***=p < 0.005. https://doi.org/10.7554/eLife.42295.008 Figure 3—source data 1 Source data for Figure 3B–C. https://doi.org/10.7554/eLife.42295.009 Download elife-42295-fig3-data1-v2.xlsx To decouple Trp limitation from iron limitation and assess their relative contribution to regulating a critical pathway for responding to IFN-γ-mediated stress, we monitored the transcript expression of the trpRBA operon under brief Trp or iron starvation by RT-qPCR. Here again, we analyzed changes in transcript levels at the 18 hpi time-point and between the 12 hpi and 12 hpi + 6 hr time-points. This allowed us to determine if differences in expression could be accounted for by reduced, maintained or induced expression relative to 12 hpi. When starved for Trp for 6 hr, we observed that the expression of trpR, trpB and trpA were all significantly induced greater than 5.18-fold relative to 12 hpi (p=0.0040, 0.020 and 0.0036, respectively; Figure 3B). All three ORFs were also significantly elevated relative to the equivalent mock-treated time-point (p=0.0039, 0.019 and 0.0035, respectively). This result demonstrated that a relatively brief duration of Trp starvation was sufficient to induce trpRBA transcription and highlights the highly attuned sensitivity of C. trachomatis to even moderate changes in Trp levels. We then performed the same RT-qPCR analysis on the expression of the trpRBA operon in response to 6 hr of iron limitation via Bpdl treatment (Figure 3C). While we observed that the transcript expression of all three ORFs was significantly elevated at least 2.1-fold relative to the equivalent mock-treated time-point (p=0.015, 0.00098 and 0.0062, respectively), we made the intriguing observation that only the expression of trpB and trpA was significantly induced relative to 12 hpi (p=0.00383 and 0.0195, respectively). 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 (Brinkworth et al., 2018). Our results suggested that while the trpRBA operon is responsive to iron limitation, trpBA may have a more complex mode of regulation given the additional induction observed relative to trpR, which only maintained expression between 12 hpi and 12 hpi +6 hr Bpdl time-points. Taken together, these findings demonstrate that an important stress response pathway, the trpRBA operon, is regulated by the availability of both Trp and iron, consistent with the notion that the pathway may be cooperatively regulated to respond to various stress conditions. Notably, iron-dependent regulation of Trp biosynthesis has not been previously documented in other prokaryotes. Specific iron-regulated expression of trpBA originates from a novel alternative transcriptional start site within the trpRBA intergenic region We hypothesized that the specific iron-related induction of trpBA expression relative to trpR expression may be attributable to an iron-regulated alternative transcriptional start site (alt. TSS) 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 (Carlson et al., 2006). However, a parallel" @default.
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• W2983571208 title "Author response: A bipartite iron-dependent transcriptional regulation of the tryptophan salvage pathway in Chlamydia trachomatis" @default.
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