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Department of Microbiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614-1700, USA
Correspondence
Brian D. Dill
dillbd{at}ornl.gov
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ABSTRACT |
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250 radiolabelled protein species visualized, densitometric analysis revealed 25 proteins that increased in expression under iron restriction compared to iron-sufficient control samples; ten protein species identified by mass spectrometry are involved in the oxidative damage response (alkyl hydroperoxide reductase, 6-phosphogluconolactonase and acyl carrier protein synthase), transcription (RNA polymerase subunit alpha and transcription anti-termination factors NusA and NusG), protein modification (peptide deformylase and trigger factor), and virulence (Chlamydia protein associating with death domains, CADD). Transcript-level expression patterns of ahpC, devB, cadd, fabF and ct538 were measured by quantitative RT-PCR throughout the developmental cycle, and each gene examined demonstrated a significant but small mid-cycle increase in transcript level in iron-restricted cultures compared to iron-replete controls. Taken together, these data suggest that the primary response of chlamydiae to reduced iron availability is to increase expression of proteins involved in protection against oxidative damage via iron-catalysed generation of reactive oxygen species and adaptation to stress by increasing expression of transcriptional machinery and other stress-responsive proteins.
Present Address: Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory, PO Box 2008, MS6131, Oak Ridge, TN 37831-6131, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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); in 2006, more than one million new cases were reported to the Centers for Disease Control and Prevention in the USA alone (CDC, 2007). While genital infections caused by the non-invasive serovars D–K are commonly asymptomatic and limited to the lower genital tract, an ascending infection marked by inflammation-induced damage can develop and lead to severe sequelae including endometritis and pelvic inflammatory disease.
Chlamydiae exhibit a biphasic developmental cycle marked by conversion between the extracellular, infectious form termed the elementary body (EB) and the intracellular, metabolically active reticulate body (RB). The developmental cycle can be interrupted by induction of persistence, in which division of the RB ceases and conversion to the EB is prevented. Identified inducers of persistence include interferon-
, penicillin exposure and nutrient deficiency. Although chromosomal replication continues, cellular division ceases, RBs become enlarged, and transcription and protein expression profiles are altered; in addition, persistent chlamydiae are refractory to killing by some antibiotics and expression of antigens continues during the prolonged development cycle (Hogan et al., 2004). Persistence has become an issue of interest in chlamydial pathogenesis due to the potential role in establishing chronic infection, which can result in the development of more severe disease outcomes.
Iron is required as a cofactor in numerous ubiquitous cellular metabolic processes in both bacterial and eukaryotic cells, including cytochromes, ribonucleotide reductase, RNA polymerase III, amino acid hydroxylases, superoxide dismutase, catalase and peroxidase (Andrews, 2000; Wooldridge & Williams, 1993). Free-iron concentrations are kept low in biological systems because unbound iron catalyses free oxygen radicals through the Fenton reaction, which results in damage to lipids, proteins and nucleic acids (Masse & Arguin, 2005; Van Ho et al., 2002; Wooldridge & Williams, 1993). In mammals, iron-binding proteins such as transferrin, lactoferrin and ferritin chelate free iron, and transport of iron into cells is tightly regulated in response to intracellular iron level (Levenson & Tassabehji, 2004; Oates & Ahmed, 2007). In addition, control of intracellular iron level plays a role in innate immunity through general limitation of iron to prevent infection and increased limitation in response to infection (Kadner, 2005; Markel et al., 2007; Sritharan et al., 2006). Indeed, all examined microbial organisms have an absolute requirement for iron, except lactobacilli (Archibald, 1983; Weinberg, 1997) and Borrelia burgdorferi (Posey & Gherardini, 2000), which use alternative transition metal cofactors, including cobalt and manganese. Furthermore, reduced iron availability is known to induce virulence factors in pathogens, such as the Shiga toxin in Shigella dysenteriae, the Shiga-like toxin in Escherichia coli, exotoxin A in Pseudomonas aeruginosa, and diphtheria toxin in Corynebacterium diphtheriae (Hantke, 2001; Litwin & Calderwood, 1993).
Iron has been shown to be necessary for normal chlamydial development, and iron restriction leads to chlamydial persistence. When intracellular iron is chelated by deferoxamine mesylate (Desferal) in vitro, the developmental cycle of C. trachomatis arrests at the RB stage and cell division ceases; also, enlarged aberrant chlamydial forms are visible by transmission electron microscopy (Raulston, 1997). It is clear that iron restriction induces persistence rather than killing the chlamydiae, as these effects on the chlamydiae are reversible upon addition of an iron source. Similar observations in response to iron restriction have been made in Chlamydophila pneumoniae (Al-Younes et al., 2001; Freidank et al., 2001) and Chlamydophila psittaci (Goellner et al., 2006).
The first investigation to describe the chlamydial requirement for iron identified several iron-responsive protein species by separation of radiolabelled protein from purified EB (Raulston, 1997). Notably, two proteins demonstrated in that study to be iron responsive have attracted interest due to their immunogenicity in Chlamydia-infected patients: cHsp60-2 (LaRue et al., 2007) and YtgA (Raulston et al., 2007). During menstruation, iron levels in endometrial cells fluctuate, and thus bacteria infecting these cells encounter an additional level of iron limitation (Anderson et al., 2001; Andrews, 2000; Cohen et al., 1987; Kelver et al., 1996); therefore, the response of chlamydiae to reduced iron availability has dramatic health implications, especially considering that the highest incidence of chlamydial infections occurs in females between 15 and 24 years of age (CDC, 2007).
While the chlamydial requirement for iron is well established, the identification of processes and pathways upregulated under iron restriction have been hindered by the lack of a direct system for genetic manipulation in Chlamydia and the lack of identifiable homologues to proteins involved in conserved iron responses in other bacteria, such as pathways for siderophore production. The goal of the present work was to begin to identify proteins increasingly expressed in metabolically active chlamydiae growing under iron restriction, followed by analysis of the corresponding transcripts for a subset of these proteins, in order to better elucidate the chlamydial response to iron restriction.
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METHODS |
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For iron restriction, HEC-1B cells were incubated 24 h prior to infection in medium containing 5 % FBS plus or minus 50 µM of the iron chelator Desferal (deferoxamine mesylate, Sigma-Aldrich). Cultures were incubated with a C. trachomatis serovar E inoculum titrated to infect 80 % of host cells. After a 1 h adsorption step at 35 °C, inoculated HEC-1B cells were returned to medium plus or minus Desferal containing 0.5 µg cycloheximide ml–1, conditions that have previously been shown to induce persistence in C. trachomatis (Raulston, 1997). In some protein analysis experiments, Percoll-purified EBs, prepared using a standard protocol, were used. HEC-1B and C. trachomatis stocks were routinely tested for mycoplasma contamination by the VenorGem PCR test (Sigma-Aldrich).
Host-cell iron-level determination via ferritin assay.
The Spectro Ferritin enzyme immunoassay (Ramco Laboratories) was used to measure ferritin levels in infected HEC-1B protein samples collected at 24 h post-infection (p.i.), according to the manufacturer's instructions. All samples were assayed in triplicate, and ferritin levels were normalized for variations in protein concentration (BCA assay, Pierce). A two-tailed Student's t-test was used to determine significance of a change in ferritin concentration.
Protein radiolabelling.
Protein radiolabelling was conducted in Chlamydia-infected HEC-1B cells between 22 and 24 h p.i. Cultures were incubated in D-MEM lacking methionine and cysteine and supplemented with 5 % FBS, 2 mM GlutaMax, 40 µg cycloheximide ml–1, plus or minus 50 µM Desferal, and 100 µCi ml–1 (3.7 MBq ml–1) 35S-labelled methionine and cysteine (Redivue PRO-MIX, GE Healthcare) for 2 h at 35 °C. Following labelling, monolayers were washed with PBS and harvested in deionized water containing 1 % CHAPS and a protease inhibitor cocktail (Pierce) and stored at –20 °C.
Sample preparation and protein separation.
Radiolabelled protein samples collected from iron-restricted or normally grown C. trachomatis-infected HEC-1B cells were separated via 2D-PAGE. Briefly, following desalting using Zeba Desalt spin columns (Pierce), samples were assayed for protein concentration by the BCA assay (Pierce) and the degree of radioisotope incorporation for each sample was determined by scintillation counting of TCA-precipitated protein from each sample. Protein samples were solubilized in Bio-Rad rehydration/sample buffer (8 M urea, 50 mM DTT, 2 % CHAPS, 0.2 % Bio-Lyte 3/10 carrier ampholytes, 0.001 % bromophenol blue). Protein loads for quantitative gels were equalized to 106 c.p.m. and 300 µg total protein; preparative gels for mass spectrometry identification were loaded with 2 mg total protein from unlabelled Percoll-purified EB and spiked with 250 000 c.p.m. from a labelled protein sample to facilitate matching of EB protein spots with radiolabelled proteins of interest.
First-dimensional isoelectric focusing (IEF) was conducted on a PROTEAN IEF cell with 17 cm isolated pH gradient (IPG) strips with a pH range of 4–7 (Bio-Rad). The pH range of 4–7 was selected because this range encompassed the majority of labelled proteins and allowed for better resolution compared to a wider-range separation. The focusing conditions were as follows: 50 µA limit per IPG strip; 250 V maximum for 15 min, 10 000 V maximum for 2 h, and 10 000 V maximum for 60 000 V h.
Following IEF, strips were equilibrated for 20 min in equilibration buffer 1 (6 M urea, 2 % SDS, 375 mM Tris/HCl, 2 % DTT, 30 % glycerol), followed by 20 min in equilibration buffer 2 (same formulation except 2.5 % iodoacetamide substituted for DTT). Separation in the second dimension was done using PROTEAN II 10 % Tris/HCl Ready Gels (Bio-Rad). Gels were run at 24 mA per gel for approximately 5 h.
Gels for quantitative analysis were fixed in 25 % methanol and 10 % acetic acid, incubated with Amplify fluorographic reagent (GE Healthcare) for 30 min, vacuum dried, and exposed to phosphor screen for 14 days. Phosphor screens were scanned with the Bio-Rad Molecular Imager FX using Quantity One software (Bio-Rad). Preparative gels were fixed in 50 % methanol and 7 % acetic acid, stained with GelCode Blue Coomassie (Pierce), vacuum dried, and exposed to X-ray film for 14 days. Two replicate gels were run from each of four biological replicates.
Protein spot quantification and identification.
Protein spots were matched and analysed for differential expression using Dymension software (Syngene). Protein spot intensities were normalized to the total valid spot intensity for each gel. Proteins were deemed iron responsive if (i) the mean density of the protein spot from iron-restricted samples showed a twofold or greater increase over control samples and (ii) the P value was <0.05 using Student's two-way t-test.
Protein spots demonstrating a significant increase in density between the iron-replete and iron-restricted samples were excised from preparative gels and submitted for identification via liquid chromatography-tandem mass spectrometry (LC-MS/MS). True matches between detected peptides and protein identities were affirmed by a Mowse score indicating P<0.05 and by comparing predicted versus observed pI and molecular mass.
Quantitative PCR analysis.
Transcript levels of ahpC, devB, cadd, fabF, ct538, ompA, euo and omcB were measured using quantitative reverse transcription PCR (qRT-PCR). HEC-1B cells were grown in six-well plates and iron-starved 24 h prior to infection as described above. Following infection, monolayers were collected at 6, 12, 18, 24, 36 and 48 h p.i., along with uninfected control samples. After washing with PBS, each monolayer was scraped with a rubber policeman into RLT lysis buffer (Qiagen), passed through a 20-gauge needle 10 times, and stored at –80 °C. Samples were collected at least in triplicate on separate occasions. Collected samples were split into two aliquots for separate RNA and DNA purifications. RNA was purified using the RNeasy Mini kit, including an on-column DNase step (Qiagen), and genomic DNA (gDNA) was purified in parallel using the QIAamp Blood DNA Mini kit (Qiagen). RNA integrity was verified using an Agilent 2100 Bioanalyser (Agilent Technologies), and RNA and DNA concentrations were determined by spectrophotometer reading at 260 nm. An equal amount of total RNA for each sample was reverse transcribed using random hexamers and SuperScript II Reverse Transcriptase (Invitrogen), following the conditions recommended by the manufacturer.
qRT-PCR was conducted on samples in triplicate using the Bio-Rad iCycler. Reaction mixtures contained 1x Platinum Quantitative PCR Supermix-UDG (Invitrogen), 0.8x SYBR green (Cambrex BioScience), 5 mM MgCl2, 300 nM concentrations of each primer, and 1 µl of sample in a total volume of 50 µl. Primer sequences for each gene are given in Table 1, except for euo and omcB (Belland et al., 2003a) and ompA (Gomes et al., 2005). The specificity of each primer set was confirmed by sequencing of the amplicons. To correct for loading variations, chlamydial chromosome copy numbers were normalized to total DNA. Assay validation and quantitative analyses were performed using a standard curve method as described previously (Guseva et al., 2007). The amplification efficiencies of all assays ranged from 94 to 102 %. Controls included (i) uninfected samples to demonstrate specificity of primers for chlamydial targets and (ii) RT minus samples, in which the reverse transcription was omitted in order to determine chlamydial gDNA contribution to the qRT-PCR signal for corresponding RT plus samples. Chlamydial genome copy number for each experimental sample was determined by qRT-PCR using the ct538 primer set against gDNA. Transcript levels are presented as copy number per chlamydial genome.
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RESULTS |
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). Ferritin levels in Desferal-exposed samples averaged 119 ng ml–1compared to 241 ng ml–1 in mock-exposed samples. This twofold difference between Desferal-exposed and unexposed samples was statistically significant, thereby validating this model for iron restriction of Chlamydia-infected cells. In addition, exposure of uninfected cells to Desferal as above resulted in a similar drop in ferritin level (data not shown).
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), which coincides with an increased demand for micronutrients such as iron. Host cells were starved for iron 24 h prior to infection to ensure that chlamydiae did not obtain iron stores early in infection. At 22 h p.i., medium for each sample was switched to the radiolabelling medium, the infection was allowed to continue for 2 h of labelling, and protein samples were collected, processed, and separated via 2D-PAGE. This methodology for generation of proteomic maps in C. trachomatis was adapted from the pioneering chlamydial proteomic studies by Vandahl et al. (2004).
Approximately 250 protein spots were visible via radiography (Fig. 2). Using Dymension 2D-PAGE analysis software, 25 proteins were determined to increase in expression during iron-restricted growth relative to iron-sufficient growth, with a threshold of 
2-fold expression difference and a P<0.05 given by Student's two-way t-test (Fig. 3).
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; Fig. 3
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Two chlamydial proteins with putative roles related to protein modification, i.e. RopA (trigger factor or peptidyl prolyl cis-trans isomerase) and Def (peptide deformylase), were found to be iron responsive, with 3.7-fold and 3.8-fold increased levels in iron-starved samples compared to control samples. Three proteins involved in transcription were also identified. Indeed, in response to iron restriction, RpoA (RNA polymerase subunit alpha) increased 3.1-fold, while the transcription elongation proteins NusA and NusG increased 2.1-fold and 5.9-fold, respectively. In addition, putative virulence factor CADD (Chlamydia protein associating with death domains) expression was found to increase 3.4-fold. The uncharacterized protein CT538 demonstrated a 2.9-fold increase in expression. Of note, no significant difference in spot intensity of the major outer-membrane protein (MOMP) was found between iron-restrictive and iron-replete growth.
Transcriptional analysis of selected genes
qRT-PCR was used to examine transcript levels of a subset of the identified iron-responsive proteins, i.e. ahpC, devB, fabF, cadd and ct538. These transcripts were selected due to their potential role in the oxidative response (ahpC, devB and fabF), the putative role of cadd in chlamydial virulence, and the fact that ct538 is uncharacterized. Transcript levels of ompA, euo and omcB were used as controls for comparison, since these should not be regulated by iron availability. It is noteworthy that equivalent levels of gDNA were detected in Desferal-exposed versus mock-exposed cultures. Transcript levels were measured at 6, 12, 18, 24, 36 and 48 h p.i. in iron-restricted and in mock-exposed control chlamydiae using qRT-PCR. Analysis of transcript levels normalized to chlamydial chromosome copy number revealed that the transcript levels for all five iron-responsive genes examined peaked at 18 h p.i. (Fig. 4). It is not surprising that the level of these transcripts would be highest mid-cycle, considering the relatively high expression of these proteins during radiolabelling, 22–24 h p.i. The transcript level of ompA was highest at 24 h p.i., euo at 12–18 h p.i. and omcB at 36 h p.i., reflecting the timing of peak transcript level previously described for these transcripts, mid-, early- and late-developmental cycle, respectively (Belland et al., 2003a).
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). Transcript levels of cadd reached a higher peak level at 18 h p.i. in samples from iron-restricted cultures compared to iron-replete cultures. In contrast, ahpC, devB, fabF and ct538 transcript levels peaked at similar levels under both growth conditions at 18 h p.i.; however, in samples from iron-restricted chlamydiae, higher levels of these transcripts were detected at 24 h p.i. compared to samples from iron-replete chlamydiae. Since protein expression differences between iron-restricted and iron-replete samples were higher than transcript level differences, the duration of heightened transcript level may be more important for regulation of protein expression under iron restriction than changes in maximal transcript levels. No significant differences in transcript levels for ompA, euo or omcB between cultures grown in the presence or absence of Desferal were found at any time, validating the differences measured for the iron-responsive transcripts. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), although a putative component of an ABC metal transporter, YtgA, has been identified in C. trachomatis (Raulston et al., 2007). YtgA has previously been shown to increase in expression during iron restriction; however, in ongoing studies on expression of YtgA, differential expression does not appear to occur until 36 h p.i. (unpublished data).
The lack of obvious iron-uptake system homologues in Chlamydia suggests that these organisms (i) possess conserved iron-acquisition genes with highly divergent sequences or (ii) use alternative or novel iron-acquisition systems. The pathways by which intracellular organisms acquire iron are much less defined compared to those utilized by extracellular bacteria, but the process appears to involve alternative systems. For example, iron reductases are believed to play a role in iron acquisition by Listeria monocytogenes through the removal and solubilization of iron from iron-binding proteins such as haem, ferritin and transferrin (Deneer et al., 1995). Interestingly, a recent study has shown that reduced expression of transferrin or Rab11, a protein involved in trafficking of transferrin-positive vesicles, leads to a decrease in the production of infectious EBs (Heuer et al., 2006).
An analogue to Fur, the primary iron regulatory protein in Gram-negative bacteria, termed DcrA, was identified in C. trachomatis serovar E, and has shown the ability to bind E. coli Fur boxes (Wyllie & Raulston, 2001). Putative DcrA-binding sites have been identified in the chlamydial genome by binding of purified E. coli Fur protein (Rau et al., 2005). Notably, binding sites for DcrA have been identified upstream of genes encoding two proteins found to be iron responsive in the current study, AhpC and FabF.
Mukhopadhyay et al. (2006) conducted a proteomic analysis of Chlamydophila pneumoniae that examined modulation of protein expression by three inducers of persistence: iron restriction, heat shock and exposure to interferon-. Proteins increasingly expressed under iron restriction but not following heat shock or interferon-gamma exposure were involved in cofactor biosynthesis, cellular processes and translation, including one protein identified as iron responsive in the current study, AhpC. The close connection between the response to iron availability and oxidative stress in bacteria and eukaryotes is well established. AhpC belongs to a family of antioxidant proteins that are responsible for the reduction of peroxides and is expressed in response to oxidative stress. Decreased iron availability has been shown to modulate expression of ahpC transcription and/or AhpC protein expression in numerous organisms, including Helicobacter pylori (Merrell et al., 2003), Corynebacterium diphtheriae (Tai & Zhu, 1995), Campylobacter jejuni (van Vliet et al., 1998) and Francisella tularensis (Lenco et al., 2007).
Two chlamydial proteins involved in biosynthetic pathways, DevB and FabF, were found to be iron responsive in the present study. DevB catalyses the hydrolysis of 6-phosphogluconolactone to 6-phosphogluconate, the second step of the pentose phosphate pathway (PPP), while FabF catalyses the chain-elongation step of type II fatty-acid biosynthesis. Like AhpC, the PPP is also involved in the response to oxidative conditions, through the generation of NADPH, which serves as an electron source for reductases and antioxidants. Another PPP enzyme, glucose-6-phosphate dehydrogenase (G6PD), is regulated by the E. coli soxR system involved in oxidative defence (Tsaneva & Weiss, 1990). G6PD mutants of E. coli (Nunoshiba et al., 1995), Salmonella typhimurium (Lundberg et al., 1999) and mouse stem cells (Pandolfi et al., 1995) have extreme sensitivity to oxidative damage. FabF is also likely iron responsive due to a role in oxidative damage defence, since increased expression of other enzymes involved in fatty acid biosynthesis appear to be involved in the iron-restriction and oxidative responses in other bacteria. For example, FabH expression is involved in hydrogen peroxide resistance and is co-regulated with siderophore production in Pseudomonas syringae (Taguchi et al., 2006), while F. tularensis (Lenco et al., 2007) and Campylobacter jejuni (Holmes et al., 2005) increase expression of FabG under iron restriction.
The finding that CADD is iron responsive is significant since it has been implicated in induction of host-cell death via interaction with the death domains of Fas (Stenner-Liewen et al., 2002). CADD is one of several identified factors chlamydiae express that is involved in induction or inhibition of host-cell death. Inhibition of apoptosis has been hypothesized to promote chronic infections (i.e. persistence), while activation of cell death is thought to be the chlamydial strategy to prevent a dying host cell from entering necrosis, which is pro-inflammatory (Byrne & Ojcius, 2004). In the case of iron restriction, chlamydiae may sense this as a sign of a dying host cell or a cellular response to interferon-. Notably, a microarray study showed cadd transcript levels of C. trachomatis serovar D increased in response to interferon- exposure by 2.85-fold (Belland et al., 2003b).
In summary, the analysis of chlamydial protein-expression alterations during iron restriction at mid-developmental cycle growth shows an increase in expression of proteins involved in protection against oxidative damage and response to stress by increasing expression of transcriptional machinery and other stress-responsive proteins. Furthermore, this has been matched with an increased level of transcript encoding five of the identified proteins during mid-developmental cycle. While the mechanisms utilized by chlamydiae to obtain iron remain elusive, the concept that chlamydiae have a reliance on iron availability is clearly indicated by the demonstrated metabolic alterations associated with iron restriction.
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ACKNOWLEDGEMENTS |
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Edited by: T. P. Hatch
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Received 29 July 2008;
revised 7 October 2008;
accepted 15 October 2008.
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) or low-iron (D+, 
) conditions. hpi, hours post-infection. The y-axes indicate mRNA copies per chlamydial genome (normalized by amount of gDNA). Times at which transcripts are significantly increased in iron-restricted versus iron-sufficient growth are marked by an asterisk. Transcripts of ompA, euo and omcB, which should not be regulated in response to iron availability, are shown for reference.