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Journal of Bacteriology, October 2008, p. 6419-6427, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00431-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

DNA Supercoiling-Dependent Gene Regulation in Chlamydia{triangledown}

Eike Niehus,1 Eric Cheng,1 and Ming Tan1,2*

Department of Microbiology and Molecular Genetics,1 Department of Medicine, University of California, Irvine, California2

Received 27 March 2008/ Accepted 16 July 2008


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ABSTRACT
 
The intracellular pathogen Chlamydia has an unusual developmental cycle marked by temporal expression patterns whose mechanisms of regulation are largely unknown. To examine if DNA topology can regulate chlamydial gene expression, we tested the in vitro activity of five chlamydial promoters at different superhelical densities. We demonstrated for the first time that individual chlamydial promoters show a differential response to changes in DNA supercoiling that correlates with the temporal expression pattern. The promoters for two midcycle genes, ompA and pgk, were responsive to alterations in supercoiling, and promoter activity could be regulated more than eightfold. In contrast, the promoters for three late transcripts, omcAB, hctA, and ltuB, were relatively insensitive to supercoiling, with promoter activity varying by no more than 2.2-fold over a range of superhelicities. To obtain a measure of how DNA supercoiling levels vary during the chlamydial developmental cycle, we recovered the cryptic chlamydial plasmid at different times after infection and assayed its superhelical density. The chlamydial plasmid was most negatively supercoiled at midcycle, with an approximate superhelical density of –0.07. At early and late times, the plasmid was more relaxed, with an approximate superhelicity of –0.03. Thus, we found a correlation between the responsiveness to supercoiling shown by the two midcycle promoters and the increased level of negative supercoiling during mid time points in the developmental cycle. Our results support a model in which the response of individual promoters to alterations in DNA supercoiling can provide a mechanism for global patterns of temporal gene expression in Chlamydia.


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INTRODUCTION
 
Chlamydia is an important human pathogen that causes infections of the reproductive tract, the eyes, and the respiratory tree (44). Chlamydia trachomatis genital infections are the most common notifiable sexually transmitted disease, with more than one million newly diagnosed cases each year (8). Other serovars of C. trachomatis cause trachoma, one of the leading causes of preventable blindness in the world. Another species, Chlamydia pneumoniae, causes community-acquired pneumonia and has been associated with atherosclerotic heart disease (23). While different Chlamydia spp. and strains can produce such different disease manifestations, the essential elements of chlamydial growth and replication inside an infected cell are remarkably similar.

All Chlamydia spp. are obligate intracellular bacteria that replicate inside a eukaryotic cell via an unusual developmental cycle in which there is conversion between two stage-specific forms (18). The infectious form is the elementary body (EB), which is metabolically inert and characterized by a highly disulfide-linked protein coat and condensed chromatin. After entry into a eukaryotic host cell, the EB differentiates into a larger, metabolically active reticulate body (RB), which is able to express RNA and proteins and replicate its DNA. After many rounds of binary fission, RBs redifferentiate into EBs before release to infect new cells (22). The whole developmental cycle takes about 48 to 72 h, depending on the species.

Gene expression is temporally regulated during this chlamydial developmental cycle. Regulation appears to be at the transcriptional level with early, mid, and late classes of genes (4, 14, 34, 41, 42). For example, ompA belongs to the class of midcycle genes, which can be detected by about 12 h postinfection (hpi) for C. trachomatis (41). ompA encodes the abundant major outer membrane protein, which is an important immunogenic determinant for different serovars and subspecies (7). A number of late genes have functions related to the morphologically dramatic events that begin at about 24 hpi, when RBs are in the process of converting back to EBs (14). For instance, omcA and omcB encode two abundant cysteine-rich proteins that are found only in the outer membrane of EBs and not on RBs (19). In addition, hctA and hctB encode the Chlamydia-specific histonelike proteins Hc1 and Hc2, respectively, which mediate the condensation of DNA observed when RBs convert into EBs (2, 6). This regulated expression of late genes demonstrates the principle that gene products appear to be transcribed only at a time in the developmental cycle when they are needed.

Although this temporal pattern of gene expression appears to be well coordinated in Chlamydia, the regulatory mechanisms are incompletely understood. We have shown that a subset of late genes is transcribed by the alternative sigma factor {sigma}28 (53, 54). However, not all late genes are regulated by {sigma}28 RNA polymerase. Some of these genes are known to be regulated by the major form of RNA polymerase, which contains {sigma}66 (14). As representative early, mid, and late genes have all been shown to be transcribed by {sigma}66 RNA polymerase (4, 14, 34, 41, 42), there clearly must be additional mechanisms for the temporal regulation of global gene expression in Chlamydia.

DNA supercoiling has been shown to be a global mechanism of gene regulation in Escherichia coli and other bacteria (9, 20, 36, 47). Changes in negative supercoiling can modulate promoter activity directly by altering DNA structure and melting energy or indirectly by affecting the binding of transcription factors. In addition to studies on the supercoiling sensitivity of individual bacterial promoters, microarray studies using gyrase inhibitors have demonstrated that a large number of genes in a bacterium can be coordinately regulated by changes in negative supercoiling (17, 35).

In this report, we show that chlamydial promoters respond differently to changes in DNA supercoiling in a manner that correlates with their temporal class and that intrachlamydial DNA supercoiling varies during the developmental cycle. We found that the midcycle promoters tested were more responsive to supercoiling than late promoters. In addition, by isolating the native chlamydial plasmid and determining its superhelical density at different times during infection, we obtained evidence that the highest levels of negative supercoiling occur at mid time points in the developmental cycle. From these findings, we propose a mechanism for temporal gene regulation in Chlamydia based on the differential response of promoters to changes in DNA supercoiling during the developmental cycle.


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MATERIALS AND METHODS
 
Construction of in vitro transcription plasmids. Each promoter insert was cloned upstream of a promoterless, G-less cassette transcription template, as previously described (46). Transcription plasmids pMT1013, pMT1150, and pMT1234 with promoter sequences for C. trachomatis LGV serovar L2 ltuB (positions –132 to +1) and omcAB (positions –122 to +1) and C. trachomatis serovar D pgk (positions –267 to +1) have been described elsewhere (40, 49, 54). Plasmid pMT1185 contains the promoter sequence of hctA (positions –137 to +1) (14) amplified from C. trachomatis serovar D genomic DNA with primers T284 (5'-ACCGAATTCTTTTCTATTAACAGAGGAAAAATAACCTA-3') and T288 (5'-TTTAATTTTTAATTAGTTTGTTTGTTCAAA-3') and cloned into pMT1125 (49). Plasmid pMT1187 contains the P2 promoter sequence of ompA (positions –137 to +1) (11) amplified from C. trachomatis serovar D genomic DNA with primers T282 (5'-CCGAATTCCCTCTCATTCTCAACAATCAAC-3') and T290 (5'-TTTTTTGATTTAGCGAATAAAGCAGA-3') and cloned into pMT1125. For some experiments, transcription plasmid DNA was linearized by digestion with EcoRI and was purified by extraction with phenol-chloroform (1:1) and with chloroform, and the DNA was recovered by ethanol precipitation.

Generation of transcription plasmid topoisomers. For each transcription plasmid, a series of topoisomers were generated by using the method of Rhee et al. (38). Ten micrograms of CsCl gradient-purified plasmid DNA was treated for 3 h at 37°C with 5 U of wheat germ topoisomerase I (Promega) in 40-µl mixtures containing 50 mM Tris-HCl (pH 7.6), 0.1 mM EDTA, 1 mM dithiothreitol, 50 mM NaCl, 10% glycerol, and concentrations of ethidium bromide ranging from 0 to 40 µM. The ethidium bromide was removed by two extractions with phenol-chloroform (1:1) and one extraction with chloroform, and the DNA was recovered by ethanol precipitation and resuspended in 30 µl of TE buffer (10 mM Tris [pH 8.0], 1 mM EDTA). The DNA concentration and purity were determined using a NanoDrop ND1000 spectrophotometer. The plasmid topoisomers were resolved on 1.4% agarose gels in 1x TAE buffer (0.04 M Tris-acetate, 1 mM EDTA) with 0.02 to 0.16 µg/ml ethidium bromide at 3.5 V cm–1 for 16 to 20 h at room temperature with buffer circulation. The average linking-number difference ({Delta}LK) was determined by the band-counting method of Keller (24). The average superhelical density ({sigma}) was calculated using the equation {sigma} = –10.5{Delta}LK/N, where N is the total number of base pairs in the plasmid.

Purification of chlamydial RNA polymerase. RNA polymerase was partially purified from C. trachomatis LGV serovar L2 at 24 hpi by heparin-agarose chromatography as previously described (46).

In vitro transcription. Transcription reaction experiments were performed as described previously (46), using either 2 µl of heparin-agarose-purified C. trachomatis RNA polymerase or 0.05 µl of E. coli {sigma}70-holoenzyme (1 U/µl; Epicentre) together with 25 nM plasmid topoisomer in a 10-µl reaction mixture. The relative promoter activity was calculated by defining the maximal promoter activity for the range of superhelicities tested as 100% and normalizing the promoter activity obtained for each topoisomer. Three measurements of relative promoter activity were obtained for each topoisomer, and a mean and a standard deviation were calculated.

Plasmid extraction. Chlamydial plasmid pCT-L2 was extracted from 3 x 108 to 1.8 x 109 C. trachomatis LGV serovar L2-infected murine L929 host cells grown in suspension. A multiplicity of infection of 100 was used for plasmid isolation at 2 and 6 hpi, and a multiplicity of infection of 3 was used for all other time points. Chlamydial RBs were recovered and lysed as previously described (46). Chlamydial EBs were recovered and lysed in the same way as RBs, except that L929 host cells were lysed by sonication (three 30-s pulses; 0.125-in. microtip; Branson 250D digital Sonifier). The plasmid was extracted from the chlamydial lysate using a Qiagen plasmid midi kit or by CsCl gradient extraction.

Generation of the chlamydial plasmid topoisomer standard. Topoisomers of the chlamydial plasmid were constructed by using the method that was used for the transcription plasmid topoisomers. Five micrograms of CsCl-purified pCT-L2 plasmid DNA was used per reaction mixture together with ethidium bromide at concentrations ranging from 0 to 80 µM. The topoisomers were resolved on one- and two-dimensional chloroquine agarose gels (see below) and used as reference standards for the migration of native chlamydial plasmid topoisomers.

One- and two-dimensional chloroquine agarose gels. For topoisomer separation, 250-ng samples of the native chlamydial plasmid were resolved on a 0.7% agarose gel in 1x Tris-borate-EDTA with concentrations of chloroquine ranging from 0 to 50 µg/ml at 3.5 V cm–1 for 20 to 24 h at room temperature with buffer circulation. For the two-dimensional gels, two samples were loaded 8 to 10 cm apart and electrophoresed in the first dimension using a chloroquine concentration of 0, 0.25, 1.0 or 2.5 µg/ml. Electrophoresis in the second dimension was performed perpendicular to the electrophoresis in the first dimension with 1.0, 2.5, or 10 µg/ml chloroquine for 16 h at 2.5 V cm–1; the other electrophoresis conditions were unchanged. Prior to the second electrophoresis step, the gel was soaked for 5 h in the new concentration of chloroquine for equilibration.

Southern blotting. Agarose gels were first soaked in distilled H2O for 1 h, stained in 3 µg/ml ethidium bromide for 1 h, destained in distilled H2O for up to 2 h, and examined under UV light. Each gel was then incubated in denaturing solution (1.5 M NaCl, 0.5 M NaOH) and in renaturing solution (1.5 M NaCl, 1 M Tris) for 30 min each and transferred to a nylon membrane in 6x SSC by capillary blotting for 16 h (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The membrane was rinsed in 2x SSC-0.1% sodium dodecyl sulfate (SDS) and hybridized with {gamma}-32P-labeled oligonucleotides specific for the chlamydial plasmid (T1129 [5'-AGGGAAGGCTTGACAGTGC-3'] and T1130 [5'-GAAAGAAACTACGGAAGGGT-3']) in hybridization solution (0.5 M NaPO4 [pH 7.2], 7% SDS) at 42°C for 16 h. The membrane was washed with 0.04 M NaPO4 (pH 7.2)-1% SDS for 5 min room temperature, for 15 min 42°C, and for 2 min room temperature and then exposed to a phosphorimager plate. The plates were scanned with a Bio-Rad personal FX scanner, and the data were analyzed with Bio-Rad Quantity One software. Densitometric traces of the topoisomer populations were generated with ImageJ 1,38x (http://rsb.info.nih.gov/ij; developed by Wayne Rasband, NIH, Bethesda, MD) to determine the predominant topoisomer bands.


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RESULTS
 
Construction of transcription plasmid topoisomers with various negative superhelicities. To assess the effect of DNA supercoiling on the activities of five chlamydial promoters, we cloned each promoter into a transcription plasmid and generated a series of topoisomers for each plasmid. We produced these topoisomers with different superhelical densities by performing in vitro topoisomerase reactions using different concentrations of ethidium bromide and the method of Rhee et al. (38). Figure 1 shows the results for a representative series of topoisomers for one of the transcription plasmids. Each reaction produced a small number of topoisomers that differed from each other by one linking number (one turn of the DNA helix) that could be resolved on an ethidium bromide gel as a ladder of distinct bands. The first reaction mixture contained a totally relaxed plasmid with a superhelical density of zero and served as a reference. There was enough overlap between the ladders so that the linking number of the predominant band for each topoisomer could be easily determined and the average superhelical density could be calculated. For each promoter, the average superhelical density of the topoisomer series ranged from 0 to ≥–0.074 (Table 1). Thus, our topoisomer superhelical densities encompassed and exceeded the physiologic range of global superhelical densities in E. coli, which vary from –0.03 to –0.06 depending on the growth state (20).


Figure 1
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  		FIG. 1. Determination of the superhelical density of topoisomers by electrophoresis. Topoisomers of a 3.1-kb transcription plasmid were resolved on two 1.4% agarose gels with different ethidium bromide (EtBr) concentrations; the higher concentration of ethidium bromide was used for resolution of more supercoiled topoisomers. The negative images of the gels shown make it easier to visualize the topoisomer bands. The concentration of ethidium bromide used in the topoisomer reaction, the {Delta}LK, and the calculated superhelical density of the plasmid ({sigma}) are indicated above each lane. {Delta}LK was determined by counting the topoisomer bands between the predominant band (arrow) of the population of topoisomers in each lane. Lane 1 contained a topoisomer population containing a completely relaxed plasmid with a superhelical density of zero, which was used as the reference band. The superhelical density was determined based on {Delta}LK, as described in Materials and Methods.


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  		TABLE 1. Transcription plasmids used in this study

Chlamydial promoters respond differentially to changes in superhelical density. In initial experiments to examine if DNA supercoiling affects chlamydial promoter activity, we tested five chlamydial promoters, comparing transcription of a supercoiled template and transcription of a linearized template. The supercoiled plasmids, which were isolated from E. coli during logarithmic growth, each had a superhelical density of about –0.06, as determined by agarose gel electrophoresis along with in vitro-generated topoisomer standards (data not shown). Using an in vitro transcription assay with partially purified C. trachomatis RNA polymerase (46), we found that DNA supercoiling had a significant effect on the level of transcription from two midcycle promoters but not on the level of transcription from three late promoters. For the midcycle promoters, transcription from a supercoiled template was 57-fold higher for ompA and 26-fold higher for pgk than transcription from the same template in linearized form (data not shown). In contrast, the promoters for the late transcripts omcAB, hctA, and ltuB showed 0.7-, 1.9-, and 1.9-fold changes in activity, respectively, for a supercoiled template compared with a linearized template (data not shown).

To obtain a more detailed view of the effect of negative supercoiling on chlamydial promoter activity, we tested a series of topoisomers for each of the five promoters in individual in vitro transcription reactions. The difference in responsiveness to supercoiling between the two midcycle genes and the three late genes again was apparent (Fig. 2A). For both midcycle genes, the promoter activity was lowest for a totally relaxed transcription template; with increased negative supercoiling, transcription increased over a 50-fold range for the ompA promoter and by up to 8-fold for the pgk promoter. In comparison, the activities of the omcAB, hctA, and ltuB promoters showed only modest changes in activity, with maximum differences of 1.6-, 2.2-, and 1.7-fold, respectively, over the entire range of superhelicities tested. This difference in supercoiling responsiveness between the midcycle and late promoters was confirmed when we repeated these transcription assays with separately prepared sets of topoisomers (data not shown).


Figure 2
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  		FIG. 2. In vitro transcription of chlamydial promoters, using heparin-agarose-purified chlamydial RNA polymerase (A) or E. coli RNA polymerase (B). For each promoter, a series of topoisomers with increasing negative superhelical densities ranging from 0 to approximately –0.1 were tested, and an individual topoisomer was used in each transcription reaction. The temporal class (4, 14, 34, 41) and the maximum difference in transcriptional activity over the range of superhelical densities tested are shown for each promoter.

To see how promoter activity varied with increasing negative supercoiling, we calculated the relative promoter activity by defining the maximum promoter activity for the range of superhelicities tested as 100% and normalizing the promoter activity obtained for each topoisomer. Figure 3A shows that for the midcycle genes, pgk and ompA, the promoter activity from a relaxed template was low, and a minimal superhelical density (–0.01 to –0.02) was required for a relative promoter activity of ≥50%. In contrast, the relative activity for the late promoters, omcAB, hctA, and ltuB, was ≥50% over the entire range of superhelical densities. Thus, in contrast to the midcycle promoters, the late promoters were generally insensitive to changes in DNA supercoiling and could be transcribed at high levels even if the template was completely relaxed.


Figure 3
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  		FIG. 3. Relative promoter activity as a function of superhelical density for midcycle and late chlamydial promoters transcribed by C. trachomatis RNA polymerase (A) or E. coli RNA polymerase (B). Relative promoter activity was expressed as a percentage of the maximal activity for the range of superhelical densities tested. The trend line was fitted by second-order polynomial regression. A relative promoter activity of 50% is indicated by a dashed line. Reactions were performed in triplicate, and standard deviations are indicated by error bars.

The relative responsiveness of the chlamydial promoters to supercoiling is the same with chlamydial and E. coli RNA polymerases. To determine if the difference in responsiveness to supercoiling is intrinsic to the individual promoters or due to chlamydial RNA polymerase, we also transcribed the promoter topoisomers with E. coli RNA polymerase. We performed in vitro transcription reactions with E. coli {sigma}70 polymerase as previously described (50) but were unable to test the ompA promoter because it is not transcribed by this heterologous RNA polymerase (12). Transcription of supercoiled and linearized templates by E. coli RNA polymerase showed the same promoter-specific differences that we observed with chlamydial RNA polymerase. DNA topology had a large effect on the activity of the pgk midcycle promoter (26-fold range) but made little difference to the activities of the omcAB (1.3-fold), hctA (0.7-fold), and ltuB (1.2-fold) promoters (data not shown).

We observed the same differential response to supercoiling for a midcycle chlamydial promoter versus a late chlamydial promoter when we transcribed a topoisomer series with E. coli polymerase. The pgk promoter showed a 17-fold range in promoter activity (Fig. 2B), and a minimal superhelicity of approximately –0.025 was required for ≥50% relative promoter activity (Fig. 3B). In contrast, the late omcAB promoter showed little variation in promoter activity (1.4-fold) (Fig. 2B), and the relative promoter activity was ≥50% over the entire range of superhelical densities tested (Fig. 3B).

Chlamydia supercoiling-responsive promoters have a high G or C content in the –10 element and the spacer region. To identify features of chlamydial promoters that might account for the observed differences in supercoiling responsiveness, we aligned the five chlamydial promoters that were tested in this study (Fig. 4). The ompA promoter has an unusual –10 element with a G or C at three of the six positions (11), which is quite different from the A- or T-rich sequence of the preferred chlamydial {sigma}66 (39, 45) or consensus E. coli {sigma}70 –10 promoter element (21). In fact, E. coli RNA polymerase is unable to transcribe this promoter (12), even when it is tested over a wide range of different superhelical densities (data not shown). Our two supercoiling-sensitive midcycle promoters both have an unusually high G or C content in the spacer (41 and 47% for ompA and pgk, respectively), although a high G or C content was not found in the discriminator region that is between the –10 element and the transcription start site. In contrast, the G or C contents in the spacer of the late promoters for omcAB and ltuB are 0 and 6%, respectively, although the G or C content is 28% for the third late promoter, hctA. Thus, at least for the promoters tested, there was a correlation between high G or C content in the promoter spacer and the –10 element and responsiveness to supercoiling.


Figure 4
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  		FIG. 4. Alignment of promoter sequences for the promoters tested. Sequences are aligned at position 1, and upstream sequences to position –50 are shown for each promoter. The –35 and –10 elements of an optimal chlamydial {sigma}66 promoter (39, 45) and the tested promoters are indicated by bold uppercase letters. The ompA promoter is ompA P2 (11). A promoter was considered supercoiling responsive if its relative promoter activity dropped below 50% over the range of superhelical densities tested.

The superhelical density of the chlamydial plasmid is higher in RBs than in EBs. To determine how the supercoiling state changes during chlamydial growth, we isolated the cryptic chlamydial plasmid pCT-L2 (28) at 24 hpi, when chlamydiae are mostly in the RB stage, and at 46 hpi, when most RBs have converted to EBs. We then compared the migration of the native plasmid to the migration of topoisomer standards of the chlamydial plasmid on chloroquine agarose gels. Chloroquine gels were used rather than ethidium bromide gels because they gave better resolution of the 7.5-kb chlamydial plasmid (data not shown). For these studies, we compared the plasmids on gels at different chloroquine concentrations, because no single concentration resolved all the topoisomers. Plasmid bands were then detected by Southern blotting.

At 10 µg/ml chloroquine, the topoisomer standards showed that a completely relaxed plasmid topoisomer migrated considerably faster than a more supercoiled species (Fig. 5A, left panel). Compared to these standards, the native plasmid isolated at 24 hpi was highly supercoiled and the plasmid isolated at 46 hpi was more relaxed (Fig. 5A, right panel).


Figure 5
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  		FIG. 5. Southern blots of topoisomer gels to determine the supercoiling level of C. trachomatis cryptic plasmid pCT-L2. The right panels show the results for the native chlamydial plasmid harvested at 24 or 46 hpi and electrophoresed on an agarose gel in the presence of 10 µg/ml chloroquine (A) or 1 µg/ml chloroquine (B) or in the absence of chloroquine (C). Each plasmid was resolved as a population of topoisomers, and the predominant band is indicated by an asterisk. The panels on the left show topoisomer standards over a range of supercoiling densities from relaxed to highly negatively supercoiled for each concentration of chloroquine.

At 1 µg/ml chloroquine, the migration pattern of the topoisomer standards was a little more complicated, but it allowed better resolution of moderately and highly negatively supercoiled topoisomers (Fig. 5B, left panel). On the gels, totally relaxed plasmid (lane 1) still migrated faster, but so did highly supercoiled plasmids (lanes 5 and 6). In contrast, topoisomers with low and intermediate superhelical densities migrated slower (lanes 2 to 4). Compared to these standards alone, the fast migration of the 24-h native plasmid was consistent with either a highly relaxed form or a supercoiled form (Fig. 5B, right panel), but based on this finding, together with the migration pattern at 10 µg/ml chloroquine (Fig. 5A, right panel), we concluded that the 24-h chlamydial plasmid had a highly negatively supercoiled topology. The 46-h native plasmid produced a pattern with a predominant band that migrated slowly, consistent with a low to intermediate superhelical density (Fig. 5B, right panel).

In the absence of chloroquine, the completely relaxed plasmid (Fig. 5C, left panel, lane 1) could be clearly resolved as a more slowly migrating band that was different from topoisomers with any level of negative supercoiling (lanes 2 to 6). The migration of both the 24- and 46-h native plasmids was fast, which indicates that neither plasmid had a completely relaxed state (Fig. 5C, right panel).

To calculate the level of supercoiling, we resolved the native plasmids and a completely relaxed topoisomer of the chlamydial plasmid using two-dimensional topoisomer gels. Figure 6 shows representative two-dimensional gels in which electrophoresis in the first dimension occurred in the presence of 1 µg/ml chloroquine and electrophoresis in the second dimension occurred in the presence of 10 µg/ml chloroquine. With these results and the results for additional one- and two-dimensional topoisomer gels with different chloroquine concentrations (data not shown), we were able to count the differences in the numbers of topoisomer bands for the 24-h, 46-h, and completely relaxed plasmid populations (i.e., the {Delta}LK). This information allowed us to calculate the superhelical densities of the two native plasmid populations. Compared to the relaxed plasmid, which had a superhelical density of zero, the 24-h plasmid consisted of a population of topoisomers with superhelical densities ranging from –0.063 to –0.077 and the 46-h plasmid had superhelical densities ranging from –0.028 to –0.035. These values correspond well to the superhelical densities in E. coli (–0.06 during mid-log growth and –0.03 in stationary phase).


Figure 6
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  		FIG. 6. Southern blots of two-dimensional agarose gels to estimate the difference in supercoiling between C. trachomatis plasmid pCT-L2 isolated at mid and late times in infection. Each gel was first electrophoresed with 1 µg/ml chloroquine and then electrophoresed in the second dimension with 10 µg/ml chloroquine, as indicated by arrows. (A) Native chlamydial plasmid harvested at 24 or 40 hpi or artificially relaxed by treatment with topoisomerase I. (B) The three gels shown in panel A were merged to allow the number of topoisomer bands for the 24-h, 40-h, and relaxed plasmids to be counted for calculation of the {Delta}LK. The predominant band for each topoisomer population is indicated by an asterisk.

The supercoiling level of the Chlamydia plasmid varies with the developmental cycle. As the global supercoiling state of the chlamydial plasmid changed more than twofold between 24 and 46 hpi, we isolated the native plasmid at additional time points during the developmental cycle to determine the temporal pattern of DNA supercoiling in Chlamydia. We electrophoresed the plasmids on agarose gels at different chloroquine concentrations to best resolve the topoisomer bands. Figure 7 shows representative gels. At 1.5 µg/ml chloroquine, we were able to show that the plasmid isolated at 18 h and the plasmid isolated at 24 h were more supercoiled than the plasmid isolated at an earlier time point (2 or 6 h) and the plasmid isolated at a later time point (46 h), both of which were less supercoiled (Fig. 7A). Using an agarose gel with 5 µg/ml chloroquine, we were able to look at the plasmids isolated at mid to late time points and demonstrate that supercoiling was greatest at 18, 24, and 28 h and that the plasmid was relatively relaxed at 40 and 46 h (Fig. 7B).


Figure 7
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  		FIG. 7. Southern blots of topoisomer gels to determine the supercoiling level of C. trachomatis plasmid pCT-L2 isolated at different times during the developmental cycle. The agarose gels were electrophoresed in the presence of 1.5 µg/ml chloroquine (A) or 5 µg/ml chloroquine (B). The time in the developmental cycle when the plasmid was isolated is indicated above each lane. The expected migration patterns of lower or higher supercoiled plasmids at the corresponding chloroquine concentration are shown for each gel, as previously determined with topoisomer standards. The predominant band for each topoisomer population is indicated by an asterisk.


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DISCUSSION
 
Although there is ample evidence that transcription is temporally regulated during the chlamydial developmental cycle (4, 14, 34, 41, 42), the mechanisms for this temporal regulation have not been well defined (29, 45). The alternative sigma factor {sigma}28 appears to have a specific role in regulating a subset of late genes, including hctB, which encodes the histonelike protein Hc2 (53, 54), and CT441, which encodes a tail-specific protease (26). A role for a second alternative sigma factor, {sigma}54, in temporal gene regulation has not been established, although this factor has been proposed to function in midcycle gene expression, based on its likely regulation of two midcycle genes (31). As the major RNA polymerase, containing {sigma}66, transcribes early, mid, and late genes (4), there must be additional mechanisms for the selective transcription of temporal classes of {sigma}66-dependent genes, such as regulation by specific transcription factors. The transcriptional activator ChxR has been proposed to be a chlamydial midcycle regulator based on its own temporal expression pattern (25). IHF is another potential regulator in Chlamydia, as it has been shown to bind upstream of the omcAB promoter, inducing a sharp bend and modestly increasing promoter activity (56). These proposed regulators, however, do not appear to sufficiently account for the patterns of temporal gene expression in Chlamydia.

In this report, we provide evidence that the response of individual promoters to changes in DNA supercoiling can be utilized as a mechanism of gene regulation in Chlamydia during its unusual developmental cycle. Mathews and Stephens previously noted that the C. trachomatis ompA promoter was sensitive to supercoiling changes induced by coumermycin treatment when it was transcribed in vivo by E. coli expressing C. trachomatis {sigma}66 (30). We demonstrate here that representative promoters belonging to two different temporal classes of chlamydial promoters respond differently to changes in DNA supercoiling. Furthermore, our results show that there is a correlation between the supercoiling responsiveness of promoters for the midcycle genes, ompA and pgk, and the higher levels of intrachlamydial DNA supercoiling at mid time points in the developmental cycle. The role of DNA supercoiling in the regulation of early genes has not been defined yet as the supercoiling responsiveness of these promoters has not been determined.

DNA supercoiling is a global mechanism for regulating gene expression in other bacteria, and in fact, it has been proposed to be the highest level in the hierarchy of prokaryotic gene regulation (20). Global studies of E. coli and Haemophilus influenzae have shown that changes in supercoiling can alter the expression of hundreds of genes (17, 35). Some bacterial promoters, such as E. coli topA (48), gyrA and gyrB (33), and ilvYC (38) and Salmonella enterica serovar Typhimurium leu-500 (37), are very responsive to supercoiling, while others are relatively insensitive.

We propose that the differential sensitivity to supercoiling displayed by chlamydial promoters is due to differences in the promoter sequence or structure. In support of this hypothesis, we found that individual chlamydial promoters showed the same differential response to supercoiling whether they were transcribed by C. trachomatis or E. coli RNA polymerase. In other bacteria, promoter features, such as nonconserved sequences in the promoter elements, the G or C content of the discriminator region, or the length and sequence of the spacer, have been associated with sensitivity to supercoiling (9, 20, 47). Spacer length did not appear to be a major determinant in our study as it was 17 bp for the two midcycle promoters and 17 or 18 bp for the nonresponsive late promoters. Our observations suggest that a high overall G or C content in the –10 element and spacer may be a feature of a supercoiling-sensitive promoter in Chlamydia. This hypothesis is testable, although it would require identification of additional active chlamydial promoters (45). For example, we attempted to transcribe additional midcycle promoters, including plasmid promoters, but we were not able to obtain sufficient levels of transcription to perform the topoisomer studies.

Our studies show that the chlamydial cryptic plasmid had the highest levels of negative DNA supercoiling at mid and mid-to-late times in the chlamydial developmental cycle and was relatively relaxed at early and late time points. These results are consistent with the results of Barry et al., who found that the chlamydial plasmid isolated late in an infection (36 hpi) was more relaxed than the plasmid purified from an early-to-mid time point (12 hpi) (3). However, Solbrig et al. detected additional plasmid topoisomers, including what appeared to be a higher supercoiled species in EBs, but not RBs, which may have been due to the presence of copurified proteins (43). In these studies the superhelical density of the chlamydial plasmid was not determined.

We propose that the superhelical density of the chlamydial plasmid at different time points can be used as an estimate of global superhelical density during the developmental cycle. At the least, we believe that the changes in plasmid supercoiling that we measured reflect the direction and trends of global supercoiling. Based on our measurements we propose a model in which the global supercoiling level changes more than twofold during the chlamydial developmental cycle and is highest in midcycle and lowest at early and late time points. The average superhelical density of the chlamydial plasmid was –0.07 in midcycle and –0.03 at late time points, and these values are similar to the values measured for E. coli (–0.06 during logarithmic growth and –0.03 during stationary phase, respectively). Reporter plasmids have been used in E. coli, Helicobacter pylori, and hyperthermophilic Archaea to detect changes in global DNA supercoiling that are dependent on growth phase (1, 27, 52). In addition, the use of plasmid supercoiling as a surrogate marker of global superhelical density has been validated in other bacteria by studies showing a correlation between the activity of a supercoiling-sensitive promoter on a reporter plasmid and the chromosome (5, 15, 51). In one study, however, it was reported that the effective superhelical density of the chromosome in the cellular microenvironment may be less than one-half the value determined with the reporter plasmid (55). Nevertheless, changes in plasmid linking number have been directly correlated to supercoiling changes in vivo (32, 55), and the commonly used values for global superhelical density in E. coli are based on reporter plasmid supercoiling.

A number of factors known to affect global DNA superhelicity in other bacteria may regulate DNA supercoiling in chlamydiae. Global supercoiling homeostasis in a bacterial cell is regulated by DNA topoisomerases (13). In all available genome sequences for Chlamydia spp. there are homologues for the main antagonistic topoisomerases, topoisomerase I and gyrase, which remove and introduce negative supercoils, respectively. As gyrase activity depends on the [ATP]/[ADP] ratio (16), DNA supercoiling can be altered by intracellular energy charge variation during bacterial growth. In addition, specific environmental stimuli, such as temperature, osmotic stress, anaerobiosis, or nutrient availability, have been shown to affect DNA superhelicity (10, 13). Barry et al. have shown that low-level expression of the chlamydial histonelike protein Hc1 can decrease superhelical density and modulate transcription of heterologous supercoiling-sensitive promoters (3). Our studies did not measure the effect of Hc1, Hc2, or other DNA-binding proteins on DNA topology and supercoiling as we had removed proteins from our chlamydial plasmid DNA in order to resolve the topoisomers on chloroquine gels.

In summary, our results support the hypothesis that there is a mechanism of temporal regulation during the developmental cycle of Chlamydia based on the differential response of promoters to alterations in DNA supercoiling. DNA supercoiling may be particularly important for genes that are upregulated during midcycle, as we found that two midcycle genes showed the greatest response to supercoiling and we also measured the highest levels of intrachlamydial negative superhelicity at mid time points. In contrast, DNA supercoiling may not play a large role in the activation of late genes as we found that three {sigma}66-dependent late promoters were largely insensitive to changes in superhelicity. Thus, while a subset of late genes is regulated by {sigma}28, additional mechanisms are necessary to account for the temporal regulation of {sigma}66-dependent late genes. Nonetheless, the different response to superhelicity of late genes may still be important as the insensitivity of these genes to supercoiling may ensure that they are transcribed at low superhelical densities when supercoiling-sensitive promoters are relatively inactive. It remains to be seen whether DNA supercoiling is a general mechanism for temporal gene regulation in Chlamydia or one of an expanding repertoire of mechanisms for the regulation of specific subsets of genes.


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ACKNOWLEDGMENTS
 
We thank Kinrin Yamanaka for excellent technical assistance and Elizabeth DiRusso Case, Johnny Akers, Allan Chen, Ayako Hasegawa, and Kirsten Johnson for critical reading of the manuscript and helpful advice.

This work was supported by grant AI 44198 from the NIH. M.T. was supported by NIH Independent Scientist Award AI 057563.


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FOOTNOTES
 
* Corresponding author. Mailing address: B240, Med Sci, Department of Microbiology & Molecular Genetics, University of California, Irvine, CA 92697-4025. Phone: (949) 824-3397. Fax: (949) 824-8598. E-mail: mingt{at}uci.edu Back

{triangledown} Published ahead of print on 25 July 2008. Back


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Journal of Bacteriology, October 2008, p. 6419-6427, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00431-08
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