A Developmentally Regulated Two-component Signal Transduction System in Chlamydia*

Ingrid Chou KooDagger and Richard S. StephensDagger §

From the Dagger  Division of Infectious Diseases, School of Public Health, University of California, Berkeley, California 94720-7360 and the § Francis I. Proctor Foundation, University of California, San Francisco, California 94143

Received for publication, December 1, 2002, and in revised form, February 20, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two-component systems allow bacteria to adapt to changing environmental conditions and may induce developmental changes necessary for survival. Chlamydia trachomatis alternates between two distinct developmental forms, each optimized for survival in a separate niche. Transcriptional regulation of development is not understood. The C. trachomatis genome sequence revealed a single pair of genes (ctcB-ctcC) predicted to encode proteins with sequence conservation to bacterial two-component systems. Sequence analysis revealed that the sensor kinase, CtcB, possessed an energy-sensing PAS domain and phosphorylation site. The response regulator, CtcC, had homology to sigma 54 activators, possessing conserved receiver and ATPase domains and phosphorylation site, but lacked the C-terminal DNA-binding domain. ctcB and ctcC were expressed late in the developmental cycle, and both proteins were detected in EB lysates. Recombinant CtcB and CtcC were purified from denatured Escherichia coli inclusion bodies and refolded. CtcC was found to aggregate as dimers and tetramers in solution. In vitro phosphorylation assays showed that CtcB autophosphorylated in the presence of Mg2+, Mn2+, and Fe2+ and transferred the phosphoryl group in the presence of CtcC. Collectively, these results show that CtcB and CtcC function as a two-component system and are likely responsible for transcriptional regulation by sigma 54 holoenzyme during late-stage chlamydial development.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two-component signal transduction systems are ubiquitous in bacteria, representing the primary mechanism of sensing and adapting to environmental changes such as pH, temperature, and osmolarity. The sensor histidine kinase autophosphorylates upon receiving a signal and transfers the phosphoryl group to the response regulator, thereby activating it to initiate or repress transcription of appropriate target response genes (1). In the NtrC family of response regulators, the phosphorylated response regulator activates transcription from sigma 54 promoters by oligomerizing at an upstream enhancer-like element of DNA (2, 3), interacting with sigma 54 holoenzyme via DNA looping (4), and hydrolyzing ATP to initiate open complex formation (5). Genes controlled by this subfamily of sigma 54 activators include those regulating nitrogen, pili synthesis, and development (6).

Several bacterial species with unique developmental stages have been shown to require two-component systems and minor sigma  factors for regulation of stage-specific gene expression. Bacillus subtilis sporulation is regulated by its cascade of sigma  factors and two-component regulatory systems (7). Likewise, both Caulobacter crescentus (8) and Myxococcus xanthus (9) specifically require activation of transcription by two-component systems and sigma 54 holoenzyme for differentiation processes in response to cell cycle signals and fruiting body formation, respectively. In fact, most bacterial species possess two-component systems, with an apparent correlation between the number of systems and adaptability to changing environments.

Chlamydiae are obligate, intracellular bacterial pathogens that adapt morphologically and metabolically according to two distinct environments, extracellular and intracellular. Their unique, biphasic developmental cycle consists of the infectious elementary body (EB)1 and the vegetative reticulate body (RB). The infectious EB are often described as "spore-like" because of their highly disulfide cross-linked outer membranes and lack of metabolic activity. Upon infecting the eukaryotic host cell and residing in a membrane-bound vesicle, called an inclusion, the EB differentiates into the metabolically active RB. The RB actively undergoes division by binary fission, but can only survive within the inclusion. After 18 h postinfection, RBs reorganize into EB within the growing inclusion, the host cell lyses, and EB are released to infect new cells. This developmental cycle appears to be tightly regulated so that dividing organisms may parasitize the host cell for energy and nutrients and yet be protected from the hostile extracellular environment. Because Chlamydia has optimized survival by adapting to two distinct environments, its development is likely controlled through recognition of environmental cues or intracellular conditions followed by subsequent regulation of gene expression.

Due to the lack of a system for stable transformation of Chlamydia, little is known about transcriptional regulation of its developmental cycle. The genome sequence of C. trachomatis (11) revealed several genes whose predicted protein products had sequence conservation to bacterial transcription factors. These included a pair of proteins with similarity to the histidine kinase response regulator pairs of two-component systems, as well as the sigma factor (sigma 54) it is predicted to activate. Interestingly, whereas many bacteria possess several two-component systems for adapting to various environmental changes, the NtrB/AtoS-NtrC/AtoC orthologs represent the only complete two-component system identified in the chlamydial genome.

Because only one sensor kinase response regulator pair has been identified in the genome, we hypothesized that the chlamydial two-component system is functional and, together with sigma 54, mediates the transcriptional regulation of stages of its developmental cycle. Our results show that Chlamydia has a functional two-component system, capable of autophosphorylation and phosphotransfer reactions, and whose expression occurs during the transition from RB to EB. Now that function has been demonstrated, we have named these genes ctcB and ctcC for chlamydial two-component system.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains-- C. trachomatis serovar D was propagated in monolayers of HeLa 229 cells, and serovar L2 was propagated in L929 cells in spinner flasks. RPMI 1640 tissue culture medium (Invitrogen) was supplemented with 5-10% fetal bovine serum and 50 µg/ml vancomycin. Chlamydial EB were isolated by sonic treatments of cell suspensions and purified by ultracentrifugation over 30 and 30/44% renograffin gradients as described by Koehler et al. (12). Aliquots were frozen at -80 °C in sucrose-phosphate-glutamate buffer.

Construction of C. trachomatis Two-component System Expression Vectors-- The pET system (Novagen, Inc.) was the expression system used for overexpression of cloned CtcB and CtcC. ctcB from C. trachomatis serovar D was amplified by polymerase chain reaction (PCR) using primers 5'-GAATTCGATGCCAAAAATCGACACTTG-3' and 5'-CTCGAGAGCGGGAGTCCATAG-3', and ctcC was amplified using primers 5'-GAGCTCCATGTCGATAGAACACATTCTTATTATTGACGATGATCCC-3' and 5'-CTCGAGTAAGAGAGCGAGCATAGAAGGAGTGATCGC-3' (Operon). The PCR products were cloned into TOPO vector (Invitrogen). The ctcB PCR product was digested with EcoRI and XhoI, and the ctcC PCR product was digested with SacI and XhoI. Each fragment was subcloned into pET21b expression vector.

The CtcC-D54A mutant was prepared using the Stratagene QuikChange site-directed mutagenesis system according to manufacturer's instructions (Stratagene). Primers selected were 5'-TCGATCTGATTATTTCCGCTATGAATATGCCTGATGGTTC-3' and 5'-GAACCATCAGGCATTTCATAGCGGAAATAATCAGATCGA-3'.

Expression and Purification of CtcB and CtcC-- The pET21b constructs were transformed into E. coli strain BL21(DE3). Bacterial cultures were grown at 37 °C in 100 ml of Luria broth with 100 µg/ml ampicillin. At OD600 of 0.6, cultures were induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 2 h. Overexpressed recombinant CtcB and CtcC were found in insoluble inclusion bodies and purified under denaturing conditions. Induced E. coli were pelleted by centrifugation at 8000 × g for 10 min and resuspended in 50 ml of wash buffer (10 mM HEPES, pH 7.2, 5 mM EDTA, and 0.1% Triton X-100), sonicated briefly on ice, and centrifuged. The pellet was washed three times with wash buffer and dissolved in 5 ml of phosphate buffer, pH 6.8, with 6 M guanidine hydrochloride for 1 h on ice. After a final centrifugation of 39,000 × g for 20 min, supernatant containing denatured recombinant protein was loaded on a nickel resin column according to manufacturer's instructions (Novagen, Inc.). Denatured protein was refolded by dialysis at 4 °C against refolding buffer (30 mM Tris-HCl, pH 7.6, 200 mM KCl, 1 mM EDTA, 5 mM dithiothreitol, 10% (v/v) glycerol) containing 6 M urea (13). The buffer was exchanged for decreasing concentrations of urea (3, 2, 0.5, 0, 0 M) for at least 4 h per buffer exchange. Refolded protein was divided into 50-µl aliquots and stored at -20 °C.

RT-PCR-- C. trachomatis serovar L2 was used to infect 8 × 105 cells/ml L929 cells at an multiplicity of infection of 1 in a 1-liter spinner flask. Total RNA was extracted from infected cells at 0, 4, 10, 24, and 48 h postinfection using TRIzol reagent (Invitrogen) according to manufacturer's instructions, followed by DNase I treatment to remove contaminating DNA. Primer pairs were designed according to the predicted open reading frames from the C. trachomatis serovar D genome and used to amplify 400-bp regions within the ctcB and ctcC coding sequences. euo and omcB were used as early and late expression controls, respectively. Primer pairs used were as follows: ctcB 5'-TCTCACGACTTCCTCTTCCT-3' (forward) and 5'-CAGGATCTATAGAGCGCTGT-3' (reverse), ctcC 5'-CCAGCGCTACAACAAGACA-3' (forward) and 5'-TGGCTTGCAAATGGATCGGA-3' (reverse), euo 5'-GCAGCAAGAAGAGAATGCTG-3' (forward) and 5'-TGCGCTCAGCTTCCTTCT-3' (reverse), and omcB 5'-TGCAACAGTATGCGCTTGTC-3' (forward) and 5'-AAGACCAATCTGCTCCTGCA-3' (reverse). cDNA synthesis was conducted using Moloney murine leukemia virus-reverse transcriptase (Invitrogen) and 1 µg of RNA at 37 °C for 1 h. PCR reactions were conducted using Amplitaq polymerase (PerkinElmer Life Sciences) and consisted of 30 cycles of 95 °C denaturation, 52-55 °C annealing, and 72 °C extension. Controls with no RT were used for each PCR reaction, and chlamydial DNA controls were used for each primer pair.

Gel Electrophoresis and Western Blot-- Purified C. trachomatis serovar D EB were pelleted and lysed with lysis buffer (0.05 M Tris-HCl, pH 7.4, 0.15 M NaCl, 0.01 M EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 0.5% Triton X-100). Genomic DNA was digested with DNase I. Protein samples were resolved on 12% SDS-PAGE gels and stained with Coomassie Blue or transferred to nitrocellulose. CtcB and CtcC were detected using mouse sera raised against recombinant proteins.

Seminative gel electrophoresis was conducted according to Cullen et al. (14). CtcC was separated on 10% polyacrylamide gels with SDS present only in the running buffer and not in the sample buffer or gel.

Phosphorylation Assays-- Autophosphorylation assays were carried out with reaction volumes of 10 µl of 12 µM recombinant CtcB in TEDG buffer (50 mM Tris-HCl, pH 7.3, 0.5 mM EDTA, 2 mM dithiothreitol, 50 mM KCl, and 10% (v/v) glycerol) supplemented with 5 mM MgCl2, MnCl2, CaCl2, FeCl2, CuCl2, or ZnSO4. CtcB was preincubated at 25 °C for 2 min before addition of 0.2 µM [gamma -32P]ATP (6000 Ci/mmol, final concentration, Amersham Biosciences) to the reaction mixture and allowed to incubate for indicated times. Reactions were stopped with 2× SDS-PAGE sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 1.42 M beta -mercaptoethanol, 20% glycerol, and 10 µg of bromphenol blue) and loaded on a 10% SDS-PAGE gel, which was dried and exposed to a PhosphorImager.

Phophotransfer assays were conducted by allowing 6 µM CtcB to autophosphorylate for 30 min, followed by the addition of purified 6.65 µM CtcC in 20 µl of TEDG buffer, pH 6.5, with 5 mM MgCl2. Reactions were stopped at indicated times. Controls included a reaction mixture with CtcB~P alone and a reaction mixture with CtcC and [gamma -32P]ATP. Reactions were separated on a 12% SDS-PAGE gel, which was dried and exposed to a PhosphorImager.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sequence Analysis of CtcB and CtcC-- ctcB(CT467) is comprised of a 1059-bp sequence, predicted to encode a 352-amino acid protein with a molecular mass of 39.9 kDa. A sequence similarity search in GenBankTM showed that CtcB was homologous to several histidine kinases, having the highest similarity scores with E. coli AtoS (15), M. xanthus PilS (16), and NtrB of several bacterial species. However, the similarity scores against these sequences were relatively low (22% identity, 41% similarity to E. coli AtoS). Alignments of the sequences (Fig. 1) showed that CtcB possesses most of the conserved motifs characteristically associated with histidine kinases. These motifs include blocks H (containing the His161 autophosphorylation site), N, G2, and F (1). Block G1, which comprises one of the glycine-rich, nucleotide-binding sites, is not conserved in CtcB. The CtcB sensor domain contains regions with homology to PAS domains (amino acids 28-96), which are sensors of overall energy levels of a cell (17). CtcB does not possess an outer membrane leader sequence and is predicted to be cytosolic.


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Fig. 1.   Alignment of predicted amino acid sequence of C. trachomatis CtcB with sequences of several histidine kinases. Identical and conserved residues are boxed. From top to bottom, E. coli AtoS (15), M. xanthus PilS (16), and S. typhimurium NtrB (18). Predicted membrane spanning and leader signal sequences have been deleted in AtoS and PilS. Conserved phosphorylation site (His160) is indicated by asterisk. Separate domains and conserved block motifs (H, N, G1, F, and G2) are indicated by brackets.

ctcC (CT468) is comprised of a 1161-bp sequence, predicted to encode a 386-amino acid protein with a molecular mass of 43.2 kDa. A sequence similarity search in GenBankTM showed good correlation with sigma 54 activator sequences. The highest similarity scores (40% identity, 62% similarity) were found with E. coli AtoC (18), Pseudomonas aeruginosa PilR (19), and NtrC from several bacterial species. Alignment of the sequences (Fig. 2) revealed that CtcC has all of the conserved residues found to be necessary for function in other response regulators, including the Asp54 phosphorylation site, and Asp10, Asp11, Lys104, and Thr82, which, together with Asp54, are predicted to constitute the active site in the receiver domain (amino acids 1-115) (20). In addition, the ATPase domain (amino acids 161-305) has high sequence similarity to the AAA+ family of ATPases, characteristic of NtrC-related transcriptional regulators. This domain includes the Walker A box, which has an ATP-binding motif (GESGCGKE), and the predicted sigma 54 interaction site (GAFTGA) (21). However, the CtcC sequence is lacking the C-terminal helix-turn-helix motif that is responsible for binding to DNA upstream of the sigma 54 promoter (22, 23). If functional, CtcC represents the only sigma 54 activator identified lacking the C-terminal DNA-binding domain.


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Fig. 2.   Alignment of predicted amino acid sequence of C. trachomatis CtcC with sequences of several response regulators. Identical and conserved residues are boxed. From top to bottom, E. coli AtoC (15), P. aeruginosa PilR (19), and S. typhimurium NtrC (18). Conserved phosphorylation site (Asp54) is indicated by the asterisk. Important residues in the catalytic domain (Asp10, Asp11, Lys104, Thr82) are indicated by open circles, and separate domains are indicated by brackets.

Expression of CtcB and CtcC-- Because CtcB and CtcC sequence analysis revealed that they are unique in comparison to other two-component systems, we first determined whether the genes are transcribed and translated during the developmental cycle. To determine the expression pattern of chlamydial ctcB-ctcC, with respect to the developmental cycle, RT-PCR was used to detect transcripts of ctcB and ctcC at various time points postinfection (Fig. 3). Time points selected (0, 4, 10, 24, and 48 h postinfection) were representative of temporal classes of transcription during the chlamydial developmental cycle (24). euo (25) and omcB (26) represented as early and late expression controls, respectively (data not shown). Both ctcB and ctcC transcripts were first detected at 24 h postinfection (Fig. 3), indicating late expression in the developmental cycle, correlating with RB to EB reorganization.


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Fig. 3.   RT-PCR analysis of ctcB and ctcC during developmental cycle. Total RNA was extracted from C. trachomatis-infected cells at indicated time points postinfection. Control reactions without RT accompanied each time point, and chlamydial DNA was used for positive PCR controls (data not shown).

To detect translated gene products in C. trachomatis, mouse antisera were raised against recombinant proteins (see below) and used to immunoblot lysates of purified EB (Fig. 4). Antisera against CtcB recognized a distinct band of Mr ~40,000, corresponding to the calculated mass of CtcB. Antisera against CtcC detected a distinct band at Mr 45,000, corresponding approximately to the calculated mass of CtcC. Neither protein was detected in chlamydial RB (data not shown). The presence of CtcB and CtcC in EB lysates is consistent with the developmentally late expression profiles for each gene.


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Fig. 4.   Western blot of CtcB and CtcC in EB lysates. Lysates of C. trachomatis EBs were separated on a 12% SDS-PAGE gel and transferred to nitrocellulose. Antisera raised against CtcB detected a band at 40 kDa, and antisera raised against CtcC detected a band at 45 kDa.

Purification of Recombinant Proteins-- Having established that ctcB and ctcC are transcribed and translated, we next characterized the function of the chlamydial two-component system by producing recombinant CtcB and CtcC for in vitro functional assays. Recombinant CtcB and CtcC were overexpressed in E. coli with His tags to facilitate purification. Coomassie Blue staining and immunoblotting of SDS-PAGE gels with E. coli lysates expressing CtcB or CtcC showed overexpressed bands at Mr 44,000 and Mr 47,000, respectively (Fig. 5). These sizes corresponded approximately to the calculated masses of recombinant protein with His tags. However, both CtcB and CtcC were found in insoluble fractions of the lysates (data not shown). To obtain soluble proteins, purified inclusion bodies were denatured in 6 M guanidine hydrochloride and refolded in decreasing concentrations of urea. The refolded proteins maintained solubility and were used in in vitro functional assays. Coomassie Blue staining of SDS-PAGE gels showed a single distinct band for each purified protein (Fig. 5). Immunoblotting with antisera against CtcB detected only the full-length purified protein (Fig. 5A), and antisera against CtcC detected the full-length purified protein, as well as some smaller minor bands that probably represent degradation products (Fig. 5B).


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Fig. 5.   Overexpression and purification of CtcB and CtcC from E. coli. Recombinant CtcB (A) and CtcC (B) were purified from insoluble E. coli inclusion bodies using a nickel column. Cell lysates and purified protein were analyzed by SDS-PAGE and Western blot. Lanes 1 and 4, E. coli/pET21b (no insert); lanes 2 and 5, E. coli expressing recombinant protein after induction with isopropyl-1-thio-beta -D-galactopyranoside; lanes 3 and 6, purified recombinant protein. Antisera raised against CtcB and CtcC were diluted 1:10,000 for Western blot detection.

Oligomerization of CtcC-- Sequence analysis revealed that ctcC was missing the C-terminal DNA-binding domain that is found in sigma 54 activators of other organisms. Because DNA binding is believed to facilitate oligomerization of NtrC-like activators in proximity to sigma 54 (3), we hypothesized that if CtcC is functional it must be capable of oligomerization, despite the absent C-terminal domain. Therefore, purified CtcC was resolved on a seminative PAGE gel and detected it by immunoblot. Bands were detected at 51, 110, and 220 kDa, corresponding approximately to the expected sizes of CtcC monomers, dimers, and tetramers, respectively (Fig. 6).


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Fig. 6.   Oligomerization of CtcC. Purified CtcC (7.5 µg) was resolved on a seminative 10% polyacrylamide gel with 1% SDS in the running buffer only. Western blot of CtcC using anti-His(C-term) antibody (1:5000), and ECL detection showed monomer, dimmer, and tetramer complexes of protein.

Autophosphorylation of CtcB-- Two-component systems possess a unique phosphotransfer mechanism in which a sensor kinase autophosphorylates a histidine residue using ATP as a phosphodonor and then transfers the phosphate to an aspartate residue in the response regulator. CtcB has the conserved His161 that is autophosphorylated in histidine kinases, but is missing one of the glycine-rich motifs (G1) predicted to be responsible for nucleotide binding. Therefore, it was important to determine whether CtcB was capable of autophosphorylation using ATP as a phosphodonor.

In vitro phosphorylation assays were conducted to determine CtcB autokinase activity. Because histidine kinases are known to have different divalent cation requirements for autophosphorylation, we varied the reaction conditions by adding Mg2+, Mn2+, Ca2+, Fe2+, Zn2+, or Cu2+ to the reaction buffer (Fig. 7A). CtcB became labeled in the presence of Mg2+ and Mn2+, and, to a lesser degree, in the presence of Fe2+. To measure the temporal process of the reaction, the phosphorylation reaction was stopped at various time points after addition of [gamma -32P]ATP (Fig. 7B). CtcB became labeled after 2 min, and the autophosphorylation reaction continued for at least 2 h.


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Fig. 7.   Autophosphorylation of CtcB. Purified CtcB was labeled with [gamma -32P]ATP, resolved by SDS-PAGE, and the dried gel was exposed to a PhosphorImager. A, divalent cations necessary for CtcB autophosphorylation include Mg2+, Mn2+, or Fe2+, but not Ca2+, Zn2+, or Cu2+. B, time course experiment shows autophosphorylation by 1 min and increase in label for up to 2 h.

Transfer of Phosphate from CtcB-P to CtcC-- If CtcB-CtcC comprise a functional two-component system, it should have both autophosphorylation and phosphotransfer activity. CtcB was shown to have autokinase activity and should, therefore, be capable of transferring the labeled phosphate to CtcC. Purified, recombinant CtcC was added to autophosphorylated CtcB, and samples were taken at various time points (Fig. 8A). Between 1 and 10 min, CtcB-32P lost its label, but only in the presence of CtcC. Autophosphorylation of CtcB continued in the absence of CtcC, indicating the necessity of CtcC for removing the label. Dephosphorylation of CtcB occurred more rapidly than the autophosphorylation reaction, indicating a high turnover of CtcC phosphorylation. To show specificity of phosphotransfer to CtcC, we generated a mutant whose predicted phosphorylation site was changed to an alanine (CtcC-D54A). Addition of CtcC-D54A to CtcB-32P did not cause a depletion of label in CtcB-32P, demonstrating that phosphotransfer is specific for CtcC containing the Asp54 phosphoacceptor (Fig. 8B).


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Fig. 8.   Phosphotransfer from CtcB-32P to CtcC. A, CtcB-32P was added to CtcC and allowed to react for indicated times. CtcB-32P became dephosphorylated over time, but only in the presence of CtcC. Controls included CtcB-32P without CtcC (lane 1) and addition of [gamma -32P]ATP to CtcC alone (lane 6). B, CtcC-D54A was added to CtcB-32P and allowed to react for indicated times. CtcC-D54A did not cause dephosphorylation of CtcB-32P. Reaction mixtures were resolved by SDS-PAGE, and the dried gel was exposed to a PhosphorImager.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sequence analysis of CtcB and CtcC revealed some important differences from other two-component systems. First, CtcB had relatively low sequence similarity to other histidine kinases and was missing the signature G1 motif in the ATP-binding cleft. Since other functional histidine kinases have been identified with missing block motifs (20), the missing G1 motif in CtcB should not ablate its function as an autokinase, but likely alters the kinetics of the autophosphorylation reaction (1). The response regulator, CtcC, had highly conserved receiver and catalytic domains and should, therefore, be capable of catalyzing the phosphotransfer reaction from CtcB. CtcB and CtcC had autophosphorylation and phosphotransfer activity in vitro, but both reactions were weaker than those of enteric NtrB and NtrC (data not shown). The weaker activity could be attributed to several possibilities. First, because CtcB is missing part of its nucleotide-binding cleft, it likely binds the ATP phosphodonor less efficiently than orthodox histidine kinases. Second, while CtcB-P maintained stable phosphorylation for up to 2 h, CtcC-P appears to have a very short half-life, as has been described for some other response regulators (1). Alternatively, because both recombinant proteins were refolded, the percentage of proteins folded to native conformation is unknown. However, when we purified soluble CtcB as a maltose-binding protein fusion protein, the autophosphorylation rate was limited as well (data not shown), suggesting that refolding was not a significant factor in reducing its activity. Because cytosolic conditions of Chlamydia are unknown, the in vitro conditions of the assays may not have been optimal for chlamydial proteins.

That chlamydial CtcB-CtcC autophosphorylation and phosphotransfer reactions were weak was not surprising, given that Chlamydia seems to acquire mildly deleterious mutations at a relatively high rate (27). As an obligate intracellular pathogen that does not encounter other organisms in its metabolically active form, Chlamydia undergoes lower selective constraints and higher mutation rates that become fixed in the population, analogous to bacterial endosymbionts (28). As a result, chlamydial proteins and enzymes tend to function less efficiently and have weaker catalytic activity than enzymes from free-living bacteria (29, 30).

CtcC is the only sigma 54-activator homolog identified that lacks a C-terminal DNA-binding domain. NtrC of enteric bacteria dimerizes in solution, and higher ordered oligomerization is facilitated by phosphorylation and binding of DNA upstream of the promoter (3). While the major dimerization determinant of enteric NtrC is found in the C-terminal domain (31), truncation studies have shown that other sigma 54 activators, such as DctD and NifA of Rhizobium, do not require this domain for activation of sigma 54 (32, 33). Since oligomerization of NtrC-like response regulators is essential for activation of sigma 54, we hypothesized that CtcC would be capable of aggregating in solution despite its missing DNA-binding domain. Our experiments confirmed that CtcC was able to dimerize and tetramerize in the absence of DNA. Therefore, CtcC likely activates transcription by oligomerizing in proximity to sigma 54 holoenzyme, thereby bypassing the DNA binding requirement.

The sigma 54 holoenzyme mode of transcription requires long stretches of intergenic DNA for binding the transcriptional activator to sequences upstream of the promoter. The Chlamydiae are very distantly related to other eubacteria, based on rRNA sequences (34). That CtcB-CtcC had significant differences from other two-component systems has important evolutionary implications, especially in light of the fact that other pathogens, including Neisseria spp., have abandoned their requirement for sigma 54-regulated transcription, based on the presence of rpoN (sigma 54) pseudogenes and non-functional sigma 54 promoters (35). The sigma 54 holoenzyme mode of transcription is advantageous to bacteria because it provides tight control of gene expression; activators of sigma 54 allow physiological or environmental conditions to govern whether genes are silent or highly expressed. However, a disadvantage is that this mode of transcription requires long stretches of intergenic DNA, thereby dictating an additional requirement for a larger chromosome. Therefore, the pressures that select for compact bacterial genomes may also select against sigma 54 holoenzyme-mediated transcription (36). The chlamydial genome is very small in comparison to other eubacteria (11), and the missing DNA-binding domain of CtcC suggests that binding to an upstream enhancer-like element is not necessary for sigma 54 activation. Therefore, without the requirement for DNA binding by CtcC for activation of sigma 54 holoenzyme, it is likely that selective pressures have enabled the development of a more compact genome for Chlamydia.

In addition to showing that CtcB and CtcC comprise a functional two-component system, it was important to show that Chlamydia expressed these genes to rule out the possibility that they were pseudogenes. The chlamydial ctcB-ctcC transcripts were detected at 24 h, about the time of transition from RB to EB. Consistent with the late gene expression profile, antisera raised against recombinant proteins detected CtcB and CtcC in EB lysates. While the RT-PCR data distinctly showed late transcription of these genes, it is unclear whether the presence of the CtcB in EB is functionally significant or results from residual undegraded proteins no longer necessary for transcriptional regulation. However, as many other two-component systems are expressed constitutively for efficient adaptation to changing environments, the late developmental expression of the chlamydial two-component system has implications for having a functional role in late developmental gene regulation. Therefore, CtcB and CtcC likely activate a subset of late genes or other transcription factors required for reorganization of RB to EB.

Although, through genomic analysis, no obvious role has been assigned to the chlamydial two-component system, we speculate that it may be necessary for regulation of the unique developmental cycle of Chlamydia. sigma 54 promoters have been identified for two chlamydial genes of unknown function (38, 39), but DNA binding by sigma 54 has not been demonstrated. Sequence analysis showed that CtcB possesses a PAS domain, which is a known sensor of energy levels or redox state (17). Earlier studies have shown that both energy levels and redox state are important determinants in the chlamydial developmental cycle (40-42). Entry of EB into host cells results in a reduction of disulfide-linked outer membrane proteins, resulting in increased membrane flexibility, relaxation of major outer membrane protein porin size, and increased uptake of essential nutrients. Likewise, upon decrease of available energy sources or reducing agents, RB development is hindered, resulting in a retardation of metabolic activity and oxidation of sulfhydryl groups of outer membrane proteins (43). As developmentally late-expressed proteins containing a redox sensing domain, the chlamydial two-component system likely has a role in late gene activation, perhaps in regulation of differentiation from RB to EB.

    ACKNOWLEDGEMENTS

We thank Dr. S. Kustu (University of California, Berkeley, CA) for her critical insight and helpful advice and Dr. E. Nicholson (University of California, Berkeley, CA) for his technical assistance in the CtcC-D54A mutant construction.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant AI42156.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Division of Infectious Diseases, School of Public Health, 140 Warren Hall, Berkeley, CA 94720-7360. E-mail: rss@uclink4.berkeley.edu.

Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M212170200

    ABBREVIATIONS

The abbreviations used are: EB, elementary body; RB, reticulate body; RT, reverse transcriptase.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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10. Deleted in proof
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