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INTRODUCTION |
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
54 promoters by oligomerizing at an upstream
enhancer-like element of DNA (2, 3), interacting with
54 holoenzyme via DNA looping (4), and hydrolyzing ATP
to initiate open complex formation (5). Genes controlled by this
subfamily of
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
factors for
regulation of stage-specific gene expression. Bacillus
subtilis sporulation is regulated by its cascade of
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
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 (
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
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.
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EXPERIMENTAL PROCEDURES |
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-
-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 [
-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
-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 [
-32P]ATP.
Reactions were separated on a 12% SDS-PAGE gel, which was dried and
exposed to a PhosphorImager.
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RESULTS |
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.
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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
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
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
54 promoter (22, 23). If functional, CtcC
represents the only
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.
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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).
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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.
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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- -D-galactopyranoside;
lanes 3 and 6, purified recombinant protein.
Antisera raised against CtcB and CtcC were diluted 1:10,000 for
Western blot detection.
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Oligomerization of CtcC--
Sequence analysis revealed that
ctcC was missing the C-terminal DNA-binding domain that is
found in
54 activators of other organisms. Because DNA
binding is believed to facilitate oligomerization of NtrC-like
activators in proximity to
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.
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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 [
-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 [ -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.
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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
[ -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.
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DISCUSSION |
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
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
54 activators, such as DctD and NifA of
Rhizobium, do not require this domain for activation of
54 (32, 33). Since oligomerization of NtrC-like response
regulators is essential for activation of
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
54 holoenzyme, thereby bypassing the DNA
binding requirement.
The
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
54-regulated transcription, based on the
presence of rpoN (
54) pseudogenes and
non-functional
54 promoters (35). The
54
holoenzyme mode of transcription is advantageous to bacteria because it
provides tight control of gene expression; activators of
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
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
54
activation. Therefore, without the requirement for DNA binding by CtcC
for activation of
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.
54 promoters have been identified
for two chlamydial genes of unknown function (38, 39), but DNA binding
by
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.