Download Chlamydia trachomatis RNA polymerase major sigma subunit

THE JOURNAL OF BIOLOGICAL CHEMISTRY
0 1990 by The American Society for Biochemistry
Chlumydia
and Molecular
trachomatis
Vol. 265, No. 22, Issue of August 5, pp. 13206-13214,199O
Biology, Inc.
Printed in U.S.A.
RNA Polymerase Major Q Subunit
SEQUENCE AND STRUCTURAL COMPARISON OF CONSERVED AND UNIQUE REGIONS WITH ESCHERZCHIA
COLI 2’ AND BACILLUS
SUBTILIS
u43*
(Received
Jane
E. Koehler$#,
Richard
R. BurgessV,
Nancy
E. ThompsonV,
and Richard
for publication,
January
24, 1990)
S. Stephens/**
From the $Department
of Medicine,
Divisions
of Infectious
Diseases
and Clinical Pharmacology
and Experimental
Therapeutics,
11Departments
of Laboratory
Medicine
and Pharmaceutical
Chemistry,
and the Francis
I. Proctor
Foundation,
University
of
California.
San Francisco.
California
94143-0412.
and TMcArdle
Laboratory
for Cancer Research,
University
of WisconsinMa&son,
‘Madison,
Wiscdnsin’53706
and sequenced
the gene for the ChlaRNA polymerase
major u subunit.
The gene encodes a 66,141-dalton
protein
(u’“), intermediate
in size between
the major Q subunits
of Escherichia coli (u’“) and Bacillus subtilis (u”“). The C.
trachomatis
u’* subunit
had extensive
amino acid homology
with the U” and a4’. The u subunit
regions
purportedly
involved
in core enzyme binding
and DNA
promoter
recognition
were also highly
conserved,
despite the lack of a DNA promoter
consensus
sequence
between
E. coli and C. trachomatis
promoters
and the
inability
of E. coli holoenzyme
to specifically
transcribe chlamydial
genes. Compared
with E. coli u”,
there were some major differences
in the chlamydial
u6’ sequence,
including
a gap of 63 amino acids and an
additional
16 amino acids at the carboxyl
terminus,
which
may play some role in modifying
the U-DNA
interaction,
such that a promoter
sequence
unique
to
C. trachomatis
is recognized.
Monoclonal
antibodies
specific
for E. coli U” were used to probe for homologous structures
between
a” and ues; only one of seven
antibodies
bound specifically
to u6(j, suggesting
minimal conservation
of antigenic
sites. The chlamydial
uea
was present
in elementary
bodies and was expressed
throughout
the developmental
cycle, which
implied
that this gene encodes the major vegetative
u subunit.
Because the ability
to study the genetics
of C. trachomatis is currently
limited,
this work provides
a tool
for more detailed
study of chlamydial
promoter
structure and of coordinate
gene expression
during
the developmental
cycle.
We identified
mydia
trachomatis
Chlamydia
trachomatis
is a significant ocular and reproductive tract pathogen; infection by this organism is associated
with staggering worldwide cost, both monetary and in terms
of reproductive and ophthalmologic morbidity (1). Very little
is known about genetic regulatory functions of Chlamydiu, a
*This
research
was supported
by funds
from the John D. and
Catherine
T. MacArthur
Foundation
and National
Institutes
of
Health
Grants
EY 07757, AI 21912, GM 28575, and CA 23076. The
costs of publication
of this article
were defrayed
in part by the
payment
of page charges.
This
article
must
therefore
be hereby
marked
“aduertisement”
in accordance
with 18 U.S.C. Section
1734
solely to indicate
this fact.
The nucleotide
sequence(s)
reported
in thispaper
has been submitted
to the GenBankTM/EMBL
Data
Bank
with
accession
number(s)
505546.
f Supported
by National
Research
Service Award 5 T32 GM 07546
from the National
Institutes
of Health.
** Research
Award recipient
from Research
to Prevent
Blindness.
eubacterium and obligate intracellular parasite with no known
close phylogenetic relatives. Chlumydia is incapable of generating ATP and thus parasitizes the host ATP to sustain
metabolic activity, as well as the host ribonucleoside triphosphate pool for RNA synthesis (2). Because of their unique
developmental life cycle, the chlamydiae have been assigned
to their own order, Chlamydiales (3). In the initial stages of
host cell infection by the chlamydial elementary body (EB),’
differentiation to the metabolically active reticulate body
(RB) occurs. After 20 to 25 h of vegetative growth, differentiation from RB to infectious EB occurs prior to release from
the host cell. Under some conditions, persistent infection is
noted, both clinically and in tissue culture, although the
mechanism by which this occurs is unknown (4).
Several gene products that are temporally regulated and
associated with RB:EB differentiation have been characterized. We have previously shown that differential transcription
of the chlamydial major outer membrane protein gene, ompl,
occurs over the time course of the developmental cycle: transcription of this gene occurs both early and late in the cycle
and is initiated independently from two promoter regions that
have little sequence homology (5). These data suggest that
temporal regulation of some chlamydial gene families may be
controlled or modulated by substitution of alternate u subunits, as occurs in Bacillus subtilis during endospore formation
(6).
Development of a genetic or transcriptional expression
system
in Chlamydia
has been unsuccessful, due to unique
difficulties inherent in working with Chlumydia. Unsuccessful
attempts have been made to transcribe chlamydial genes in
Escherichia
coli, to culture Chlamydia outside host cells, and
to amass large enough quantities of Chlamydia for RNA
polymerase purification (2, 7). Defining the structure of chlamydial RNA polymerase and the characteristics of a major u
subunit, in addition to any minor u subunits, is essential for
the study of transcriptional regulation and promoter structure
of Chlamydia.
The composition of DNA-dependent RNA polymerase was
first described for E. coli in 1969 (8,9). There are four subunits
in the core enzyme of eubacteria ((Y&Y); the addition of a
sigma (u) subunit creates the holoenzyme form of RNA polymerase and confers specificity on the process of transcription through promoter recognition. The gene sequence for
1 The abbreviations
used are: EB, elementary
body; RB, reticulate
body; 0, 0 subunit
of RNA polymerase;
c”, major Q subunit
of E. coli
RNA polymerase;
u43, major (T subunit
of B. subtilis RNA polymerase;
SDS-PAGE.
sodium
dodecvl
sulfate-polvacrylamide
gel electropho_
resis; kb, kilobase(
ORF, open reading frame;
us, major (r subunit
of C. trachomatis
RNA polymerase.
13206
Chlamydia
trachomatis
RNA Polymerase Major a@ Subunit
each of the E. coli subunits
has been determined,
in addition
to the
gene
organization
and
function
for several
of the
subunits
(10).
The
e subunit
has been
studied
intensively
because
of its role in the regulation
of transcription
and its
potential
for controlling
differentiation
and cellular
responses
during
adverse
environmental
conditions.
The gene sequence
for the major
u subunit
of several
procaryotic
organisms
has
been determined,
and striking
homologies
have
been
noted
among
diverse
organisms
(11, 12).
To elucidate
the subunit
composition
of RNA
polymerase
of C. trachomatis,
we analyzed
immunoblots
utilizing
monoclonal
antibodies
specific
for different
regions
of the E. coli
major
c subunit
(u70). Specific
reactivity
occurred
with
one
anti-a
antibody
which
was previously
reported
to bind to E.
coli holoenzyme
and to inhibit
transcription
(13).
To determine
the
gene
sequence
of the chlamydial
u subunit,
we
synthesized
oligonucleotides
that
code for conserved
amino
acid sequences
from
E. coli ur” and the B. subtilis major
a
subunit
(u”~) (14) and used the oligonucleotides
as primers
for
DNA
amplification
of the C. trachomatis
u gene homolog
(15).
The amplified
DNA
was used to probe
a C. trachomatis
DNA
library,
and the chlamydial
major
u gene was identified
and
sequenced.
The
C. trachomatis
gene encoded
a u protein
of
66,141
daltons,
intermediate
in size between
the major
u
subunit
of E. coli (70,000
daltons)
(16) and B. subtilis (43,000
daltons)
(17).
Regions
previously
reported
to be highly
conserved
among
diverse
organisms
were also highly
conserved
in C. trachomatis,
despite
a lack of consensus
promoters
and
a highly
divergent
evolutionary
origin
compared
with
other
organisms.
Identification
of the subunit
composition
and sequence
of chlamydial
RNA
polymerase
provides
an essential
tool
for the study
of promoter
structure
and
function,
in
addition
to temporal
gene expression
in C. trachomatis.
EXPERIMENTAL
PROCEDURES
Bacterial
Strains-C.
trachomatis
strain L2/434/Bu
was grown in
an L929 mouse fibroblast
cell line in spinner
flasks. RPM1 1640 tissue
culture
medium
(Flow
Laboratories,
Inc.) was supplemented
with
10% fetal bovine
serum,
0.1 mg/ml
vancomycin,
and 0.1 mg/ml
streptomycin.
Host L929 cells (at a cell density
of approximately
7 x
105/ml)
were infected
with chlamydial
EBs (lo9 inclusion
forming
units).
EBs were harvested
at 48 h by centrifuging
at 3,000 rpm in a
GSA rotor
(Sorvall
RC-5B).
The resuspended
pellet was sonicated
for a total of 45 s on high setting
(Braun-Sonic
2000 Sonicator).
The
lysate was centrifuged
at 1,200 rpm, and the supernatant
was withdrawn
and centrifuged
at 16,000 rpm for 30 min. The pellet was
resuspended
and puritied
by ultracentrifugation,
first over 30% Renografin
(E. R. Squibb
& Sons) (18) and then over a discontinuous
Renografin
gradient
to separate
EBs from RBs and any remaining
host material.
The purified
EBs were used to prepare
protein
lysates
and DNA.
E. coli Q359 was used to prepare
the bacteriophage
X1059 library
(19); E. coli XLI-blue
and bacteriophage
Ml3 were used in sequencing
(20). B. subtilis ATCC
strain 6633 was used for blotting
procedures,
and E. coli strain TBl was used in immunoblots
and Southern
blots.
Immunoblot
Analysis
of C. trachomatis
L2 RNA
PolymeraseGradient-purified
EBs were diluted
1:lO in 10 mM Tris buffer
(pH 8)
and sonicated
for 15-30 s. Sample buffer
containing
P-mercaptoethanol (P-ME)
and sodium dodecyl
sulfate
(SDS) was added to give a
final concentration
of 5% and 2%, respectively.
E. coli and B. subtilis
were prepared
by centrifuging
a 250-ml
overnight
suspension
in a
GSA rotor for 5 min at 5,000 rpm (Sorvall
RC-5B)
and resuspending
the organisms
in 5 ml of 25 mM Tris buffer
(pH 7.5). Sample buffer
containing
P-ME and SDS was added to give a final concentration
of
5% B-ME and 2% SDS. All samples were sonicated
for 15 s and boiled
for 3 min just before loading
onto a 12% SDS-polyacrylamide
gel.
After electrophoresis,
proteins
were transferred
to nitrocellulose
paper by the method
of Towbin
et al. (21). The nitrocellulose
paper was
blocked
overnight
in 1% bovine serum albumin
at 4 “C. The following
mouse monoclonal
antibodies
against
E. coli RNA polymerase
subunits were tested:
u70-specific
(lH6,
3D3, 2D4, 2D1, lS4, 2F8, and
2GlO) (Ref. 13), a-specific
(3RA1,
4RA1, 5RA1, and 4RA2),
and a
single monoclonal
antibody
against the fl subunit
(4RBl).
The monoclonal
antibodies
were diluted
1:500 or l:l,OOO and incubated
with
the nitrocellulose
for 30 min at 25 “C. A goat anti-mouse
IgG, alkaline
phosphatase-conjugated
antibody
(1:7,500
dilution)
was added, and
immunoreactivity
was detected
using nitro blue tetrazolium
and 5bromo-4-chloro-3-indolyl
phosphate
color
development
substrates
(Promega),
as described
previously
(22). The molecular
weight
(M*)
of immunoreactive
bands was estimated
by relative
mobility
compared with the M, of empirically
determined,
prestained
M, standards:
phosphorylase
b, 130,000; bovine serum albumin,
75,000; ovalbumin,
50,000; carbonic
anhydrase,
39,000; soybean trypsin
inhibitor,
27,000;
and lysozyme,
17,000 (Bio-Rad
Laboratories).
Time Course of 5 Gene Expression-L929
host cells were grown in
l-liter
spinner
flasks to a density
of approximately
7 x lo5 cells/ml
and infected
with lo9 inclusion
forming
units of purified
EBs. Samples
were withdrawn
at the time of infection
(time 0) and at subsequent
hours after infection
as indicated.
The infected
cells were then centrifuged
at 1200 rpm for 5 min in a Sorvall tabletop
centrifuge
(model
TSOOOB),
the supernatant
was discarded,
and the pellet was stored
immediately
at -70 “C. Two ml of loading
buffer
containing
P-ME
and SDS (5% and 2% final concentrations,
respectively)
were added
to each sample, and the sample was boiled and sonicated
prior to the
loading of the 10% SDS-polyacrylamide
gel. The proteins
were transferred to nitrocellulose
paper and probed with the 2GlO monoclonal
antibody,
as described
above.
c Gene Amplification-Synthetic
oligonucleotides
containing
5’terminal
sequences
for BamHI
and EcoRI
restriction
endonucleases
were prepared
using an automated
synthesizer
(Biosearch
8600 or
Applied
Biosystems,
Inc. 380B).
The amino acid sequences
of two
regions
highly
conserved
in E. coli and B. subtilis
0 subunits
were
reverse-translated
into oligonucleotide
sequences,
with an attempt
to
give preference
to codons
consistent
with known
chlamydial
codon
bias (23). The sequence
for the 5’ end mimer
was B’GGT
GGA TCC
GAY
CCN
GTN
CGN ATG
TAY3’;
with Y corresponding
to a
deeenerate
olieonucleotide
mix containine
C and T. R containing
A
and G, and N iontaining
G, A, T, and C. The nucleotide
sequencg
of
this primer
corresponded
to conserved
regions of E. coli and B. subtilis
amino
acids 96-101
and 100-105,
respectively
(underlined
above),
with the addition
of a BamHI
restriction
site. The sequence
for the
3’ end primer
corresponded
to the reverse complement
of E. coli and
B. subtilis amino acids 593-601 and 352-360, respectively
(underlined),
with an EcoRI
restriction
site added (5’ACC
GAA TTC NGG RTG
NCN
NAR YTT
NCG NAR NGC YTTB’).
The chlamydial
0 gene
homolog
was amplified
using these synthetic
oligonucleotides
and
Thermus
aquaticus
polymerase
in a Perkin-Elmer
Cetus DNA Thermal Cycler, with amplification
for 35 cycles, using 94 “C for melting,
37 “C for annealing,
and 72 “C for polymerization,
allowing
an extension time of 3 min (24). The amplified
gene product
of approximately
1300 base pairs was cloned into pUC18 after restriction
endonuclease
digestion
(pEl-L2).
Southern
Hybridization
Analysis-C.
trachomatis
L2/434/Bu,
E.
coli, B. subtilis, and L929 genomic
DNA was digested
with EcoRI, and
DNA restriction
fragments
were separated
by electrophoresis
in a 1%
agarose
gel. Following
separation,
the DNA was denatured
and the
gel was neutralized
prior
to the transfer
of DNA
fragments
to a
synthetic
transfer
membrane
(GeneScreenPlus,
Du Pont-New
England Nuclear)
by capillary
action.
Following
transfer
to the membrane, the DNA was denatured
and the membrane
was neutralized
and dried
at room
temperature.
The chlamydial
c amplification
product
pEl-LP
was labeled with [cy-32P]dCTP
by the random
primer
method
using commercially
prepared
reagents
(Bethesda
Research
Laboratories).
The
hybridization
solution
contained
the labeled
probe,
1% SDS, 10% dextran
sulfate,
50% formamide,
100 fig/ml
denatured
salmon
sperm
DNA,
and 1 M sodium
chloride
and was
carried
out at 42 “C for 18 h. The membrane
was washed
with 2 x
SSC (1 X SSC contains
0.15 M sodium
chloride
and 0.015 M sodium
citrate)
at room temperature
for 10 min, then 2 X SSC with 0.1%
SDS at 65 “C for 60 min, and finally,
0.1 x SSC at room temperature
for 60 min.
Selection
and Analysis
of X1059 Recombinant
Phage-The
library
of C. trachomatis
L2/434/Bu
genomic
DNA in h1059 was described
previously
(23). Phage DNA was isolated
using standard
procedures
(25). Labeled pEl-LB
(see above) was used to probe plaques produced
by transfection
of E. coli Q359 with h1059 recombinants,
as previously
described
(26,27).
Plaques producing
strong signals were selected and
purified
by reinfecting
6359. DNA
was isolated
from the selected
pbage,
digested
with
restriction
endonucleases
and analyzed
by
Southern
hybridization,
using standard
procedures
(25). Fragments
13208
Chlamydia
trachomatis
RNA Polymerase Major a66 Subunit
of interest
were cloned into pUC18,
and were mapped
by restriction
endonuclease
digestion.
Sequence of 0 Subunit
Gene-The
gene was sequenced
after ligation
into M13, using the dideoxy
sequencing
method
of Sanger et al. (28)
and modified
T7 DNA polymerase
(Sequenase,
United
States Biochemical
Corp.).
The reaction
used either fluorescent
primers,
with
the sequence
analyzed
by an Applied
Biosystems
Automatic
Sequencer (Model
370A), or [a-“‘S]dATP,
with the sequence
read following
autoradiography.
Synthetic
oligonucleotide
primers
were used when
necessaryto continue sequencing.The sequencewas confirmed by
analysis
of the complementary
strand.
Where analysis
of the complementary
strand
was not done, the sequence
was unequivocal
and
determined
by two or three independent
reactions.
RESULTS
Immunoblot
Analysis of C. trachomatis L2 RNA Polymerme-The monoclonal antibodies specific for E. coli a7’, LYand
,6 subunits, were used to probe immunoblots of C. trachomatis
EB, RB, E. coli, and L929 mouse fibroblast host cell protein
lysates. With E. coli /3-specific monoclonal antibody 4RB1, no
specific band was observed with the L929 host cell lysate or
chlamydial EB lysate (data not shown). No specific band was
noted with the four E. coli a-specific monoclonal antibodies
on immunoblots with C. trachomatis protein lysate (data not
shown).
Seven monoclonal antibodies specific for E. coli u7’ subunit
were screened. Although each antibody bound E. coli u7’ in
immunoblots, only one, 2G10, recognized a protein in the
chlamydial lysate, detecting a band at M, = approximately
70,000 (Fig. 1A). Of the antibodies that failed to bind, 3D3,
2F8, and lS4 map closely to 2GlO (13). The epitopes recognized by 2Dl and 2D4 map to the amino and carboxyl termini
of E. coli u7’, respectively.
In immunoblots with E. coli, C. trachomatis, and B. subtilis,
the monoclonal antibody 2GlO bound the g subunits of all
three genera, at an apparent M, appropriate for each by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) (80,000,
no,g-
-
---
tl-
b(
w-80I
i-
--
Jo39-
-95
70
. 2
A
B
FIG. 1. Immunoblot
E.
coli,
clonal
B.
70,000, and 55,000, respectively) (Fig. 1, A and B). Note that
the B subunit is an extremely acidic protein; consequently,
aberrant migration in the SDS-PAGE system overestimates
the actual molecular weight calculated from the nucleotide
sequence (10, 13). The 2GlO monoclonal binding data
prompted our efforts to isolate and sequence the chlamydial
c gene. The cross-reactivity of the 2GlO antibody with both
E. coli and C. trachomatis major u subunits precluded use of
this antibody to differentially screen expression libraries for
the chlamydial (r gene.
Time Course of (r Gene Expression-Fig. 2 demonstrates the
time course of C. trachomatis g subunit expression in Chlamy&a-infected L929 host cells. The immunoreactive band at
M, = 70,000 was detected at 12 h, which was similar to the
time of detection of the major outer membrane protein, a
product of the ompl gene known to be expressed early in the
developmental cycle (5). The omp2 gene product, an outer
membrane protein expressed late in the cycle, was detected
at 20 h in time course experiments using the same lysates
(data not shown). There was an apparent cross-reaction with
host cell material noted in the first four samples; this reaction
diminished as time progressed, probably due to Chlamydiainduced inhibition of host cell protein synthesis. This band
was not seen with purified EBs or RBs, implicating host cell
material as the source of the cross-reaction. The chlamydial
c subunit was detectable to the 72-h sample, although in
decreasing amounts in the final three samples due to cumulative host cell lysis and loss of cell-associated organisms that
occurs late in the cycle.
c Gene Amplification-The
polymerase chain reaction was
used to amplify the gene homolog for the chlamydial c subunit, with chlamydial DNA template and oligonucleotides
constructed
from
conserved amino acid sequences of E. coli
and B. subtilis 0 subunits (see “Experimental Procedures”).
Amplification of chlamydial DNA using these primers produced a single product of approximately 1300 base pairs; no
product was produced with identical oligonucleotides and
amplification of the control L929 host cell DNA (data not
subtilis,
antibodies
of protein lysates from C. truchomatis,
and host L929 cells probed with monospecific for E. coli 0”. After SDS-PAGE
of
protein
lysates,
separated
proteins
were electrophoretically
transferred to nitrocellulose
paper and probed with monoclonal
antibodies.
No immunoreactivity
to the chlamydial
EB protein
lysate was detected using monoclonal
antibody
2D4, although
a prominent
band
was observed
in the E. coli lysate (A). In contrast,
the monoclonal
antibody
2GlO recognized
a M, = 70,000 protein
in a C. trachomatis
EB lysate and a i~4, = 80,000 protein
in both an E. coli lysate and
purified
E. coli RNA polymerase
holoenzyme
(RNAP)
(A, arrows).
No immunospecific
band was observed
in the L929 host cell lysate.
The 2GlO monoclonal
antibody
also recognized
a M, = 55,000 protein
in the B. subtilis lysate (B, arrow).
Numbers
at the far right and left
indicate
M, (X lo-“).
$
0
4
8
12 16 20 24 36 48 52 72
Time (hours)
5
E
FIG. 2. Immunoblot
of protein lysates from C. truchomatisinfected host cells sampled sequentially
postinfection
and
probed with E. coli a”‘-specific
monoclonal antibody 2GlO. At
the times indicated
after infection
with C. truchomatis,
samples
were
taken from a single suspension
culture
of L929 host cells and solubilized in SDS sample
buffer.
After
SDS-PAGE,
separated
proteins
were
electrophoretically
transferred
to nitrocellulose
paper
and
probed
with E. coli cr70-specific
monoclonal
antibody
2GlO.
Arrow
indicates
a C. truchomatis-specific
band at M, = 70,000 detectable
within
12 h after infection.
The lane on the fur right contains
a
protein
lysate of E. coli. Numbers
at the fur right and left indicate
M,
(x lo-:‘).
Chlamydia
trachomatis
RNA Polymerase Major (T@Subunit
shown). The amplified product was cloned into pUC18 (pElL2).
Southern Hybridization
Analysis-A
Southern transfer containing C. trachomatis, E. coli, B. subtilis, and L929 DNA was
probed with labeled pEl-L2.
A specific band was seen only
with digested C. trachomatis DNA, localizing the chlamydial
0 gene to a 15kilobase
(kb) EcoRI restriction
fragment (Fig.
3). Although the E. coli monoclonal antibody 2GlO recognized
a homologous
epitope on the C. trachomatis
g polypeptide,
the C. trachomatis
gene sequence was sufficiently
different
from the E. coli gene sequence to preclude specific binding of
the probe to E. coli DNA using stringent hybridization
conditions.
Cloning and Mapping of C. trachomatis L2 g Subunit GeneUsing labeled pEl-L2
to screen the C. trachomatis L2/434/
Bu X1059 library, a clone (X1.2) was identified
which contained two BamHI/BamHI
fragments (15 and 10 kb) (Fig. 4).
By Southern hybridization,
the chlamydial
0 gene was identified on the 15-kb BamHI/BamHI
fragment
(data not
shown). Cleavage of the 15-kb fragment with Sac1 yielded two
SacI/SacI fragments of 2.2 and 2.1 kb, which were cloned into
pUC18 and probed by Southern hybridization.
The C. trachomatis major n subunit gene was localized to the 2.1-kb fragment (pJK-4) (data not shown).
Sequence of o Subunit Gene-The
gene sequence was determined by restriction
of pJK-4 with Hind111 and Sac1 and
sequencing of these fragments in M13. A 0.8-kb SacI/BamHI
fragment was also cloned in Ml3 (M800) to provide the 3’
sequence. The pEl-LB fragment was also sequenced. Restriction endonuclease sites and the sequencing strategy are shown
in Fig. 4. The C. trachomatis major 17subunit gene consisted
of 1,713 nucleotide pairs, beginning at the first AUG codon
of the open reading frame (ORF) (Fig. 5). As with E. coli u’”
(16), there was no strong ribosome binding site complementarity, and thus the amino terminus was not well defined. A
14-base pair dyad, followed by 7 thymidine
residues, was
identified at the 3’ end of the ORF. This structure resembled
a p-independent
terminator.
Beginning with the first methionine of the ORF, the chlamydial
g subunit had a calculated
molecular mass of 66,141 daltons (u@). Two fragments of the
cloned chlamydial
g66 gene, one encoding amino acids 101 to
330 and the other 327 to 543, were each cloned in the vector
pGEX, expressed as polypeptides
fused with glutathione
Stransferase and purified by glutathione
affinity chromatography (29). These chlamydial
a6fi gene products were immunoblotted using the E. coli US”-specific monoclonal
antibody
2GlO. There was specific immunoreactivity
to the fusion
polypeptide
comprised of amino acids 327 to 543 and no
1
FIG.
3. Southern
hybridization
major
o subunit
gene. C.
analysis
of
the
C.
trucho-
trachomatis, E. coli, B. subtilis, and
cell 1,929 DNA was digested with EcoRI restriction endonuclease.
DNA fragments were separated by agarose gel electrophoresis, transferred to a synthetic membrane, and probed with pEl-L2 labeled with
matis
host
[n-“P]dCTP.
Only
one band was observed,
binding
to a restriction
fragment
of 15,000
EcoRI-digested
DNA from Chlamydia.
1,,1,,,,,,,,,,,,,,,,,,,,11
2
4
representing
base pairs
6
specific
(arrow), in
10
8
12
14
16
sac1
Sac I
sac1
18
u)
ECORI
Sac I
<
Sac I
t
24
kb
EWRI
sac1
ECORI sac1
EkoBl
Barn HI
Barn HI
Barn HI
22
JY
Hind III
xbal
I
I I
Hind III
I
+-=e---
sac1
pJK-4
a
tt
Sac1
I
+
Hind III
I
I
-
sac I
Sac I
I 1
I
HindIII
I
BZSllI-II
I
MS00
es--pEl-L2
@‘CR)
%F=
chain reaction
WCR)
4. Restriction
map of the C. truchomatis
IJ” gene. The labeled polymerase
product
pEl-L2
was used to isolate a recombinant
X1059 clone containing
the C. trachomatis
0” gene from a
librarv
of C. trachomatis
L2/434/Bu
eenomic
DNA. A clone which contained
two BamHIIBnmHI
fragments
(15
and li kb (hb)) was identified
(Xi.2). ?‘he chlamydial
c gene was identified
on the 15-kb BamHI/BamHf[
fragment,
and the gene further
localized
to a 2.1-kb SacI/SacI
subclone
of the X1.2. This subclone
was ligated into pUC18
(PJK-4).
pJK-4 was cleaved with Hind111 and SacI, and the resulting
fragments
were cloned in Ml3 and sequenced.
The pEl-L2
fragment
was also sequenced.
A 0.8-kb
SacI/BamHI
fragment
was also cloned in Ml3 (M800)
to
provide the 3’ sequence.
The scale at the top represents
the number
of kilobases,
and the BamHI/BamHI
fragment
below the scale is a schematic
representation
of a restriction
endonuclease
map of the clone X1.2. The open reading
frame (ORF)
of the C. trachomatis
0” gene is indicated
by the open box between
13 and 15 kb. Arrows below
restriction
fragments
indicate
the sequencing
strategy
for the clones named at the fur right of the figure.
FIG.
13210
Chlamydia trachomatis
RNA Polymerase Major u@ Subunit
Lys Asp Gln Gly Phe Ile Thr 'I'yr Glu Glu Ile Am Glu Ile Leu Pro
AAG~cAAax;~ATcpM;TATcAAGAAATcFAT~AIlT~ccTccI~~~T~ccA~cFLj~GATcAAGITTTA~180
Pro
Ser
Phe Asp
Thr
Pm
Glu
Gin
Ile
Asp
Gin
Val
Leu
Ile
BD
Phe Leu Ala Gly bkt Asp Val Gin Val Leu Asn Gln Ala Asp Val Glu Arg Gin
~Cn;GcGGvLATGGATGITcAA~Cn;AAccAAocA~~cAGcGAcpGAwLGAAAGAAAAApGcAAGcAAAAGAGcpAGAAaX;270
Lys
Glu
Arg
Lys
Lys
Glu Ala
Lys
Glu
Leu Glu
Gly
4)
Leu Ala Lys Arg Ser Glu Gly Thr Pro Asp Asp Pro Val Arg
~A~AAG~~GAA~Aa;ccA~T~ccI~GITa;r~T~AwL~ATGcGAFccGITccPcTA~~AGAGAG~360
Gly
Thr Val
Pro
Ieu
Leu Thr
Arg
Glu
Glu
120
Phe kg
Tyr
Ser
Thr
Lys
Glu
Ala
Val
150
I&t
Tyr
Leu Lys
Glu
b&t
Glu Val Glu Ile Ser Lys Aq Ile Glu Lys Ala Gln Val Gln Ile
~~~~TcTAAGpGAATAGAAAAA~cAAGITcAAATA~FGA~AIlT~cGc~cQ:TAT~AcAApG~o=T~450
Glu
Aq
Ile
Ile
Leu Aq
Ser Ile Ala Gin Tyr Leu Ile Am Gly Lys Glu Arg Phe Asp Lys Ile Val
TCGA~GCGCPGTAT~An:AATGGTAAAGAAa;Am~APGATCGITTCTCAAAAG~GIIAGAA~T~~CAC?TCCTI~AATO
Ser
Glu
Lys
Glu Val
Glu
Asp
Lys
Thr
His
Phe Leu Asn
180
Leu Leu Pro Lys ku Ile Ser Leu Ieu Lys Glu Glu
~~~AAACK:A~T~TT~AT~G~~~G~LT~~T~~GP~;~~CT~~~~~T~~~;~~~~~O
Glu Arg
Leu
ku
Ala
Leu
Lys Asp
Pro
Ala
Ieu
210
Gln
Ile
AsnAspLeu
Lys
AlaAqAlaGlu
Ieu
Asp
Ala
'Ihr Glu Asp Phe GlyGluVal
Val Phe LysAlaTyr
Asp Ser
~cAA~T~a;A~~~~ApGa;TTAT~TTcA~Tn;BG~AGAA~cAAATcAATGAT~AAAGa;a;AGcp~810
Tyr
Leu Glu
Phe LeuGln
Leu
GluGln
Lys
Arg
Glu Val
Ala
Ala
Gly
Arq
Thr
Lys Lys AspValArgIvk?tleu
GlnArg
TrpM&
Asp Lys Ser Gin Glu
AAAAAA~T~ccGILTGTpAcAG~TGGATG~AAGIux:cpGcAAGccAAAAAA~An;~cAA~AAcTpAar;~GTA~990
ALa Lys
Lys
Glutit
Val
Glu
Ser
Am
Leu Aq
Ser Ile Ala Lys Lys Tyr Thr Asn Arg Gly Leu Ser Phe Leu Asp
TccATcGcG~~TAT~AAc~ax;crc;Tcr~TII;~T~A~~cJIA~~TAn;ax:~A~~~~GAGApG~O~O
Ile
Glu
Gly Am
l&t
Gly
Ieu
&t
AlaVal
ThrArg
Ala
Ile
Aq Am Lys Phe Ala Ala Ala Lys Leu Ala Ala Ala Arg Arg Lys Leu His
pGAAATApG~GccGcAGcGAAAcTTGcpGcGGcGcGGa;AApG~cA(3ApGa=rcpG~AGcG~a;Aax:AcTcTpGAA~~~
Leu
Gin
PheGlu
TyrAryArgGly
Tyr Lys Phe Ser Tnr Tyr Ala Thr TrpTrp
IleArqGln
mGPI;~TCGTACJLGCATA(:AAGTIT~~T~Aa;TGGTGG~CGTCPGCCTGIT~CGGGCT~Ga;GATCAAGCAa;A1170
Thr Ile Arg Ile Pro Val His Wt Ile Glu Thr Ile Am Lys Val
~~oX~~GITcAcAn;ATAGwLpIx~AATAAAGpGcTpa=rGGA~AAAAAA~ATGATGGAA~GGGAAA~~
Leu Ary
Ser
Lys
270
Glu
Glu
Phe
300
L.euVal
Ile
330
Lys Ala
Val
Lys
360
AlaAsp
GlnAla
Aq
390
Glu
Gly
Ala
Lys
Lys
Leu f-&t
Met Glu
Thr
Gly
Lys
Glu
Pro
420
1260
Pro
Ile
Ser
Ieu
Thr Pro Glu Glu Ieu Gly Glu Glu Ieu Gly Phe Thr Pro Asp At-g Val
~OCGGAA~~a;A~~~GGCm~ccACAT~GIT~CJIAfLTCTATAAAATAGCPCAACP13CCGA~~~CAG1350
Arg
Glu
Ile
!&r
Lys
Ile
Ala
Gln
Gln
450
Ala Glu Val Gly Asp Gly Gly Glu Ser Ser Phe Gly Asp Phe Ieu Glu
GCA~~GCACAT~GGGGAGPM:~mGGACAT~~CAACAT~a3TGIT~TCTa3AaY;GAGGCA~CGG~TTCC1440
Asp
Thr
Ala
Val
Glu
Ser
Pro
Ala Glu Ala Ihr Gly Tyr Ser
480
Met Leu Lys Asp Lys Met Lys Glu Val Leu Lys Thr Leu Thr Asp Arg Glu
ATGTPAAAACdTAAAATGAAA~GIT~AFAIU3GCITPM;GAL:~GPGCGTmGIT~~CATCGGmcc;rcrrcrr~TCZ=r1530
Arg
Phe Val
tiu
Ile
His
Arg
Phe Gly
Leu Ieu
Asp
Gly
510
Glu Arg
Ile
Aq
Gin
Ile
Glu
Ala
Lys
Ala
ku
Arg
Lys
St
540
Glu
Glu
Lys
Ile
Gly
Ser
Gly
Lys
Ile
Lys
Ser
'I@
Lys
570
Arq Pro Lys Thr Leu Glu Glu Val Gly Ser Ala Phe Asn Val Thr
o=rccCAAG~TpG~cpG~GGT~GcATK:AAT~~a;AGAGcGGA~cGccAAIL?TGAA~AAA~~ccA~ATG1620
Aq
Ary His Pro Ile Arg Ser Lys Gin ku Arq Ala Phe Ieu Asp Leu Ieu
cGI~T~~~TccAAAcAG~cGAGQ;~cllAcATcTA~GAAcAAvLAAAAFlITGGI~TcGGGTAA?TAAA~TATAAA~710
Glu
His
FIG. 5. Gene sequence
of the C. trackomatis
L2 D subunit
RNA
polymerase
gene. The gene consisted
of 1,713 nucleotide
pairs, beginning
at the first AUG
codon of the ORF. A 14-base
pair dyad, followed
by 7
thvmidine
residues
at the 3’ end of the open reading frame is underlined.
This structure
resembled
a p-independent
teiminator.
The deduced
amino acid sequence
is shown above the nucleotide
sequence.
Using the first methionine
of the ORF. the chlamvdial
(T subunit
had a calculated
molecular
mass of 66,141 daltons
(a?.
The nucleotide
sequences
oi the polymkase
chain reaction
primers
used to amplify
chlamydial
genomic
DNA to produce
pEl-L2
are boned.
reaction
with the control fusion polypeptide
representing
amino acids 101 to 330 (data not shown). This was consistent
with the location of the 2GlO binding domain on u7’ (Fig. 6).
This demonstrated
that the C@ gene codes for a polypeptide
with the same immunoreactivity
as the polypeptide
detected
in EBs. The monoclonal
antibody binding data, in addition
to the detection
of a single c gene homolog by Southern
hybridization,
suggested that the cloned a66 gene encodes the
Chlamydia
trachomatis
RNA
Polymerase
Major
o66 Subunit
13211
ct 44
EC 30
as 30
C!t 86
EC so
as so
FIG. 6. Comparative
amino
acid
sequences
of C. trachomatis
CT”‘, E.
coli c”, and B. subtilis
u43. The predicted amino acid sequences
of the major
v subunit
of C. trachomatis, E. co& and
B. subtilis are aligned to show sequence
homology
(14).
The
numbers
of the
amino acids of the corresponding
genus
are indicated
at the far right. Boxed regions show areas of amino acid homology. Asterisks denote
gaps introduced
into the sequence
to improve
the alignment. The B. subtilis sequence
has 254
fewer amino acids than E. coli, and a gap
was introduced
between B. subtilis amino
acids 130 and 131 to accomodate
this
difference.
Regions
l-4
correspond
to
those described
by Gribskov
and Burgess
(11) and reviewed
by Helmann
and
Chamberlin
(30).
The
lightly
shaded
amino acid sequence
(E. coli amino acids
361-390)
represents
the core binding
site
proposed
by Lesley
and Burgess
(39).
The darkly shaded amino acid sequence
(E. coli amino acids 456-496)
represents
the proposed
binding
site of the E. coli
anti-UT0 monoclonal
antibody
2GlO (13).
This monoclonal
antibody
also bound C.
trachomatis ae6 and B. subtilis u43 by
immunoblot
analysis
(see Fig. 1). In the
region between
regions 1 and 2, no alignment was attempted
due to lack of homology in the E. coli and C. trachomatis
sequences.
ct 131
EC 126
as 130
ct 181
EC 176
ct 231
EC 226
m 281
EC 276
Ct 284
EC 326
ct 318
EC 376
as 135
ct 368
EC 426
es 185
ct 418
EC 476
as 235
Ct 468
EC 526
as 285
ct 518
EC 576
as 335
4.2
*,
c-t 571
EC 613
es 371
M, = 70,000 protein
identified
by the monoclonal
antibody
2GlO in chlamydial
EB lysates.
Comparative Amino Acid Sequence of u Subunit Gene-Fig.
6 compares the amino acid sequence of C. trachomatis ufi6 with
E. coli u7’ and B. subtilis u43. The percentage
of conserved
amino acid sequence in the entire chlamydial
u6’j polypeptide,
compared with E. coli u7’ and B. subtilis u43, was 35 and 32%,
respectively.
Alignment
of the conserved
amino acid sequences of u subunits has led to the designation
of regions 1
to 4, with further division into subregions (reviewed by Helmann and Chamberlin
(30)). The amino acid homology of the
individual
regions of C. trachomatis P with E. coli u”’ and B.
subtilis u43 was: region 1, 27 and 21%; region 2, 93 and 86%;
region 3,46 and 52%; and region 4,67% and 78%, respectively.
Of these regions, region 1 of C. trachomatis
u66 showed the
least homology with E. coli u7’ and B. subtilis u43. The role of
this region in u7’ is currently unknown.
The central region between the two conserved regions 1
and 2 has not been given a numerical
designation,
but there
were major differences noted among the sequences of E. coli
u7’, B. subtilis u43, and C. trachomatis
ufi6 subunits in this
region. Compared with E. coli u7’, the B. subtilis u43 has a 245amino acid gap, and the chlamydial
P polypeptide
had a gap
of 63 amino acids. The rest of this region was retained in the
chlamydial
a@; however, there was no apparent conservation
of these amino acids relative to the E. coli u7’ sequence. The
function of this region is unknown,
but it is likely that at
least some portion is not essential, because an in-phase mutation (rpoD800) deleting 14 amino acids in this region does
not affect
E. coli u7” function
(31).
The
rpoD800 mutation
does confer temperature
sensitivity, and it has been suggested
that this region may play a role in the stability or integrity of
the u subunit (32). Region 2 was the most highly conserved
in C. trachomatis u6’ compared with the E. coli u”” amino acid
sequence. When compared with E. coli, 6 of the amino acids
of the B. subtilis region 2 sequence differ, and 5 of the
chlamydial
u66 amino acids differed from E. coli.
Region 3 designates a 45-amino
acid sequence which is
absent in many of the smaller u subunits and weakly conserved, when present. The sequence is 65% conserved in B.
subtilis u43 relative to E. coli u7’; the C. trachomatis
u6‘j sequence was only 46% and 52% conserved relative to E. coli
uTo and B. subtilis u4’, respectively. The function of this region
is not known, although genetic studies of mutations localized
to this region are consistent with a role in structural integrity
(30). Region 4 was also highly conserved, notably region 4.2
where only 4 of 28 amino acids differed. Surprisingly,
the P
had 16 amino acids at the carboxyl terminus which were not
present in either B. subtilis u4’ or E. coli u7’.
DISCUSSION
Transcription
DNA-dependent
of specific genetic sequences is catalyzed by
RNA polymerase in procaryotes.
The tran-
13212
Chlamydia
trachomatis
RNA Polymerase Major a66 Subunit
scriptional
apparatus plays a central role in the regulation of
gene expression,
often during the initiation
step, through
interactions
between a g subunit of RNA polymerase and the
cognate promoter sequence. Extensive homology is present in
the amino acid sequences of the major u subunits of E. coli
and B. subtilis (u7’ and u43, respectively),
in addition to homology among the largest subunits of eucaryotic and procaryotic RNA polymerases
(14, 33). C. truchomatis
is very distantly related to other eubacteria
whose RNA polymerase
subunits have been studied. A comparison of chlamydial
16 S
rRNA sequences with other procaryotic
organisms by Weisburg et al. (34) confirmed the identification
of Chlamydia as
a eubacterium,
without a close relative among the more than
400 partial 16 S rRNA eubacterial
sequences studied. The
transcriptionally
active form of Chlamydia exists only intracellularly,
after host cell infection
(35). The composition
of
RNA poiymerase in Chlamydia has not been elucidated previously, and the study of gene expression and developmental
regulation
has been difficult because of the obligate intracellular habitat, lack of a genetic system, and the difficulty
in
growing sufficient quantities
of Chlamydia for conventional
RNA polymerase purification.
Early studies of Chlamydia demonstrated
that a rifampicinsensitive transcriptional
apparatus is present in the elementary body, which is capable of RNA synthesis immediately
following
entry into host cells (36, 37). In the absence of
specific data about the chlamydial
transcriptional
apparatus
or isolation
of RNA polymerase,
initial
experiments
were
directed toward expressing chlamydial genes in other bacterial
systems. However, expression of chlamydial
genes in E. coli
has met with limited, or no, success. When E. coli is transformed with chlamydial
genomic DNA inserted into vectors
lacking E. coli expression signals, and the recombinants
are
screened with antichlamydial
antibodies, very few identifiable
expression products are obtained (7, 38). Palmer and Falkow
(7) utilized cloned chlamydial
DNA sequences in complementation studies with E. coli mutants known to be deficient in
various amino acid biosynthetic
pathways, but were unable to
demonstrate
genetic complementation.
Identification
of consensus promoter sequences in E. coli
and B. subtilis has greatly enhanced the study of DNA-RNA
polymerase interactions
and transcriptional
regulation.
Chlamydial promoters appear to differ from consensus sequences
identified for E. coli; even among chlamydial
DNA sequences
a consensus has not been established
(5, 38). The lack of
similarity
to E. coli promoters
is not surprising,
given the
apparent inability of E. coli to recognize chlamydial promoters
effectively, but identification
of a consensus sequence will be
an important
first step in studying transcriptional
regulation
in this organism. We have identified
and sequenced the major
chlamydial
RNA polymerase (r subunit, which will facilitate
identification
of promoter
sequences by DNA footprinting
studies, and isolation of the vegetative chlamydial
RNA polymerase c subunit for use in reconstitution
studies and in
uitro systems with E. coli.
The first evidence for a chlamydial
RNA polymerase, with
a 0 subunit homologous to the major c subunit of E. coli (a7’),
was provided by the specific monoclonal
antibody binding.
Interestingly,
the E. coli anti?
monoclonal
antibody which
recognized a specific protein in C. trachomatis
also showed
immunoreactivity
with the major B. subtilis (r subunit (racy),
indicating
that the chlamydial
c subunit shares a highly
conserved region with both of these distantly related genera.
This was confirmed
by the gene sequence obtained subsequently,
which showed that regions homologous
in other
organisms are also highly conserved in the C. trachomatis
u
subunit. Considering
the minimal
evolutionary
relatedness
between C. trachomatis and other eubacteria, including E. coli
and B. subtilis, the amount of amino acid sequence conservation was striking. The high degree of conservation
was unexpected because only one of seven monoclonal
antibodies
bound the chlamydial
u@. Expression of the cloned chlamydial
u@ gene as fusion polypeptides,
followed by immunoblotting,
demonstrated
the same pattern of monoclonal
antibody binding as seen with chlamydial
EB lysates. This demonstrated
that the translated protein product of the cloned ue6 gene is
antigenically
indistinguishable
from the protein detected by
the E. coli u?specific
monoclonal
antibody
in chlamydial
lysates. The immunoreactive
monoclonal
antibody,
2G10,
which bound to chlamydial
ue6 as well as B. subtilis 8, is
believed to bind to the amino acids from approximately
456
to 496, on the basis of epitope mapping of E. coli Jo peptide
proteolytic
fragments (13). These amino acids are located in
the intervening
region between regions 2 and 3 and in the
amino half of region 3 (Fig. 6). Although
there were two
groups of six amino acids in the chlamydial
u@ region corresponding
to the 2GlO binding domain
which were 100%
conserved compared with E. coli, the overall conservation
of
this domain in C. truchomatis was only 49%. It is somewhat
surprising
that the E. coli u7’ monoclonal
antibody,
which
cross-reacted
with chlamydial
P,
maps to one of the less
conserved regions and to a region without a predicted common
function. Functional
studies with the 2GlO monoclonal
antibody, however, demonstrate that this antibody can effectively
bind and remove E. coli u7’ and holoenzyme from solution, in
addition
to markedly
inhibiting
transcriptional
activity by
RNA polymerase
in vitro (13). Thus, the epitope recognized
does not appear to be involved in a:core binding, is localized
to an external region of the holoenzyme,
and appears to be
important
in the transcriptional
process, although it is unclear
what stage of transcription
is affected and whether this effect
is through direct or indirect interaction.
On the basis of biochemical
parameters, Chlumydia shares
some characteristics
such as Gram-negative
staining and common lipopolysaccharide
antigens with free living, Gram-negative organisms (35), and thus might appear to be phenotypically closer to E. coli than B. subtilis. When estimated by 16
S rRNA, the evolutionary
relationship
between E. coli and B.
subtilis is closer than that of C. trachomatis
with either of
these two organisms
(34). The extensive u amino acid sequence homology among these three genera would indicate
that this important
u subunit structure probably originated
before the very early point at which chlamydial
divergence
occurred; it would seem unlikely that such extensive homology
could be the result of a convergent evolution.
The complex biochemical functions attributed to u subunits
include binding to core enzyme, promoter
recognition,
and,
possibly, DNA melting and inhibition
of nonspecific
transcription (30). Functional
studies of E. coli u7’ activity involve
both biochemical
and genetic approaches, including
the use
of monoclonal
antibodies and functional analysis of point and
deletion mutations.
Two of the four regions of E. coli u7’
designated by Gribskov and Burgess (11) are believed to be
involved in core binding and promoter interaction.
We compared the amino acid sequences of these regions of E. coli u7’
and B. subtilis a43 with C. trachomatis
CT%,in an attempt to
understand
any functional
differences of us6 and to explain
the inability
of E. coli to efficiently
recognize chlamydial
promoters.
The site for binding of the u subunit to core enzyme was
proposed to involve region 2 initially
(Fig. 6), on the basis of
the conservation
of this region in the majority of sequenced u
Chlamydia
trachomatis
RNA Polymerase Major a@ Subunit
subunits (11) and analysis of binding of tryptic fragments of
E. coli u7’ to core enzyme (10). The chlamydial
C? was highly
conserved in this region, including
subregion 2.2, which is
comprised of 15 amino acids at the center of this possible
core-binding
region (30), as well as within the “rpoD box”
described by Tanaka et al. (12), where the conservation
was
100%. A more recent study by Lesley and Burgess (39) indicates that E. coli u7’ mutants with deletions through most of
region 2, including
the highly conserved subregion 2.2 and
entirely through region 3, are still capable of binding to core.
On the basis of these data, the core binding region in u7’ is
proposed to begin just before region 2 and include amino acids
361-390. Of these 30 amino acids in the region just before
region 2 and extending
through the first 7 amino acids of
region 2.1, 17 were conserved in C. trachomatis &j6. Although
this region had less homology than that of 2.2, there may be
sufficient conservation
to constitute a homologous domain for
core and u interaction
among all three genera. If one of these
highly conserved regions is the region most important
to the
u:core enzyme interaction,
and given that the E. coli Jo
functions
with B. subtilis core enzyme and vice versa (40),
reconstitution
experiments
with C. truchomatis
uG6 and core
from E. coli or B. subtilis should be feasible. The ability to
reconstitute
chlamydial
uG6 with E. coli core would have important application
for facilitating
the study of chlamydial
promoters and regulation of gene expression. Despite the high
degree of homology in this region, it is possible that there are
factors in addition to binding in this region, such as structural
interactions
at other sites, which may preclude C. truchomatis
u@ from binding E. coli core enzyme.
The amino acid sequence of region 4 is highly conserved
among diverse u subunits and was highly conserved in a@.
This region is predicted to form a helix-turn-helix
configuration, the classic motif observed in double-stranded
DNA
binding proteins. This has lead to the proposal of this region
as a site of interaction
between u and the -35 consensus
promoter region (ll),
which is supported by study of mutations mapped to this region (41, 42). Recent studies also
identify involvement
of region 2, specifically subregion 2.4, in
the recognition
of the -10 consensus region in E. coli promoters, by analysis of rpoD mutants (42). Of the 19 amino
acids in this region of uG6, only a single amino acid change
was observed among C. trachomatis, B. subtilis, and E. coli. It
is paradoxical
that despite the significant
amount
of u’j6
sequence homology in the purported
-10 and -35 consensus
promoter
recognition
regions, chlamydial
promoters
do not
appear to share consensus sequences with E. coli promoters
or be recognized by E. coli u7’. Several explanations
may
account for this. 1) The promoters for the six chlamydial gene
sequences to date do not represent the cognate sequence for
the ufi6 (i.e. they represent the promoters for other chlamydial
u factors). 2) There is a different conformation
achieved when
u66 interacts with the core and/or DNA, such that promoter
sequences different from those of E. coli are recognized in C.
trachomatis. 3) Additional
factors in chlamydiae may be necessary to enable u‘j6 to efficiently
recognize authentic
promoters. It is interesting
that the chlamydial
u66 sequence had
an extra 16 amino acids at the carboxyl terminus, contiguous
with the purported
-35 recognition
site. Perhaps some interaction with this highly lysine-rich
region modulates promoter
specificity. Other unique regions which may change the promoter sequence recognized by uG6 include region 1, parts of
region 3 (which had substitutions
at locations
highly conserved between E. coli u7’ and B. subtilis Use), and the sequence
between regions 1 and 2, where the 63-amino
acid gap occurred; otherwise, the C. trachomatis u66 polypeptide
showed
remarkable
homology with E. coli u7’ and B. subtilis u43.
The chlamydial
u’j6 was present early and throughout
the
developmental
cycle, and thus was the analog of the vegetative, major c factors in E. coli and B. subtilis. Immune detection of u6’j in the metabolically
inert EB form demonstrated
that EBs contain preformed RNA polymerase u subunit. The
earliest transcribed
genes, whose products are essential for
EB:RB differentiation,
are likely to be recognized by the major
u@ subunit. In B. subtilis, there is evidence that a series of
sequentially
expressed minor u subunits are involved in the
regulation
of gene expression during sporulation,
as initially
proposed by Losick and Pero (43). In Chlumydia, the developmental cycle involves a sequence of differentiation
events,
during which coordinate
expression of genes occurs. Given
the lack of promoter consensus sequence among genes transcribed at different developmental
times, it is likely that one
or more u factors are present concomitantly
with the major
chlamydial
Us, and that other u factors are present in a
sequential pattern, as necessary for the coordinate expression
of gene families required for differentiation
in C. trachomatis.
Isolation and/or expression of the chlamydial
u@ will provide detailed analysis of chlamydial
promoter
structure, including information
about the differences
currently
noted
among chlamydial
promoters and whether these differences
are related to coordinate gene expression during the developmental cycle. The isolation and sequencing of the C. trachomatis u6‘j provides an important
first step in the study of gene
expression
during differentiation
of this microbiologically
unique and important
human pathogen.
Acknowledgmenb-We
technical
assistance
nucleotides.
thank
Leanne
and Anne Cummings
Cornel
for her excellent
for synthesizing
the oligo-
Note Added in Proof-The
sequence
for the Chlamydia
trachomatis
human
biovar
RNAP
sigma subunit
(a%) described
in this paper is
corroborated
by the recently
published
sequence
for the mouse pneumonitis
biovar
(44). The phvlogenetic
distance
(30-60%
DNA
homology
(35)) between
these two lbiovars
is reflected
by 14 amino acid
differences
between
the two RNAP
sigma subunit
sequences.
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