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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). 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