Vol. 172, No. 12

JOURNAL OF BACTERIOLOGY, Dec. 1990, p. 6959-6972

0021-9193/90/126959-14$02.00/0 Copyright © 1990, American Society for Microbiology

Heat Shock Response of Murine Chlamydia trachomatis JOANNE N. ENGEL,l.2* JONATHAN POLLACK,1'3 EVE PERARA,1 AND DON GANEM1'2

Departments of Microbiology and Immunology,1 Biochemistry and Biophysics,3 and Medicine,2 University of California Medical Center, San Francisco, California 94143 Received 19 June 1990/Accepted 13 September 1990

We have investigated the heat shock response in the mouse pneumonitis strain of Chlamydia trachomatis. The kinetics of the chlamydial heat shock response resembled that of other procaryotes: the induction was rapid, occurring over a 5- to 10-min time period, and was regulated at the level of transcription. Immunoblot analysis and immunoprecipitations with heterologous antisera to the heat shock proteins DnaK and GroEL demonstrated that the rate of synthesis, but not the absolute amount of these two proteins, increased after heat shock. Using a general screen for genes whose mRNAs are induced by heat shock, we identified and cloned two of these. DNA sequence analysis demonstrated that one of the genes is a homolog of dnaK. Further sequence analysis of the region upstream of the dnaK gene revealed that the chlamydial homolog of the grpE gene is located just adjacent to the dnaK gene. The second locus encoded three potential nonoverlapping open reading frames. One of the open reading frames was 52% homologous to the ribosomal protein S18 of Escherichia coli and thus presumably encodes the chlamydial homolog. Interestingly, this ribosomal protein is not known to be induced by heat shock in E. coli. S1 nuclease and primer extension analyses located the start site of the dnaK transcript to the last nucleotide of the grpE coding sequence, suggesting that these two genes, although tandemly arranged, are transcribed separately. No promoter sequences resembling the E. coli consensus heat shock promoter could be identified upstream of either the C. trachomatis dnaK, grpE, or S18 gene. The induction of the dnaK and S18 mRNAs by heat shock occurred at a transcriptional level; their induction could be blocked by rifampin. The mechanisms of induction for these two loci were not the same, however; they were differentially sensitive to chloramphenicol. Whereas the induction of dnaK mRNA required de novo protein synthesis, the induction of the S18 mRNA did not. Thus, C. trachomatis utilizes at least two different pathways to induce the transcription of mRNAs encoding proteins induced in the heat shock response.

Chlamydia trachomatis is a medically important gramnegative bacterium that causes a large array of sexually transmitted diseases as well as ocular infections (trachoma). This obligate intracellular parasite of eucaryotic cells replicates via a unique developmental cycle that involves the serial alternation of two distinct forms within the cytoplasm of the infected cell (for reviews, see references 3, 32, and 33). The life cycle commences when the extracellular form of chlamydia, the sporelike, metabolically inactive elementary body (EB), is taken up by the host eucaryotic cell. Upon binding to the host cell membrane and subsequent internalization into a host-derived endosome, the EB undergoes a striking morphologic transformation into the intracellular vegetative form, the reticulate body (RB). The RB replicates by binary fission while enclosed within this cytoplasmic vacuole and subsequently redifferentiates into EBs. The EBs are released from the host cell, thus completing the developmental cycle. Chlamydiae are important human pathogens (32). Acute genital infection with C. trachomatis leads to urethritis and cervicitis, whereas primary ocular infection leads to a selflimiting mucopurulent conjunctivitis. Chronic genital infection can result in fallopian tube scarring and resultant infertility, whereas the end result of repeated ocular exposure is blinding trachoma. In fact, chlamydia is the major cause of preventable infertility in well-developed countries and the primary cause of preventable blindness in the third world. Although the pathogenetic events that lead to the debilitating sequelae of blinding trachoma and infertility are unknown, studies of chlamydial inclusion conjunctivitis (a *

Corresponding author.

model system for chlamydial infections) support the hypothesis that an immunological mechanism is involved. Early studies in humans and other primates suggested that prior vaccination with killed chlamydiae frequently resulted in more severe trachoma upon reinfection (4, 14, 39, 40, 41). Morrison and co-workers have recently demonstrated that a chlamydial 57-kDa outer membrane protein elicited an ocular hypersensitivity response characterized by a mononuclear macrophage and lymphocyte cellular infiltrate in guinea pigs previously immunized with C. trachomatis (25). Cloning of the gene for this 57-kDa protein revealed that it encodes the chlamydial homolog of the heat shock protein GroEL, and the recombinant protein itself elicited an ocular delayed hypersensitivity response in immune guinea pigs (24). Thus, products of the heat shock response may play a role in the pathogenesis of chlamydial disease in humans. The heat shock response is a universal process whose components are among the most highly conserved and abundant proteins found in nature (22). In Escherichia coli, a shift from 30 to 42°C, as well as exposure to a variety of environmental stresses such as ethanol, results in the induction of approximately 20 new proteins whose synthesis peaks within 5 min and then declines rapidly to new steadystate levels that are characteristic of the new ambient temperature (26). Initiation of the heat shock response is regulated transcriptionally. Specifically, a new u factor ((J32) associates with E. coli core RNA polymerase enzyme. This alternative holoenzyme transcribes only heat shock genes, which have promoter sequences that differ from those transcribed by holoenzyme containing &70, the normal vegetative initiation factor. The end result is the coordinate expression of all of the heat shock proteins (15, 16). The regulation of (F32 itself is complicated: it is under the control of at least 6959

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ENGEL ET AL.

three promoters and is present at low levels during vegetative growth of E. coli. The heat shock event leads to a stabilization and increased steady-state level of the aj32 protein (34) through a pathway that is yet to be elucidated. The amino acid sequences of several of the heat shock proteins in distantly related organisms, such as Drosophila melanogaster and Homo sapiens, are remarkably similar to those in E. coli, suggesting that the heat shock response is of ancient origin and may be of fundamental importance to cellular physiology. The function of the heat shock proteins, however, is unclear. It has recently been shown that they may play roles in the assembly and disassembly of macromolecular complexes (GroEL and GroES [19]), intracellular transport (SaccharoMyces cerevisiae Hsp7O [6, 8, 19]), transcription (u70 [36]), proteolysis (lon protease [13]), translation (lysyl tRNA synthetase [38]), and the host autoimmune response (42). Furthermore, several of the heat shock proteins in E. coli, such as GroEL, GroES, DnaK, and GrpE, are synthesized during normal vegetative growth, and mutations in these genes exhibit a phenotype during vegetative growth (1, 2, 12). Little is known about the chlamydial heat shock response. Although two of the genes encoding chlamydial homologs of E. coli heat shock proteins have been recently cloned (2a, 7), the actual heat shock response of this intracellular parasite has never been characterized. It is not clear that this intracellular parasite would exhibit a detectable response to a heat shock stress; this phenomenon might be obscured by the continuous synthesis of heat shock proteins in response to the hostile intracellular environment that chlamydia must experience when replicating intracellularly in the host cell cytoplasm. In this paper we analyze the heat shock response of the murine strain of C. trachomatis, the mouse pneumonitis (MoPn) agent.

MATERIALS AND METHODS Reagents. Products were obtained from the following sources and were used according to the manufacturer's specifications: restriction enzymes, bacterial alkaline phosphatase, and T4 DNA ligase, New England BioLabs, Inc. (Beverly, Mass.); T4 polynucleotide kinase, Boehringer Mannheim Biochemicals (Indianapolis, Ind.); DNA polymerase I, Pharmacia Fine Chemicals (Piscataway, N.J.); 32P-containing radioisotopes, Amersham Corp. (Arlington Heights, Ill.); [35S]methionine, ICN (Irvine, Calif.); Seaplaque and Seakem agarose, FMC Bioproducts (Rockland, Maine); ampicillin, rifampin, protein A-Sepharose CL-4B, aprotinin, phenylmethylsulfonyl fluoride, leupeptin, and DNase I, Sigma (St. Louis, Mo.); and protein molecular weight markers and murine leukemia virus reverse transcriptase, Bethesda Research Laboratories (Bethesda, Md.). Growth of chlamydia and in vivo labeling of chlamydial proteins. The MoPn strain of C. trachomatis wag grown in monolayers of HeLa or J774 cells (31). For labeling of chlamydial proteins at various times during infection, chlamydia-infected HeLa cells were incubated in Dulbecco modified medium lacking methionine and cysteine in the presence of cycloheximide (50 t.g/ml, to block host protein synthesis) for 30 min and then pulse-labeled with [355]methionine (100 pXCi/ml) for 10 to 30 min. For the heat shock experiments, the flasks containing the chlamydiainfected monolayers were floated in a 450C water bath for 5 to 10 min before being labeled for 10 to 30 min with [355]methionine. After extraction with lysis buffer (10% glycerol, 50 mM Tris chloride [pH 7.5], 150 mM NaCi, 0.2%

J. BACTERIOL.

Triton X-100, 1 pug aprotinin per ml, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin), samples were electrophoresed on 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels. Two-dimensional electrophoresis was carried out as described previously (27). Immunoblot analysis and immunoprecipitations. Immunoblots were carried out as described previously (37) with 2% gelatin as a blocking agent. The GroEL antiserum, a kind gift of P. Bavoil (University of Rochester, Rochester, N.Y.), was used at a dilution of 1:200. The antiserum against DnaK of E. coli, a gift of Hatch Echols (University of California, Berkeley), was used at a dilution of 1:800. Alkaline-phosphatase conjugated goat anti-rabbit immunoglobulin G or anti-mouse immunoglobulin G (Promega, Madison, Wis.) was used at a dilution of 1:7,500 for the second antibody reaction. Immunoprecipitations were performed as previously described (17), with the following modifications. Cells were labeled as described above and then removed from the dishes by gentle agitation with 2 ml of lysis buffer; the supernatant was centrifuged at 12,000 x g. The supernatant was added to 20 to 50 ,ul of protein A-Sepharose CL-4B beads along with immune antiserum (1 to 5 1LI of serum per 10 1.l of beads) and rocked for 12 to 18 h at 40C. The protein A-Sepharose beads were washed once with buffer (50 mM Tris chloride [pH 7.5], 0.3 M NaCl, 20 mM EDTA, 0.2% Triton X-100, 0.05% SDS, 1% deoxycholate) and then three times with phosphate-buffered saline. The bound antigen was eluted in Laemmli sample buffer (22) containing 5% (vol/vol) P-mercaptoethanol and electrophoresed on 12.5% SDS-polyacrylamide slab gels. The radiolabeled product was visualized by fluorography. Nucleic acid preparation and analysis. Chlamydial DNA from the MoPn strain of chlamydia was prepared as described previously (10). Chlamydial RNA was prepared from RBs of chlamydia-infected cells. Either total RNA (host cell plus chlamydial RNA) or partially purified RB RNA (18) was isolated by using the guanidinium thiocyanate method (23) with the following modifications. Total RNA was harvested by collecting the cells from T-150 flasks by using glass beads then centrifuging at low speed to pellet the chlamydia-infected cells. The cell pellet was lysed with 4 M guanidinium thiocyanate, phenol extracted, and ethanol precipitated. The pelleted nucleic acids were treated with RNase-free DNase (Promega) and then applied (50 mM MOPS [morpholinepropanesulfonic acid] [pH 7.0], 15% ethanol, 0.4 M NaCl) to a Quiagen column (Stratagene, La Jolla, Calif.) under conditions in which the RNA could be selectively eluted separately from protein and DNA (50 mM MOPS [pH 7.0], 15% ethanol, 1.1 M NaCl, 2 M urea). The RNA was collected by ethanol precipitation. Partially purified RBs were isolated from chlamydia-infected cells as follows. The chlamydia-infected cells were removed from T-150 flasks with glass beads and were Dounce homogenized ca. 30 times, a manipulation which breaks open the host cell but leaves the RBs metabolically intact (18; J. Engel, unpublished observations). Cellular debris was removed by centrifugation at low speed, and the supernatant was then spun at 18,000 x g to pellet the RBs. The RBs were then lysed in 4 M guanidinium thiocyanate, and RNA was prepared as described above. Standard recombinant DNA methods were used for nucleic acid preparation and analysis (23). Restriction fragments were subcloned into a pGEM7Zf (Promega) plasmid vector. Southern blotting and Northern RNA blotting were carried out as described previously (10), with Hybond paper (Amersham). Radioactive DNA probes were labeled by nick translation (23). SP6 and T7 polymerase

HEAT SHOCK RESPONSE OF MURINE CHLAMYDIA TRACHOMATIS

VOL. 172, 1990

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FIG. 1. Effect of heat shock on chlamydial protein synthesis. HeLa cells grown at 370C were infected with the MoPn strain of C. trachomatis; cycloheximide was added, and the cells were simultaneously starved for methionine and cysteine for 30 min at 0 (lanes 1 and 2), 8 (lanes 3 and 4), or 20 (lanes 5 and 6) hpi. The cultures were then incubated for an additional 10 min at 370C (lanes 1, 3, and 5) or shifted to 45TC for 10 min (lanes 2, 4, and 6) and then pulse-labeled with [35S]methionine for 10 mmin at either 37TC (lanes 1, 3, and 5) or 45TC (lanes 2, 4, and 6). Lysates of the cells were electrophoresed on a 12.5% SDS-polyacrylamide gel and then subjected to fluorography. Molecular mass markers in kilodaltons are shown to the right.

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Preparation of a chlamydial DNA library and isolation of heat-shock-specific genes. Chlamydial DNA was digested with EcoRI and cloned into a pGEM7Zf vector previously cleaved with EcoRI and dephosphorylated with bacterial alkaline phosphatase. DNA from randomly selected individual clones was prepared by the minilysate method (23) and radiolabeled by nick translation. Approximately i07 cpm were hybridized to a Northern blot of chlamydial RNA isolated at various times during infection or after a 10-min heat shock at 18 h postinfection (hpi). Clones that annealed preferentially with the heat shock RNA were selected for further study. DNA sequencing. The dideoxy-chain termination method of DNA sequencing (29) was carried out on double-stranded fragments cloned into pGEM7Zf with the Sequenase kit (U.S. Biochemical Corp., Cleveland, Ohio). Sequencing reactions were primed with oligonucleotides homologous to the T7 and SP6 promoters (Promega) flanking the cloned inserts in the pGEM7Zf vector. Exonuclease III digestions were carried out as previously described (9). Si nuclease and primer extension studies. S1 nuclease digestions and primer extension studies were carried out as described previously (10). A 20-bp oligonucleotide primer (GAGAACGCAGAGACATTACC) complementary to nucleotides 57 to 76 of the dnaK gene was employed for the primer extension experiment (see Fig. 5).

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FIG. 2. Two-dimensional electrophoresis of proteins synthesized by C. trachomatis in response to heat shock. HeLa cells were infected with MoPn for 18 hpi and pulse-labeled with [355]methionine at 370C (A; non-heat-shocked control) or 450C (B; heat shock) for 30 min as described in the legend to Fig. 1. Cell extracts were electrophoresed on isoelectric focusing gels and then electrophoresed on SDS-polyacrylamide gels. Protein molecular mass markers in kilodaltons are shown to the left.

RESULTS

Characterization of the heat shock response. We initially studied the heat shock response of chlamydia by examining the pattern of chlamydial protein synthesis before and after a shift from 37 to 450C, as detailed in Materials and Methods. One-dimensional SDS-polyacrylamide gel electrophoretic analysis of the products revealed significant increases in the synthesis of several species, including proteins of 20, 27, 43, 55, and 70 kDa (Fig. 1). The full spectrum of the stress response was better seen with two-dimensional gel electrophoresis (Fig. 2). Figure 2B shows a fluorograph of a two-dimensional electrophoresis of lysates from chlamydiainfected HeLa cells at 18 hpi that had been pretreated with cycloheximide for 30 min, shifted from 37 to 450C for 10 min, and then pulse-labeled with [35 ]methionine for 30 min. This experiment demonstrates that the synthesis of many proteins was altered by a 10-min temperature shift as compared with

6962

ENGEL ET AL.

J. BACTERIOL.

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4 5 6 7 8 9 10 11 2 13l 141 516 17 18 19 20 21 22 23 24 A Bi C FIG. 3. Immunoprecipitation of GroEL and DnaK. HeLa cells were infected with MoPn for 0 (lanes 1, 2, 9, 10, 17, and 18), 8 (lanes 3, 4, 11, 12, 19, and 20), 14 (lanes 5, 6, 13, 14, 21, and 22), and 18 (lanes 7, 8, 15, 16, 23, and 24) h and pulse-labeled with [35Slmethionine for 10 min after growth at 370C (lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23) or after a 10-min heat shock to 450C (lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24). (A) Lysates from these infections electrophoresed on 12.5% SDS-polyacrylamide gels. (B) Immunoprecipitation of these lysates with antiserum prepared to purified GroEL from a human strain of C. trachomatis. (C) Immunoprecipitation of these lysates with antiserum prepared to purified DnaK from E. coli. K, DnaK; G, GroEL; M, 40-kDa protein; + and -, presence or absence of exposure to heat shock, respectively.

3

protein synthesis by chiamydia not exposed to a heat shock stress (Fig. 2A). Again, increased amounts of the ca. 20-, 27-, 40-, 55-, and 70-kDa species, as well as other proteins, were visible. Additionally, the level of some proteins appeared to decrease in response to heat shock. The heat shock response could be elicited by shifting the chlamydiainfected cells from 370C to temperatures between 42 and 450C; at 500C, chlamydial protein synthesis was completely inhibited (data not shown). As little as 5 min of heat shock was sufficient to induce the response (data not shown). The heat shock response was blocked by pretreatment with rifampin, an inhibitor of bacterial transcription (data not shown). The heat shock response could be elicited early in infection (8 hpi; Fig. 1, lanes 3 and 4) as well as later in infection (14 to 24 hpi; Fig. 1, lanes 5 and 6, and data not shown). Furthermore, the heat shock response, as judged by one-dimensional protein gel electrophoresis, could also be induced when a tissue culture macrophage cell line, J774, was infected with chlamydia (data not shown). Temperature shift increases the rate of heat shock protein synthesis but not necessarily the total amount of protein present. We next examined the effect of a heat shock stress on the synthesis of two proteins, DnaK and GroEL, whose synthesis in other procaryotes is known to increase in response to heat shock. We reasoned that since these proteins are highly conserved among organisms ranging from bacteria to mammals, they would likely be conserved in chlamydia and would be components of its heat shock response. By using a rabbit polyclonal antiserum prepared to GroEL purified from a human strain of C. trachomatis, lysates from chlamydia-infected HeLa cells that were pulselabeled for 10 min with [35S]methionine after a 10-min heat

shock at 8, 14, or 18 hpi were immunoprecipitated. Figure 3A is a fluorograph of the pulse-labeled lysate (used in the subsequent immunoprecipitations) electrophoresed through an SDS-polyacrylamide slab gel. As described above, the induction of three prominent proteins of 43, 55, and 70 kDa was readily observed in the lysates derived from chlamydiainfected cells exposed to a 10-min heat shock (Fig. 3a, lanes 4, 6, and 8). Figure 3B shows an immunoprecipitation of these lysates with the antiserum raised against GroEL from a human strain of C. trachomatis. Both early and late in infection, heat shock led to an increase in the rate of newly synthesized GroEL protein as compared with the rate in non-heat-shocked controls (Fig. 3B, lanes 11 through 16). On the original gel, immunoprecipitation of GroEL could be detected in the sample from 8 hpi (Fig. 3B, lanes 11 and 12). It should be noted that, in addition to immunoprecipitating a 55-kDa protein, the GroEL antiserum also immunoprecipitated a slightly faster migrating protein species whose rate of synthesis increased after heat shock. This protein may represent a degradation product or a modified form of the GroEL protein. Identical experiments were carried out with a rabbit polyclonal antiserum made to the DnaK protein from E. coli. Figure 3C shows an immunoprecipitation, with antiserum raised to DnaK purified from E. coli, of lysates from chlamydia-infected HeLa cells that were pulse-labeled for 10 min with [35S]methionine after a 10-min heat shock at 8, 14, or 18 hpi. The rate of newly synthesized DnaK-reactive species increased after a 10-min heat shock (Fig. 3C, lanes 22 and 24) as compared with the rate the non-heat-shocked controls (lanes 21 and 23). Again, on the original autoradiograph, the DnaK antiserum showed a similar pattern of immunoprecip-

HEAT SHOCK RESPONSE OF MURINE CHLAMYDIA TRACHOMATIS

VOL. 172, 1990

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FIG. 4. Immunoblot of heat-shocked C. trachomatis with GroEL antiserum. (A) HeLa cells were grown at 37°C, uninfected (lanes 1 and 2) or infected with the MoPn strain of C. trachomatis (lanes 3 and 4), cycloheximide was added, the cells were simultaneously starved for methionine and cysteine for 30 min, and the cultures were incubated for an additional 10 min at 37°C (lanes 1 and 3) or shifted to 45°C (lanes 2 and 4) for 10 min followed by pulse-labeling with [35S]methionine for 10 min at 37°C (lanes 1 and 3) or 45°C (lanes 2 and 4). Lysates of the cells were electrophoresed on a 12.5% SDS-polyacrylamide gel and then subjected to fluorography. (B) Similar gel immunoblotted to the GroEL antiserum.

itation, including an increased rate of newly synthesized DnaK-reactive species in the lysates prepared from HeLa cells infected with chlamydia for 8 hpi. Interestingly, this antiserum also immunoprecipitated a 55-kDa protein species that comigrated with the GroEL-reactive protein (Fig. 3B and data not shown) as well as a ca. 40-kDa protein species;

6963

nonimmune serum failed to immunoprecipitate any protein (data not shown). This 40-kDa species, whose synthesis did not appear to increase in response to heat shock, could be the chlamydial major outer membrane protein; this observation is commented on in the Discussion. An immunoblot of lysates from non-heat-shocked MoPninfected HeLa cells probed with the GroEL antiserum revealed a 55-kDa protein species in chlamydia-infected cells that was absent in uninfected HeLa cells. This polypeptide was present throughout most of the life cycle. It increased in amount between 8 and 20 hpi, reflecting the expected increase due to replication of chlamydial organisms that occurs during this time (data not shown). Figure 4B demonstrates the surprising result that exposing the chlamydiainfected cells to a 10-min heat shock stress did not further increase the total amount of GroEL-reactive protein (compare lanes 3 and 4). The control experiment, showing that a heat shock response had occurred, is shown in Fig. 4A, a fluorograph of the immunoblots. These samples were derived from chlamydia-infected cells that had been pulselabeled with [35S]methionine for 10 min after a 5-min heat shock. An immunoblot of lysates from MoPn-infected cells probed with the heterologous antiserum to DnaK demonstrates that the total amount of DnaK-reactive protein also did not increase after a 10-min heat shock at 8, 14, or 18 hpi (data not shown). Given the results above, we conclude that the rate of the synthesis of at least two chlamydial heat shock proteins, DnaK and GroEL, increases in response to heat shock, but that the steady-state levels of these two proteins fail to increase after heat shock. The significance of this finding will be considered further in the Discussion. Characterization of heat-shock-specific genes. During a screen for developmentally regulated genes, we identified two EcoRI restriction fragments (2.0 and 1.5 kb) that hybrid-

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500 bp FIG. 5. Transcription initiation site of dnaK mRNA. (A) An oligonucleotide primer (107 cpm; end labeled with polynucleotide kinase) complementary to the dnaK coding region (Fig. 6A, Materials and Methods) was hybridized to 10 ,ug of heat shock RNA (isolated from HeLa cells infected with MoPn for 18 h at 37°C and then shifted for 10 min to 45°C; lane 1) or control RNA (isolated from uninfected HeLa cells exposed to a 10-min heat shock at 45°C; lane 2). Primer extension reactions were carried out as described previously (10), and the products of the reaction were electrophoresed on a sequencing gel next to a sequencing ladder of the 2.0-kb EcoRI fragment sequenced with the same oligonucleotide primer. The transcription start site of the mRNA complementary to this primer maps to the 28th and 29th nucleotides preceding the initiating AUG of the dnaK coding region. (B) Restriction map of the 2.0-kb EcoRI fragment. Salient restriction enzyme sites are shown above the map. The two ORFs, grpE and the 240 amino acids of the dnaK coding region, are illustrated. Below the restriction map is diagrammed the strategy for the S1 nuclease experiment described in the text. Shown above the restriction map is the DNA sequence surrounding the transcription initiation site of the dnaK transcript.

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ENGEL ET AL.

J. BACTERIOL. GGAGCACTTGGCCGACCGACCCTAGATGTTCACAAAAAC 1

CAGCAGAGTTGACTCGACGTTCTTCTATAAAATTATTACCAGC2Aa=TAAC

Met Thr Glu Thr ATG ACA GAA ACC 94

Pro Asn Thr Ser Ser Glu Glu Ile Gln Thr Ser Glu Pro Ser Ser Asp Asn Glu CCC AAT ACC TCG TCA GAA GAA ATT CAG ACT AGC GAG CCT TCG TCT GAT AAC GAA

Leu Gln Thr Leu Gln Gln Glu Asn Ala Asn Leu Lys Ala Glu Leu Lys Glu Lys CTC CAA ACG CTT CAA CAA GAG AAC GCT AAT TTA AAA GCC GAA CTG AAA GAA AAA Asn Asp Arg Tyr Leu Met Ala Leu Ala Glu Ala Glu Asn Ser Arg Lys Arg Leu AAT GAC CGT TAT CTT ATG GCC CTT GCT GAA GCG GAA AAT TCA AGA AAA CGT CTA

Gln Lys Glu Arg Thr Glu Met Met Gln Tyr Ala Val Glu Asn Ala Leu Leu Asp CAG AAA GAA CGT ACA GAA ATG ATG CAA TAT GCT GTA GAA AAT GCT CTT TTG GAT

Phe Leu Pro Pro Met Glu Ser Met Glu Lys Ala Leu Gly Phe Ala Ser Gln Thr TTC CTC CCT CCA ATG GAA AGT ATG GAA AAA GCC TTG GGG TTC GCT TCT CAA ACC Ser Asp Glu Val Lys Asn Trp Ala Ile Gly Phe Gln Met Ile Leu Gln Gln Phe TCT GAT GAA GTG AAA AAT TGG GCC ATA GGG TTC CAA ATG ATC TTA CAA CAA TTT

Lys Gln Val Phe Glu Asp Lys Gly Val Val Glu Tyr Ser Ser Lys Gly Glu Leu AAA CAA GTA TTC GAA GAT AAG GGT GTT GTC GAA TAC TCT TCA AAA GGA GAA TTA

Phe Asn Pro Tyr Leu His Glu Ala Val Glu Ile Glu Glu Thr Thr Asp Ile Pro TTC AAC CCT TAT CTG CAT GAA GCT GTA GAA ATC GAA GAA ACC ACA GAT ATT CCG Glu Gly Thr Ile Leu Glu Glu Phe Thr Lys Gly Tyr Lys Ile Gly Asp Arg Pro GAA GGG ACT ATC TTG GAG GAA TTT ACA AAA GGT TAT AAG ATA GGA GAC CGT CCT

Ile Arg Val Ala Lys Val Lys Val Ala Lys Phe Pro Thr Lys Gly Asn Asn Asp ATT CGT GTA GCT AAA GTG AAA GTA GCT AAA TTT CCT ACT AAA GGA AAT AAC GAC Ser Asn Glu Glu Lys Glu AGT AAC GAA GAA AAA GAA

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Thr Thr Pro Ser Ile Val Ala Phe Lys Gly Ser Glu Thr Leu Val Gly Ile Pro ACT ACT CCT TCT ATC GTT GCT TTT AAA GGT AGC GAA ACT CTC GTG GGG ATC CCT

Ala Lys Arg Gln Ala Val Thr Asn Pro Glu Lys Thr Leu Ala Ser Thr Lys Arg GCA AAA CGT CAA GCA GTA ACG AAC CCA GAA AAA ACA TTA GCT TCT ACT AAA CGA

Phe Ile Gly Arg Lys Phe Ser Glu Val Glu Ser Glu Ile Lys Thr Val Pro Tyr TTC ATT GGC AGA AAA TTC TCT GAA GTC GAA TCT GAA ATC AAA ACA GTT CCC TAT

Lys Val Ala Pro Asn Ser Lys Gly Asp Ala Val Phe Glu Val Glu Asn Lys Leu AAA GTA GCT CCT AAC TCC AAA GGA GAC GCT GTT TTT GAA GTA GAA AAC MG CTG

FIG. 6. DNA sequence of C. trachomatis grpE and dnaK. The DNA sequence and derived amino acid sequence of a portion of the 2.0-kb

EcoRI fragment is shown. The protein-coding region of grpE commences at nucleotide 94 and terminates at nucleotide 663. The protein-coding region of dnaK commences at nucleotide 692. The start sites of the dnaK transcript are indicated by asterisks. Only the first

725 nucleotides of the dnaK gene have been sequenced. Putative ribosome-binding sites preceding the grpE and dnaK coding regions are underlined. The region complementary to the primer used in the primer extension sequences, nucleotides 57 to 76 of the dnaK coding region, is in italics.

ized on Northern blots to mRNAs that were induced by heat shock (see Materials and Methods). The restriction map of the 2.0-kb EcoRI fragment is shown in Fig. SB. A probe made by nick translation of this 2.0-kb DNA fragment hybridized on Northern blots to a 2.3-kb mRNA isolated

from chlamydia subjected to heat shock (see Fig. 9A). The DNA sequence of most of the 2.0-kb EcoRI fragment was determined, and a portion is shown in Fig. 6. Two potential open reading frames (ORFs) were identified. Beginning at bp 692 and extending to the 3' EcoRI site of the restriction

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6965

Tyr Thr Pro Glu Glu Ile Gly Ala Gin Ile Leu Met Lys Met Lys Glu Thr Ala TAC ACT CCA GAA GAA ATT GGA GCT CAA ATC CTC ATG AAA ATG AAG GAA ACA GCA

Glu Ala Tyr Leu Gly Glu Thr Val Thr Glu Ala Val Ile Thr Val Pro Ala Tyr GAA GCT TAT CTT GGA GAG ACT GTA ACT GAA GCT GTC ATC ACT GTA CCT GCT TAT Phe Asn Asp Ser Gln Arg Ala Ser Thr Lys Asp Ala Gly Arg Ile Ala Gly Leu TTT AAC GAC TCT CAA AGA GCC TCT ACA AAA GAT GCT GGA CGC ATT GCA GGA CTC Asp Val Lys Arg Ile Ile Pro Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly Ile GAT GTT AAA CGC ATT ATT CCT GAG CCA ACA GCT GCT GCT CTT GCT TAT GGT ATT

Asp Lys Glu Ala Asp Lys Lys Ile Ala Val Phe Asp Leu Gly Gly Gly Thr Phe GAC AAA GAG GCA GAT AAA AAA ATT GCC GTC TTT GAC TTG GGA GGA GGA ACT TTC Asp Ile Ser Ile Leu Glu Ile Gly Asp Gly Val Phe Glu Val Leu Ser Thr Asn GAT ATC TCT ATC TTA GAA ATT GGT GAT GGA GTC TTT GAA GTT CTC TCA ACA AAT

Gly Asp Thr His Leu Gly Gly Asp Asp Phe Asp Glu Val Ile Ile Asn Trp Met GGT GAC ACT CAC TTG GGT GGA GAT GAT TTC GAT GAA GTA ATC ATC AAC TGG ATG Leu Gly Glu Phe TTA GGT GAA TTC

1417

FIG. 6-Continued

fragment is an ORF capable of encoding a 242-amino-acid protein fragment that begins with a methionine residue. Assuming that the full-length DnaK protein of C. trachomatis is similar in size to the E. coli homolog, the minimumsized RNA necessary to encode the complete protein would be 1,950 nucleotides. Comparison to the protein data base of the predicted amino acid sequence derived from the DNA of this truncated ORF revealed that this sequence encodes the N-terminal portion of a homolog of the E. coli DnaK heat shock protein. This N-terminal fragment was 63% identical to the DnaK protein of E. coli over the 242-amino-acid region sequenced (Fig. 7). The chlamydial homolog has an N-terminal extension of 6 amino acids when compared with the E. coli protein. The C. trachomatis DnaK N-terminal fragment was 50 to 55% identical to the D. melanogaster, Caenorhabditis elegans, human, and maize hsp70 homologs (data not shown). Danilition et al. (7) recently isolated a chlamydial dnaK homolog by antibody screening of a lambda library of a human strain of C. trachomatis. In the first 220 amino acids of coding region that we sequenced, the two genes were 87% identical on the DNA level and 98% identical on the protein level (data not shown). Upstream of the dnaK gene lies a second ORF, beginning with a methionine residue at bp 94, which is capable of specifying a 190-amino-acid protein that ends at nucleotide 663 (Fig. 6). A protein homology search revealed that this ORF encodes the homolog of another heat shock protein of E. coli, GrpE. The chlamydial homolog was 30% identical to the E. coli protein over the 172-amino-acid overlap (Fig. 8). Notably, no mRNA of a size that might be predicted to encode this 190-amino-acid protein was detected on Northern blots of MoPn heat shock mRNA hybridized to a probe made from the 2.0-kb EcoRI fragment (Fig. 9A). Mapping the dnaK mRNA transcription initiation site. To determine whether the grpE and dnaK genes form part of an operon or are transcribed separately, S1 nuclease and primer extension studies were carried out. S1 nuclease experiments with the 5' EcoRI-BamHI fragment (Fig. SB) 5' labeled at the BamHI site protected a 239- to 244-bp fragment, localizing

the mRNA to approximately 27 to 33 nucleotides upstream of the initiator AUG of the dnaK gene (data not shown). The mRNA start site was more precisely determined by carrying out a primer extension experiment with a primer made to nucleotides 57 to 76 of the dnaK coding region. Two products, 104 and 105 bp in length, were observed (Fig. 5A, lane 1) that were absent when the primer extension reaction was carried out on control (uninfected HeLa cell) RNA (lane 2). Coelectrophoresis of this primer extension product next to a sequencing ladder of the 2.0-kb EcoRI fragment primed with the same oligonucleotide localized the exact transcription start sites to 28 and 29 bp upstream of the initiator ATG of the dnaK gene (Fig. SB and 6). Notably, this transcription initiation site overlapped the TAA termination residue of the C. trach

20 30 40 50 10 MSEKRKSNKIIGIDLGTTNSCVSVMEGGQPKVIASSEGTRTTPSIVAF-KGSETLVGIPA

MGKIIGIDLGTTNSCVAIMDGTTPRVLENAEGDRTTPSI IAYTQDGETLVGQPA 50 40 10 20 30

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KRQAVTNPEKTLASTKRFIGRKF--SEVESEIKTVPYKVAPNSKGDAVFEVENKLYTPEE .

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KRQAVTNPQNTLFAIKRLIGRRFQDEEVQRDVS IMPFKIIAADNGDAWVEVKGQKMAPPQ 70

60

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IGAQILMKMKETAEAYLGETVTEAVITVPAYFNDSQRASTKDAGRIAGLDVKRIIPEPTA

E .coli

ISAEVLKKMKKTAEDYLGEPVTEAVITVPAYFNDAQRQATKDAGRIAGLEVKRI INEPTA 120

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230 220 210 180 190 200 AALAYGIDK-EADKKIAVFDLGGGTFDISILEI ----GDGVFEVLSTNGDTHLGGDDFDE

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AALAYGLDKGTGNRTIAVYDLGGGTFDISIIEIDEVDGEKTFEVLATNGDTHLGGEDFDS 180

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E. coli

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FIG. 7. Comparison of the predicted amino acid sequence of the first 240 amino acids of the DnaK protein of the MoPn strain of C. trachomatis with that of the E. coli homolog. Amino acid identity is indicated by a colon, and conserved amino acids are indicated by periods. The analysis was performed by using the DFASTP program.

*,2.0;' :w

J. BACTERIOL.

ENGEL ET AL.

6966

10 20 30 40 MTETPNTSSEEIQTSEP-SSDNELQTLQQENANLKAELKE----KND

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60 100 50 70 80 90 RYMALAEAEINSRKRLQKERTEMMQYAVENALLDFLPPMESMEKALGFASQTSDEVKNWA

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110 120 160 130 140 150 IGFQMILOOFKQVFEDKGVVEYSSKGELFNPYLHEAVEIEETTDIPEGTILEEFTKGYKI

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180

170

FIG. 8. Comparison of the predicted amino acid sequence of the GrpE protein of the MoPn strain of C. trachomatis with that of the E. coli homolog. Amino acid identity is indicated by a colon, and conserved amino acids are indicated by periods. The analysis was performed by using the DFASTP program.

grpE gene. This observation suggests that the transcription of the 2.3-kb mRNA initiated 3' to this gene. Since we did not observe hybridization of the 2.0-kb EcoRI fragment to any other mRNA species on Northern blot analysis, we presume that the grpE mRNA is a rare or unstable species whose concentration is below the limits of detection of Northern blot analysis. Alternatively, the grpE and dnaK genes could be transcribed as a polycistronic RNA, followed by rapid processing and degradation of the grpE portion of the polycistronic transcript. L

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FIG. 9. Northern blot analysis of the dnaK and S18 transcripts. Total RNA (1 ,ug) isolated from uninfected HeLa cells (lane 1) and from MoPn-infected HeLa cells 4 hpi (lane 2), 7 hpi (lane 3), 12 hpi (lane 4), 18 hpi (lane 5), and 21 hpi (lane 6) and 18 hpi that were exposed to a 10-min heat shock to 450C (lane 7) was electrophoresed through a 1% agarose-formaldehyde gel (23), transferred to Hybond paper, and hybridized to 107 cpm of a nick-translated probe made to the 2.0-kb EcoRI fragment encoding DnaK (A) or the 1.5-kb EcoRI fragment encoding S18 (B). The size of the mRNA that hybridizes to the probe is indicated to the right and was determined by comparison with RNA size markers coelectrophoresed on the gel.

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.O* Sequencing strategy FIG. 10. Restriction map of p328. A more detailed map of the 1.4- and 1.5-kb EcoRI fragments is illustrated below it. Potential ORFs are indicated by the shaded boxes. The region of hybridization to the 1.2-kb mRNA is illustrated. This extent was determined by Northern blot analysis with subclones derived by exonuclease III digestion of the 1.4- and 1.5-kb EcoRI fragments (data not shown). Also shown is the sequencing strategy.

Examination of the DNA sequence upstream of the presumptive dnaK mRNA transcription initiation site revealed no sequences with homology to either the vegetative or heat shock promoter sequences of E. coli. This region showed 89% sequence identity to the corresponding region upstream of the human C. trachomatis dnaK gene (7). Assuming that this apparent transcription start site represents the initiation of a primary transcript rather than a processing site, it can be concluded that the promoter recognized by chlamydial RNA polymerase during a heat-shock-induced transcription of the chlamydial dnaK gene appears different from that of its E. coli homolog. Furthermore, this promoter is likely to lie within the grpE gene. A second gene induced by heat shock. The second EcoRI fragment (1.5 kb) that we isolated (pG82) hybridized to a 1.2-kb mRNA on Northern blots of heat shock RNA (Fig. 9B). The polarity of the transcript was determined by hybridizing probes generated by SP6- and T7-driven radiolabeled RNA transcripts to Northern blots of chlamydial RNA (data not shown). Further RNA blot analysis with probes derived from subcloned restriction fragments demonstrated that the transcribed region could be localized to the 5'-terminal 1-kb EcoRI-NsiI fragment (data not shown). Because this fragment was apparently shorter than the mRNA to which it hybridizes, we probed an SphI-XhoI library of cloned MoPn DNA with the 1.5-kb EcoRI fragment (pG82) to isolate an overlapping region. Figure 10A illustrates the restriction map of the SphI-XhoI fragment (p328) that hybridized to the pG82 probe. This 6.0-kb fragment contains the original 1.5-kb EcoRI fragment; it has additional upstream and downstream sequences. Probing Northern blots of heat shock mRNA with subclones from p328 demonstrated that both the 1.5-kb (pG82) and 1.4-kb EcoRI fragments hybridized to the 1.2-kb mRNA (data not shown). The coding region was further localized to an approximately 1,200-bp region by probing Northern blots with smaller subclones of the 1.4- and 1.5-kb EcoRI fragments (generated by exonuclease III digestion of the parent EcoRI fragment; data not shown). Figure 11 shows the DNA sequence and derived amino acid sequence of portions of two adjacent EcoRI fragments, encompassing the fragment extending from the XbaI site of the 1.4-kb EcoRI fragment to close to the NsiI site of the 1.5-kb EcoRI fragment (Fig. 7A). Several small ORFs could be identified. Beginning at the XbaI site of the 1.4-kb EcoRI fragment was an ORF capable

VOL. 172, 1990

HEAT SHOCK RESPONSE OF MURINE CHLAMYDIA TRACHOMATIS

of encoding 168 amino acids potentially representing the C-terminal portion of a protein. There were additional ORFs extending from nucleotides 954 to 1184, 1219 to 1473, and 1491 to 1706. This last ORF might represent the N-terminal portion of a protein. Potential ribosome-binding sites could be identified upstream of each of these ORFs. Comparison to the protein and nucleic acid data bases revealed only one of the ORFs (extending from nucleotide 954 to 1184) to be related to a known protein; it encoded the chlamydial homolog of the ribosomal protein S18 (Fig. 12). Over the 77 amino acids of the S18 coding region the chlamydial homolog showed 52% identity to E. coli, 42% identity to the liverwort (Marchantia polymorphia) chloroplast homolog, and 34% identity to the common tobacco chloroplast homolog (data not shown). Interestingly, this protein is not known to participate in the heat shock response of E. coli. No sequences resembling either the E. coli heat shock or vegetative promoter consensus sequences were found upstream of the 518 gene. Furthermore, no potential promoter sequences were common to the upstream regions of the MoPn C. trachomatis grpE, dnaK, and S18 genes. The transcript encoded by the p328 EcoRI fragment is referred to below as the SJ8 mRNA. Developmental regulation of these two heat shock operons. We further analyzed the transcription of dnaK and 518 mRNA at various times throughout the life cycle and in response to heat shock. Figure 9 illustrates a Northern blot of RNA at various times throughout the chlamydial life cycle that was hybridized to a probe made from the dnaK gene (the 2.0-kb EcoRI fragment; Fig. 9A) or from pG82 (the 1.5-kb EcoRI fragment that encodes S18; Fig. 9B). dnaK transcripts were barely detectable by Northern blot analysis during the chlamydial life cycle; they were, however, induced at least 10-fold after a 10-min exposure to heat shock (Fig. 9A, lane 7). In contrast, the 1.2-kb transcript that hybridizes to the pG82 probe was present throughout the chlamydial life cycle and was induced severalfold after a 10-min exposure to heat shock. It was detectable as early as 4 hpi (Fig. 9A, lane 2) and appeared to transiently increase at approximately 12 hpi (lane 4), roughly the time at which RB replication commences. dnaK and S18 mRNA inductions occur by two different pathways. We next asked whether the heat-shock-induced appearance of the dnaK and S18 mRNAs occurred at a transcriptional or posttranscriptional level by examining the effect of rifampin, an inhibitor of bacterial RNA synthesis, on their induction during a heat shock stress. Figure 13 shows a Northern blot of chlamydial RNA extracted at 18 hpi after a 10-min heat shock (lane 1) and after a similar heat shock in the presence of rifampin (lane 2) or chloramphenicol (lane 3). The blot was simultaneously hybridized to the DnaK and pG82 probes. Although both rifampin and chloramphenicol inhibited the induction of dnaK mRNA during heat shock, the induction of S18 mRNA by heat shock was inhibited by rifampin but not by chloramphenicol. These results suggest that the increased level of the two heat shock mRNAs is under transcriptional control. Furthermore, it can be concluded that the heat shock induction of dnaK mRNA is dependent upon prior protein synthesis, whereas that of the S18 mRNA is not. dnaK and S18 mRNAs have similar half-lives. The half-lives of the dnaK and S18 mRNAs were directly assayed by measuring the decay of their mRNAs after a 10-min heat shock, at which time rifampin was added; mRNA was extracted 2, 5, and 10 min after the addition of rifampin. The amount of each mRNA was assayed by Northern blot

6967

analysis; the mRNAs decayed with identical half-lives of approximately S min (data not shown).

DISCUSSION In this paper we have presented a systematic analysis of the heat shock response in the MoPn strain of C. trachomatis. Pulse-labeling of chlamydial proteins after exposure of chlamydia-infected HeLa cells to a temperature shift from 37 to 450C for 10 min allowed the identification of approximately 20 proteins whose synthesis is increased by heat shock. In contrast to results with another intracellular pathogen, Salmonella typhimurium (5), there were no quantitative or qualitative differences in the heat shock response between chlamydia-infected epithelial cell lines or macrophage cell lines in tissue culture. Not surprisingly, the kinetics of the chlamydial heat shock response resembled those of other procaryotes: the induction was rapid, occurring over a 5- to 10-min time period, and was regulated at the level of transcription. If the mechanism of transcriptional regulation is similar to that utilized by E. coli, then we infer that it should be dependent upon the association of a new r factor with the core RNA polymerase enzyme. Experiments to detect a chlamydial heat shock factor and its gene are underway. Unlike E. coli, however, chlamydia exhibited no detectable protein synthesis at 500C. In E. coli under these conditions, the cells rapidly become inviable and only heat shock proteins continue to be synthesized for as long as the now-dead cells can make protein (26). The ability of E. coli to synthesize heat shock proteins at this extreme temperature may be related to the continued synthesis of &32 from the rpoH gene. At least three different initiation sites of the rpoH transcript have been mapped by S1 nuclease and primer extension studies (11), presumably reflecting the presence of at least three promoters driving the transcription of this gene. The transcript from one of these promoters is abundant only at high temperature and is present after a shift to the lethal temperature of 500C, even at times when there is no detectable transcription from the other rpoH promoters. Although it is unclear what form of RNA polymerase recognizes this promoter in E. coli, the equivalent RNA polymerase may be missing in chlamydia. A likely explanation for the lack of such a system in chlamydia is that the intracellular form of chlamydia, the RB, is unlikely to encounter such extreme temperatures in viable mammalian cells. In contrast, the sporelike EB is already temperature resistant (3) and can withstand temperatures as high as 50°C. We next examined the behavior of two highly conserved heat shock proteins, DnaK and GroEL, in response to heat shock stress in C. trachomatis. The rate of synthesis of these two proteins after heat shock was studied by immunoprecipitation of pulse-labeled cells infected with the murine strain of C. trachomatis with heterologous antisera prepared to GroEL purified from a human strain of C. trachomatis and to DnaK purified from E. coli. These heterologous antisera were able to immunoprecipitate proteins of the expected sizes from extracts prepared from MoPn-infected cells, supporting the notion that chlamydiae synthesize homologs of these two heat shock proteins. Immunoprecipitations with either antiserum demonstrated that the rate of synthesis of each of these proteins increased in response to heat shock in the murine strain of C. trachomatis. However, surprisingly, immunoblot analysis revealed that the absolute amounts of DnaK and GroEL did not increase after heat shock, even though the steady-state levels of DnaK and GroEL increased

6968

J. BACTERIOL.

ENGEL ET AL. T 1

Leu Asp Met Leu Ala Ser Arg Phe Ser Gly Ala Phe Arg Glu Ala Pro Arg CTA GAC ATG CTG GCA TCT CGT TTC TCT GGG GCT TTT CGT GAA GCC CCT CGT

Leu Phe Ser Ser Phe Met Lys Val Glu Thr Ser Cys Gly Val Ile Val Leu Ile CTC TTT TCT TCG TTT ATG AAG GTA GAA ACT TCC TGC GGA GTT ATT GTT CTT ATT Lys Pro Ser Thr Tyr Val Asn Leu Thr Gly Lys Ala Val Leu Ala Ala Lys Arg AAA CCC TCG ACT TAT GTG AAT CTT ACT GGC AAG GCT GTT TTG GCT GCC AAG AGG

Phe Phe Gly Val Ser Val Glu Gly Ile Leu Ile Val Ala Asp Asp Ile Asn Arg TTT TTC GGC GTT TCT GTG GAA GGT ATT TTG ATT GTA GOC GAT GAT ATC AAT CGA Glu Phe Gly Ser Ile Arg Phe Arg Gln Asp Cys Gly Ala Gly Gly His Asn Gly GAG TTC GGA TCT ATA CGT TTC CGA CAA GAC TGT GGT GCC GGT GGG CAT AAC GGG Leu Lys Asn Thr Thr Gln Val Leu Gln Ser Asn His Tyr Trp Gln Leu Arg Leu CTT AAA AAT ACC ACA CAA GTT CTG CAA TCT AAT CAT TAT TGG CAA TTG CGT CTT

Gly Val Gly Arg Pro Ser Asn Pro Glu Ser Glu Gly Gly Ala Asp Tyr Val Leu GGA GTT GGC AGG CCT TCA AAT CCG GAG TCG GAG GGG GGG GCT GAC TAT GTG TTA Ser Asn Phe Ser Phe Asn Glu Arg Lys Gly Leu Asn Gly Phe Phe Glu Lys Gly TCC AAT TTT TCT TTT AAT GAA AGA AAA GGT TTA AAT GOT TTT TTT GAA AAA GGA

Ile Glu Glu Ile Ser Pro Trp Leu Ala Phe Asn Leu Lys Gly Ile Tyr Cys Ser ATA GAA GAA ATT TCC CCT TGG TTA GCT TTT AAT TTA AAA GGG ATT TAC TGT TCA Leu Phe Glu Lys Lys Ser Ser CTT TTT GAA AAA AAA AGT TCC TGAAAAATAATCAGTTTCGATAACATGGAAGCGCTTCTTATAG

505 AGTAGCTATCCATAGTTATTGGAAGAAGCAAGGTCAATGCTTAGGAGTTTTTAATGAAAAAAAAAACAGGC CAACTTTATGAGGGAGCCTATGTTTTTAGCGTGACATTAAGTGAAGACGCTAGACGTAAGGCTTTAGAAAA

AGTTACTCTGGAATCACCAACTATGTGCGAAGTTCTGAAAATTCATGATCAGGGCGOAAAAAGTTAGCTTA CACAATTCGGGGAGCCAGAGAAGGTTATTATTACTTTATCTACTTTACAGTAGCCOCAGAAGCTATTTCAG AGTTGTGGAGAGAGTATCATTTAAATGAAAGATCTTCTTCGATTCATGACTCTTAAAGCAAGCGCTGTGAA Met Asn Arg Pro Val AAT AGA CCT GTT

AGAAGTTTTAGAATTCGCTACATTGOCAGAATAATAGTTA&GGjGAACGT ATG 954

His Asn Glu His Arg Arg Lys Arg Phe Ala Lys Lys Cys Pro Phe Val Ser Ala CAT AAT GAA CAC AGA AGG AAG CGT TTC GCG AAG AAA TGT CCT TTT GTT TCC GCG

Gly Trp Lys Thr Ile Asp Tyr Lys Asp Val Val Thr Leu Lys Arg Phe Ile Thr GGT TGG AAG ACC ATC GAT TAC AAG GAC GTT GTC ACT CTA AAA AGG TTT ATT ACG Glu Arg Gly Lys Ile Leu Pro Arg Arg Ile Thr Gly Val Ser Ser Arg Phe Gln GAA AGA GGA AAG ATC CTT CCA AGA AGA ATT ACT GGA GTT TCT TCT CGC TTC CAA

Ala Leu Leu Ala Gln Ala Val Lys Arg Ala Arg His Val Gly Leu Leu Leu Ser GCA CTA CTT GCT CAG GCT GTT AAG AGA GCT OGG CAT GTT GGG CTT TTG CTT TCG

FIG. 11. Primary DNA sequence of p328, extending from the XbaI site to close to the NsiI site, and derived amino acid sequence of the potential ORFs. Putative ribosome-binding sites in front of the ORFs are underlined.

during the chlamydial life cycle in the absence of heat shock (especially between 8 and 14 hpi; J. Engel, unpublished observations). Thus, the failure to detect an increase in these proteins in response to heat shock is not a result of gross insensitivity of the immunoblot development agent to detect a further change in the level of the protein. Another explanation, although one we consider extremely unlikely, is that the lack of detection of increased protein by Western blot analysis derives from the possibility that heat shock alters the DnaK and GroEL proteins in such a way that the epitopes recognized during immunoprecipitation of the newly synthesized protein in response to heat shock are not recognized by these antisera when the total proteins are run on a denaturing gel before immunoblotting. In E. coli, both the rates of synthesis and the steady-state

levels of DnaK and GroEL and of most other heat shock proteins increase in response to heat shock. The significance of the contrasting behavior of C. trachomatis is unclear; however, it may reflect more rapid turnover of these proteins at high temperatures. In addition, as a result of the continuous exposure to the stress of the hostile intracellular environment, this parasite may synthesize these two proteins at a higher basal level than E. coli. This higher baseline rate of synthesis may obscure the relatively small increase in accumulation resulting from a brief burst of increased synthesis in response to heat shock. The DnaK antiserum coimmunoprecipitated three proteins of 70, 55, and 40 kDa. The 55-kDa protein, whose synthesis also increased in response to heat shock, comigrated with the chlamydial protein immunoprecipitated by

VOL. 172, 1990

HEAT SHOCK RESPONSE OF MURINE CHLAMYDIA TRACHOMATIS

6%96

1184

TAGGAGAAGATTAAACTTAA

Met Lys Pro Gln Leu Leu Leu Pro Glu ATAAGG ATG AAA CCA CAA TTA CTT TTG CCA GAG

1219 Asp Val Asp Gly Leu Gly Arg Ser Gly Asp Leu Val Val Ala Lys Pro Gly Tyr GAT GTC GAT GGC TTA GGA CGT TCT GGC GAT CTT GTT GTC GCT AAA CCA GGA TAC Asn Tyr Leu Leu Pro Lys Gly Lys Ala Val Val Ala Ser Ala Gly Thr Val GTT XGA AAC TAC CTA CTT CCT AAG GGG MA GCT GTA GTA GCT AGC GCT GGA ACT Leu Arg Phe Ser Lys Gln Asn Cys Lys Ser Asn Val Cys Cys Arg Leu Pro Leu CTC CGT TTT TCC AAG CAA AAT TGC AAG AGC AAC GTT TGT TGC AGG CTG CCG CTG

Ile Lys Lys Ser Leu Phe Val Trp Leu Arg His Leu Glu Ala Ser Phe Trp Ile ATA AAG AAG AGT CTC TTC GTT TGG CTG AGA CAC TTA GAA GOC TCG TTT TGG ATT Ser Lys Phe Val Met Tyr Gly Ser Val Thr Val Asn Asp TCC AAG TTC GTG TAGATTCTGAGAACMAT ATG TAC GGT TCA GTA ACC GTG AAT GAT 1473 1491

Ile Ile Ser Val Ala Asp Gin Lys Gly Val Val Leu Thr Arg Lys Asn Phe Pro ATC ATT AGC GTT GCT GAT CAA AAA GGT GTT GTT CTT ACA CGT AAA AAT TTC CCA Arg Ala His Ser Gly Val Lys Thr Leu Gly Lys His Val Ile Gly Leu Lys Leu CGC GCT CAT AGC GGA GTT AAA ACT TTA GGG AAA CAT GTA ATT GGA TTG AAA TTA Lys Glu Gly Val Thr Ala Asp Leu His Leu Glu Val Arg Ala Asp His Glu Ile AAA GAA GGC GTG ACT GCG GAT CTT CAC TTA GAA GTT CGT GCT GAT CAC GAA ATC

Ala Glu Gln Lys Glu Leu Lys Ala GCT GAA CM AAA GAA CTC AAA GCA 1706

FIG. 11-Continued

the GroEL antiserun. It is tantalizing to speculate that the 55-kDa protein is indeed GroEL; experiments to confirm this are underway. The identity of the 40-kDa protein, which was not heat shock inducible, is unknown, although it could be the chlamydial major outer membrane protein. This coimmunoprecipitation might reflect a functional association of these three proteins, conserved epitopes between them that are recognized by this antiserum, or nonspecific binding of some or all of these three moieties to this antiserum. The absence of binding to nonimmune antiserum and the failure of the serum to detect all three proteins on immunoblots (J. Engel, unpublished observations) argue strongly against the latter. We favor the first interpretation, since similar observations were made when antiserum to DnaK from C. crescentus was used to immunoprecipitate pulse-labeled proteins after exposure of this organism to heat shock (28). Also it has

10

C.trach

recently been shown that purified GroEL displays affinity toward immobilized thiol groups (Bavoil et al., in press). These data support the notion that chlamydial GrOEL may associate with the 40-kDa major outer membrane protein of C. trachomatis and thus may be involved in the assembly of the chlamydial disulfide-rich outer membrane late in development. The finding that the presumptive degradation or alternatively modified product of GroEL, which was immunoprecipitated by the GroEL antiserum but was not immunoprecipitated by the DnaK antiserum, does not contradict this hypothesis; this alternate form may not participate in the putative DnaK-GroEL-major outer membrane protein complex. Analysis of the organization and transcription of two chlamydial heat shock genes has yielded insights into the chlamydial heat shock response and into the heat shock

20

30

40

60

50

MNRPVNEHRRKRFAKKCPFVSAGWKTIDYKDVvTTxFITERGKILPRRITGVsSRFQA

E. coli

10 C.trach

70 LLAQAVKRARHVGLLLS

E.coli

QLAFAIKRARYLSLLPYTDRHQ 60 70

20

30

40

50

:::::... ::

FIG. 12. Amino acid comparison of the predicted amino acid sequence of the S18 protein of the MoPn strain of C. trachomatis compared with that of the E. coli homolog. Amino acid identity is indicated by a colon, and conserved amino acids are indicated by periods. The analysis was performed by using the DFASTP program.

6970

J. BACTERIOL.

ENGEL ET AL.

dnaK

Si8

_

*

1

._

2 3

FIG. 13. Rifampin (Rif) and chloramphenicol (Cam) differentially inhibit the transcription of the dnaK and S18 mRNAs during heat shock. Total RNA was extracted from chlamydia-infected HeLa cells 18 hpi after a 10-min heat shock to 45°C either with no added drug (lane 1) or with rifampin (100 ,ug/ml, lane 2) or chloramphenicol (10 ,ug/ml, lane 3). After electrophoresis through a 1% agaroseformaldehyde gel, the blot was transferred to a Hybond filter and hybridized simultaneously to probes made by nick translation of the 2.0-kb EcoRI fragment encoding the dnaK gene and the 1.5-kb EcoRI fragment (pG82) encoding the S18 gene.

response in general. The sequence analysis of the EcoRI fragment, which encodes the N-terminal 242 amino acids of the murine C. trachomatis homolog of dnaK, revealed that the gene encoding the chlamydial homolog of the E. coli heat shock protein GrpE lies 29 bp upstream of the dnaK gene. Several studies in E. coli have shown that DnaK and GrpE proteins functionally associate with each other (1, 20). First, the E. coli GrpE protein binds to a DnaK affinity column. Second, both proteins can be coimmunoprecipitated with antiserum to DnaK. Third, at least one extragenic suppressor of a grpE mutation maps to dnaK. Furthermore, this mutation allows the mutant form of the GrpE protein to be coimmunoprecipitated with the protein product specified by the gene encoding the dnaK suppressor mutation. Notably, the grpE and dnaK genes are located far apart on the E. coli chromosome, whereas they are separated by a mere 29 bp in C. trachomatis. Although transcription of all the heat shock proteins is coordinately induced in E. coli, only the groEL and groES genes are known to be linked in this

genome.

To address this question of whether the grpE and dnaK form an operon and are transcriptionally linked, we mapped the transcription start site of the dnaK mRNA. Northern blots hybridized to the probe made to the 2.0-kb EcoRI fragment showed a single 2.3-kb band of hybridization. A mRNA of this size would be capable of encoding both the grpE and dnaK gene products, assuming that they are approximately the same size as their E. coli homologs. However, S1 nuclease studies and primer extension experiments mapped the transcription initiation site to the last nucleotide of the grpE gene. If the dnaK mRNA transcription initiation site that we have identified represents the start of the primary transcript, then no sequences with homology to procaryotic heat shock or vegetative promoters can be identified upstream of it. This finding is consistent with our previous observations that three other chlamydial genes, known to be constitutively expressed, do not have promoters resembling the prototype E. coli vegetative promoter (9a, 10, 30, 35). Furthermore, a chimeric gene consisting of the C. trachomatis dnaK promoter (the 5' EcoRI-BamHI fraggenes

ment) linked to the E. coli chloramphenicol acetyltransferase gene transformed into E. coli did not direct a heat-shockmediated increase in chloramphenicol acetyltransferase activity (J. Pollack, unpublished observations), suggesting that the chlamydial heat shock promoter is not functional in E. coli. If the grpE and dnaK genes are transcribed separately and the dnaK mRNA transcription initiation site that we mapped indeed represents the start site of the primary transcript, then it would be necessary to postulate that the grpE mRNA is too unstable to be detected by Northern blot analysis, since we failed to observe hybridization of a probe made with this 2.0-kb EcoRI fragment to any other mRNAs. Alternatively, the grpE and dnaK genes may be cotranscribed as part of an operon. This model has theoretical appeal, because the tandem organization of the dnaK and grpE genes in C. trachomatis would allow the coordinate transcription and translation of these two gene products. Evaluating this possibility will require mapping the 5' and 3' ends of the GrpE mRNA. Notably, no sequence resembling a rho-independent transcription terminator is identifiable after the grpE gene. Although we have not identified the start site of the grpE transcript, no sequences bearing recognizable sequence homology to E. coli heat shock or vegetative promoters are present upstream of the grpE coding region. Furthermore, no conserved sequence motifs are observed in the upstream regions of the C. trachomatis grpE and dnaK genes. The heat shock transcript derived from the p328 fragment almost certainly represents a polycistronic transcript that includes the chlamydial homolog of the small ribosomal subunit protein S18. It most likely encodes one or two additional proteins with no known homology to other procaryotic and eucaryotic proteins. As might be expected for an operon encoding a ribosomal protein, it is constitutively transcribed, as revealed by Northern blot analysis. Study of the regulation of heat shock transcription with the inhibitors rifampin and chloramphenicol has uncovered the existence of two pathways for the induction of heatshock-mediated transcription in C. trachomatis. One pathway, utilized for the induction of dnaK mRNA, is dependent upon de novo protein synthesis; the same pathway is probably utilized during heat-shock-mediated transcription of the dnaK gene in E. coli (Carol Gross, personal communication). Several models could account for this observation. Heat-shock-mediated transcription could require the synthesis of an inducer or the synthesis of an inhibitor of a repressor. Alternatively, the gene product itself could stabilize its own mRNA. Evidence in E. coli suggests that dnaK mRNA induction during heat shock requires de novo protein synthesis of a molecule that stabilizes the &32 heat shock

transcription factor. In contrast, a second, novel pathway is utilized for the heat-shock-mediated induction of the S18 mRNA. In this case, the increased transcription is not dependent upon.prior protein synthesis. The identity of the transcription factor(s) involved in this pathway remains to be elucidated. Thus, C. trachomatis utilizes at least two different pathways to induce the transcription of proteins involved in the heat shock response. ACKNOWLEDGMENTS We thank Patrick Bavoil and Hatch Echols for the kind gift of the GroEL and DnaK antisera, respectively. We are grateful to Renata Gallagher for a critical review of the manuscript.

HEAT SHOCK RESPONSE OF MURINE CHLAMYDIA TRACHOMATIS

VOL. 172, 1990

This work was supported by grants from the National Institutes of Health (Public Health Service grant A124436 to D.G.) and the Lucille P. Markey Charitable Trust (grant 88-36 to J.N.E.). J.N.E. is a Lucille P. Markey scholar. J.P. was supported by the Medical Scientist Training Program.

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