INFECTION AND IMMUNITY, Jan. 1989, p. 71-750019-9567/89/010071-05$02.00/0Copyright © 1989, American Society for Microbiology

Isolation of a Gene Encoding a Chlamydia sp. Strain TWAR ProteinThat Is Recognized during Infection of Humans

LEE ANN CAMPBELL,'* CHO-CHOU KUO,1 ROBERT W. THISSEN,1 AND J. THOMAS GRAYSTON"2

Departments ofPathobiology' and Epidemiology,2 University of Washington, Seattle, Washington 98195

Received 6 July 1988/Accepted 14 September 1988

Chlamydia sp. strain TWAR is a unique Chlamydia sp. that causes acute respiratory disease. A gene bankconsisting of TWAR isolate AR-39 DNA in pUC19 was screened with anti-AR-39 rabbit immune sera. Twopositive clones were isolated that contained 7.3-kilobase (pLC1) and 14.9-kilobase (pLC2) plasmids. Restrictionmapping and hybridization studies showed that both pLCl and pLC2 contained a common 4.2-kilobase PstIfragment. Plasmids were used as templates of in vitro transcription-translation. All three plasmids had a novelprotein product of ca. 75 kilodaltons not found in the vector alone. Western blots showed that this proteinreacted with anti-TWAR rabbit immune sera and with human immune serum from an individual who hadproven TWAR infection. Whole-cell lysates of TWAR demonstrated a protein having the same molecularweight and immunoreactivity as the recombinant gene product. This protein was also recognized by rabbitimmune serum against Chlamydia psittaci or Chlamydia trachomatis. Southern hybridizations with the clonedfragment as a probe of digests of other Chlamydia spp. showed weakly hybridizing fragments. These resultssuggest that we have isolated a gene encoding a protein recognized during human TWAR infection that containssome sequences shared among Chlamydia spp.

Chlamydia sp. strain TWAR has been shown to be animportant respiratory pathogen associated with acute respi-ratory diseases including pneumonia, bronchitis, andpharnygitis (13, 14). Serological and epidemiological evi-dence has indicated a wide geographical distribution of theorganism in both epidemic and endemic disease (13, 19). Theisolation of Chlamydia sp. strain TWAR from seropositiveindividuals has confirmed the association of this organismwith respiratory infection (13). The TWAR organism is a

member of the genus Chlamydia sharing the chlamydialgenus-specific antigen and unique developmental cycle (20).However, several characteristics distinguish Chlamydia sp.strain TWAR from Chlamydia trachomatis and the hetero-geneous species Chlamydia psittaci (7-9, 20). The organismis serologically distinct from the other Chlamydia spp. (20)and can be differentiated from strains of both species byrestriction endonuclease analysis (7). Ultrastructural studieshave shown that the morphology of the elementary body ofthe TWAR organism is distinct from that of C. trachomatisor C. psittaci (8). In determinations of DNA homology,TWAR was found to have less than 10% DNA relatedness toC. trachomatis or C. psittaci (9). This cumulative evidencehas resulted in the proposal that a new Chlamydia species,designated C. pneumoniae, be established for this group oforganisms (J. T. Grayston, C.-C. Kuo, L. A. Campbell, andS.-P. Wang, Int. J. Syst. Bacteriol., in press).Because of the difficulty associated with isolating and

growing the TWAR organism, we used recombinant DNAmethodology for identification of genes encoding immuno-reactive proteins. In this report, we describe the expressionof a TWAR protein in Escherichia coli that is recognizedduring human infection and contains genus reactivities.

MATERIALS AND METHODS

Strains. The Chlamydia strains studied included the fol-lowing: (i) Chlamydia sp. strain TWAR isolates AR-39,LR-65, AR-388, and TW-183 (13, 20); (ii) C. trachomatis B/

* Corresponding author.

TW-5/OT (22); and (iii) C. psittaci 6BC (12) and strainscausing meningopneumonitis (Mn strain) (11), feline pneu-monitis (FP strain) (2), guinea pig inclusion conjunctivitis(GPIC strain) (27), and sheep abortion (OA strain; a localisolate obtained from P. Dilbeck, Washington State Univer-sity, Pullman, Washington). All Chlamydia strains wereadapted to grow in HeLa 229 cells (22). Infected cells were

harvested after 3 days of incubation followed by centrifuga-tion at 500 x g for 10 min to remove cell debris. Theorganisms were recovered by centrifuging at 30,000 x g for20 min. Elementary bodies were purified by pelletingthrough a 30% meglumine diatrizoate cushion (16) (Hy-paque-76; Winthrop-Breon Laboratories, New York, N.Y.)and banding in a 30 to 65% linear gradient of megluminediatrizoate at 45,000 x g for 90 min. The organisms were

collected, washed with Hanks balanced salt solution, sus-

pended in either sucrose-phosphate glutamate buffer or

N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid,and frozen at -75°C until used.

Antisera. Rabbit immune sera were prepared againstTWAR strains AR-39 and TW-183 grown in HeLa cells bythe method of Kenny (18). Rabbit anti-C. trachomatis (B/TW-5/OT; grown in chicken embryo yolk sacs) and anti-C.psittaci (Mn, grown in chicken embryo allantoic cavities)antisera were those prepared in an earlier study (21). Themonoclonal antibodies used were Chlamydia genus-specificCF-2, FC-5, RR-405, and TT-304 and TWAR-specific RR-402. These monoclonal antibodies were prepared from fu-sions with C. trachomatis (CF-2 and FC-5) or with TWAR(RR-402, RR-405, and TT-304) as described previously (29).The genus-specific monoclonal antibodies CF-2, RR-405,and TT-304 reacted with lipopolysaccharide in the immuno-blot. We used CF-2, FC-5, and RR-402 in earlier studies withTWAR (20). Human sera used were from the isolationproven TWAR case AR-277 (13) and from normal personswho had no microimmunofluorescence antibody (31) or

complement fixation antibody against TWAR, C. trachoma-tis, or C. psittaci.

Chemicals and enzymes. Restriction enzymes were ob-

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72 CAMPBELL ET AL.

tained from Pharmacia Fine Chemicals (Piscataway, N.J.).T4 DNA ligase was obtained from Bethesda Research Lab-oratories, Inc. (Gaithersburg, Md.). All chemicals werereagent grade and obtained from Sigma Chemical Co. (St.Louis, Mo.) or J. T. Baker Chemical Co. (Phillipsburg,N.J.).Gene bank construction. Chlamydia sp. strain TWAR

DNA was isolated as described previously (7). Chromo-somal DNA was digested with PstI and ligated to similarlydigested pUC19 by a standard protocol (26). pUC19 wastreated with calf intestinal phophatase to prevent self-liga-tion. After CaCl2 transformation into Escherichia coli TB1(26) by the method of Hanahan (15), transformants wereplated onto LB agar containing 50 ,ug of ampicillin per ml and40 jig of 5-bromo-4-chloro-3-indoyl-,-D-galactoside per ml.Colonies containing inserts were replicated onto nitrocellu-lose and lysed by standard methods (26). For screening ofcolonies, nitrocellulose was blocked with Tris-buffered sa-line containing 1% bovine serum albumin by incubating atroom temperature for 1 h. Then filters were incubated withanti-TWAR rabbit immune serum (1:300 dilution) for l h,washed six times (5 min each) in Tris-buffered saline-0.05%Tween 20, incubated with goat anti-rabbit serum conjugatedto horseradish peroxidase (diluted 1:1,000; Cooper Biomed-ical, Malvern, Pa.), and washed with Tris-buffered saline-0.05% Tween 20 six times (5 min each), followed by twowashes (5 min each) with Tris-buffered saline. Color wasdeveloped by the addition of hydrogen peroxide and 4-chloro-1-naphthol.Western blot (immunoblot) analysis. Whole cell lysates of

recombinants, host cells containing vector only, and TWARelementary bodies were analyzed by discontinuous sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis bythe method of Laemmli (23) with 13% polyacrylamide gels.Approximately 20 pug of protein, as determined by themethod of Lowry et al. (24), was loaded into the sample well.Molecular weight markers were from the Pharmacia electro-phoresis calibration kit, which includes phosphorylase b (94kilodaltons [kDa]), bovine serum albumin (67 kDa), ovalbu-min (43 kDa), carbonic anhydrase (30 kDa), soybean trypsininhibitor (20.1 kDa), and lactalbumin (14.4 kDa). Proteinswere transferred to nitrocellulose by the method of Towbinet al. (30). Molecular weight markers were stained withAurodye (Janssen Life Science Products, Piscataway, N.J.).The immunoassay employed was as described above, excepta 1:250 dilution of rabbit immune serum, a 1:20 dilution ofhuman immune serum, or a 1:50 dilution of the appropriatemonoclonal antibody was used as dictated by the experi-ment. Anti-E. coli reactivities were removed by adsorbing tocyanogen bromide-activated Sepharose 4B (Pharmacia) towhich E. coli TB1 lysates had been coupled according to thedirections of the manufacturer. Subsequent experimentsshowed that the use of nonadsorbed sera did not influencethe detection of the chlamydial gene product on Westernblots. Thus, the adsorption step was omitted unless statedotherwise. Goat anti-rabbit, goat anti-human, or goat anti-mouse immunoglobulin serum conjugated to horseradishperoxidase were used at a dilution of 1:500. The extent ofprotein transfer was monitored by amido black stain.

In vitro transcription-translation. Plasmid preparationswere purified by CsCl density gradient ultracentrifugation(26) and used as a template for protein synthesis with theprocaryotic DNA directed translation kit (Amersham Corp.,Arlington Heights, Ill.) as directed by the manufacturer.[35S]methionine-labeled protein products were identified byfluorography of SDS-polyacrylamide gels. "'C-labeled mo-

lecular weight markers (Bethesda Research Laboratories)were simultaneously run for molecular weight determina-tions.DNA analysis. Restriction digestions were done as recom-

mended by the manufacturer. Fragments were analyzed on0.8 to 1.5% agarose gels, depending on the experiment.For hybridizations, fragments were transferred to nitro-

cellulose by the method of Southern (28). The cloned TWARinsert was purified by electroelution (26) and nick translatedby using [32P]dATP and [32P]dGTP to a specific activity of108 cpm; 107 cpm was added per hybridization. Hybridiza-tion reactions and washes were done under stringent condi-tions as previously described (7). The hybridization reactionbuffer included 50% formamide, 5x Denhardt reagent, 5xSSPE (lx SSPE is 180 mM NaCl, 10 mM NaH2PO4 [pH7.4], and 1 mM EDTA [pH 7.4]), 10 g of salmon sperm DNAper ml, and 0.1% SDS. After hybridization for 18 h at 42°C,three washes were done in O.lx SSC (lx SSC is 0.15 MNaCl and 0.015 M sodium citrate) and 0.1% SDS for 15 mineach at 52°C, followed by three washes with O.1x SSC.

RESULTS

Isolation and characterization of recombinants. Twoclones, containing plasmids designated as pLC1 and pLC2,were identified by their reactivity with anti-AR-39 rabbitimmune serum. pLC1 was ca. 7.3 kilobases (kb), and pLC2was ca. 14.5 kb. Restriction endonuclease analysis demon-strated a common 4.2-kb PstI fragment. In addition, pLC1contained a 400-base-pair PstI fragment, and pLC2 had a6-kb PstI fragment. The 4.2-kb insert was isolated by digest-ing pLC2 with PstI and electroeluting. This fragment wasused as a probe in Southern hybridizations of PstI-, PstI-HindlIl-, or PstI-EcoRI-digested pLC1 and pLC2 DNA. Inall cases, the hybridization patterns for both clones demon-strated that the 4.2-kb fragments were identical (data notshown). pLC3 was constructed by digesting pLC2 with PstI,religating, and screening for transformants containing onlythe 4.2-kb insert. The restriction map of this fragment isshown in Fig. 1.

Analysis of gene products. Whole-cell lysates of E. coliTB1 containing pLC1, pLC2, or pLC3 were subjected toSDS-polyacrylamide gel electrophoresis; a novel weak pro-tein band was observed at ca. 75 kDa (data not shown).Western blot analysis with anti-AR-39 rabbit immune serumdemonstrated an immunoreactive protein product in E. coliTB1 harboring pLC1 or pLC2 that was not seen in whole-celllysates ofTB1 containing vector alone or of HeLa cells (Fig.2A). Identical results were obtained with anti-TW-183 rabbitimmune serum. This immunoreactive protein was also ob-served in whole-cell lysates of E. coli TB1 containing pLC3(data not shown), indicating that the 4.2-kb fragment en-codes the reactive protein product. Immunoblots were alsodone with human sera. The gene product expressed in E. coliwas recognized by the human immune serum against TWAR(Fig. 2B). No reactivity was observed with whole-cell ly-sates of E. coli containing pLC3 or whole-cell lysates ofTWAR elementary bodies against normal serum. Whole-celllysates ofTWAR demonstrated a protein of the same appar-ent molecular weight as the recombinant protein that wasrecognized by both anti-TWAR rabbit serum and serum froma TWAR-infected individual (Fig. 2B). The same immunore-activities were observed with two other TWAR isolates,LR-65 and TW-183 (data not shown).The clones expressing TWAR proteins were tested for

reactivity with genus-specific (CF-2, FC-5, RR-405, and

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CLONING OF A CHLAMYDIA SP. STRAIN TWAR GENE 73

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FIG. 1. Restriction map of 4.2-kb Pstl TWAR fragment. bp, Base pairs.

TT-304) and TWAR-specific (RR-402) monoclonal antibod-ies. No reactivity was observed with any of the monoclonalantibodies tested.

Protein products were further characterized by in vitrotranscription-translation. All recombinant plasmids encodeda protein of ca. 75 kDa, which was the same apparentmolecular size as the immunoreactive protein observed inWestern blots (Fig. 3).

Demonstration of shared antigens. To test any cross-

reactivity of the expressed protein product with immune seraprepared from immunization with C. trachomatis or C.psittaci, Western blots were done with anti-TW-5 rabbitserum and anti-Mn rabbit serum. Both sera reacted stronglywith the cloned TWAR insert protein product (Fig. 4). Thisreactivity was not observed in E. coli containing the vectoralone. Each serum also reacted with a protein of similarmolecular weight found in C. trachomatis, C. psittaci, andTWAR.Based on these results, we hypothesized that if similar

epitopes are recognized, sequences found in the clone mightbe shared among the Chlamydia spp. Chromosomal PstIdigests of C. trachomatis serovar B and various strains of C.psittaci were probed with the 4.2-kb TWAR insert. Understringent hybridization conditions, a 4.2-kb PstI fragment

1 2 3 4 1 2 3 4

reacted in all TWAR isolates tested (Fig. 5). The higher-molecular-weight reactivities in TWAR appear to be due toincomplete digestions. Hybridizing bands ranging from 0.6to 2.9 kb were observed in the C. psittaci strains, and aweakly hybridizing 4.7 kb fragment was observed in C.trachomatis serovar B. Under less stringent conditions anadditional reactive C. trachomatis fragment was observed(data not shown).

DISCUSSION

A 4.2-kb PstI fragment encoding an immunoreactive pro-tein of 75 kDa has been isolated from the human respiratorypathogen Chlamydia sp. strain TWAR. Immunoreactiveproteins of similar apparent molecular weight were alsoidentified in whole-cell lysates of TWAR, C. trachomatis,and C. psittaci. These results demonstrate that the TWAR-encoded protein contains genus reactivities. In addition tothe recognition of the antigenic cross-reactivities, the TWARfragment encoding this protein was shown to share sequencehomology with the respective fragments of C. trachomatisand C. psittaci.

All members of the genus Chlamydia share a commonlipopolysaccharide antigen that has been shown to be iden-tical to 2-keto-3-deoxyoctanoic acid (6, 10). The results ofour study showed that there are shared antigenic sites on a

1 2 3 4 5

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FIG. 2. Immunoblots demonstrating a 75-kDa protein encodedby the cloned fragment that reacts with anti-TWAR rabbit andhuman sera. Whole-cell lysates were separated by SDS-polyacryl-amide gel electrophoresis, electrophoretically transferred to nitro-cellulose, and reacted with immune sera as described in Materialsand Methods. Lanes: 1, TB1(pLC1); 2, TB1(pLC2), 3 TWAR isolateAR-39; 4, TB1(pUC19). (A) Reactivity with rabbit immune serum;(B) reactivity with serum from an individual with TWAR infection.

FIG. 3. In vitro transcription-translation analysis of cloned pro-tein products. When used as templates for in vitro transcriptiontranslation, all recombinants encoded a 75-kDa protein (arrow).Lanes: 1, pLC1; 2, pLC2; 3, pLC3; 4, pUC19.

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74 CAMPBELL ET AL.

1 2 3 4 5 6

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FIG. 4. Immunoblots demonstrating the 75-kDa fusion proteins(arrow). Lanes: 1, TB1(pLC3); 2, TWAR isolate LR-65; 3, TW-5; 4,Mn; 5, TB1(pUC19); 6, HeLa. (A) Anti-TW-5 (C. trachomatis)rabbit immune serum; (B) anti-Mn (C. psittaci) rabbit immuneserum.

ca. 75-kDa protein among the Chlamydia spp. Although thisprotein could represent a high-molecular-weight lipopolysac-charide aggregate or precursor, the nonreactivity of genus-specific monoclonal antibodies with the clone or the proteinfrom Chlamydia elementary bodies suggests that an epi-tope(s) found on this protein represents another sharedchlamydia epitope. The recognition of other genus antigenicreactivities is in agreement with recent reports of antigenicanalysis of C. trachomatis have identified genus reactivity inthe major outer membrane protein (1, 25) and 70-, 75-, and100-kDa proteins (25). However, Chlamydia sp. strainTWAR was not included in these studies.

Immunoreactive proteins with molecular weights similarto that of our recombinant protein have been observed instudies done with C. trachomatis and C. psittaci. In a recent

1 2 3 4 5 6 7 8 9 1011

study analyzing the humoral response of guinea pigs toinfection with C. psittaci, an immunoreactive protein ofsimilar molecular weight was observed in serum samplestaken 21 to 41 days after intravaginal infection with guineapig inclusion conjunctivitis (3). For C. trachomatis, antibod-ies against a 74-kDa protein antigen were found in sera frominfected patients, regardless of the infected serovar (32).Subsequently, a C. trachomatis serovar L2 gene encoding animmunoreactive protein of ca. 74 kDa was cloned andexpressed in E. coli (17). However, this protein was deter-mined to represent a species-specific antigen because therewas no reactivity with anti-Mn serum (17). Under stringenthybridization conditions, the C. trachomatis cloned frag-ment did not react with undigested C. psittaci strain MnDNA. Under reduced stringency, Mn chromosomal DNAdid react with the cloned serovar L2 fragment (17). Inanalyzing the human serologic response to chlamydial infec-tion to C. trachomatis antigens, Brunham et al. demon-strated that antibodies to a 75-kDa protein were prevalentand induced antibodies associated with protection againstascending infection (4, 5). In subsequent studies, they de-scribed genus specific-monoclonal antibody directed againstthe 75-kDa protein. Rabbit hyperimmune sera were preparedagainst the 75-kDa protein and were shown to have neutral-izing activity (25). Whether the immunoreactive proteins ofsimilar molecular weight described in the literature are thesame as those in the 75-kDa immunoreactive protein de-scribed in this paper needs further investigation.

This is the first report of the isolation of genes encodingimmunoreactive proteins of the novel Chlamydia sp. strainTWAR pathogen and of a gene encoding a protein havinggenus reactivities that is not a lipopolysaccharide. Becauseof the limited DNA relatedness of the Chlamydia species,the demonstration that the cloned TWAR sequence encodesa genus antigenic reactivity and has DNA homology withsequences of C. trachomatis and genetically diverse strainsof C. psittaci (9) suggests that this protein may have animportant antigenic or biologic function. The recognition ofthis protein during human TWAR infection and other chla-mydial infections (3-5, 32) further supports this hypothesis.Current studies are aimed at addressing the functions of thisprotein during TWAR infection and more precisely identify-ing the shared immunoreactive site(s).

ACKNOWLEDGMENTS

This study was supported by Public Health Service grants Al21885 and EY 00219 from the National Institutes of Health and by agrant from the Edna McConnell Clark Foundation.

FIG. 5. Southern blot of chromosomal PstI digests of variousChlamydia strains probed with the 4.2-kb TWAR insert understringent hybridization conditions. Lanes: 1, LR-65; 2, TW-183; 3,AR-388; 4, blank; 5, TW-5; 6, Mn; 7, FP; 8, OA; 9, 6BC; 10, GPIC;11, HeLa. The arrow marks the 4.2-kb reactive fragment of theTWAR isolates.

LITERATURE CITED1. Baehr, W., Y.-X. Zhang, T. Joseph, H. Su, F. E. Nano, K. D. E.

Everett, and H. D. Caldwell. 1988. Mapping antigenic domainsexpressed by C. trachomatis major outer membrane protein(MOMP) genes. Proc. Natl. Acad. Sci. USA 85:4000-4004.

2. Baker, J. A. 1944. A virus causing pneumonia in cats andproducing elementary bodies. J. Exp. Med. 70:159-171.

3. Batteiger, B. E., and R. G. Rank. 1987. Analysis of the humoralimmune response to chlamydial genital infection in guinea pigs.Infect. Immun. 55:1767-1773.

4. Brunham, R. C., I. W. Maclean, B. Binns, and R. W. Peeling.1985. Chlamydia trachomatis: its role in tubal infertility. J.Infect. Dis. 152:1275-1282.

5. Brunham, R. C., R. Peeling, I. Maclean, J. McDowell, K.Persson, and S. Osser. 1987. Postabortal Chlamydia trachomatissalpingitis: correlating risk with antigen specific serologic re-sponses and with neutralization. J. Infect. Dis. 155:749-755.

6. Caldwell, H. D., and P. J. Hitchcock. 1984. Monoclonal antibody

1 2 3 4 5 6".eas*a1t

_

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from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 12

Nov

embe

r 20

21 b

y 11

7.14

6.51

.220

.

CLONING OF A CHLAMYDIA SP. STRAIN TWAR GENE 75

against a genus-specific antigen of Chlamydia species: locationof the epitope on chlamydial lipopolysaccharide. Infect. Immun.44:306-314.

7. Campbell, L. A., C-C. Kuo, and J. T. Grayston. 1987. Charac-terization of the new Chlamydia agent, TWAR, as a uniqueorganism by restriction endonuclease analysis and DNA-DNAhybridization. J. Clin. Microbiol. 25:1911-1916.

8. Chi, E. Y., C.-C. Kuo, and J. T. Grayston. 1987. Uniqueultrastructure in the elementary body of Chlamydia sp. strainTWAR. J. Bacteriol. 169:3757-3763.

9. Cox, R. L., C.-C. Kuo, J. T. Grayston, and L. A. Campbell.1988. Deoxyribonuclic acid relatedness of Chiamydia sp. strainTWAR to Chlamydia trachomatis and Chlamydia psittaci. Int.J. Syst. Bacteriol. 38:265-268.

10. Dhir, S. P., G. E. Kenny, and J. T. Grayston. 1971. Character-ization of the group antigen of Chlamydia trachomatis. Infect.Immun. 4:725-730.

11. Francis, T., Jr., and T. 0. Magill. 1938. An unidentified virusproducing acute meningitis and pneumonia. J. Exp. Med. 68:147-150.

12. Gordon, F. B., and A. L. Quan. 1978. Occurrence of glycogen ininclusions of the psittacosis-lymphogranuloma venereum tra-choma agents. J. Infect. Dis. 115:186-196.

13. Grayston, J. T., C.-C. Kuo, S.-P. Wang, and J. Altman. 1986. Anew Chlamydia psittaci strain, called TWAR, from acute respi-ratory tract infection. N. Engl. J. Med. 315:161-168.

14. Grayston, J. T., C.-C. Kuo, S.-P. Wang, M. K. Cooney, J.Altman, T. J. Marrie, J. G. Marshall, and C. M. Mordhorst.1986. Clinical findings in TWAR respiratory tract infection, p.336-337. In D. Oriel, G. Ridgway, J. Schachter, D. Taylor-Robinson, and M. Ward (ed.), Chlamydial infection. CambridgeUniversity Press, Cambridge.

15. Hanahan, D. 1983. Studies on transformation of Escherichia coliwith plasmids. J. Mol. Biol. 166:557-580.

16. Howard, L., N. S. Orenstein, and N. W. King. 1974. Purificationon Renografin density gradient of Chlamydia trachomatis grownin the yolk sac of eggs. Appl. Microbiol. 27:102-113.

17. Kaul, R., and W. M. Wenman. 1985. Cloning and expression inEscherichia coli of a species-specific Chlamydia trachomatisouter membrane antigen. FEMS Microbiol. Lett. 27:7-12.

18. Kenny, G. E. 1967. Heat-lability and organic solubility ofmycoplasma antigens. Ann. N.Y. Acad Sci. 143:676-681.

19. Kleemola, M., Saikku, R. Visakorpi, S.-P. Wang, and J. T.Grayston. 1988. Pneumonia epidemics in military trainees inFinland caused by TWAR, a new Chlamydia organism. J.Infect. Dis. 157:230-236.

20. Kuo, C.-C., H.-H. Chen, S.-P. Wang, and J. T. Grayston. 1986.Identification of a new group of Chiamydia psittaci strainscalled TWAR. J. Clin. Microbiol. 24:1034-1037.

21. Kuo, C.-C., G. E. Kenny, and S.-P. Wang. 1971. Trachoma andpsittacosis antigens in agar gel double immunodiffusion, p. 113-123. In R. L. Nichols (ed.), Trachoma and related disorderscaused by chlamydial agents. Excerpta Medica, Amsterdam.

22. Kuo, C.-C., S.-P. Wang, and J. T. Grayston. 1977. Growth oftrachoma organism in HeLa 229 cell culture, p. 328-336. In D.Hobson and K. K. Holmes (ed.), Nongonococcal urethritis andrelated infections. American Society for Microbiology, Wash-ington, D.C.

23. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

24. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

25. Maclean, I. W., R. W. Peeling, and R. C. Brunham. 1988.Characterization of Chlamydia trachomatis antigens withmonoclonal and polyclonal antibodies. Can. J. Microbiol. 34:141-147.

26. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1983. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

27. Murray, E. S. 1964. Guinea pig inclusion conjunctivitis virus. I.Isolation and identification as a member of the psittacosislymphogranuloma-trachoma group. J. Infect. Dis. 114:1-16.

28. Southern, E. 1975. Detection of specific sequences among DNAfragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517.

29. Stephens, R. S., M. R. Tam, C.-C. Kuo, and R. C. Nowinski.1982. Monoclonal antibodies to Chlamydia trachomatis: anti-body specificities and antigen characterization. J. Immunol.128:1083-1089.

30. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

31. Wang, S.-P., and J. T. Grayston. 1970. Immunologic relation-ship between genital TRIC, lymphogranuloma venereum andrelated organisms in a new microtiter indirect immunofluores-cence test. Am. J. Ophthalmol. 70:367-374.

32. Wenman, W. M., and M. A. Lovett. 1982. Polypeptide antigensof Chlamydia trachomatis cell walls, p. 51-55. In P.-A. Mardh,K. K. Holmes, J. D. Oriel, P. Piot, and J. Schachter (ed.),Chlamydial infection. Elsevier Biomedical Press, Amsterdam.

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