Vol. 61, No. 2INFECTION AND IMMUNrrY, Feb. 1993, p. 470-4770019-9567/93/020470-08$02.00/0Copyright © 1993, American Society for Microbiology

Cloning and Sequencing of Coxiella burnetii OuterMembrane Protein Gene coml

LAURA R. HENDRIX,l2* LOUIS P. MALLAVIA,2 AND JAMES E. SAMUEL1MicroCarb Inc., Gaithersburg, Maryland 20879,1 and Department ofMicrobiology,

Washington State University, Pullman, Washington 991642

Received 5 June 1992/Accepted 19 November 1992

The gene for an approximately 27-kDa outer membrane-associated, immunoreactive protein was clonedfrom the rickettsial pathogen CoxieMla burnetii. The gene, designated comr for CoxieUla outer membrane protein1, was expressed in Escherichia coli, presumably by its own promoter. The complete nucleotide sequence of thegene was determined. The deduced amino acid sequence of 252 residues includes a putative leader sequence.The leader sequence is recognized in and removed by E. coli on the basis of the difference in the molecular massof the protein produced in an in vitro transcription-translation system (27.6 kDa) and that of the proteinimmunoprecipitated from an iodinated E. coli clone (25.7 kDa). The Coml protein expressed in E. coli wasproteinase K sensitive in nondisrupted cells and soluble in 1% Sarkosyl, suggesting a loose association with theouter membrane. While the complete predicted sequence of the Coml protein does not show any overallsimilarity to those of previously described proteins, a region which includes the only two cysteines in Coml ishomologous to the catalytic site of protein disulfide oxidoreductases.

Coxiella burnetii is an obligate intracellular rickettsialpathogen which normally causes an acute, flulike illness inhumans. Other, more serious conditions which may resultfrom a primary infection or following the activation of latentor inapparent infections include pneumonia, hepatitis, andchronic endocarditis (1). C. bumetii isolates from patientswith chronic endocarditis differ in plasmid type (32), inlipopolysaccharide (LPS) type (12), and in their chromo-somal DNA (16) from isolates which originate from patientswith acute diseases. While LPSs of different C. bumetiistrains differ in structure (presumably as variants of the 0side chain) (12), a smooth-to-rough LPS phase transition in agiven isolate is the only characterized phenotypic variationthat relates to virulence (38). This has been demonstratedpreviously by an absence of seroconversion in guinea pigsfollowing infection with as many as 108 phase II (rough-typeLPS) organisms, in contrast to seroconversion followinginfection with as few as 2 to 4 phase I (smooth-type LPS) C.bumetii organisms from either acute or chronic strains (25).The only detectable difference between acute- and chron-

ic-disease isolates in an animal model is their abilities tocause fever in the guinea pig. More than 105 endocarditisstrain organisms are required to produce fever, while only 4organisms are required with an acute-disease strain (25).Since purified LPS from either strain will not induce fever inrabbits except at large doses (25), the role of phase I LPSvariations between strains in the pathogenesis of infectionhas not been resolved (12). Therefore, factors other thanendotoxin are likely candidates for causing this difference indisease manifestation between the strains.There are no obvious differences in proteins between the

strains, as seen by sodium dodecyl sulfate (SDS)-polyacryl-amide gel electrophoresis (PAGE) (12), and only a fewsurface proteins have been isolated from and described forC. bumetii. A gene expressing a 55-kDa protein encoded ona plasmid unique to one group of chronic-disease isolates hasbeen sequenced. This protein was surface iodinated and

* Corresponding author.

immunoprecipitated in the chronic-disease C. burnetii strain(23, 24). A 29.5-kDa protein which can be surface iodinatedand immunoprecipitated with immune sera has been purifiedby high-performance liquid chromatography from detergentextracts of C. burnetii (36). This protein was closely associ-ated with the cell wall and with the peptidoglycan (43). Aprotein of 27 kDa is the major labelled peptide in a mem-brane fraction of C. burnetii cells harvested from the tissueculture medium of infected BHK cells (naturally released C.burnetii). This protein is not found in rickettsiae harvestedfrom disrupted, infected BHK cells (29). One group ofresearchers reported using a highly purified, 27-kDa C.burnetii protein as a vaccine for guinea pigs, mice, and cattle(34). This protein was recognized in guinea pigs early in thecourse of infection and following vaccination (33).We have approached the isolation of surface-exposed

putative virulence factors through the cloning of immunore-active surface proteins, since the identification of virulencecomponents by the isolation of mutants is difficult because ofthe obligate intracellular nature of C. bumetii. This studypresents the cloning and sequence analysis of a gene, desig-nated coml, which encodes an approximately 27-kDa C.bumetii protein containing sequence homology to the cata-lytic site of protein disulfide oxidoreductases. This protein isthe first outer membrane-associated immunoreactive proteinfound in both acute- and chronic-disease strains to be clonedfrom this rickettsial pathogen.

MATERIALS AND METHODS

Bacterial strains, phage, plasmids, and growth conditions.The C. burnetii Nine Mile RSA493 acute-disease isolate wasobtained from M. G. Peacock, Rocky Mountain Laborato-ries, National Institute of Allergy and Infectious Diseases,Hamilton, Mont. The H WSU101 chronic-disease isolatewas cultured at the Washington State University Biocontain-ment Laboratory, Pullman, Wash. (16). C. burnetii isolateswere propagated as previously described (14). The X EMBL3bacterial host, Escherichia coli P2392 (Stratagene, La Jolla,Calif.) was grown in TB medium (1% Bacto Tryptone, 0.5%

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NaCl, 0.2% maltose, 10 mM MgSO4 [pH 7.4]) at 370Covernight with shaking. Prior to their use in phage propaga-tion, the cells were pelleted and suspended at 0.4 times theoriginal volume in 10 mM MgSO4. The bacteria were ad-justed to an A6. of 0.5 (3 x 108/ml) in phage dilution buffer(0.58% NaCl, 0.2% MgSO4, 0.05 M Tris-HCl [pH 7.5], 0.01%gelatin). E. coli P2392 cells were infected with the bacterio-phage X EMBL3 vector (Stratagene) and grown in topagarose (0.6% agarose in H2O) on NZY agarose plates (1%NZ-amine A [ICN Biochemicals, Cleveland, Ohio], 0.5%NaCl, 0.5% yeast extract, 0.2% MgSO4, 1.4% agarose [pH7.5]). Bacteriophage plaques were removed with Pasteurpipettes, transferred to phage dilution buffer, and stored at4°C. E. coli DH5a and HB101 (Bethesda Research Labora-tories, Gaithersburg, Md.) containing plasmids were grownat 37°C in Luria-Bertani medium (22) under antibiotic selec-tion with ampicillin (100 .g/ml) for pLRH26, chloramphen-icol (25 jig/ml) for pLRH22 and pLRH23, or tetracycline (10jig/ml) for pLRH24 and pLRH25.DNA cloning and sequencing. Chromosomal DNA from C.

bumnetii Nine Mile RSA493 was isolated as previously de-scribed (31) and partially digested with MboI. Fragments of10 to 20 kb were collected from agarose gels (22) and ligatedinto BamHI-cut X EMBL3. The DNA was packaged intoGIGA-PACK (Stratagene) and transfected into E. coliP2392. Plaques were absorbed onto nitrocellulose, blockedin Tris-buffered saline (20 mM Tris, 500 mM NaCl [pH 7.5])with 3% gelatin, and incubated in rabbit polyclonal anti-serum prepared by injecting animals with formalin-killed C.burnetii Nine Mile RSA493 whole cells. The antiserum waspreabsorbed with both sonicated and intact E. coli P2392cells. Positive plaques were visualized by an enzyme-linkedimmunosorbent assay (ELISA) with horseradish peroxidase-conjugated goat anti-rabbit and color development reagents(Bio-Rad Laboratories, Richmond, Calif.). Twenty-threepositive plaques out of approximately 1,000 screened weredetected, and each positive plaque was subsequently puri-fied twice. Phage DNA was extracted from each of thepurified, positive plaques by scraping the top agarose fromplates containing nearly confluent plaques and vortexing theagarose in phage dilution buffer with 10 ,u of chloroform perml added. After 3 h of elution at room temperature, theagarose and bacterial cells were pelleted by centrifugation.The supernatant was added to the top of glycerol stepgradients made by pipetting 3 ml of 5% glycerol on top of 3ml of 40% glycerol. The gradients were centrifuged for 1 h at4°C at 35,000 rpm in an SW41 rotor (Beckman Instruments,Fullerton, Calif.). The bacteriophage pellets were suspendedin phage dilution buffer to which 1 ,ug of both DNase I andRNase A per ml were added. After being incubated at 370Cfor 30 min, they were extracted once with phenol, once with1:1 (vol/vol) buffered phenol-chloroform (22), and once withchloroform. The DNA was precipitated in isopropanol con-taining 0.3 M sodium acetate and centrifuged in order topellet the DNA. The DNA was precipitated a second time inabsolute ethanol containing 2.5 M ammonium acetate. Thepelleted DNA was suspended in 10 mM Tris-HCl-1 mMEDTA, pH 8. Sall, EcoRI, and HindIII-SmaI fragments of XEMBL3 clone 7 were separated by electrophoresis through0.7% agarose gels, excised from the gels, and purified withGeneclean (Bio 101, Inc., La Jolla, Calif.). Purified DNAwas then cloned by standard methods (22) into pACYC177and pACYC184 (7). E. coli DHSa or HB101 competent cellswere transformed with the ligation mixtures. Plasmid DNAwas isolated from E. coli by alkaline extraction (4) or withQIAGEN columns (QIAGEN, Studio City, Calif.). DNA

sequencing of the coding region for the coml gene was doneby using double-stranded DNA from plasmids pLRH22, -23,-24, and -26 and Sequenase version 2.0 (United StatesBiochemical, Cleveland, Ohio). Oligonucleotide primers (19-to 21-mers) were synthesized by using an Applied Biosys-tems DNA synthesizer. Both strands of DNA in the codingregion were sequenced. Sequence data were analyzed byusing MacVector (IBI, New Haven, Conn.).DNA hybridization. Southern blot analysis was used to

map the X EMBL3 clone 7 DNA insert and to detect itspresence in C. burnetii DNA. DNA fragments of X EMBL3clone 7 were isolated and labelled with [a-32P]dCTP byrandom primer extension (Pharmacia LKB, Piscataway,N.J.). The DNA to be probed was digested to completionwith restriction enzymes, electrophoresed in 0.7% agarose,and then denatured and transferred to GeneScreen mem-branes (NEN Research Products, Boston, Mass.) (37).Membranes were prehybridized for 1 h at 680C in 6x SSC(lx SSC is 150 mM NaCl plus 15 mM sodium citrate) and1Ox Denhardt's solution (0.2% bovine serum albumin[BSA], 0.2% Ficoll, 0.2% polyvinylpyrrolidone) and thenhybridized for 4 h at 68°C in 5 x SSC-5 x Denhardt'ssolution-20 mM Tris-HCl (pH 8)-50 ,g of salmon spermDNA per ml-0.1% SDS-5 mM EDTA. Probe (106 cpm/ml)was added for incubation overnight, and then membraneswere washed four times at high stringency for 30 min each at68°C with 1x SSC containing 5 mM EDTA and 0.2% SDS.Gene expression and immunoblot detection. Proteins pro-

duced by the positive A plaques were purified by scraping thetop agarose from plates containing nearly confluent plaquesand vortexing the agarose in phage dilution buffer. After a3-h elution period, the agarose was pelleted at 10,000 x g for15 min at 4°C, and proteins in the supernatant were precip-itated with 10% cold trichloroacetic acid overnight at 4°C.The proteins were pelleted at 10,000 x g for 20 min at 4°C,washed twice in phosphate-buffered saline (PBS), suspendedin Laemmli sample buffer (19), and subjected to SDS-PAGE.Phage and E. coli clone protein profiles were analyzed forthe expression of C. burnetii proteins by Western immuno-blot analysis (39) with the ELISA used to screen plaques. C.bumetii protein expressed in E. coli subclones of A EMBL3clone 7 was also detected in an in vitro transcription-translation system (Promega Corp., Madison, Wis.). Todetect the presence of the 27-kDa protein in C. burnetii cells,antiserum enriched for antibodies to this protein was pre-pared by the method of Oaks et al. (27). Rabbit anti-Cburnetii polyclonal antiserum was incubated with nitrocellu-lose to which clone 7 plaques had first been absorbed andblocked as described above. After a 3-h incubation period,the nitrocellulose was washed first in Tris-buffered salinewith 0.05% Tween 20 added, then in Tris-buffered saline,and finally in 0.15 M NaCl before the bound antibody waseluted in 0.2 M glycine plus 0.15 M NaCl, pH 2.8. Theantibody solution was immediately neutralized with 0.75 MTris, pH 8.8, and used at a three- to fourfold dilution inWestern blots or undiluted in immunoprecipitations.

Radioimmunoprecipitation. Radioimmunoprecipitationswere done to determine the location of the Coml protein inboth C. burnetii cells and in E. coli containing pLRH26.Iodogen (Pierce, Rockford, Ill.) was resuspended in chloro-form at 1 mg/ml and dried under a stream of nitrogen gas tocoat the bottom of a glass tube. To each tube was added 100,ul of 0.3 M sodium phosphate [pH 6.8], 500 ,ul of bacteria inPBS (5 x 108 E. coli cells or 1 mg [dry wt] of C. bumetiicells), or membrane preparations made from an equivalentquantity of bacteria and 10 pu (0.5 mCi) of 125I. After 3 to 5

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min of incubation on ice with frequent shaking, the mixturewas quenched by transferring it to a tube containing 500 p.l ofTris-BSA (50 mM Tris-HCl, 150 mM NaCl, 1% BSA [pH7.8]) and centrifuging it for 10 min in a microcentrifuge(14,000 x g) to pellet the iodinated cells or membranes. Thecells or membranes were washed once more with Tris-BSAand suspended in PBS. Approximately 7.5 x 106 dpm ofeach preparation was pelleted and resuspended in lysisbuffer (20 mM Tris-HCl [pH 7.5], 0.85% NaCl, 0.1% SDS,0.1% deoxycholic acid, 2% Triton X-100, 10 mM EDTA, 20,ug of aprotinin per ml, 200 ,ug of phenylmethylsulfonylfluoride per ml) for 30 min at 4°C. The lysed cells werepelleted in a microcentrifuge for 20 min at 4°C. Supernatantlysates were precleared by incubating them in Pansorbin(Calbiochem, La Jolla, Calif.) for 1 h at 4°C with constantrotation. After the removal of Pansorbin by pelleting in amicrocentrifuge, the lysates were incubated overnight at 4°Cin an equal volume of X EMBL3 clone 7-specific antiserum.GammaBind Plus (Pharmacia) was added to each sample,rotated for 30 min at 4°C, and pelleted for 30 s in amicrocentrifuge. The GammaBind Plus pellets were washed3 or 4 times in 1 ml of lysis buffer and eluted in 100 ,ul ofLaemmli sample buffer for 15 min at room temperature.Samples were boiled for 5 min, and 50 pul was subjected toSDS-PAGE. Gels were dried under a vacuum and exposedto X-ray film.Membrane preparations. E. coli cells containing pLRH26

were suspended in 10 mM HEPES (N-2-hydroxymethylpi-perazine-N'-2-ethanesulfonic acid) (pH 7.4) with proteaseinhibitor cocktails containing 20 pug (each) of leupeptin,chymostatin, and pepstatin per ml, 40 pug of antipain per ml,200 ,ug of benamidine per ml, and 15 pug of aprotinin per mland sonicated six times on ice for 30 s each at a setting of 4with a Bronson Sonicator. The disrupted organisms werecentrifuged at 10,000 x g for 10 min at 4°C. The supematantwas centrifuged at 100,000 x g for 1 h at 4°C. The resultingpellet was referred to as the membrane preparation. Solubi-lization of the plasma membrane was carried out by incubat-ing the membrane preparation in 1% Sarkosyl (Sigma Chem-ical Co., St. Louis, Mo.) for 30 min at room temperature andpelleting the outer membranes at 100,000 x g for 1 h at 4°C.To aid determination of the location of the cloned Comlprotein in E. coli cells containing plasmid pLRH26, cellswere either treated with 500 pug of proteinase K per ml for 1h at 55°C and then boiled in Laemmli sample buffer; firstboiled in sample buffer, treated with proteinase K as de-scribed above, and boiled again; or not treated with protein-ase K, but simply boiled in sample buffer and subjected toSDS-PAGE. Gels were analyzed by immunoblotting to vi-sualize the cloned C. bumetii protein or stained withCoomassie blue for total protein.

Nucleotide sequence accession number. The GenBankaccession number for the coml nucleotide sequence isM88613.

RESULTS

Cloning of the comr gene. A gene bank of C. bumnetii NineMile RSA493 DNA was<. constructed in X EMBL3 andtransfected into E. coli P2392 cells. Plaques were screenedfor the production of C. bumetii immunoreactive proteins.Twenty-three positive plaques out of approximately 1,000screened were visualized by ELISA. After the positiveplaques were purified, proteins released into the plaqueswere purified from the top agarose and subjected to SDS-PAGE. Phage-encoded proteins were analyzed for the pres-

Kb

23.9 -

9.9 -6.6 -

4.3

2.22.0 -

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

_~~~~~~wol

=** ! -~~41

4.

A B CFIG. 1. Southern hybridization of restriction enzyme digestion

fragments homologous to C. bumnetii-specific DNA in X EMBL3clone 7. (A) Agarose gel containing HindIII-digested X DNA (lane1), Sall-digested X EMBL3 clone 7 (lane 2), HindIII-SmaI-digestedpLRH26 (lane 3), HindIll-digested C. burnetii genomic DNA fromNine Mile RSA493 (lane 4), HindIll-digested C. bumetii genomicDNA from H WSU101 (lane 5), and HindlIl-digested E. coligenomic DNA from DH5a (lane 6). Also shown are autoradiographsof the Southern blot prepared from the agarose gel in panel Ahybridized with a HindIII-SmaI fragment probe from X EMBL3clone 7 DNA (B) and a A EMBL3 probe (C).

ence of C. burnetii proteins by Western immunoblot analy-sis. All A EMBL3 clones produced an approximately 60-kDaimmunoreactive protein, while one, termed X EMBL3 clone7, expressed an additional immunoreactive protein of ap-proximately 27 kDa (15). The 60-kDa protein cross-reactedstrongly with an E. coli protein of a similar size despiteextensive adsorption of the antisera with E. coli cells andsonic lysates. Because we were interested in cloning immu-noreactive surface proteins unique to C. bumetii, our effortswere focused on characterizing the clone expressing the27-kDa protein.To confirm that X EMBL3 clone 7 contained C. bumetii

DNA, a Southern blot analysis was done. Figure 1B showsthat a 9.5-kb restriction enzyme fragment probe of X EMBL3clone 7, encompassing the majority of the insert DNA (Fig.2), hybridizes with DNA from two isolates of C. burnetiirepresenting two strains (Fig. 1B, lanes 4 and 5). This probedid not hybridize with A or E. coli genomic DNA (Fig. 1B,lanes 1 and 6, respectively). The two C. bumnetii strains,which have significant differences in their restriction enzymebanding patterns (16), hybridize with the probe at restrictionenzyme fragments of an identical size. A X EMBL3 probe,used as shown in Fig. 1C, hybridizes only with A DNA (lanes1 and 2).

Subcloning and localization of the comr gene. X EMBL3clone 7, which produced the 27-kDa protein, was mapped byrestriction enzymes and Southern blot analysis (Fig. 2), andsubclones of the cloned fragments in X EMBL3 clone 7 weremade in the vectors pACYC177 and pACYC184 (7). Proteinsproduced by these subclones were subjected to SDS-PAGEand tested by Western immunoblot analysis for the produc-tion of a 27-kDa immunoreactive protein. The protein wasexpressed by only one subclone, pLRH26, which contains aHindIII-SmaI fragment of clone 7 (Fig. 2). These resultssuggested that the coding region required for expression ofthe 27-kDa protein must span the central EcoRI and SalIsites, since subclones which did not span these sites did notproduce the 27-kDa protein.DNA sequence analysis. To determine the nucleotide se-

quence of the open reading frame (ORF) encoding the Comlprotein, clones pLRH22 and pLRH23 were sequenced in

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C. BURNETII OUTER MEMBRANE PROTEIN 473

S Sm 1 2 3 4 5 6 7 8 MSm S E Sm H

subclone 27kDa

pLRH22 -

pLRH23 -

pLRH24

SH E E

s

S E SmE E

pLRH25 -

S E

N EEpLRH26 +

S E Sm

I kb

FIG. 2. Subcloning strategy of coml gene with restriction en-

zyme maps of EMBL3 clone 7 and subclones. The presence (+) or

absence (-) of an immunoreactive 27-kDa protein produced by eachsubclone is indicated as determined by Western blot analysis withC. bumetii whole-cell-specific antibody. Sm, SmaI; S, Sail; H,HindIII; E, EcoRI.

opposite directions beginning from the central site of thethree SaII sites in clone 7, as shown in Fig. 2. An ORF longenough to encode an approximately 27-kDa protein beginswith a GTG codon 339 bp upstream from the central EcoRIsite and ends 257 bp downstream from the central SalI site.The sequence of the 756-bp ORF was confirmed by sequenc-

ing the opposite strand and by sequencing across the centralSalI site with pLRH26.As indicated in the nucleotide sequence in Fig. 3, this ORF

begins with a GTG codon at position 289 and ends with a

TAA termination codon at position 1045, followed immedi-ately by a second in-frame TAA stop codon at position 1048.Beginning 6 bases upstream of the GTG start codon, theORF is preceded by a polypurine-rich sequence of AAGGGG representing a potential Shine-Dalgarno ribosome bindingsequence (35). A predicted promoter sequence, TTCGAA-N15-AATAAT, occurs between positions 238 and 264 (30).The G+C content of the coding region was 42 mol%, quitesimilar to the value of 43 mol% for C. bumnetii total genomicG+C (42).

-43

-29

-18.4

-14.3FIG. 4. In vitro transcription-translation of plasmid DNA from

E. coli subclones of A EMBL3 clone 7. The [35S]methionine-labelledproteins were separated by SDS-PAGE (12% acrylamide) andexposed to X-ray film. The autoradiograph is shown. Lanes: 1,control DNA pGEM3GAL expressing ,-galactosidase and ,B-lacta-mase; 2, pLRH22; 3, pLRH23; 4, pLRH24; 5, pACYC184; 6,pACYC177; 7, _LRH26 expressing Comi, as indicated by thearrowhead; 8, 1 I-labelled E. coli/pLRH26 immunoprecipitatedwith polyclonal rabbit antibody to C. burnetii enriched for Coml-specific antibodies (the Coml protein is indicated by the arrow); M,

14C-labelled molecular mass markers (in kilodaltons).

Protein structure. The ORF encodes a protein of 252amino acid residues, as deduced from the nucleotide se-

quence (Fig. 3). The N-terminal amino acid sequence hascharacteristics typical of a bacterial leader sequence (41),with 2 basic amino acids at the N terminus followed by a

region of hydrophobic and neutral amino acids. Severalpotential sites for cleavage by leader peptidase exist in the Cterminus of the leader sequence, but the most likely cleavagesite follows an Ala-Ile-Ala sequence (amino acids 19 to 21)before an Ala-Pro sequence (28). The primary translationproduct of the cloned gene has a deduced molecular mass of27,591 Da. Cleavage by leader peptidase would result in a

protein of 231 amino acids with a predicted molecular massof 25,509 Da. Processing of Coml by E. coli was assessed bySDS-PAGE (Fig. 4). The unprocessed protein produced by

101

201

GTCAATTATCTTGAAGACAAACGTTACAACCCAGCCCTGCAATTGGAACGAAGCAAkTTGGGCCGATGATCAGGAGCATCGATTGCACAACGCAATGCAATCGATGGATGATCGAAGTCGAGATATTTATATCAGCGTTGGTTAAGTGMA.AATCAACGCTTCATGAATTAGCAGCACAATACGCTGTTTCTGCCGAA

-35 -10 rbs V K N RCGTATTCGGCAGCTTGAAAAAAACGCCATGCAGAAAA &&CTGCTATGGCAGATGAZ 0&AITTTTAACTCTG mATAGATCGTGAAGAACCGT

5 L T A L F L A G T L T A G V A I a sA P S Q F S F S P Q Q V K D I Q S301 TTGACTGCACTATTTTTAGCCGGAACCTTGACCGCAGGCGTGGCGATAGCCGCCCCCTCTCAATTCAGTTTTTCTCCTCAACAAGTCAAAGACATACAAA39 I V H H Y L V N H P E V L V E A S Q A L Q K K T E A Q Q E E H A Q

401 GCATCGTCCACQTTATTTAGTCAACCACCCAGAAGTTTTAGTAGAAGCATCCCAAGCATTGC GACAGAAGCGCAACAAGAAGAACACGCTCA

72 Q A I K E N A K K L F N D P A S P V A G N P H G N V T L V E F F D501 ACAAGCAATTAAAGAAAATGCAAAGAAATTATTTAACGACCCTGCATCACCAGTGGCAGGCAATCCTCATGGCAATGTTACATTGGTTGAATTTTTCGAT105 Y Q C G H C K A N N S V I Q A I V K Q N K N L R V V F K E L P I F G601 TATCATGTGGCCATTGCAAAGCCATi T=GTTATTCAAGCTATCGTGAAACAAAATAAAAACCTCCGCGTTGTCTTCAAAGAACTGCCCATTTTTG

ZcoRI139 G Q S Q Y A A K V S L A A A KQ G K Y Y A F H D A L L S V D G Q L701 GCGGCCAATCGCAATACGCTGCCAAAGTATCATTAGCAGCCGCTAAACAAGGAAAATATTATGCTTTCCACGACGCGCTGCTCAGTGTCGaCGGCCAATT

SalI172 S E Q I T L Q T A E K V G L N V A Q L K K D M D N P A I Q K Q L R801 ATCAGAACAAATCACCCTTCAAACCGCAGAAAAAGTAGGATTAAATGTTGCTCAGCTCAAAAAAGACATGGATAATCCTGCTATCCAAAAACAACTGCGT205 D N F Q L A Q S L Q L A G T P T F V I G N K A L T K F G F I P G A T901 GATAACTTCCAATTAGCTCAATCGTTACAGCTAGCAGGCACCCCGACGTTCGTCATTGGTAAT AGSc=ATCGGTTTTATACCCGGCGCCA

Rpai239 S Q Q N L Q K E I D R V E K

1001 CCTCACAACAAAACCTTCAAAAAGAAATCGACCGGGTAGAAAAG:CA&ATCCTCACTGATACGACCTCTTCCGCCCAAACGGGAGAGGTCAAGAGGCG1101 CTCTAAGCCTCACCCTATCATTACCACGCCCCTCGTCATCCC

FIG. 3. Nucleotide and predicted amino acid sequences of the coml gene from C. bunetii. Putative promoter regions (-35 and -10), theribosome binding site (rbs), and the leader peptidase cleavage site ( I ) are indicated.

A EMBL3 clone 7 SH E E

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Coml (96-113) W Q C G i C K

PDI (24-41) WY1FYaPWCG C:KAIthioredoxin (21-38) g a i

F W a e W C G PC K ml

DsbA (38-55) a p qV S fL PCpCy qf

FIG. 5. Comparison of the catalytic sites of disulfide oxidoreduc-tases with Coml. Sequences are from the first active site of ratprotein disulfide isomerase (PDI) (8), E. coli thioredoxin (20), E. coliDsbA (3), and Coml. Amino acids which are identical or highlysimilar to those of the Coml sequence are shaded. Residue numbersare shown in parentheses.

an in vitro transcription-translation extract had an estimatedmolecular mass of 27,600 Da (Fig. 4, arrowhead in lane 7).The mature protein immunoprecipitated from "25I-labelled E.coli pLRH26 (Fig. 4, arrow in lane 8) has an estimated sizeof 25,700 Da. It is interesting that plasmid pLRH26 alsoappears to encode an approximately 36-kDa nonimmunore-active protein (Fig. 4, lane 7) which mapped to a region 5' tothe coml locus (lane 2). This protein is approximately thesame size as the product of the pACYC184 tetracyclineresistance gene expressed in plasmid pLRH24 (Fig. 4, lane4).By using the nucleotide sequence and the deduced amino

acid sequence of the coml gene, a computer search of theGenBank and National Biomedical Research Foundationdata bases was conducted. While no significant DNA or

amino acid homologies to the complete sequence were

identified, a region of Coml (Fig. 5) containing the onlycysteines (amino acids 107 and 110) displays homology toregions encompassing the catalytic sites of disulfide oxi-doreductases from eukaryotes and bacteria (3, 8, 20).

Surface expression of Coml protein in E. coli and C.burnetii. Radioimmunoprecipitations were performed to de-termine the location of Coml protein in E. coli/pLRH26 andin C. bumetii. A protein of 25.7 kDa was immunoprecipi-tated from iodinated C. bumnetii Nine Mile whole cells (Fig.6, lane 1) and from E. coli DH5a whole cells and membranepreparations containing plasmid pLRH26 (Fig. 6, lanes 2 and3, respectively) but not from E. coli DH5a whole cellscontaining pACYC177 (Fig. 6, lane 4). This analysis alsodemonstrates that the mature Coml protein expressed by E.coli is identical in size to the Coml protein expressed by C.burnetii.To support the indication of its surface location, the

sensitivity of Coml to proteolytic attack on whole C. bur-netii cells was determined (Fig. 7). Both 50 (lane 5) and 500(lane 4) ,ug of proteinase K per ml completely eliminated an

immunoreactive 27-kDa product seen on untreated C. bur-netii cells (lane 1) and untreated E. coli/pLRH26 (lane 7).Additionally, the protein expressed by E. coli/pLRH26 wasshown to be sensitive to proteinase K treatment, both withcells disrupted in Laemmli sample buffer containing SDS and2-mercaptoethanol (Fig. 8, lane 3) and with nondisruptedcells (Fig. 8, lane 2). Results of these experiments suggestthat the Coml protein is exposed on the cell surface in C.burnetii and in the E. coli clone. However, when membranepreparations of the E. coli clone were extracted in 1%Sarkosyl, Coml protein was found to be soluble in Sarkosyl,a common property of proteins associated with either thecytoplasmic membrane or nonintegral outer membrane pro-teins (Fig. 9, lane 4). Hydrophilicity and surface probabilityplots of the predicted amino acid sequence of Coml (Fig. 10)

M 1 2 3 4 5 6 7

43

29-

18.4 .

14.3- ~

FIG. 6. Radioimmunoprecipitation of Coml protein. Whole cellsof C. bumnetii, E. coliIpLRH26, and E. coli/pACYC177 and amembrane preparation of E. coliIpLRH26 were labelled with "~I.Lysates of these preparations were immunoprecipitated with poly-clonal rabbit antibody to C. burnetii enriched for antibodies toComl, as shown in lanes 1 to 4; lysates prior to antibody precipita-tion are shown in lanes 5 to 7. Lanes: 1 and 5, C. buetii Nine MileRSA493 whole cells; 2 and 6, F. coli/pLRH26 whole cells; 3, F.coliIpLRH26 membrane preparation; 4 and 7, F. coliIpACYC177whole cells; M, 14C-labelled molecular mass markers (in kilodal-tons).

revealed three strongly hydrophilic regions. This analysissuggested several possible membrane integration regions.

DISCUSSION

This article presents the results of cloning and sequenceanalysis of a C. burnetii gene, designated coml, whichencodes an immunoreactive outer membrane protein and isexpressed in E. coli. Cloning of the coml gene was accom-plished by the detection of immunoreactive plaques froma C. burnetii genomic bank with polyclonal rabbit antiserumto formalin-killed C. bumn:etii whole cells. Twenty-threeplaques out of approximately 1,000 plaques screened reactedwith the antiserum. Yet by Western blot analysis, only oneimmunoreactive phage clone, clone 7, contained a proteindifferent from the approximately 60-kDa protein produced

RSA43 whle clls 2 3n 6, E. 6oipR2whl7els,E

- 106- 80

-49.5

-32.5

-18.5

FIG. 7. Immunoblot of SDS-PAGE depicting proteinase K sen-sitivity of Coml in C. bumnetii with antisera enriched for antibodiesto Coml as described in Materials and Methods. Lanes: 1, untreatedwhole cells ofCR buetii Nine Mile RSA493; 2 and 3,Co bumnetiicells boiled in Laemmli sample buffer prior to treatment withproteinase K at final concentrations of 500 and 50 j±g/ml, respec-tively; 4 and 5, proteinase K-treated whole cells of C. bumnetii withfinal concentrations of proteinase K of 500 and 50 p.g/ml, respec-tively; 6, molecular mass markers (in kilodaltons); 7, untreatedwhole cells of E. coliepLRH26.

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M 1 2 3

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FIG. 8. Immunoblot of SDS-PAGE depicting proteinase K sen-

sitivity of Coml in E. coli/pLRH26 with rabbit polyclonal antiserumto C. bumetii Nine Mile RSA493. Lanes: M, molecular massmarkers (in kilodaltons); 1, untreated whole cells of E. coli/pLRH26;2, proteinase K-treated whole cells of E. coli/pLRH26; 3, E.coli/pLRH26 cells boiled in Laemmli sample buffer prior to beingtreated with proteinase K.

by all other positive clones. In the screening of a second C.burnetii gene bank made in X EMBL3 with DNA from achronic-disease strain, all positive clones produced an ap-proximately 60-kDa protein on Western blot analysis (datanot shown). The 60-kDa protein may be either HtpB, apreviously characterized C. bumnetii GroEL analog (40), anovel C. burnetii protein, or a cross-reactive E. coli protein.The restriction enzyme banding patterns of DNA obtainedfrom clones expressing the 60-kDa protein did not identify acommon fragment. More selective antibody is required inorder to determine whether these clones express the same ordifferent proteins. The apparent inability of the polyclonalsera used for screening to select other surface proteins of C.burnetii has led us to produce a series of monoclonalantibodies to acute- and chronic-disease C. burnetii strains(unpublished data).

Subcloning of the X EMBL3 clone 7 insert which encodesthe 27-kDa protein into several pMBl/ColEl replicon-con-taining vectors was not successful after numerous attempts.Although clone 7 DNA was detected in the recombinants bycolony blot analysis, inserts of the correct size could not berecovered. Whether the inability to clone this DNA intopMBl/ColEl replicon-containing vectors was due to theincompatibility of the DNA or to lethality due to highplasmid copy numbers was not determined. Subclones were

M 1 2 3 4 5 6 7

110-8447-

33-24

16-

FIG. 9. Immunoblot of SDS-PAGE depicting Sarkosyl solubilityof Coml in E. coli/pLRH26. Blots were prepared as described in thelegend to Fig. 8. Lanes: M, molecular mass markers (in kilodaltons);1 to 3, E. coli/pLRH26 whole cells, membrane preparation, andSarkosyl insoluble membrane preparation, respectively; 4, Sarkosylsoluble supernatant from E. coli/pLRH26 membrane preparation; 5to 7, E. coli/pACYC177 whole cells, membrane preparation, andSarkosyl insoluble membrane preparation, respectively.

easily obtained with the P15A replicon-containing vectorspACYC177 and pACYC184. The subclones made in thepACYC vectors allowed for deletion analysis of the codingregion of the coml gene.A putative ribosome binding site at position 275 of the

nucleic acid sequence is different from others seen in C.bumnetii (21) but is the same as a Shine-Dalgarno sequencereported for the Legionella micdadei mip gene (2). Thespacing of the -35 and -10 promoter sequences and thesequences themselves are similar to what has been reportedpreviously for other C burnetii genes (21). The GTG initia-tion codon at position 289 represents the first putativenon-ATG start codon reported for C. bumetii. GTG is usedas an initiation codon in 5 to 10% of E. coli genes (17).The ORF for the coml gene encodes a predicted protein of

27.6 kDa, which agreed with the results seen with SDS-PAGE following in vitro transcription-translation of the E.coli subclone pLRH26. The protein seen in iodinated wholecells of E. coli/pLRH26 or C. burnetii had a molecularweight of 25,700. A putative leader sequence, which wouldbe cleaved by leader peptidase in whole cells but not in an invitro transcription-translation system, could account for thedifference in size between the unprocessed and matureproteins. Leader sequences have been reported for proteinsdestined for the outer membrane, the cytoplasmic mem-brane, or the periplasmic space (41). The Coml protein waspresent in a membrane preparation, indicating that it isprobably not localized in the periplasmic space. The solubil-ity of the Coml protein in 1% Sarkosyl is a property mostcommonly seen with cytoplasmic membrane proteins (9).However, some outer membrane proteins are easily re-moved from the membrane with Sarkosyl or other milddetergents (26). Coml was not seen in previous Westernblots of C. bumetii whole cells immunoblotted with antiseraraised against a C. burnetii membrane preparation preparedby extraction with 1% Sarkosyl (15). The ability to surfacelabel Coml with 125I in both C. burnetii cells and the E.coli/pLRH26 clone and the sensitivity of Coml to proteinaseK in nondisrupted cells indicate that it is most likely an outermembrane protein.The amino acid sequence deduced from the coding region

predicts a hydrophobic leader sequence followed by a ma-ture protein with a pI of 8.8. While no clear membrane-spanning or strongly hydrophobic region(s) is seen in themature protein by Kyte-Doolittle plots (18) (Fig. 10), thereare several possible membrane integration regions. Threestrongly hydrophilic regions exist. One is close to theN-terminal region, with the other two at or near the Cterminus. No significant homology exists between Coml andpreviously described major outer membrane proteins ofother gram-negative bacteria such as porins, lipoproteins, orproteins involved in specific diffusion processes, such asLamB. The inferences on the structure of Coml on the basisof amino acid sequence data are consistent with the experi-mental evidence that Coml is loosely attached to the mem-brane as opposed to being an integral membrane protein.

Previously described C. burnetii proteins of approxi-mately 27 kDa share some characteristics with Coml. The27-kDa protein labelled in C. burnetii cells naturally releasedfrom infected BHK cells was secreted or easily removedfrom the membrane (29), which corresponds to the presentresults indicating that the protein is soluble in 1% Sarkosyl.The other reported 27-kDa protein was an immunogenicprotein used as a trial vaccine in cattle, guinea pigs, andmice. The researchers reported on the nature of the antibodyresponse to this protein following vaccination and following

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476 HENDRIX ET AL.

Hydrophilicity Window Size = 11 Scale = Kyte-Doolittle

00-

oo~~~~~~~~~~--- ---- -- ------ -----

oo~~~~~~~~~~~~~~~~~~~~~~~~~~~- - -- - --- - -- -- ----

0-

00-50-

70

40 -1

30

90-

0

50 100 150 200 250FIG. 10. Hydrophilicity and surface probability plots of C. burnetii Coml. Plots were generated by the program MacVector with a span

setting of 11 amino acids. Positive values on the y axes represent predicted hydrophilic regions with a greater than 50% chance of surfaceprobability. Values on the x axes represent numbered residues in the amino acid sequence.

natural infection (33, 34). The procedure used to clone thecoml gene resulted in the selection of immunoreactiveproteins. However, the serum used for selection was notobtained from natural infections, so it is not possible to inferthe importance of these proteins in the immune response toC. burnetii.While the homology between Coml and disulfide oxi-

doreductases (Fig. 5) may provide clues to its function, therole of such a protein in pathogenesis is not obvious. Proteindisulfide oxidoreductases catalyze thiol-disulfide inter-change reactions, promoting protein disulfide formation,reduction, or isomerization, depending on the redox poten-tial and the polypeptide substrate (10, 11, 13). C. burnetiigrows in the phagolysosome of eukaryotic cells (5, 6), anenvironment which differs significantly from most bacterialgrowth conditions. Coml may be released from the surfaceof C. bumnetii under such conditions to alter the redoxpotential of the phagolysosome, affecting the surface pro-teins of C. burnetii or proteins in the phagolysosome.Determining an enzyme capability for Coml will allow fortesting of models describing the possible role of the comlgene in pathogenesis.

ACKNOWLEDGMENTSThis work was supported in part by grant A120190 (L.P.M.) from

the National Institutes of Health (NIAID).We thank Debbie L. Weinstein for helpful suggestions and Susan

Turcovski and Shirley Schmitt for their technical expertise.

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