Download - Cloning and nucleotide sequence comparison of the groE operon of Pseudomonas aeruginosa and Burkholderia cepacia

APMIS 103: 113-123, 1995 Printed in Denmark . All rights reserved

Copyright 0 A P M I S 1995

ALWUS ISSN 0903-4641

Cloning and nucleotide sequence comparison of the groE operon of Pseudbmonus aeruginosa

and Burkholderia ceDacia

PER JENSEN,' ANDERS FOMSGAARD,2 NIELS H01BY',2 and PETER HINDERSSON3

'Institute of Medical Microbiology and Immunology, 2Department of Clinical Microbiology, National University Hospital, Rigshospitalet, and 3M & E Aps, Copenhagen, Denmark

Jensen, P, Fomsgaard, A., H~riby, N. & Hindersson, P Cloning and nucleotide sequence comparison of the groE operon of Pseudomonas aeruginosa and Burkholderia cepacia. APMIS 103: 113-123, 1995.

By alignment of GroEL amino acid sequences from four distantly related bacteria two highly conserved domains were identified. Two oligonucleotides complementary to the conserved domains were designed based on the preferred Pseudomonas aeruginosa codon usage. The primers were used in the PCR to amplify a 900-base fragment of the P. aeruginosa groEL gene. The fragment was sequenced and the partial GroEL sequence was expanded by vectorette PCR upstream and downstream to cover the complete P. aeruginosa groE operon. The same technique was used to sequence the Burkholderia cepacia (formely Pseudomonas cepacia) groE operon and the region immediately upstream of groES. The B. cepacia groE operon is preceded by typical -10 and -35 heat shock expression signals. A total of 2041 and 2139 bp was sequenced from P. aeruginosa and B. cepacia respectively. Each revealed two open reading frames encoding two proteins with a predicted molecular mass of 10 and 57 kDa, corresponding to GroES and GroEL respectively. The GroEL proteins show an interspecies amino acid homology of 71%, and 73% with E. coli GroEL. Both GroEL proteins are 52% homologous to the corresponding human mitochondria1 GroEL protein. The sequence data confirm the existence of highly conserved structures, which could be functionally important for the concerted action of GroEL and GroES in the folding and assembly of other proteins, and possibly in the initiation of autoimmune diseases.

Key words: Pseudomonas aeruginosa; Burkholderia cepacia; Pseudomonas cepacia; groE operon; heat shock proteins; autoimmunity.

Per Jensen, Institute of Medical Microbiology and Immunology, Panum Institute 24.1.9, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.

Chronic bacterial lung infection is the major factor responsible for the severity of illness and mortality of patients with cystic fibrosis (CF). Pseudomonas aeruginosa and Burkholderia cepacia are the most frequent causes of degenerative chronic lung infection in CE P. aeruginosa is the most prevalent etiological agent worldwide. In the United States, Canada and some Euro-

Received June 10, 1993. Accepted November 17, 1994.

pean countries B. cepacia seems to be an in- creasing problem. Association of autoimmune diseases such as arthritis, episodic arthropathy and diabetes mellitus with CF has been reported (2, 4, 21, 25). Whether these autoimmune conditions are related to the fundamental biochemical defect in CF or are associated with the sec- ondary microbial infection is still unclear.

Bacterial GroEL heat shock proteins, also designated Hsp60-65 or common antigen (27, 29), may constitute a link between infectious diseases and autoimmunity, as indicated by a

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variety of experimental data (20, 23, 35). Thus, bacterial GroELs may initially be a key factor in autoimmune diseases associated with C E This idea is supported by several observations. The mycobacterial GroEL homologue Hsp65 is implicated in autoimmune arthritis in Lewis rats. Two T-cell lines which recognize the mycobacterial Hsp65 protein are able to induce or protect against adjuvant arthritis (32, 33). Co- hen and co-workers have shown that the development of autoimmune diabetes in non-obese diabetic (NOD) mice is associated with T-cell reactivity to the mycobacterial Hsp65 protein. T-cell clones recognizing an epitope of the human GroEL homologue, referred to as H- hsp65, mediated insulitis and hyperglycaemia (8, 9). Such T-cell clones can be attenuated and used as therapeutic vaccines to abrogate the diabetonic process (9). A strong immune response to bacterial GroELs has been observed as a result of frequent bacterial lung infections in patients with C F (15, 16).

Sequence data of the phylogenetically conserved GroEL proteins might provide further insight into the role of autoimmunity and aid detailed studies of the cellular and humoral immune response to Pseudornonas GroELs, es- pecially in cases of autoimmune diseases associated with C E

This paper describes the use of PCR techniques to clone the groESL genes of the clinical strains P. aeruginosa P1118 and B. cepaciu ATCC 17559. The inter- and intraspecies vari- ation in the bacterial groE operon of P. aeruginosa and B. cepacia, and the recently published groESL DNA sequences of P. ueruginosa ATCC 27853 (30) was examined. The nucleotide sequence similarity of the groE operons and the homology between the deduced amino acid sequences with the human mitochondria1 GroEL homologue P1 (1 8) were analysed.

MATERIALS AND METHODS

E.utraction of genomic DNA B. cepaciu ATCC 17559 and P. ueruginosu PI 118

0 : 3 (10, 17) were used. A previously described method (22) was employed for purification of genomic DNA with some modifications. Briefly, the bacterial culture was grown overnight in Luria-Bertani (LB) broth with aeration at 37°C. The culture was harvested and the bacterial pellet resuspended in ST

buffer (25% sucrose (wtivol, Merck, Darmstadt, Ger- many), 50 mM Tris-HCI (Sigma, St. Louis, MO, USA), pH 8.0). The material was incubated for 15 min on ice with 1.0 mg/ml lysozyme (Sigma), whereafter 0.1 M ethylenediaminetetraacetic acid (EDTA. BDH Chemicals, Poole, UK) and 2% sodium dodecyl sulfate (SDS, Sigma) were added to the mixture. Thorough shaking was followed by the addition of 1 M sodium perchlorate (Sigma) and one volume of chloroform-isoamyl-alcohol (24: 1, Merck). The material was centrifuged at 10,000 g for 10 min, and the supernatant was quickly added to 99'% ethanol (Merck), supplemented with 1 M ammonium acetate (Merck). DNA, collected with a sterile glass rod and transferred to distilled water, was treated with 0.25 mgiml RNase (Boehringer Mannheim, Germany) in 10 mM Tris-HCI, 15 mM NaCl (Merck), pH 7.5, for 1 h at 37"C, followed by incubation with 0.5 mgiml proteinase K (Boehringer Mannheim) for an ad- ditional 1 h at 37°C. The process was repeated from the addition of sodium perchlorate. whereafter DNA threads were collected and transferred to TE buffer (10 mM Tris-HCI, 1 mM EDTA, pH 8.0).

PCR amplification und cloning All primers used for DNA sequencing and PCRs

were prepared on a DNA synthesizer (381A, Applied Biosystems, Foster City, CA, USA). Synthetic primers were phosphorylated using a 5' end labelling kit and lambda T4-polynucleotide kinase (Boehring- er Mannheim). PCRs were performed with 0.25 pM primers, 0.25 unit Taq DNA polymerase (Boehringer Mannheim) and 0.16 pg purified P. uerugznosa PI 1 18 genomic DNA under standard PCR conditions of 94°C for 1 min, 50°C for 1 min and 72°C for 2 min with 30 cycles (DNA Thermal Cycler, Perkin Elmer Cetus, Norwalk, CO, USA). Vectorette PCR was performed using phosphorylated vectorette I1 primers and ClaI vectorette I1 (Cambridge Research Bio- chemicals, Cheshire, UK), as described by the manufacturer. The vectorettes are partially double-strand- ed pieces of DNA which can be ligated onto fragments of DNA pre-cut with the corresponding restriction enzyme.

After PCR amplification, DNA was treated with Klenow polymerase (Boehringer Mannheim) and iso- lated from a 0.7%) agarose gel (SeaKem GTG, FMC BioProducts, Rockland, ME, USA) using a Spin-X centrifuge filter unit (Costar, Cambridge, MA, USA). The DNA was precipitated with ethanol, ligated to SmaI cut, dephosphorylated pUC 18 vector (Pharma- cia, Sweden), and finally transformed into competent E. coli JM 105 (Pharmacia, Sweden). Recombinants were selected on LB agar plates containing 24 pg/ ml isopropyl-beta-D-thiogalactopyranoside (IPTG, Sigma), 0.1 mg/ml 5-bromo-4-chloro-3-indolyl-beta- D-gdlactopyranoside (X-GAL, Sigma) and 0.2 mg/ml ampicillin (Sigma).

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Plusmid extraction and sequencing procedure Selected colonies were grown in LB broth supple-

mented with 0.2 mg/ml ampicillin at 37°C with aeration overnight. The plasmids were extracted using the boiling mini-prep protocol (Stratagene, La Jolla, CA, USA). All sequencing was performed using the dideoxynucleotide chain termination method (26) with dATP (specific activity 650 Ci/mmol; Amersham International, Buckinghamshire, UK) and the Sequenase kit, version 1.0 (United States Biochemical Corporation, Cleveland, OH, USA). Universal primer and synthetic oligonucleotide primers were used for the sequencing procedures and the processes were repeated until all fragments were sequenced in both directions. DNA sequences were analysed using the Pustell Sequence Analysis Pro- gram, version 2.03 (International Biotechnologies, CT, USA) and the Genetics Computer Group Se- quence Analysis Software Package, version 6.0 (7).

E.xpression and purijication of recombinant I? aeruginosa 60 kDa GroEL

The groEL gene of P. aeruginosa was cloned into the expression vector pET16b (Novagen, Madison, WI, USA) based on the assembled groEL DNA sequence of P. aeruginosa strain P1118. Two PCR primers, PR5203 and PR5204 (Table l ) , were synthesized. Each primer included one recognition site for the restriction enzyme NdeI or BamHI (underlined). These two primers covered the entire coding sequence of the P. aeruginosa PI118 groEL gene. DNA amplification was performed with 0.25 pM of each primer and 0.16 pg purified P. aeruginosa P1118 DNA under the following conditions: at 94°C for 1 min, 60°C for 1.5 min, and 72°C for 2 min (40 cycles). The PCR product (-1.7 kbp) was purified from an 0.7% agarose (SeaKem GTG) using Spin-X centri- fugation. The purified PCR product was cut with NdeI and BamHI, and ligated into pET16b vector (Novagen). The ligated material was transformed into competent E. coli NovaBlue (Novagen) and selected on LB agar plates containing 0.2 mg/ml ampicillin overnight at 37°C. Plasmids were purified using the boiling mini-prep protocol (Stratagene) and

transformed into competent E. coli B121 (DE3) (No- vagen). Cells were placed on LB agar plates containing 0.2 mg/ml carbenicillin (Sigma) and incubated overnight at 37°C.

Purification of recombinant protein was performed as recommended by the manufacturer with some modifications. Briefly, selected clones were grown in 30 ml LB broth with 0.2 mg/ml carbenicillin at 37°C until optical density (OD) at 600 nm reached 0.8. The cells were chilled to 4"C, harvested at 5,000 g for 5 min, and grown in 600 ml LB broth supplemented with 0.2 mg/ml carbenicillin at 37°C until OD600 reached 1.0. The culture was induced with 1 mM IPTG for 3 h. Induced cells were harvested at 4°C in binding buffer ( 5 mM imidazole (Sigma), 0.5 M NaCl and 20 mM Tris-HCL, pH 7.9) supplemented with 0.1'X) Triton X-100 (Sigma), sonicated for 3x45 s, centrifuged at 48,000 g for 20 min at 4"C, and 0.2 pm filter sterilized. The sonicate was loaded onto an Ni2+ charged His-Bind-Resin column, washed in 60 mM imidazole, 0.5 M NaCl and 20 mM Tris-HCI, pH 7.9, and eluted in 1 M imidazole, 0.5 M NaCl and 20 mM Tris-HC1, pH 7.9. The eluted fractions were analysed by SDS-PAGE and stained with Coomassie brilliant blue (Sigma). Fractions containing recombinant protein were pooled, dialysed against isotonic phosphate-buffered saline (PBS, pH 7.4), and concen- trated against polyethylene glycol 3000 (PEG; Merck) at 4°C. Protein concentration was measured by Bio-Rad protein assay (range 0.2-1.4 mg/ml; Bio- Rad Laboratories, Hercules, CA, USA) with bovine serum albumin (BSA, Sigma) as standard. The protein concentration was adjusted to 4 mg/ml in 100 mM NaCI, 50 mM Tris-HC1 and 1 mM CaCl, (Sigma), pH 8.0. The recombinant GroEL protein was cut with factor Xa (restriction protease factor Xa, Boehringer Mannheim). The conditions for cut- ting were 7.5 pg factor Xa/mg protein in 100 mM NaCI, 50 mM Tris-HCI, 1 mM CaCI2 in 1 M urea (Merck), pH 8.0, and incubation overnight at room temperature. The material was 10-fold diluted in binding buffer and loaded a second time on the His- Bind-Resin column. The protein material not bound by the column was collected, dialysed and adjusted

TABLE 1. Synthetic oligonucleotides used for cloning and vectorette PCRs PR2348: GCC GGC GAC GGC ACC ACC ACC GCC AC-3'; PR2351: TTG AT (GC) PR2462: TAG CCC AGG TAG GCA CCA TCT CCG-3'; PR2464: TTG ATC TAG AAT GAA GCT TCG TCC TC-3'; PR2468: GAA AAC ACC ACC ATC ATC GAT (3-3' PR2775: TTA CAT CAT GCC GCC CAT GCC GCC CAT GCC GCC C-3'

ACG GC (GC) ACG CCG CCG GC-3';

PR5277: CCA CTT CAC CGC GGT TCG GCT TC-3' PR6062: GAA GGG GTA CAA CGA GGA GC-3' PR2656: CTT CGC CCT GGT CCG GC-3' PR5448: CAA CGC AGC GAC GGT TCG ACT ACG-3' PR5203: GAT TTA PR5204: CCA ACC ACA GGG GCC GGA TCC TTA CAT CAT GCC GCC C-3'

GAG GAA AGA GCA TAT GGC TGC CAA AGA AG-3'

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SalI NarI SalI Fig. 1. Restriction map and HSfI HqeI AatISp$ Kp$1 AatI I S p N cloning strategy of (A) the P. A

n

aeruginosa groE operon and -1 GroES H GroEL I (B) the B. cepacia groE operon. t I 5801 The overlapping PCR frag- I I

ments of P. aeruginosa groE were designated 5801-03 with an estimated size of 900. 1450

B GroES M M l and 1250 bases respectively. 1

The two fragments of B. cepac-

and 6081 with an estimated size

I I 6081 ia groE were designated 6009 I I 6009

of 1800 and 1200 bases respectively.

to 0.5 mg protein/ml in 100 mM NaCI, 50 mM Tris- HCl, pH 8.0, and stored at -20°C until used.

Antigen absorption For testing of P. aeruginosa GroEL in the recom-

binant protein preparation, polyclonal rabbit anti-E. coli 021:H27 (15) antisera were absorbed with sonicated P. aeruginosa P1 1 1 8 antigen or the recombinant purified protein. The absorption was performed by incubating 400 p1 rabbit serum with 7.2 mg sonicated P. aeruginosa P1118 antigen (1 8 mg/ml) or 1.6 mg recombinant protein (4 mg/ml). The material was incubated for 1 h at 37°C under rotation, overnight at 4"C, centrifuged for 1 h at 20,000 g , and stored at -20°C until use.

Western blotting and gold staining The SDS-PAGE separation, Western blotting and

immunostaining were performed essentially as described previously (1 3, 17). The nitrocellulose paper was prior to immunostaining blocked by Western blot washing (WBW) buffer (0.6 M NaCl, 0.125 M Tris base and 0.6% Tween 20, pH 7.4) for 10 min at room temperature. Hereafter followed incubation for 2 h with rabbit anti-E. coli antibodies (absorbedhn- absorbed) diluted 1 :200 or mouse monoclonal antibody C2-F41 diluted 1:500 or patient serum diluted 1 : 1000 in WBW buffer. The nitrocellulose was washed three times with WBW buffer and incubated for 1 h with 1: 1000 diluted peroxidase-labelled swine anti-rabbit Ig antibodies (P217, Dako A/S, Glostrup,

Denmark) or rabbit anti-mouse Ig antibodies (P260, Dako A / S ) or rabbit anti-human Ig antibodies (P214, Dako NS) in WBW buffer at room temperature. It was then again washed three times with WBW buffer, and finally developed with 2.5 mM tetramethyl benzi- dine (TMB, Merck) in dimethyl sulfoxide (DMSO, Merck), 4.5 mM dioctyl sodium sulfosuccinate (DONS, Merck) and 0.05% (vol/vol) hydrogen per- oxide (H202, Merck) in phosphate-citrate buffer (54 mM citric acid (Sigma), 0.1 M Na2HP04 (Merck), pH 5.0). Staining was stopped with 4.5 mM DONS in water. Gold staining of transferred proteins was performed by blocking the nitrocellulose paper in WBW buffer for 10 min, washing briefly in water, and incubating for 30 min in citric acid buffer (55 mM citric acid, pH 3.0) at room temperature. The blot was developed for 2 4 h with 75% (vol/vol) gold solution (25 mM gold chloride (Sigma), 0.1 mM sodium citrate (Merck) in water) in citric acid buffer supplemented with 0.1% (vol/vol) Tween 20 (Merck).

Laser scanning densitometry Laser scanning densitometry was used to evaluate

the immunostaining assay. Photographic repro- ductions of immunoblots were examined in a helium- neon laser scanner (UltroScanXL, LKB 2222, Bromma, Sweden). The geometric peak area of inten- sity (measured by OD at 633 nm) was integrated automatically using JAUXmm, where AU is the abs-absbaseline in units (baseline determined by the 16

Fig. 2. Nucleotide sequences of the groE operon of 1) P. aeruginosa and 2) B. cepacia. The open reading frames of the groES gene extend from nucleotide no. 1-294, and from 3441987 for the groEL gene. The predicted amino acid sequences of P. aeruginosa are for each open reading frame listed above the corresponding nucleotide sequences. The P. aeruginosa clinical isolate P1 1 1 8 showed minor differences in the DNA sequences to the previously published P. aeruginosa ATCC 27853 groE operon (30): nt 132 C to T; nt 601 T to C; nt 619 G to C; nt 710 C to G; nt 721 C to T; nt 757 G to C; nt 758 C to G; nt 759 G to C, and nt 775 C to A. Of these substitutions, nucleotide no. 757-759 resulted in two amino acid changes (aa 138 W to C and aa 139 R to A). The B. cepacia groE - 10 and - 35 heat shock expression signals are underlined.

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lowest points in the scan) and mm is the peak width at baseline. The peak area was presented in units.

RESULTS

By aligning GroEL sequences from E. coli (12), Legionella micdadei (1 3), Coxiella burnetti (34) and M. tuberculosis (28), conserved amino acid sequences were identified within these proteins. Two highly conserved sequences were used to predict the corresponding amino acid sequence of the P. aeruginosa GroEL gene. The preferred P. aeruginosa codon usage was calculated and a primer pair based on conserved sequences was designed for PCR. A PCR reaction with P. aeruginosa DNA as template and a primer pair (PR2348 and PR2351, Table) amplified a frag-

ment of approximately 900 bases (5801; Fig. 1 A), which corresponds to the expected size calculated by counting the codons between the two conserved domains. This fragment was cloned into the SmaI site of the vector pUC18 and sequenced. The cloned fragment contained an open reading frame with striking homology to previously published GroEL sequences and a codon usage characteristic of P. aeruginosa.

In many bacteria, e.g. E. coli, L. micdadei, C. burnetti and M. tuberculosis, a gene encoding a 10-15 kDa “GroES” protein is found immediately upstream of the groEL gene. We assumed a similar arrangement of the genes in P. aeruginosa and aligned four known GroES amino acid sequences. A highly conserved GroES amino acid domain, 10 amino acid residues long, was identified which included the GroES methionine

Fig. 3. Alignment of the predicted amino acid sequences of B. cepucia and P. ueruginosu GroES with the GroES of E. coli (12), and of B. cepaciu and P. ueruginosu GroELs with E. coli GroEL (12), L. pneumophilu HtpB (14), M . tuberculosis Hsp65 (28) and the human mitochondria1 P1 (18). (-) indicates identical amino acid sequence between the aligned proteins and (.) indicates deletions.

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start codon. A set of PCR primers was con- structed (PR2464 and PR2462) based on the conserved GroES sequence and the already sequenced part of the groEL gene. These primers were used to obtain an overlapping fragment (5802; Fig. 1A) which was cloned and sequenced. Finally, a primer was predicted corresponding to a conserved Gly-Gly-Met repeat at the carboxy terminus of the groEL gene. This primer, PR2775, was combined with PR2468 to amplify a third PCR fragment (5803; Fig. 1A) which was used to obtain the remaining carboxy-terminal part of the P. aeruginosu groEL sequence. The DNA sequences further upstream and downstream of the P. aeruginosa groE operon were determined using the vectorette PCR technique, which allows PCR ampli-

27- C d

Fig. 4. Immunostaining of E. coli. P. ueruginosrr and recombinant 60 kDa GroEL of P. rrcruginosu. Lane 1: E. coli B121(DE3) sonicated antigen, lane 2: P. ueruginosu PI I18 sonicated antigen. lane 3: 0.1 pg crude recombinant protein, and lane 4: 0.1 pg purified recombinant protein. (a) Polyclonal rabbit anti- E. coli 021:H27 antibodies; (b) as in a, but absorbed with P. aeruginosa PI 118 sonicated antigens; (c) as in a, but absorbed with recombinant protein; (d) mouse monoclonal anti-P. aeruginosa 60 kDa GroEL-specific antibody C2-F41. MW, prestained molecular weight markers (low range, Bio-Rad).

fication of DNA between a known primer and a restriction enzyme site located outside the known DNA sequence. P. cieruginosri DNA was completely digested with the restriction enzyme Cia1 and the fragments were ligated to the CIaI vectorette. This material was used as template in a PCR reaction with primer PR5277 located inside the groES gene and a primer specific for the vectorette. The specific primers used in vectorette PCR were PR5277 and PR6062 for the up- and downstream regions respectively. The complete DNA sequence, assembled from the three overlapping PCR products and the vectorette PCR products, is given in Fig. 2. The assembled sequence consists of 2041 bp and con- tains two open reading frames of 294 and 1643 nucleotides. These correspond to the groES and groEL genes respectively.

The B. cepacia groE operon was cloned and sequenced using essentially the same technique as for the P. aeruginosa groE operon. Genomic B. cepuciu DNA was used as template in PCR, with primers PR234WPR2775 and PR235 I / PR2464 (Fig. IB). Although these two sets of primers were designed based on the preferred P. aeruginosu codon usage, they were able to amplify fragments corresponding to the B. cepuciu groEL sequence. The entire B. cepciciu groE op-

mw kDa

Fig. 5. Gold staining of 0.1 pg recombinant P. ueruginosu 60 kDa GroEL, cut with restriction protease factor Xa. MW, prestained molecular weight markers (low range).

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kD8 1 2 3 4 5 6 7 8 9 x )

0.20 1

* 2

0.00 - 4 - 3 - 2 - 1 0 1

T h (y.JrS)

Fig. 6. One patient, a 17-year-old CF girl who developed diabetes mellitus at age 13 years, was evaluated for serum IgG antibodies to recombinant jyP. ueruginosu 60 kDa GroEL determined by immunostaining. Lanes 1: 8/7/87, 2: 15/8/88, 3: 3/5/90, 4: 816190, 5: 1110190, 6: 4/12/90, 7: 2811191, 8: 14/3/91, 9: 1614191, and 10: 2/7/91. The patient developed diabetes 22/3/91 with a blood glucose level of 14.6 mM, determined by an oral glucose tolerance test (21). An immunoblot scanning assay revealed, a 43.8% increase in IgG anti-P. aeruginosa GroEL antibodies during the period 3/5/90 to 1/10/90 5 months prior to onset of diabetes.

eron was obtained from two overlapping PCR fragments, 6081 and 6009, as illustrated in Fig. 1 B.

To obtain information about the regulatory expression signals upstream of the B. cepacia groE operon, we used the vectorette PCR technique. B. cepacia DNA was completely digested with the restriction enzyme ClaI and the fragments were ligated to the ClaI vectorette. This material was used as template in a PCR reaction with primer PR2656 and a primer specific for the vectorette. A product of approximately 940 bases was obtained, which was cloned and sequenced partially in the region immediately upstream of the groES gene. The assembled B. cepacia sequence of 21 39 nucleotides contained the groES gene of 294 bases and the groEL gene of 1640 bases.

The groES genes of P. aeruginosa and B. cepacia are 58% homologous. The groEL genes showed 73% identity. The calculated molecular mass of the GroES from P. aeruginosa and B. cepacia is 10,353 and 10,461 Da respectively. The calculated molecular mass of the GroEL from P. aeruginosa and B. cepacia is 57,049 and

57,245 Da respectively, which corresponds to previous estimates obtained by immunostaining (17). In the DNA sequence upstream of the B. cepacia groES gene, a typical - 10 and - 35 heat shock expression signal was identified (Fig. 2).

The groEL gene of P. aeruginosa and B. cepucia has a nucleotide acid sequence similarity within the coding region of 73% and an amino acid homology of 71%. A comparison of the amino acid sequence of P. aeruginosa and B. cepacia GroEL with other remotely related GroEL sequences confirmed the well-known GroEL structure with several extremely conserved sequence domains flanking more variable structures. The alignment of P. aeruginosa, B. cepacia, E. coli, L . pneumophila and M. tuberculosis GroELs with the human homologue P1 (1 8) clearly illustrates the distribution of conserved and variable domains (Fig. 3).

For further analysis of the 60 kDa GroEL protein of P. aeruginosa, recombinant GroEL was produced using PET vector 16b and His- Bind-Resin purification. The GroEL preparation was analysed by immuno- and gold staining. Immunostaining of E. coli, P. ueruginosa and recombinant GroEL of P. aeruginosa with rabbit anti-E. coli antibodies is shown in Fig. 4a (lanes 1 4 ) . This staining revealed the profile of E. coli and P. aeruginosa sonicated antigens (lanes 1-2), and the recombinant protein preparations in lanes 3 4 . A small amount of con- tamination was observed in the crude protein preparation (lane 3), but none in the final protein preparation (lane 4). Examination of the recombinant protein preparations using anti-E. coli antibodies absorbed with P. aeruginosa sonicate (Fig. 4b) or recombinant protein (Fig. 4c) showed that the recombinant purified protein was of P. aeruginosa origin. Finally, examination of the recombinant protein preparation with the monoclonal antibody C2-F41, which is specific for P. aeruginosa GroEL and does not react with either E. coli or B. cepacia GroELs (1 7, unpublished observations), clearly demon- strated that the recombinant purified protein was P. aeruginosa GroEL (Fig. 4d). The recombinant P. aeruginosa GroEL (lane 4) was slightly larger (-2 kDa) than the original P. aeruginosa GroEL (lane 2) which was estimated to be 58 kDa. Gold staining of the recombinant GroEL preparation is shown in Fig. 5. This staining revealed trace amounts of partially de-

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graded recombinant protein in the range 45-55 kDa.

We have examined the IgG anti-P. aeruginosa 60 kDa GroEL antibody response in one patient with CF for a period of 3.5 years during the development of diabetes mellitus (Fig. 6). The patient, a 17-year-old girl who developed diabetes when aged 13, was evaluated for IgG anti-P. aeruginosa GroEL antibodies by immunostaining for a period of from 3.2 years before to 4 months after onset of disease. An immunoblot scanning assay revealed a 43.8%) increase in IgG anti-P. aeruginosa GroEL antibodies during a 5-month period (3/5/90 to 1/10/90) 5 months prior to onset of diabetes.

DISCUSSION

The antigenic similarity of Pseudomonas Gro- ELs to the corresponding protein of many other microorganisms has previously been studied in our laboratory using modified crossed immuno- electrophoresis (15, 16, 17). By in situ absorption techniques it was possible to obtain semi- quantitative measures of the similarity of Gro- ELs from a wide range of microorganisms. A comparison of the predicted amino acid compo- sition of P. aeruginosa and B. cepacia GroEL proteins shows an amino acid homology of 71%. This conforms well with the antigenic similarity of 75% measured by immunochemical techniques (17). The function of the GroEL protein is to aid folding, assembly and translo- cation of proteins (3, 6, 11). GroES and GroEL interact during these processes (3 1). The highly conserved domains might represent functionally important surface-exposed structures which help GroEL interact with unfolded polypeptides, but the exact mechanism by which unfolded polypeptides are recognized by GroEL is not known. Also, the precise cellular localiz- ation of GroEL has yet to be resolved.

Recently, we have presented data which indicate the existence of two forms of P. aeruginosa GroEL, one of which is soluble and one of which is associated with lipopolysaccharide (17). The latter most likely represents a cell- wall-associated GroEL. The two different forms of Pseudomonas GroELs show a reaction of partial identity in crossed immunoelectro-

phoresis. Which of the two GroELs we have cloned here and whether association of GroEL with lipopolysaccharide is the only difference between these two Pseudomonas GroEL forms is an unresolved question. Two distinctly different copies of the groEL gene in M. tuberculosis have been identified by low stringency hybridi- zation. These forms showed a homology of only 60% (24). It will be interesting to investigate whether other groEL genes exist in Pseudomon- as or whether the partial identity observed (17) is a result of the association of GroEL with lipopol ysaccharide.

A striking feature of the carboxyl-terminal end of the GroEL protein is the repeating Gly- Gly-Met sequence (Fig. 3). This repeat (or a very similar structure) is found in most bacterial GroELs and in the human homologue P1 (1 8). The Gly-Gly-Met repeat is absent from all chloroplast GroELs and from GroELs of pho- tosynthetic bacteria. The function of the Gly- Gly-Met repeat at the molecular level is still ob- scure. In Chlamydia trachomatis it has been shown that the serological response to GroEL is directed predominantly against the carboxy- terminal part of the molecule (5 ) . This is con- sistent with the antibody response to M. tuberculosis Hsp65, in which species-specific and the majority of immunodominant epitopes are located within the carboxyl-terminal region of the molecule (33).

The groEL-groES sequence of P. aeruginosa was recently determined independently by Sipos et al. (30). The sequence shows very few discre- pancies and only two amino acid residues differ (Fig. 3). We found that the GroEL protein of P. aeruginosa and B. cepacia contained one cysteine residue at position aa 138. One cysteine group is located in exactly the same position in E. coli, L. pneumophila, L. micdadei and C. burnetti GroELs. This conserved residue identified in many bacterial GroELs, is located in a non- conserved domain, suggesting an important function. However, this residue is not present in the mycobacterial GroELs (28) or in the human P1 (18), which could indicate a species-specific function. In the homologous GroEL protein of C. trachomatis, amino acid sequence analysis has revealed four cysteine residues, and it has been suggested that this protein is membrane- associated and involved in the assembly/disas- sembly of the outer membrane and maintenance

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of outer membrane proteins in their monomeric dissociated state ( I ) .

The GroEL protein is immunogenic in man, and anti-GroEL antibodies have been identified in sputum immune complexes from chronically P. aeruginosa-infected C F patients (19). Inter- estingly, GroEL of P. aeruginosa and B. cepacia is 52% homologous to the human PI, and it is tempting to speculate that cross-reactivity between human and bacterial GroEL is a factor which can trigger the autoimmune processes associated with CF. However, the GroEL epitope recognized by the T-cell clone A2 involved in adjuvant arthritis is found in a non-conserved region (32, 33). Also, the T-cell clone C9 able to induce insulin-dependent diabetes in NOD mice recognizes a non-conserved epitope (8,9). Thus, molecular mimicry between bacterial and mam- malian GroEL seems to be the least obvious molecular basis for the observed autoimmune phenomena. However, the specific T cells in both models are able to transfer autoimmunity to naive animals. Therefore T-cell receptorlpep- tide interaction is indeed a crucial event. The mouse diabetonic T-cell clone C9 recognizes a 12 amino acid epitope, which closely resembles the corresponding region of the mouse GroEL. But this epitope shows only 50% homology with the mycobacterial GroEL epitope, also recognized by C9. The corresponding epitope in P. aeruginosa and B. cepacia and in other bacterial GroELs shows a similar level of homology. The finding that T cells can be stimulated by GroEL peptides with such as low level of homology is surprising. One might imagine that a normally functioning immune system is able to downreg- ulate such cross-reactive T cells of broad speci- ficity. But a repeated and intensive stimulation with bacterial GroEL, as seen in chronic pul- monary infections, could result in a counterpro- ductive immune response and abrogation of tolerance. The combination of highly conserved amino acid domains and less conserved structures found in GroEL might pose a special problem with respect to the maintenance of tolerance in the immune system.

The increase in anti-P. aeruginosa GroEL antibodies in a C F patient before onset of diabetes is interesting, and a possible influence of bacterial GroELs on the development of diabetes in patients with C F needs extensive study. The sequence data presented here will allow a

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detailed investigation of the cellular and humoral immune response to the P. aeruginosa and B. cepacia GroEL proteins, which could be of value in the elucidation and prevention of autoimmune phenomena associated with CE

The skilful technical assistance of Ditte Arnt is greatly appreciated. The monoclonal antibody C2- F41 was kindly provided by Dr Selmer (Novo Nord- isk AIS, Bagsvzrd, Denmark). The Danish Biotechno- logical Database (BioBase, Aarhus, Denmark) gave access to the GCG Sequence Analysis Software Pack- age. This work was in part supported by the Danish Cystic Fibrosis Association.

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