The Escherichia coli K99 periplasmic chaperone FanE is a monomeric protein

FEMS Microbiology

Letters 138 ( 1996) 18% 189

The Escherichia coli K99 periplasmic chaperone FanE is a monomeric protein Olaf Mol, Heleen Fokkema, Bauke Oudega

*

Department of Molecular Microbiology, Institute of Molecular Biological Sciences, BioCentrum Amsterdam, Faculty of Biology, Vrije Uniuersiteit, De Boelelaan 1087, IO81 HV Amsterdam, The Netherlands Received 29 January

1996: accepted

14 March 1996

Abstract The monomeric or dimeric nature of the K99 periplasmic chaperone FanE was examined. The gene encoding FanE was subcloned in a pINIIIA1 derivative expression vector. A complementation experiment showed that the subcloned FanE was biologically functional. The protein was purified from the periplasm of cells harbouring the constructed plasmid. Automated Edman degradation experiments confirmed the predicted N-terminal amino acid sequence of FanE. A polyclonal mouse

antiserum was raised against the FanE chaperone. The monomeric or oligomeric nature of the protein in the periplasm was studied by gel filtration, immunoblotting and chemical cross-linking experiments. The results indicated that FanE is a monomeric protein, in contrast to the K88 periplasmic chaperone. Keywords: Periplasmic

chaperone;

Fimbriae

biosynthesis;

K99 fimbriae;

1. Introduction The biosynthesis of fimbriae requires a transport and assembly machinery [1,2]. This machinery consists of a periplasmic chaperone and a molecular usher located in the outer membrane. The periplasmic chaperone interacts with most of the fimbrial subunits, prevents premature polymerisation, protects these subunits against proteolytic degradation in the periplasm, assists in folding and probably targets these subunits to the molecular usher. The usher is involved in the ordered translocation of subunits across the outer membrane and the polymerisation of the subunits into a growing fimbrial structure.

* Corresponding author. Tel. + 31 (20) 444 7176; Fax: (20) 444 7123; E-mail: [email protected] 0378-1097/96/$12.00 0 1996 Federation PII SO378-1097(96)00104-S

of European

+ 31

Microbiological

Monomer;

Dimer; Escherichia coli

K99 fimbriae are often found on enterotoxigenic Escherichia coli strains that cause neonatal diarrhoea in calves, lambs and piglets [3]. The biosynthesis of K99 fimbriae requires two regulatory genes [4] and six genes that encode structural fimbrial proteins (FanC-H, see also Fig. 1) [5-81. FanE and FanD are the periplasmic chaperone and the molecular usher, respectively [5.9]. FanE is a member of the large family of periplasmic chaperones. The three-dimensional structure of one of the members of this family (PapD) has been solved [lo]. The protein consists of two immunoglobulin like domains and a substrate-binding cleft between these two domains [l 11.Comparison of the primary structure of several fimbrial chaperones revealed a high degree of structural similarity between these proteins. The overall topology of PapD appears to be conserved in all members of this family [12]. Societies. All rights reserved

186

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The PapD protein is present in the periplasmic space as a monomer and the chaperone-subunit complexes contain one chaperone molecule and one subunit polypeptide [ 131. The K88 chaperone FaeE forms trimeric complexes in the periplasmic space with most K88 subunit proteins [ 14,151. These complexes consist of one subunit molecule and two chaperone polypeptides. In contrast to PapD, the FaeE chaperone is present in the periplasm as a dimer [14]. Nothing is known about the possible oligomeric nature of the K99 chaperone FanE. To analyse this protein, the gene encoding FanE was subcloned in an expression vector. The monomeric or oligomeric nature of the protein in the periplasm was studied by gel filtration, immunoblotting and chemical crosslinking experiments.

2. Materials and methods

2. I. Bacterial strains and culture conditions E. coli K12 strain C600 e 14- fmcrA) supE44 thi-I thr-I leuB6 lacYI tonA was used as a host for plasmid constructions. E. coli K12 strain HB 101 supE44 aral4 galK2 lacy1 proA rspL20 ~1-5 mtl-I recAl3 A(mcrC-mrr) HsdS- (r- m ) was routinely used as a host for expression of proteins and for the isolation of proteins encoded by the various plasmids described below. E. coli K12 strain MC4100 araD139 A(argF-lac)Ul69 rpsL150 relAl jlbB.5301 deoC1 ptsF25 rbsR was used for the expression of K99 fimbriae from pFK99 and pOM99-1. YT medium [ 161 was routinely used for the culturing of E. coli strains. When required, ampicillin (100 pg/ml) and/or chloramphenicol (30 pg/ml) was added to the culture medium. Strain MC4100 was used for the expression of K99 fimbriae and was

Letters

138 (19961 185-189

cultured in Minca medium [4]. Isopropyl-/3-Dthiogalactopyranoside (IPTG, 0.5 mM) was used for induction of plasmid encoded gene products. 2.2. Plasmids Plasmid pOM88-E, encoding the K88 chaperone FaeE, has been described before [ 1.51. Plasmid pOM99-E was constructed by cloning the ScaI-Sphl DNA fragment of pFK99 [17], containing the jknE gene, into the SmaI-SphI sites of pJL22-(SphI). Plasmid pJL22-(SphI) [ 181 is a derivative of the expression vector pINIIIA1. Plasmid pFK99-Eis a pBR322 derivative plasmid containing the K99 operon with a frameshift mutation in ,fanE [7]. To facilitate the introduction of both pOM99-E and a K99(,fanE) operon into one cell, the BamHI fragment of pFK99-Em, containing the K99(,&E) operon. was cloned into the corresponding site of pACYC 184. This plasmid was designated pOM99- 1. All basic recombinant DNA procedures were carried out as described elsewhere [19]. 2.3. Protein techniques SDS-PAGE was carried out essentially as described by Laemmli 1201. Following gel electrophoresis, proteins were transferred onto nitrocellulose filters as described by Krone et al. [21]. The ELISA for the semi-quantitative measurement of K99 has been described before [4,6-81. Isolation of periplasmic proteins, automated Edman degradation, gel filtration of proteins and the protein cross-linking technique used have been described [ 14,151. For the preparation of a polyclonal mouse antiserum against the periplasmic chaperone FanE, the periplasmic protein fraction of E. coli HB 101 (pOM99-E) was isolated. The proteins were sub-

P T

I-

IAIIBI II

11.7

Regulalion

I c

II

16.5

Adhesin Major Component

D 84.5

Usher

1 E

j

23.2

Periplasmic Chaperone

F 33.9

I’i

G 16.9

1 H 16.7

Minor Components

Fig. I. Genetic organisation of the K99 gene cluster. Genes are represented by box.\. the designation of the genes is given in the boxes and the relative molecular mass of the mature proteins is given in kDa. A short description of the function of the different proteins is given, P. promoter. T, terminator. Arrows indicate direction of transcription.

0. Mel et al. / FEMS Microbiology

jetted to preparative SDS-PAGE, blotted onto nitrocellulose filter paper and stained with Ponceau-S [22]. The protein band corresponding to FanE was cut out and destained in PBS (0.15 M sodium chloride, 0.1 M sodium phosphate, pH 7.0). This material was used to immunise mice. 2.4. Purification

of FanE

3. Results and discussion and expression

30-

4

- FaeE -FanEi

20-

Cells of E. coli HB 101 harbouring pOM99-E were grown in YT to a culture turbidity of 0.8. Protein synthesis was then induced with IPTG. Cells were allowed to grow for 1 h at 37°C and periplasmic protein fractions were isolated [15]. The FanE protein was purified by Fast Protein Liquid Chromatography (FPLC) using a Mono S HR 5/5 column (Pharmacia). The protein was released from the column by elution with a linear gradient of O-300 mM NaCl in 50 mM Tris .HCl (pH 8.0) at a flow rate of 1 ml/min. The FanE protein eluted at 200 mM NaCl.

3.1. Subcloning

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Letters I38 (1996) 185-189

of FanE

To study whether the FanE chaperone is a monomer like PapD or functions as a dimer like FaeE, the gene encoding this protein was cloned into pJL22(SphI). The gene was cloned under the control of the IPTG-inducable lpp/fac promoter/operator system of pJL22(SphI). This plasmid encoding fanE was designated pOM99-E. The expression of the subcloned gene was studied in E. coli cells. FanE is synthesised with an N-terminal signal sequence and is secreted into the periplasm [9]. Therefore, the periplasmic fraction of cells harbouring pOM99-E was isolated and analysed by SDS-PAGE (Fig. 2A). E. coli cells harbouring pOM99-E expressed a protein of about 24 kDa (Fig. 2A, lane 1). This protein was not detected in cells containing the empty expression vector (see Fig. 2, lane 1). Cells harbouring pOM88-E did not express a 24 kDa protein. These cells expressed a 27 kDa protein, namely the FaeE periplasmic chaperone (Fig. 2A, lane 2) [14]. The 24 kDa protein was identified as the periplasmic chaperone FanE upon determina-

I 1

1

2

2

3

aFanE! Fig. 2. Expression and detection of subcloned gene products. (A) SDS-PAGE of periplasmic fractions of E. co/i HBlOl harbouring various plasmids. The gel was stained with Coomassie brilliant blue. The plasmids used are indicated above the lanes. In lane 3, the purified FanE was applied. The equivalent of 0.2 ODWanm units of cells was applied to each lane. (B) Immunoblotting of periplasmic factions of E. coli HBlOl harbouring various plasmids. The plasmids used are indicated above the lanes. The blot was developed with the FanE antiserum. The position and size &Da) of the molecular mass markers and the position of FaeE and FanE are indicated.

tion of the N-terminal amino acid sequence by Automated Edman degradation. The experimentally determined sequence was identical to the N-terminal amino acid sequence of the mature FanE protein as predicted by the DNA sequence [9]. Complementation experiments were carried out to investigate whether the subcloned gene product was biologically functional. Plasmid pOM99- 1 carries the K99 gene cluster with a frame shift mutation in fanE. Cells harbouring this plasmid do not produce K99 fimbriae and the fimbrial subunit proteins are rapidly degraded in the periplasm [17]. Complementation of cells of E. coli (pOM99-1) with pOM99-E restored K99 timbriae production to a normal level, as judged by an enzyme-linked immuno-sorbent assay (ELISA, data not shown). These experiments showed that the subcloned fanE gene was expressed and that the FanE protein was biologically active in the periplasm of Escherichia coli cells. 3.2. Purification

of FanE

and production

of poly-

clonal mouse antiserum

The FanE protein was purified SDS-PAGE, the purified protein

by FPLC. Upon migrated at the

3.3. The FanE chaperone

is a monomeric

protein

The K88 periplasmic chaperone FaeE is a dimer. This dimer forms trimeric complexes with the subunits FaeG, FaeH and FaeI 114,151. To analyse whether FanE is present in the periplasm as a monomer or as a dimer, gel filtration and cross-linking experiments were carried out. The calculated molecular mass of FaeE and FanE, as deduced from the DNA sequence, is 24.8 kDa and 23.2 kDa, respectively. In a previous study it was shown that, upon gel filtration, the K88 periplasmic chaperone FaeE eluted as a protein with a molecular mass of about 50 kDa [14]. The purified FanE eluted as a protein with a molecular radius of a protein with an estimated molecular mass of about 24 kDa (Fig. 3). Comparable results were obtained when a periplasmic fraction of E. coli HBlOl (pOM99-E) was

FanE

FaeE

same position as periplasmic FanE (Fig. 2A, lane 3). Subsequently, this purified protein fraction was subjected to preparative SDS-PAGE for the immunisation of mice. About 80 pg of purified FanE was used. The antiserum obtained specifically recognised FanE (Fig. 2B, lane 2). The antiserum did not recognise the K88 periplasmic chaperone FaeE (Fig. 2B, lane 3).

67-

BS3

-

+

-

+

Fig. 1. lmmunoblotting of periplasmic fractions of t: co/i containing FaeE or FanE after cross-linking with BS3. The addition of the cross-linker and the position and size (kDa) of the molecular mass markers are indicated. The FaeE blot was developed with a FaeE monoclonal antiserum. The FanE blot was developed with a polyclonal FanE antiserum.

analysed by gel filtration (data not shown). This experiment strongly suggested that FanE protein is a monomer. Cross-linking experiments were carried out to further analyse whether FanE is present in the periplasmic fraction as a monomer. For this purpose, the chemical cross-linker BS’ was used. The FaeE dimeric chaperone was used as a control [ 141. Under conditions where FaeE is cross-linked to a dimer, the FanE chaperone could not be cross-linked (Fig. 4). When the concentration of cross-linker was increased, the amount of FaeE cross-linking product

0.3

-it 11

12

Elution &me

(ml)

14

15

16

Fig. 3. Gel filtration of purified FanE. A calibrated Superose 12HR IO/30 column and a standard FF’LC system were used. 200 mM phosphate buffer (pH 8.0) was used as an elution buffer at a flow rate of 0.4 ml/mm The figure presented is a compilation of several separate, independent gel filtration runs. The positions of the marker proteins and their relative molecular mass are indicated. The insert shows a graphic analysis of the molecular mass of the FanE protein in relation to the various marker proteins used.

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also increased, but no FanE cross-linking product could be detected. When the BS3 concentration was even further increased, the reaction became aspecific, resulting in large aggregates (data not shown). These results indicated that the FanE chaperone, in contrast to the FaeE chaperon, is present in the periplasmic fraction as a monomeric protein.

Acknowledgements This work was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research @IWO). The technical assistance of Roe1 van der Schors (automated Edman degradation) was greatly appreciated.

References [l] Hultgren, S.J., Abraham, S., Caparon, M., Falk, P., St. Geme III, J.W. and Normark, S. (1993) Pilus and nonpilus bacterial adhesins: assembly and function in cell recognition. Cell 73, 887-901. [2] De Graaf, F.K. and Gaastra, W. (1994) Fimbriae of enterotoxigenic Escherichia co/i. In: Fimbriae: Adhesion, Genetics, Biogenesis, and Vaccines (Klemm, P., Ed.), pp. 53-83, CRC Press, Boca Raton, FL. [3] Gaastra, W. and De Graaf, F.K. (19821 Host-specific fimbrial adhesins of non-invasive enterotoxigenic Escherichia coli. Microbial. Rev. 46, 129- 161. [4] Roosendaal, E., Boots, M. and De Graaf, F.K. (1987) Two novel genes, fanA and funB involved in the biogenesis of K99 fimbriae. Nucl. Acids Res. 15, 5973-5984. [5] Roosendaal. B. and De Graaf, F.K. (1989) The nucleotide sequence of the fanD gene encoding the large outer membrane protein involved in the biosynthesis of K99 fimbriae. Nucl. Acids Res. 17, 1263. [6] Roosendaal, B., Gaastra, W. and De Graaf, F.K. (1984) The nucleotide sequence of the gene encoding the K99 subunit of enterotoxigenic Escherichia coli. FEMS Microbial. Lett. 22, 253-258. [7] Simons, B.L., Willemsen, P.T.J., Bakker, D., Roosendaal, B., De Graaf, F.K. and Oudega, B. (1990) Structure, localization and function of FanF, a minor component of K99 librillae of enterotoxigenic Escherichia coli. Mol. Microbial. 4, 20412050. [8] Roosendaal, E., Jacobs, A.A.C., Rathman, P., Sondermayer, C., Stegehuis, F., Oudega, B. and De Graaf, F.K. (1987) Primary structure and subcellular localization of two timbrial

[9]

[lo]

[I 11

[12]

[13]

[14]

[15]

[ 161 [17]

[18]

1191

[20]

[21]

1221

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subunit-like proteins involved in the biosynthesis of K99 fibrillae. Mol. Microbial. 1, 211-217. Bakker, D., Vader, C.E.M., Roosendaal, B., Mooi, F.R., Oudega, B. and De Graaf, F.K. (1991) Structure and function of periplasmic chaperone-like proteins involved in the biosynthesis of K88 and K99 fimbriae in enterotoxigenic Escherichiu coli. Mol. Microbial. 5, 875-886. Holmgren, A. and Branden, C.-I. (1989) Crystal structure of chaperone protein PapD reveals an immunoglobulin fold. Nature 342, 248-25 1. Kuehn, M.J., Ogg, D.J., Kihlberg, J., Slonim, L.N., Flemmer, K., Bergfors, T. and Hultgren, S.J. (1993) Structural basis of pilus subunit recognition by the PapD chaperone. Science 262, 1234-1241. Holmgren, A., Keuhn, M.J., Brlnden, C-I. and Hultgren, S.J. (1992) Conserved immunoglobulin-like features in a family of periplasmic pilus chaperones in bacteria. EMBO J. 11, 1617-1622. Striker, R., Jacob-Dubuisson, D., Frieden, C. and Hultgren, S.J. (1994) Stable fiber-forming and nonfiber-forming chaperone-subunit complexes in pilus biogenesis. J. Biol. Chem. 269, 12233-12239. Mol, 0.. Visschers, R.W., De Graaf, F.K. and Oudega, B. (19941 Escherichia coli periplasmic chaperone FaeE is a homodimer and the chaperone-K88 subunit complex is a heterotrimer. Mol. Microbial. 11, 391-402. Mol. 0.. Oud, R.P.C., De Graaf, F.K. and Oudega, B. (1995) The Escherichia coli K88 periplasmic chaperone FaeE forms a heterotrimeric complex with the minor limbrial component FaeH and with the minor fimbrial component FaeI. Microb. Pathog. 18, 115-128. Miller, L.H. (1972) Experiments in Molecular Genetics Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. De Graaf, F.K., Krenn, B.E. and Klaasen, P. (1984) Organization and expression of genes involved in the biosynthesis of K99 fimbriae. Infect. Immun. 43, 508-514. Luirink, J., Duim, B., De Gier, J.W.L. and Oudega, B. (1991) Functioning of the stable signal peptide of the pCloDFl3-encoded bacteriocin release protein. Mol. Microbiol. 5, 393-399. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Krone, W.J.A., De Vries, P., Koningstein, G., De Jong, A.J.R., De Graaf, F.K. and Oudega, B. (1986) Uptake of cloacin DF13 by susceptible cells: removal of immunity protein and fragmentation of cloacin molecules, J. Bacterial. 166, 260-268. Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.