Mol Gen Genet (1991) 229:285-291 002689259100307P © Springer-Verlag 1991

Deletion and duplication of specific sequences in the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli Per Amstrup Pedersen* and Lene Nonboe Andersen Department of Microbiology,Build. 221, The TechnicalUniversityof Denmark, DK-2800 Lyngby,Denmark

Summary. Small, defined in-flame deletions and inframe duplications of specific sequences were made within the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coIi. The cellular localization and proteolytic stability of the different mutated fimbrial subunit proteins were determined, and compared with those of the wild-type protein. Based upon these results, we predict a functional role of specific structures in the K88ab fimbrial subunit protein in subunit-subunit interactions as well as in interactions between FaeG and the other proteins encoded by the K88ab operon. The results obtained were further compared with results obtained from operon deletions, linker insertion mutagenesis and the current model for biogenesis of K88 fimbriae. One of the mutated fimbrial subunit genes was used to construct a secreted in-frame fusion between FaeG and a characterized epitope (lacking cysteine) from the Hepatitis B pre-S2 protein. Such fusion proteins might be useful in the design of recombinant vaccines. Key words: K88 fimbriae- Protein secretion assembly - Escherichia coli

Organelle

Introduction K88 fimbriae are filamentous protein polymers present on the outer surface of particular Escherichia coli strains (Orskov et al. 1961). They mediate adhesion to porcine intestinal epithelium, and allow K88-positive bacteria to colonize the small intestine (Jones and Rutter 1972). About 100 identical fimbrial subunit molecules and a small amount of at least one minor component are assembled into a single fimbrial structure (Oudega et al. 1989). The K88 antigen has been found to exist in at least Offprint requests to: P. Pedersen * Present address: AugustKrogh Institute,Universityof Copenhagen, Universitetsparken 13, DK-2100 CopenhagenOE, Denmark

three serologically distinct variants, designated K88ab, K88ac and K88ad (Orskov etal. 1964; Guinee et al. 1979). The primary structures of various K88 subunit proteins are known (Klemm 1981; Gaastra et al. 1981, 1983; Josephsen et al. 1984; Dykes et al. 1985). The molecular weight of the 264 amino acid residue K88ab subunit protein is 27.54 kDa, as calculated from the amino acid sequence (Klemm 1981). A DNA fragment involved in biosynthesis of K88ab fimbriae has been cloned and shown to contain six genes designated faeC to f a e H (Mooi and de Graaf 1979b; Mooi et al. 1981) (fae, fimbrial adhesion eighty-eight) (Fig. 1). T h e f a e G gene encodes the K88ab fimbrial subunit protein, while the other genes encode morphopoietic proteins necessary for secretion and assembly of fimbrial subunits and minor components (Oudega et al. 1989; Mooi et al. 1982). FaeC is a minor component located on the tip of the fimbrial structure (Oudega et al. 1989), while FaeD is an outer membrane protein (Van Doorn et al. 1982). The FacE and FaeF proteins are located in the periplasmic space (Van Doom et al. 1982). The localization of the FaeH protein has not been determined. A model has been proposed for the secretion of the fimbrial subunit proteins based upon the cellular localHm EI II pPAP171 b c P l l J G

El II O1 H

H~E] pPAP183 lacP In I J a I O1 G H' HE pPAP5?4 P l l ~ , C

D

I

• E

El k14 F G'

H~ I 02 lkbp

Fig. 1. Genetic maps of plasmids pPAP171 and pPAP183 used for mutagenesisexperimentsand pPAP574 used for complementation. Abbreviations: C-H represent the genesfaeC-faeH present on the cloned DNA; HI, HindlII; EI, EeoRI; lacP, the lae wild type promoter; P1, promoter P1 from pBR322; O1, the pMB1 origin of replication; 02, the pI5A origin of replication

286

~

C G G

C o OM

PS IM mRNA

Fig. 2. Modifiedversion of the model for secretion of K88 fimbrial subunits proposed by Mooi and de Graaf (1985). Abbreviations: C-H, faeC-faeH; IM, inner membrane; OM, outer membrane; PS, periplasmic space; R, ribosome. The FaeC protein is translocated to the outer surface of the cell, probably in order to initiate secretion of FaeG proteins. After entering the periplasmic space FaeG associates with FacE. FaeF now binds to the FaeG-FaeE complex, probably in order for FaeG to obtain an export-compatible conformation. FaeG is transported to the surface of the cell via FaeD, which may constitute an FaeG, FaeC specificpore

ization of the proteins encoded by the K88 operon (Fig. 2) (Mooi and de Graaf 1985) and the identification of periplasmic FaeE-FaeG and FaeE-FaeF-FaeG complexes in K88 producing strains (Mooi et al. 1983). We have studied the biosynthesis of K88ab fimbriae in E. coli and especially the structure/function relationship of the subunit protein by means of internal in-frame deletions and duplications in the faeG gene and have compared the cellular localization of the corresponding mutated proteins with that of the wild-type protein. Two of the mutants constructed were used to make in-frame fusions between a mutated FaeG protein and a characterized epitope from the Hepatitis B pre-S2 protein.

Materials and methods

Bacterial strains and culture conditions. The E. coli strains used in this study were NF1830 (MC1000 recA harbouring an F'lacl q Tn5) (N. Fiil) and the derivative PAP574 (NF1830 harbouring the plasmid pPAP574) (Pedersen 1991). Cells were grown on solid LB medium (Bertani 1951) or in liquid LB medium supplemented with the appropriate antibiotics. These were 100 gg/ml ampicillin, 20 gg/ml kanamycin and 8 gg/ml tetracycline. DNA techniques. Isolation of plasmid D N A was carried out as described by Del Sal et al. (1989). Restriction enzymes and D N A modifying enzymes were purchased from New England Biolabs, Boehringer Mannheim and United States Biochemicals, and used according to the manufacturers' specifications. Analysis of D N A fragments, ligation and transformation were carried out as described by Maniatis et al. (1982). The oligo-nucleotide encoding a Hepatitis B epitope was synthesized on an Applied Biosystems D N A synthesizer and purified on a 20% polyacrylamide-urea gel according to Maniatis et al. (1982).

Construction of plasmids. The plasmids pPAP171, pPAP183, pPAP263, pPAP370, pPAP371, pPAP373, pPAP374, pPAP376, pPAP380, pPAP382, pPAP582, pPAP574 and pPAP654 have been described previously (Pedersen 1991). Plasmid pAL1 was constructed by ligation of the 4.2 kb HindIII-SmaI fragment from pPAP263 to the 190 bp HindIII-SmaI fragment from pPAP371. Plasmid pAL7 contains the 190 bp HindIII-SmaI fragment from pPAP371 ligated to the 3.7 kb HindIII-SmaI fragment from pPAP376. For plasmid pAL24 the 4.2 kb HindIIISmaI fragment from pPAP371 was ligated to the 250 bp HindIII-SmaI fragment from pPAP376. Plasmid pAL31 bears the 3.6 kb HindIII-SmaI fragment from pPAP373 next to the 430 bp HindIII-SmaI fragment from pPAP370. In plasmid pAL43 the 400 bp HindIIISinai fragment from pPAP382 is adjacent to the 3.7 kb HindIII-SmaI fragment from pPAP370. Plasmid pAL48 was constructed by ligating the 250 bp HindIIISinai fragment from pPAP376 to the 4.2 kb HindIIISmaI fragment from pPAP263. Plasmid pAL17 carries the 210 bp HindIII-SmaI fragment from pPAP263 and the 3.8 kb HindIII-SmaI fragment from pPAP376. Plasmids pAL212 and pAL401 were constructed by inserting a 10 bp BglII linker and the Hepatitis B oligonucleotide, respectively, into SmaI-digested pAL17. Plasmid pAL419 bears the Hepatitis B oligonucleotide inserted into Sinai-digested pAL24. Plasmid pPAP160 was constructed by digestion of pPAP171 with BssHII. The 5' extension was filled in using the Klenow fragment of E. coli D N A polymerase I, followed by ligation to an 8 bp SmaI linker. Plasmid pPAP408 was synthesized by ligating the 920 bp HindIII-SmaI fragment from pPAP382 to the 4.2 kb HindIII-SmaI fragment from pPAP371. Plasmid pPAP421 contains an 8 bp J(baI linker inserted into HindIl-digeste~ pPAP171. For plasmid pPAP488, the 250 bp HindIII-SmaI fragment from pPAP376 was ligated to the 3.7 kb HindIII-SmaI fragment from pPAP373. Plasmid pPAP456 was constructed by ligation of the 430 bp HindIII-SmaI fragment from pPAP370 to the 3.9 kb HindIII-SmaI fragment from pPAP374. In plasmid pPAP477 the 780 bp HindIII-SmaI fragment from pPAP380 is inserted near to the 3.5 kb HindIII-SmaI fragment from pPAP382. Plasmid pPAP480 carries an 8 bp J(baI linker inserted into Eco47III-digested pPAP171. Plasmids pPAP497 and pPAP543 were constructed by inserting an 8 bp BgllI linker into Sinai-digested pPAP448 and pPAP456, respectively. Plasmid pPAP564 was constructed by ligation of the 920 bp HindIII-SmaI fragment from pPAP382 to the 3.9 kb HindIII-SmaI fragment from pPAP376. To generate plasmid pPAP610 the 920 bp HindIII-SmaI fragment from pPAP382 was ligated to the 3.3 kb HindIII-SmaI fragment from pPAP582. For plasmid pPAP616, the 810 bp HindIII-SmaI fragment from pPAP582 was inserted into the 3.3 kb HindIII-SmaI fragment from pPAP380. Plasmid pPAP624 was constructed by ligation of the 810 bp HindIII-SmaI fragment from pPAP582 to the 3.5 kb HindIII-SmaI fragment from pPAP382.

287

Plasmid pPAP662 bears a 10 bp BglII linker in the

SmaI site of pPAP160. Plasmid pPAP674 was constructed by inserting a 10 bp BglII linker into Sinai-digested pPAP610. Plasmid pPAP694 was synthesized by ligation of the 760 bp HindIII-SmaI fragment from pPAP654 to the 3.7 kb HindIII-SmaI fragment from pPAP160. Plasmid pPAP720 carries an 8 bp XbaI linker in the Sinai site of pPAP624.

DNA sequence analysis. DNA sequences were determined by the dideoxy method (Sanger et al. 1977) using denatured plasmids. Primers were synthesized on an Applied Biosystems DNA synthesizer, and were constructed so as to hybridize to thefaeG gene 50-100 bases from the region of interest.

Determination of protein stability. 100 gg/ml chloramphenicol was added to cells growing exponentially at 37°C in LB medium (Bertani 1951) at OD~5o= 1.0 in order to terminate protein synthesis. 1.5 ml samples were transferred to ice-cold tubes containing the serine protease inhibitor phenyl-methyl-sulphonyl-fluoride (PMSF) at a final concentration of 2 mM at 0, 10, 20 and 30 min after the addition of chloramphenicol. Cells were lysed by sonication and Microtiter wells were coated with 50 gl sonicated cells and incubated overnight at 4 ° C. The amount of fimbrial subunits present in the cell extracts was determined in an ELISA assay (Gaastra 1984).

#-lactamase assay. The amount of fl-lactamase present in the growth medium was measured according to the method of Andrup et al. (1988).

Immunization and serum. Female albino rabbits were immunized with purified K88ab fimbriae, after emulsification in Freund's incomplete adjuvant (0.2 mg protein per injection). Rabbits were immunized three times at intervals of 2 weeks, and bled after 2 months. The antisera obtained were routinely adsorbed with sonicated PAP574 in order to remove nonspecific antibodies.

Induction offimbrial biosynthesis. 100 ml LB (Bertani 1951) containing the appropriate antibiotics was inoculated with an overnight culture to OD45o =0.05. Fimbrial biosynthesis was induced at OD~5o=0.4 by the addition of isopropyl-fl-thio-galacto-pyranoside (IPTG) to a final concentration of 10 mM. Growth was followed until 0D¢5o = 1.5.

In situ detection of mutatedfimbriae. A 1.0 ml sample of IPTG-induced cells was collected at OD45o = 1.5 and resuspended in 50 gl 0.9% NaC1. A 10 gl sample was mixed with 10 lal adsorbed anti-K88ab serum on glass slides. The presence of K88 fimbriae caused agglutination of the cells. Whole cell ELISA tests were performed with an antiK88ab serum in order to determine whether any fimbrial subunits were present on the outer surface of the various mutants. A 1.5 ml sample of IPTG-induced cells was collected at OD4so = 1.5 and resuspended in 600 gl 0.9% NaC1. 50 lal samples were transferred to Microtiter vessels and incubated overnight at 4° C. The ELISA test was performed as described by Gaastra (1984).

Results and discussion

Construction of deletions and duplications in the faeG gene Ten different plasmids (Pedersen 1991) (Fig. 3) each containing a unique Sma! site in the faeG gene were used to construct a series of mutations in which specific sequences of thefaeG gene were either deleted or duplicated. In all plasmids, expression of the mutatedfaeG gene is regulated by the lac wild-type promoter/operator system. The principle used for mutagenesis is indicated in Fig. 4, and the deletions and duplications actually constructed are shown in Fig. 5. In some cases the creation of a deletion or a duplication changed the reading frame of the faeG gene. To regain the original reading frame a 10 bp BglII linker or an 8 bp XbaI linker was introduced into the unique Sinai site present in all constructions. An oligonucleotide (Fig. 6) encoding a characterized 14-residue antigenic determinant from the Hepatitis B pre-S2 protein (Galibert et al. 1979; Neurath et al. 1984) was inserted into Sinai-digested pAL7 and pAL24. The resulting plasmids were designated pAL401 and pAL419. The composition of the proteins encoded by pAL401 and pAL419 is illustrated in Fig. 6. The host strain used for transformation was NF1830, which, due to overproduction of the lac repressor, allows controlled expression from the constructed plasmids.

Protein characterization and Western immunoblotting. Fimbriae were purified as described by Mooi and de Graaf (1979a) from cells induced with IPTG at OD~5o = 0.4 and grown until OD45o = 1.5. The proteins present in the growth medium were precipitated in 5% trichloroacetic acid and resuspended in 0.5 ml water. Cells were fractionated according to Osborn et al. (1972). Protein preparations were subjected to one-dimensional SDSpolyacrylamide gel electrophoresis (SDS-PAGE) performed in 12% slab gels as described by Laemmli (1970). Western blotting was performed as described by KyhseAndersen (1984) with an adsorbed anti-K88ab serum.

371 460 376 EVP R H~I SS S LACPI I

373 370 E A SS

464 N S

'

654. 380 582 SPH R SSS ' ' '

382 E47 S I

lOObp

Fig. 3. The positions of the Sinai sites introduced at ten different positions in thefaeG wild-type gene. The plasmid number is given for each allele of the faeG gene above the restriction site used for Sinai linker insertion. Abbreviations: EV, EcoRV; P, PstI; R, RsaI; E, EcoRI; A, AluI; N, NsiI; S, SspI; H, HindII; HIII, HindIII ; E47, Eco47III; LACP, the lactose wild-type promoter

288 P R

Hm

A,~/A

WT

G Hgl

HIII

A

H

G

H

PS/R

I vH.l I

I

R

'

~//A

'

I

TAT TTT CCG GCT GGC GGC ATC GAT AGA TCT 3' Tyr Phe Pro A[a Gly Gty I[e Asp Arg Ser

Fig. 6. The composition of the two in-frame Hepatitis B preS2FaeG fusion proteins constructed. A, FaeG(A 12-30+Hep); B, FaeG(D 12-30+Hep). Numbers represent the positions of amino acids in the mature FaeG protein, starting from the amino-terminal end. The nucleotide sequence of the Hepatitis B derived oligonucleotide and the primary structure of the corresponding peptide used for making preS2-FaeG in-frame fusions are also given

I

H

,

20aa 5' AGA TCT CAG GAT CCG CGT GTT CGT GGT CTG Arg Ser Gin Asp Pro Arg Vat Arg G[y Leu

1

S G

H

B

G

I V//~\\\\\\ki

I 12 30 12 30

P S/R

1

13

12 30

,

G

Fig. 4. The principles applied to make deletions (A) and duplications (D) between two restriction sites, e.g. PstI(P) and RsaI(R) in the faeG gene. To delete or duplicate the dark region between P and R a clone designated 2, containing a SmaI linker in the PstI site (indicated by P/S) and a clone designated 1, containing a SmaI linker in the RsaI site (indicated by R/S) is used. A deletion (A) is constructed by exchanging the small HindIII-SmaI fragment from clone I with the small HindIII-SmaI fragment from clone 2. A duplication (D) is constructed by exchanging the small HindIII-SmaI fragment from plasmid 2 with the small HindIIISmaI fragment from plasmid 1. Abbreviations: HIII, HindIII; P, PstI; R, RsaI; S/R, the insertion of a SmaI linker in the RsaI site; S/P, the insertion of a SmaI linker in the PstI site; G, faeG; H, faeH; A, a plasmid containing a deletion; D, a plasmid containing a duplication

The D N A sequence around the mutagenized site was confirmed by D N A sequencing. Plasmid pPAP477 was found to contain a frame shift mutation at codon 208 and not the deletion expected. Thus, plasmid pPAP477 encodes a protein consisting of the 208 amino-terminal residues of the FaeG wild-type protein fused to a tetrapeptide (marked ' a ' in Fig. 5 and Table 1). The primary structure of this tetrapeptide is Pro-Gly-Leu-Gly, starting f r o m the amino-terminal end.

Expression o f mutated fimbrial subunit genes

G r o w t h experiments were performed with PAP574 harbouring the mutated plasmids to determine the effect of fimbrial gene expression on growth. PAP574 constitutively expresses the genes necessary for secretion and assembly of the fimbrial subunit protein (Pedersen 1991). Expression of the mutated fimbrial genes after induction of the lac p r o m o t e r with 10 m M I P T G did not have any effect on the growth rate compared with growth of the positive controls PAP574(pPAP171) and PAP574(pPAPI83), and the negative control PAP574

Since it is impossible to select directly for bacteria accumulating mutated F a e G proteins in the secretion and assembly pathway, and since it is troublesome to screen for the presence or absence of mutated fimbrial subunit proteins, all constructions were screened at the D N A level. Plasmid D N A was prepared f r o m a number of transformants and m a p p e d by restriction enzyme digestion to verify the construction of a deletion, a duplication or the insertion of the Hepatitis B derived oligonucleotide.

/~

12 18 30 I I I pAL1 pAL7 pPAP497 pAL212

79 90 I

129

I

I

pPAP543

155 I

180 I

202207219 I

q

]

255 I

pPAP661 pPAP477 pPAP674 pPAP480

1219 30 I I I pAL24 pAL48 D

79 91 I

q

129 I

pAL.___31 pPAP408 pPAP564

155 I

180 I

202208219

pPAP694 - -

II

255

I

I

p.PAP616

10aa

Fig. 5. The in-frame deletions (A) and in-frame duplications (D) constructed as shown in Fig. 4. Numbers represent amino acids in the mature FaeG protein, starting from the amino-terminal end. Lines represent sequences deleted or duplicated. The letter ' a ' represents a frame shift mutation after residue 208. The numbers of the plasmids encoding the mutations are also given

289 Table 1. Characterization of the mutated FaeG proteins

Plasmid

Mutation

Aggl

pPAP171 pPAP183 pPAP574 pALl pAL7 pAL24 pAL31 pAL48 pAL212 pAL401 pAL419 pPAP408 pPAP477 pPAP480 pPAP497 pPAP543 pPAP564 pPAP616 pPAP661 pPAP674 pPAP694

Wild type Wild type A12-18 A12-30 D12-30 D79-91 D19-30 A18-30 A12-30+Hep D l ~ 3 0 +Hep D12-254 A207-264a A255-264 A30-79 A90-129 D30-254 D208-219 A155-180 A219-254 D 180-202

+ + + + + + ÷ + + + + + + -

ELISA

Fire

Peri

Med

+ + + ---

----+ ----

+ + + +

>216 >216 0

+ + -

÷

>216

--

0 >216 >216

+ ÷ ÷ -+

+ + ÷

>216 0 0

÷ ÷

>216 > 216 29 0 >216 213

--

2 ~4

--

21~ 0 213 0

--

ND

+ + + + + + ÷ + + + + + + + +

Class III III I II I III II III I I III II I I II II II II I II I

The results of the agglutination, ELISA and cell fractionation experiments using a mixture of two adsorbed anti-K88ab sera are shown. All plasmids were tested in PAP574, which encodes the helper proteins. Abbreviations: AX-Y and DX-Y represent a deletion and a dublication, respectively, of residue X-Y in the mature FaeGwt protein starting from the amino-terminal end; Aggi, agglutination; - , +, + +, + + + indicate no agglutination, a low level, an inter-mediate level and a high level of agglutination respectively; ELISA, enzyme linked immunosorbent assay; the ELISA values are represented as the dilution of serum at which discrimination between the mutant studied and PAP574 was no longer significant; Fim, fimbriae; + and - indicate the presence or absence of fimbrial subunits in heat shock extracts; Peri, the periplasmic space; + and - indicate the presence or absence of fimbrial subunits in the isolated periplasmic space; Med, concentrated growth medium; ÷ and - indicate the presence or absence of fimbrial subunits in the growth medium; ND, not detected; + indicates the absence of fimbrial subunits in the compartments investigated; Class, the roman numerals I, II and III indicate to which class a particular mutation belongs

(data n o t shown). P A P 5 7 4 ( p P A P 1 7 1 ) a n d P A P 5 7 4 ( p P A P 1 8 3 ) p r o d u c e the w i l d - t y p e f i m b r i a l subunit, desi g n a t e d F a e G w t , in a d d i t i o n to F a e C , F a e D , F a e E a n d F a e F , while P A P 5 7 4 o n l y p r o d u c e s the latter proteins. T h e / M a c t a m a s e activity p r e s e n t in the g r o w t h mediu m at the end o f each g r o w t h e x p e r i m e n t was determ i n e d in o r d e r to c o n f i r m t h a t severe cell lysis h a d n o t t a k e n place. E l e v a t e d a m o u n t s o f / % l a c t a m a s e were n o t f o u n d in any o f the s u p e r n a t a n t s c o m p a r e d with the c o n t r o l strains (data n o t shown).

Detection of fimbrial subunits and cellfractionation F r o m each g r o w t h e x p e r i m e n t with P A P 5 7 4 h a r b o u r i n g m u t a n t plasmids, samples were t a k e n for a g g l u t i n a t i o n tests a n d E L I S A assays. A m i x t u r e o f two a d s o r b e d a n t i - K 8 8 a b sera was used in these experiments. T h e results are p r e s e n t e d in Table 1. P r o t e i n s released by h e a t s h o c k (which liberates wild type f i m b r i a e f r o m the cell surface) a n d the p r o t e i n s present in the p e r i p l a s m i c space were isolated f r o m the v a r i o u s m u t a n t s after e a c h g r o w t h e x p e r i m e n t . T h e p r o -

reins p r e s e n t in the g r o w t h m e d i u m were c o n c e n t r a t e d by T C A p r e c i p i t a t i o n . T h e h e a t s h o c k p r e p a r a t i o n s , the p e r i p l a s m i c fractions a n d the c o n c e n t r a t e d g r o w t h m e d i a were subjected to o n e - d i m e n s i o n a l S D S - P A G E , a n d the s e p a r a t e d p r o teins visualized with a m i x t u r e o f two a d s o r b e d antiK 8 8 a b sera. W e s t e r n blots o f the cell fractions t h a t were f o u n d to c o n t a i n f i m b r i a l subunits are s h o w n in Fig. 7 and the results are s u m m a r i z e d in Table 1. Based u p o n E L I S A e x p e r i m e n t s a n d W e s t e r n b l o t t i n g the p h e n o types o f thefaeG m u t a t i o n s w e r e divided i n t o three classes. Class I m u t a n t s did n o t carry any f i m b r i a l subunits on their surface, i.e. Class I h e a t s h o c k extracts were n e g a t i v e in Western blots a n d Class I m u t a n t s were negative in a w h o l e cell E L I S A . T h e Class II m u t a n t s carried fimbrial s u b u n i t s o n their surface, but these w e r e n o t liberated f r o m the h o s t cell by heat shock t r e a t m e n t , i.e. the Class II h e a t shock extracts were n e g a t i v e in Western blots b u t the Class II m u t a n t s w e r e positive in a w h o l e cell E L I S A . T h e Class III m u t a n t s c a r r i e d fimbrial subunits o n their surface a n d these m u t a t e d subunit p r o t e i n s were l i b e r a t e d f r o m the cells by the h e a t shock p r o c e d u r e . Table 1 shows the class to w h i c h indiv i d u a l m u t a t i o n s belong.

290 a

b

c

d

e

f

g

h

i

j

43 43 30

20

• •

30

20

Fig. 7. Western blotting of heat shock extracts isolated from: a, PAP574; b, PAP574(FaeG D 12-30); c, PAP574(FaeG D 19-30); d, PAP574(FaeG D 12-30+Hep); e, PAP574(FaeGwt); and periplasmic fractions from: f, PAP574(FaeGwt); g, PAP574(FaeG D 12-30); h, PAP574(FaeG D 12-30+Hep); and concentrated growth media from: i, PAP574(FaeGwt);j, PAP574(FaeG A 208264a), using a mixture of two adsorbed anti-K88ab sera. Molecular weight markers are shown in kilodaltons. The positions of FaeG proteins are indicated with arrowheads

Class I mutations, resulting in a non-fimbriated phenotype The non-fimbriated phenotype of the Class I mutants was found to result from proteolytic degradation of the mutated fimbrial subunit proteins. Protein stability assays were performed in exponentially growing cultures at 37 ° C after inhibition of protein synthesis with chloramphenicol. The kinetics of protein degradation were measured in an ELISA assay using sonicated cell extracts. All Class I proteins were found to be present in very low amounts in exponentially growing cells and to be degraded with half-lives of between 5 and 10 min. At least two explanations can be envisaged for FaeG protein instability: incorrect interactions between the mutated FaeG proteins and the FaeE chaperone protein or proteolytic degradation resulting from improper folding of FaeG proteins. Wild-type fimbrial subunits are known to be rapidly degraded in an faeE mutant (Mooi et al. 1982) and incorrectly folded proteins are wellknown targets for proteolytic enzymes (Gottesman 1989). The reason for the Class I protein instability requires further investigation. Class H mutations, resulting in a low amount of fimbrial subunits on the cell surface Class II proteins were secreted to the surface of the bacterial cell, as judged from the ELISA experiments (Table 1). Western blotting of the isolated heat shock extracts, periplasmic fractions and concentrated growth media from the Class II mutants did not however reveal the presence of fimbrial subunits. From these experiments we conclude that the Class II proteins were secreted to the surface of the bacteria although in smaller amounts than wild-type fimbrial proteins. The reduced amount of fimbrial subunit proteins present on the sur-

face of the Class II mutants was found to result from proteolytic degradation of mutated fimbrial subunit proteins, presumably for reasons analogous to those proposed for the degradation of Class I proteins. The kinetics of Class II fimbrial subunit degradation were determined as described for the Class I proteins and the Class II fimbrial subunits were found to have half-lives of between 25 and 30 min, i.e. slightly longer than the observed generation time (data not shown). FaeG(D 79-90), FaeG(D 208-219), FaeG(D 219254), FaeG(D 30-254) and FaeG (D 12-254) were found to be located on the surface of the bacterial cell as judged by the ELISA experiments (Table 1). These proteins contain 12, 12, 36, 225 and 243 residues, respectively, in addition to the 264 residues present in the mature wildtype FaeG protein. These results show that the helper proteins are able to secrete fimbrial subunits substantially larger than the wild-type protein without interfering with cell growth. Four deletion derivatives of the wild-type FaeG protein also belong to Class II: FaeG(A 12-30), FaeG(A 30-79), FaeG(A 90-129) and FaeG(A 219-254). These results indicate that the deleted residues are not vital for interaction with the morphopoietic proteins FaeC, FaeD, FaeE or FaeF. The finding that FaeG(A 12-18) was located on the bacterial surface as judged by the ELISA experiments, while FaeG(A 12-30) and FaeG(A 18-30) were not, suggests that residues 18-30 constitute a topogenic structure essential for secretion of fimbrial subunit proteins. The Class III phenotype ." mutations not interfering with fimbrial secretion and assembly The Class III FaeG proteins were secreted in approximately wild-type amounts and presumably assembled into a ' normal' fimbrial structure, as they were liberated from the cell surface by heat shock treatment (Fig. 7 and Table 1). The observation that large parts of the amino-terminal region can be repeated without interfering with secretion and assembly shows that the size of a fimbrial subunit is not critical for secretion and assembly. The FaeG(D 12-30), FaeG(D 19-30) and FaeG(D 12-30+ Hep) fusions, containing an additional 18, 11 and 41 residues, respectively, were still secreted and presumably correctly assembled. The detection of FaeG(D 12--30) and FaeG(D 12-30+Hep) in the periplasmic space, in contrast to the FaeGwt protein, shows that the rate of passage through the outer membrane is reduced for the two mutated subunit proteins when compared with the wild-type protein. These results are in agreement with results obtained by linker insertion mutagenesis (Pedersen 1991). The rate of passage through the outer membrane was found to be reduced for fimbrial subunit proteins with a few additional amino acids inserted at positions 17 and 29 in the mature FaeGwt protein. The observation that a frameshift mutation at codon 208 resulted in secretion of fimbrial subunits in to the growth medium indicates that amino acids proximal to

291 residue 208 are i n v o l v e d in a n c h o r i n g f i m b r i a e to the o u t e r surface o f the cell, in s u b u n i t - s u b u n i t i n t e r a c t i o n s o r in i n t e r a c t i o n s b e t w e e n s u b u n i t s a n d m i n o r c o m p o nents. K n o w l e d g e o f the p h e n o t y p e s o f the m u t a t i o n s described in this p a p e r a n d in a p r e v i o u s p a p e r ( P e d e r s e n 1991) s h o u l d a l l o w the selection o f a m i n o acids in the F a e G p r o t e i n for m o r e d e t a i l e d m u t a g e n e s i s experiments. Acknowledgements. We would like to thank Anders Ostergaard,

Kirsten Hansen and Elisabeth Andersen for technical assistance and our colleagues for helpful discussions and criticism of the manuscript. This work was partially supported by the Danish Medical Research Council, the Alfred Benzon Foundation and the Danish Center for Microbiology.

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Deletion and duplication of specific sequences in the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli.

Small, defined in-frame deletions and in-frame duplications of specific sequences were made within the faeG gene encoding the K88ab fimbrial subunit p...
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