Characterization of two genes encoding antigenically distinct type-1 fimbriae of Klebsiella pneumoniae

Gene, 64 ( 1988) 231-240

231

Elsevier GEN 02342

characterization pneumoniae

of two genes encoding

antigenically

(Hemagglutination; epitopes; nucleotide

recombinant DNA; expression sequence; limbrial subunit)

distinct

in Escherichia

type-l

fimbriae

coli; uropathogenic;

of K~e~s~eZZ~

genomic

library;

Gerald-F. Gerlach and Steven CIegg Department of Microbiology, University ofZowa, Iowa Citv, IA 52242 (U.S.A.) Received

7 October

Revised

7 December

1987

1987

Accepted

28 December

Received

by publisher

1987 19 January

1987

SUMMARY

A uropathogenic isolate of Klebsie~~a pne~moni~e was shown to exhibit a m~nose-sensitive hema~utinating phenotype and to produce type-l limbriae consisting of subunits with a different electrophoretic mobility than those previously investigated. The gene cluster encoding expression of limbriae was cloned and the genetic organization of the encoded polypeptides was determined. The gene encoding the major limbrial subunit was localized and further examined by nucleotide sequence analysis. Comparison of two K. pneumoniue limbrial genes revealed a nucleotide sequence agreement of 73 %, and amino acid sequence agreement of 84% for the mature ~mbrial subunits. Predictions of putative antigenic sites were correlated with regions demonstrating amino acid variability. In agreement with these predictions, no serological cross-reactivity between both limbrial proteins could be demonstrated using an enzyme-linked immunosorbent assay (ELISA).

INTRODUCTION

K. pneumoniae is a frequent opportunistic pathogen implicated in urinary and respiratory tract infections (Fader et al., 1979; Highsmith and Jan+, 1985; Maayan et al., 1985). For the colonization of

Correspondence 10: Dr. G.-F. biology,

University

of Iowa,

Gerlach, Iowa

Department City,

IA 52242

of Micro(U.S.A.)

Tel. (3193335-7778. Abbreviations: alkaline

ELISA, bromide;

aa, amino acid(s); Ap, ampicillin;

phosphatase;

both systems, the ability to adhere to mucosal surfaces is a preliminary step in the infective process. The role of type-l (mannose-sensitive) limbriae in facilitating this initial step is suggested by the fact that more than 80% of clinical isolates of K. pneumoniae express type- 1 fimbriae (Przondo-

bp, base pair(s);

BAP, bacterial

Cm, chloramphenicol;

enzyme-linked

immunosorbent

HA, hemagg~utination;

sensitive;

nt, nucleotidejs);

phoresis;

PBS, phosphate-buffered

sodium

0378-t ll9:881$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

dodecyl

sulfate;

PAGE,

assay;

EtdBr, ethidium

kb, 1000 bp; MS, mannosepolyacrylamide saline;

Sp, spectinomycin;

gel electro-

R, resistance;

SDS,

Tc, tetracycline.

232

Hessek

and Pulverer,

1983)

and is further

docu-

MATERIALS

AND

METHODS

mented through studies using animal models of infection (Fader et al., 1979; Maayan et al., 1985). Fader et al. (1979) demonstrated an approx. lOO-fold greater

adherence

to rat bladder

ial cells by fimbriate than K. pt~euttzotzi~te. This increase by preincubation

fimbrial

serum or by pretreatment

cells with D-mannose.

of the bacteria A different

and hemagglutina-

tion tests

epithel-

by non-fimbriate was significantly

lowered

(a) Bacteria, growth conditions,

with anti-

of the epithelial approach,

taken

K. pneutnoniue lates obtained

IA565 and IA551 are urinary

from the medical microbiology

tory of the University

of Iowa Hospitals

iso-

labora-

and Clinics,

Iowa City, IA. Fimbriation and hemagglutinating activity were detected following serial passage in

by Maayan et al. (1985), showed that after intravesicular inoculation of mice with a K. pneumoniue

broth medium

population

fimbriate,

minicell-producing

(Orndorff

et al., 1985) was used in all transformation

containing

both

non-fimbriate

and

fimbriate phenotypes, the fimbriate bacteria were selected over the non-fimbriate organisms. Furthermore, there was a significantly reduced infection rate in mice which had been inoculated with nonfimbriate K. pneumoniae that had lost the ability to undergo fimbrial phase variation. In Escherichia coli, it has been shown that an invertible DNA fragment, containing the promoter region of the fimbrial gene, plays a role in mediating phenotypic conversion due to phase variation (Abraham et al., 1985). Initial comparison of K. pneumoniae type- 1 fimbriae from various strains suggested that the fimbrial filaments are morphologically indistinguishable (Old and Adegbola, 1984), functionally identical (Duguid, 1957; Prozondo-Hessek and Pulverer, 1983) and antigenically closely related (Nowotarska and Mulczyk, 1977). However, Korhonen et al. (1983) observed differences in the electrophoretic mobility of fimbrial subunits of K. pneumoniae and K. aerogenes, with the latter species presently being classified as separate strains of K. pneumoniae (Rlrskov, 1984). In this paper we have confirmed and further investigated this difference at the nucleotide sequence and protein levels. Inverted repeats at nt -446 and -139 have the same sequence as that found flanking an invertible segment that controls phase variation of type 1 fimbriae of E. coli (Abraham et al., 1985), suggesting a possibly similar function in K. pneumoniae.

Unless

incubated

otherwise

stated,

at 37’ C for 48-h periods. the genotypically

non-

strain E. coli ORN103

experiments. Competent cells were prepared by the calcium chloride procedure of Mandel and Higa (1970). Bacteria possessing recombinant plasmids were grown on L-agar plates supplemented with antibiotics at the following concentrations @g/ml): Ap, 100; Tc, 20; Sp, 40; Cm, 25 (200 for plasmid amplification). HA tests were performed in microtiter plates by mixing equal volumes (25 ~1) of bacterial and guinea pig erythrocyte suspensions. To detect MS HA the bacterial suspension was premixed with an equal volume of %-methyl-D-mannoside in PBS. (b) Enzyme-linked

immunosorbent

assays

A competitive ELISA (Rothbard et al., 1984) was performed to determine the degree of antigenic cross-reactivity between purified fimbriae of K. pneumoniue IA565 and IA55 1. (c) Preparation of the genomic library The positive selection vector pUN121, kindly provided by B. Nilsson, was used in the preparation of the chromosomal DNA library. This vector possesses ApK and TcR determinants, the latter being under the transcriptional control of a repressor protein encoded by the phage A c1 gene (Nilsson et al., 1983). Thus, cloning into the unique EcoRI site of the c1 gene results in the constitutive expression of the TcR determinant. Chromosomal DNA was prepared from overnight broth cultures as described by Stauffer et al. (1981). Subsequently, the DNA was partially digested with the restriction endonuclease EcoRI, and DNA fragments of 12 to 15 kb

233

were separated by electrophoresis through and eiution from 0.3 % low-gelling-temperature agarose. The fragments were ligated into the EcttRI site of pUN 121 which had been treated with BAP. E. cdi HB 101 (Maniatis et al., 1982) was transformed and plated on agar containing Tc and, following incubation at 37°C all colonies were screened for MS HA activity. The presence of plasmid DNA was verified using the method described by Holmes and Quigley (1981), and such molecules were purified by SDS lysis and subsequent EtdBr-CsCl equilibrium density gradient ~entrifugation (Guerry et al., 1973).

pGG

(d) Restriction

endanuclease

digestion

DNA, agarose gel electrophoresis

of plasmid

and construction

of subclones

Restriction endonucleases were purchased from commercial sources and were used according to the manufacturers’ instructions. Restriction fragments were analysed by agarose gel efectrophoresis in Trisacetate buffer (Maniatis et al., 1982). Subcloning was performed by separation and subsequent Iigation of the appropriate DNA fragments in lowgelling-temperature agarose (Struhl, 1983).

101

pGGl02 pGG 103 pGG 105 pGG106 pGG107

Fig. I, Physical

map end genetic organization

cloning vehicle (pBK322), indicated

below the locations

of the previously derivatives is indicated

published

ofpGG101

of the appropriate

pGGlO1

and its deletion

derivatives.

K~e~sj~~~u-~~r~~~~ DNA. The moiecular

genes. The gene encoding

gene cluster ofK. pneumoniae

were constructed

by the lines.

of plasmid

and the thin Iines represent

The thick tine represents masses

the major fimbriaf subunit is designated$px.

restriction

enzyme fragments.

The DNA remaining

are

The organization

1.4551 (pBP7; Purcell and Clegg, 1983) is shown for comparison.

by removal of appropriate

DNA of the

{kL)a) of the gene products

The deletion

in these plasmids

234

(e) Purification

vehicles

of fimbriae

from K. ~n~urnoni~e IA565 and recom-

Fimbriae

pBR322

and

pACYC184.

The

plasmid constructed which retained sequences of K.pneu~to~j~e fimbriae

smallest

the coding and hemag-

binant bacteria, were prepared as described in detail elsewhere (Gerlach and Clegg, 1988). Fimbriae from

glutinin production,

which was designated

pGG 10 1

(Fig. 1), possessed

Klebsiellu-derived DNA

10.5 kb

K. pneumoniue IA55 1 (Purcell and Clegg, 1982), and

in length.

transformants prepared

expressing

this fimbrial

using the method described

and Ou (1979). The protein content parations

was quantified

et al. (1951). analysed

Purified

by McMichael of fimbrial

by the method fimbrial

by discontinuous

acrylamide,

antigen, were

of Lowry

preparations

O.l”/, SDS-PAGE

0.3 y0 bisacrylamide)

pre-

to the

method of Laemmli (1970). Samples were boiled for 30 min in a reducing SDS buffer (0.0625 M Tris pH 1.8, 3% SDS (w/v), 15 y0 glycerol (w/v), 5 “/;, 2-mercaptoethanol (v/v), 0.1% bromophenol (w/v). Following electrophoresis, the gels stained with Coomassie brilliant blue. (g) Nucleotide

and localization

encoded by the Ktebsiella cluster

of poiypeptides

pneumoniae

fimbrial gene

were (12%

according

(b) Identi~cation

blue were

Specific deletions

sequence analysis

A

The nucleotide sequence of the fimbrial structural gene was determined using the chain-te~ination method of Sanger et al. (1977) and the chemical modification method of Maxam and Gilbert (1977).

of pGG101

were constructed

by

subcloning specific restriction Fragments (Fig. 1); the subclones were used to transform E. coIi ORN 103. Subsequent analysis of the plasmid-encoded polypeptides from these deletions permitted the localization of genes encoding six polypeptides associated

67

0

B

C

-4

43

RESULTS AND DISCUSSION

(a) Cloning of the nucleotide sequences type 1 fimbriae of Klebsiella pneumoniae

encoding IA565

Of 2000 TcR transfo~~ts tested (see MATERIALS AND METHODS, section c), three possessed the Fim + MS HA+ phenotype and also reacted with antiserum raised against the purified timbriae of the wild-type strain. Plasmid DNA, prepared from each of these strains, was able to transform the genotypitally non-~nlbriate strain E. co/i ORN103 to the Fim’ MS HA+ phenotype with 100% efficiency. Following restriction by EcoRI, all three plasmids were shown to possess a 14.5-kb DNA insert. The three recombinant plasmids demonstrated identical Hind111 and BarnHI restriction profiles and one of these plasmids, designated pGG100, was used in subsequent experiments. The genes encoding type 1 fimbriae were further subcloned using the cloning

30

0

20.1 #ia@

14.4 0

-

';

Fig. 2. 0.17; SDS-12.5% PAGE of iimbriae (indicated by arrows) purified from: K.pneumoniue IA.551 (lane B) and ~.~neurno~jue IA565 (lane C). The samples were treated as described in MATERIALS AND METHODS, section f. The A4, standards (lane A) are bovine serum albumin (6 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa)l, soybean trypsin inhibitor (20.1 kDa), and z-lactalbumin (14.4 kDa).

235

with fimbrial

expression.

these gene products 8%kDa

based

on SDS-PAGE

relative locations peptides

The molecular

are 1%, 19.5,

masses

and

(not shown).

The

of the genes encoding

are shown

of

22-, 22.5,

in Fig. 1. The

(c) Nucleotide

these poly-

I%kDa

The results

poly-

limbrial

IA565

the IA565type

cultures

transformed

sonicates

by pGG106

(Fig. I) could be used to remove bodies

causing

serum, whereas

fimbrial sonicates

pGG102

antilimbrial

agglutination of pGG103,

subunit

upstream

anti-

sequences

pGG105

-139,

and

pGG107 transformants failed to do so. Therefore, the gene encoding the major ~mbrial subunit of K. pneumoniae IA565 must be located on the 1.&kb SphI fragment of pGG101 (Fig. 1).

encoding

Fig. 3. Nucleotide

sequencing

strategy

and the sequencing

described

in MATERIALS

fragments

are represented

of plasmid strategy

AND METHODS,

pGGIOi

is outlined

and the region immediately possesses

flanked by inverted-repeat + 161 and

+ 405; these

sequences (Fig. 4). In

addition a palindromic sequence 14 bp in length is present at nt position 234. The genes encoding the IA565 subunit and that encoding the IA55 1 fimbrial

and the relative

by the arrows

sequenced

three disnucleotide

-463 and -3.5, -446 and

11, 10, and 9 bp, respectively

position

underneath

of the fimbrial

structurat

the map. Nucleotide

section g. S’-end labelled DNA fragments

by circles. DNA fragments

the major

IA565 is shown in

100

area is enlarged

analysis

sequence of the gene encoding

fimbriae

at nt positions

and at

contain

fragment

from this determinant

tinct segments

from immune

sequence

of K. pneumoniae

Fig. 4. The nucleotide

of bacterial and

of the nucleotide

(Fig. 3) of the DNA

peptide possesses an electrophoretic mobility identical to that of the purified limbriae of K. pneumoniae (Fig. 2). In addition,

and amino acid sequence of fimbrial

genes

are represented

bp

gene j&r. The sequenced

sequencing by squares,

by the method of Sanger et al. (1977) are marked

was performed

as

and 3’-end labelled

with open arrowheads.

236

-450 AAT AAC GAA CAG GTC AAT TGG GGC CAA ACT GTT TAT ATC 1 r) -420 -390' ATA AAA ATG TTT TGA CAT ATT TTG CAA CTC ACT GCG CCA TAT TTC CCC AAA AAA TTA AAG AAT TAA AM

-360 -330 AAA TGG TAT CTC ATT GAT TAT TAT CTA TTT TTA AAC CTA CAA TTA CTC ACT TTT -300 -270 CTC TAA CCA TAT CTC TGC TGC ACC GGC GAT CCG TCG CCA GCG CCA TCG CTA TTC -240 -210 CTT TCG CAC TCC CTT CCA CCA CAC CAG ATT ATA TTA CCA TAA AAG CCA AAT TTG -180 GCA GAA TTT AAA ACC ACA GAA TAC CAG GTT GAC ACA AAA TAC AGA TCA TTT ATC -150 -120 CCA ACG AGT CAA AAT GGC CCC AAT CGT CTC ATC GGT TGG GCG AAA ACT GTG CAG 9 -90 -60 AGC CGG CAG CCG GAC CAC TCG GCT GAA ATC TGA AAT GCC TGA AGG CAC AAC GGC L

-30

+1 CGA TAT CGT TCA AAG GAA AAC AGT ATG AAA ATC

TGC cAA TCC GY +40 AAA ACA CTG GCA ATG ATT GTT GTG TCA GCA CTG GCA CTG AGC TCC ACG GCA GCT +70 +100 CTG GCC GAT ACC ACC ACG GTC AAC GGC GGG ACC GTG CAT TTT AAA GGA GAA GTC +130 +160 GTT AAT GCG GCC TGT GCG GTG GAT GCC GGC TCT ATC GAT CAA ACC GTT CAG PA 3 +190 +220 GGC CAG GTG CGT TCC GCG AAA CTG GCC ACG GCG GGG AGC ACC AGT TCG GCC GTC +250 GGT TTC AAC ATC CAG CTG CAT GAT TGC GAC ACT ACG GTA GCC ACC AAA GCG TCC oooo......ooooOeH~ +310 GTC GCC TTC GCC GGT ACG CCC ATC GAC AGC AGC AAT ACC ACC GTT CTG GCC CTG +340 +370 CAA AAC TCT GCC GCT GGC AGC GCA ACC AAC GTC GGC GTG CAG ATC CTC GAC AAT +430 +400 ACC GGT ACG CCA CTG GCA CTG AAC GGC GCC ACC TTC AGT GCT GCG ACC ACG CTG 3 +490 +460 AAT GAC GAC CCC AAC ATC ATC CCG TTC CAG GCA CGT TAC TAC GCG ACC GGC GCA +520 *. GCG ACG GCT GGT ATA GCG AAC GCG GAT GCC ACG TTC AAA GTG CAG TAC GAG TAA l

Fig. 4. Nucleotide

sequence

of the gene encoding

the major fimbrial subunit

start codon is labelled

+ 1. The three series of inverted-repeat

palindromic

is indicated

counted.

sequence

nucleotide

of K. pneumoniae

sequences

IA565. The first nt in the putative

are underlined

by a chain of solid circles. The last digit of the nucleotide

and labelled

numbers

ATG

1, 2, and 3. The 14.bp

is aligned

with the nucleotide

-23 -20 -10 A MET LYS ILE LYS THR LEU ALA MET ILE VAL VAL --- SER ALA LEU B MET LYS ILE LYS THR LEU ALA --- ILE VAL VAL LEG SER ALA LEU -1

+1

A ALA LEU SER SER THR ALA ALA LEU ALA ASP THR THR THR VAL ASN B !!!!?l? LEU SER SER ALA ALA ALA LEU ALA ASP THR THR THR VAL ASN t +10 +20 A GLY GLY THR VAL HIS PHE LYS GLY GLU VAL VAL ASN ALA ALA CYS B GLY GLY THR VAL HIS PHE LYS GLY GLU VAL VAL ASN ALA ALA CYS +30 A ALA VAL ASP ALA GLY SER ILE ASP GLN THR VAL GLN LEU GLY ?%i B ALA VAL ASP ALA GLY SER m ASP GLN THR VAL GLN LEU GLY GLN +40 +50 A ~~~.tikt%if.~~.~%. LEU ALA THR ALA GLY a THR SER SER ALA B VAL ARG m AL.4?i?i? LEU m ALA GLY ALA THR SER SER ALA .......................... +60 A VAL GLY PHE ASN ILE GLN ~~tf.A'3P.~~.EPS.~~~.~~ THR VAL ALA B VAL GLY PHE ASN ILE GLN LEU i...*...................... m ASP CYS ASP THR THR VAL ALA +70 .............+80 ........ A THR LYS ALA SER VAL ALA PHE ALA GLY THR ALA ILE ASP SER SER B THR LYS ALA m VAL ALA PHE jt!?! GLY THR ALA ILE m +90 .... A Bjir THR THR VAL LEU ALA LEU GLN ASN SER ALA ALA GLY SER ALA B HIS THR m VAL LEU ALA LEU GLN SER SER ALA ALA GLY SER ALA +100 +110 A THR ASN VAL GLY VAL GLN ILE LEU ASP ASN THR GLY THR PRO LEU B THR ASN VAL GLY VAL GLN ILE l LEU ASP %? THR GTY Am LEU mmmmmmmmmmmmmmmmmmm~**mmmm

+120 ............. A ALA LEU ASN GLY ALA THR PHE SER ALA ALA THR THR LEU ASN ASP B ALA LEU m GLY ALA THR PHE SER m THR THR LEU ASN ASN +140 .............+130 A ASP PRO ASN ILE ILE PRO PHE GLN ALA ARG TYR TYR ALA THR GLY B ????%? ASN %?i? ILE PRO PHE GLN ALA ARG TYR ??!?ALA THR GLY +150 A ALA ALA THR &L& GLY $& ALA ASN ALA ASP ALA THR PHE LYS VAL B ALA ALA THR PRO GLY ALA ALA ASN ALA ASP ALA THR PHJZLYS VAL

A: K. B: K. Fig. 5. Comparison sequence

of the predicted

ofthe IA55

the two subunits

are marked

below (K. pneumoniae of the signal pcptidc

of the timbrial subunits

of K. pneumoniae IA565 and IA55 1. The nucleotide

1fimbrial subunit has been published elsewhere (Purcell et al., 1987). Variations ofthe amino acid sequence between

lines, in the signal peptide which are predicted

amino acid sequences

IA565 IA551

pneumoniae pneumoniae

by solid lines. The site of cleavage

region, demonstrate

to form strong

antigenic

IA551) the relevant is labelled

-1.

the absence determinants

sequences.

of the processed

of the corresponding are represented

timbrial

subunit

is shown by the arrow;

amino acid at that position.

the dashed

The amino acid regions

by a chain of heavy dots above (K. pneumoniae IA565) or

The first aa of the mature

fimbrial

subunit

is labelled

+ 1, and the terminal

aa

235

subunit (Purcell et al., 1987) demonstrate a signilicant degree of nucleotide sequence agreement (73”/,). However, the sequences upstream from the start codons in both genes exhibit no similarity

two fimbrial proteins,

antiseraraised

against limbriae

purified from recombinant strains were used in the competitive ELISA. In the ELISA no cross-reac-

to the inverted

repeats

tivity between the two antigens was (Table I). Thus, no inhibition of serum

at nt positions -446 and -139. The amino acid sequence of the timbrial

subunit

with solid-phase antigen was observed by amounts of the heterologous antigen which were significantly

except in the regions adjacent

was derived from the nucleotide sequence and is presented in Fig. 5. The degree of amino acid

greater

sequence agreement between both timbrial subunits (IA565 and IA551) is 84%. However, the limited

demonstrate

variations are not randomly distributed the limbrial subunit but are concentrated

throughout at aa posi-

tions + 39 to +47, + 79 to + 84, + 106 to + 114, and + 126 to + 130 (Fig. 5). The predicted amino acid sequence was used to determine the hydrophilicity profile using the algorithm of Hopp and Woods (1981) which in addition can be used to predict potential antigenic determinants. These are located at aa positions 36-41, 58-63, 77-82, and 124-129 for the IA565 limbrial subunit and at aa positions 40-45, 59-64, and 104-109 for that of IA551 (Fig. 5). (d) Immunologic properties of fimbriae To investigate

the antigenic

relevance

of amino

acid variations, as predicted from the nucleotide sequences of the fimbrial subunit genes, between the

required

than

those

of the

to cause a 507,

homologous

inhibition.

that the two limbrial

immunologically

observed reactivity

antigen

These results proteins

are not

identical.

(e) Conclusions Our initial observations that the physical maps of two recombinant plasmids encoding the expression of K. pneumoniue type 1 limbriae were not identical was interesting since we have previously shown that one of these plasmids is similar to that encoding the type 1 fimbriae of E. colz’ (Clegg et al., 1985). In addition, it has already been demonstrated that the M,s of the fimbrial subunits from different isolates of K, pneumoniae are not identical (Korhonen et al., 1983; Fig. 1). Therefore, we decided to further analyze the two KlebsiellQ-delved ~mbrial gene clusters to compare genetic organization, fimbrial subunit structure and antigenicity of Klebsielh timbriae. Translation

of the

limbrial

gene

located

on

pGG 101 is thought to begin at the ATG codon labeled + 1 in Fig. 5. The evidence suggesting this TABLE

I

Competitive

ELISA

showing

antigenic

differences

between

Klebsiella pneumoniae IA565 and IA551 fimbriae _..

--.--

ELISA

isolated

solid-phase

Competitive

antigen

from

antigen ” pGGlO1

pBP7 --~

IAS654imbriae IA5Sl-fimbriae J Appropriate

> 300

2ah 11000 dilutions

of antisera

38 were preincubated.

37’ C, with equal volumes of serial two-fold dilutions gous or heterologous serum suspension

antigen.

Subsequently

was incubated

for

rabbit

was developed

antigen

values

to decrease by SO”,,.

to microtiter

using phosphatase-con;ugated

IgG and ~-nitrophenyl

’ Inhibition required

the antigen-anti-

I h at 37°C with the solid

phase antigen which had been adsorbed reaction

phosphate

are stated the reaction

for 1 h at of homolo-

wells. The goat anti-

as substrate.

as the quantity of antiserum

of antigen

(ng)

with solid phase

codon as the start codon rather than the ATG sequence at aa position + 21, is the high degree of sequence agreement with the gene encoding the IA551 fimbriae. The predicted N-terminal amino acid residues of this latter gene product are identical to those observed by amino acid sequence analysis of the purified subunit (Purcell et al., 1987). In addition, a possible Shine-Dalgarno sequence is present beginning at nt position -11. Furthermore, the ATG codon is immediately followed by a codon encoding a positively charged amino acid residue which is commonly associated with proteins which are transported across the bacterial membrane (Michaelis and Beckwith, 1982). A function for the inverted repeats and the 14 bp palindromic sequence at nt position + 234 observed in the nucleotide sequence of the subunit gene has so far not been determined. However, an invertible

239

DNA

element

that controls

identical

to that

(Abraham with the

the phase variation

in E. coli is flanked

type 1 fimbriae

at nt positions

of

-446

and

Guerry,

determinants

revealed

type 3 (MR/K) Microbial.

Highsmith,

A.K.

selected

a high

Holmes,

and

Jarvis,

to pathogenicity.

M.: A rapid boiling method plasmids.

T.P. and Woods,

Anal. Biochem.

for the

114 ( 198 1)

K.R.: Prediction

of protein

from amino acid sequences.

antigenic

Proc. Nat]. Acad.

Sci. USA 78 (1981) 3824-3828. Korhonen,

T.K., Tarkka,

E., Ranta.

role in bacterial

adhesion

to plant

Laemmli,

M.K.:

assembly

Cleavage

of structural

and

J. Bacterial.

proteins

of the head of bacteriophage

O.H.,

Protein

Rosebrough,

155

during

T4. Nature

N.J., Farr,

measurement

Chem. Maayan.

the

227 (1970) R.J.: J. Biol.

193 (1951) 265-275. M.C., Ofek, I., Medalla,

pneumoniae during

urinary

A.: Calcium

dependent

E.F. and Sambrook,

Manual.

Harbor,

in

bacteriophage Cloning.

Laboratory,

Cold

NY, 1982.

A.M. and Gilbert,

W.: A new method

for sequencing

Sci. USA 74 (1977) 560-564.

J.C. and Ou, J.T.: Structure

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cell envelope

infection

J.: Molecular

Cold Spring Harbor

DNA. Proc. Natl. Acad.

Michaelis,

tract

J. Mol. Biol. 53 (1970) 154-162.

T., Fritsch,

McMichael,

M.: Popula-

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49 (1985) 785-789.

M. and Higa,

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experimental

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A Laboratory

ACKNOWLEDGEMENTS

H. and Haahtela,

of Klebsiella sp.: molecular

3 timbriae

Maniatis,

Republic

Klebsiella pneumoniae:

that contribute

6 (1985) 75-77.

of bacterial

determinants

sites.

the Federal

116

193-197. Hopp,

prediction was confirmed by a competitive ELISA. Thus, whereas a 50% inhibition in antigen-antibody reactivity was caused by less than 50 ng of homologous timbriae, no inhibition could be induced with heterologous fimbriae even when high concentrations of antigen were used (Table I). This paper describes the comparison of two type 1 fimbrial gene clusters in K. pneumoniue with emphasis on the fimbrial structural gene. The prediction of different immunogenic epitopes on both fimbrial subunits raises the question, whether it might be possible to construct antigenically hybrid fimbriae by the modification or exchange of antigenic

program

method for the

acid. J. Bacterial.

W.R.:

factors

D.S. and Quigley,

preparation

a close correlation

is a fellow of the postdoctoral

S.: General

deoxyribonucleic

virulence

Infect. Control

(Fig. 5). Thus these variable regions appear to confer unique epitopes to the two fimbrial subunits. This

G.F.G.

Lett. (1988) in press.

D.J. and Falkow,

of plasmid

of the

of Klebsiella

limbriae

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