Vol. 56, No. 9INFECTION AND IMMUNITY, Sept. 1988, p. 2317-23230019-9567/88/092317-07$02.00/0Copyright C) 1988, American Society for Microbiology

Expression of K99 Adhesion Antigen Controlled by theEscherichia coli Tryptophan Operon Promoter

PRESTON A. BAECKER,' EARL R. SHELTON,1 HELA BURSZTYN-PETTEGREW,l FELIX H. SALAZAR,'ERIC G. OSEN,1 STEVEN D. STOUFER,lt SIMON W. LEE,2 AND HARDY W. CHAN"*

Institute of Bio-organic Chemistry' and Institute of Biological Sciences,2 Syntex Research, Palo Alto, California 94304

Received 1 February 1988/Accepted 8 June 1988

The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [OiOl:K99] has beencloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J. D. A.van Embden, F. K. de Graaf, L. M. Schouls, and J. S. Teppma, Infect. Immun. 29:1125-1133, 1980). Cellsharboring one such construction, known as pK99-64, are capable of expressing K99 antigen on the cell surface.We replaced the natural promoter sequence for the gene encoding the K99 pilus subunit with a strong,inducible exogenous promoter, the E. coli tryptophan (tip) operon promoter, to construct the plasmidpBR-TrpK99. E. coli cells harboring pBR-TrpK99 or a similar construction in the plasmid pDR540, known aspKO-TrpK99, upon induction with 3-p-indoleacrylic acid, produced about fourfold more K99 antigen than didcells bearing pK99-64 with the natural promoter. Expression of the pilus antigen was found to be under controlof the tryptophan promoter. Plasmid instability was encountered, however, in cells bearing pKO-TrpK99 whenthe tip promoter was derepressed. Introduction of the aminoglycoside 3'-phosphotransferase gene oftransposable element TnS into pKO-TrpK99 to generate pKON-TrpK99 effectively stabilized the plasmid incells grown under identical conditions in medium containing kanamycin.

The specific adherence of enterotoxigenic Escherichia colito intestinal mucosal membranes is an essential step in thedevelopment of diarrheal diseases. The bacterial surfacestructures responsible for adherence are generally referredto as adhesins or colonization factors (for a review, seereferences 1 and 18). They are composed of protein subunitswhich form extracellular filamentous structures known asfimbriae or pili. Two examples of adhesins in diarrhealdiseases are K88ab and K99 (7, 24).

Genetic determinants responsible for synthesis of fimbrialsubunits and their subsequent assembly into fimbriae aretypically encoded by complex operons found on either thebacterial chromosome or large episomal plasmids (24, 32).Many of these operons have been molecularly cloned andstudied. The complexity in their structure and regulation iswell exemplified by a model for the biosynthesis of K88 pilusprotein (14, 25-27, 31).

It has been established that immunity to pathogenic E. coliorganisms bearing adhesins can be conferred by immunizinganimals either with E. coli cells bearing pilus antigens or withpurified pilus preparations (16, 17, 28). Hence, considerableeffort has been devoted to selection of highly pilus-producingbacterial strains, as well as optimization of growth condi-tions to maximize pilus production (12).We are particularly interested in development of an E. coli

strain capable of producing large amounts of the K99 anti-gen. This report describes a procedure to increase the yieldof the K99 antigen. Our approach relies on incorporation ofa strong exogenous promoter immediately upstream fromthe coding sequence for the pilus subunit to produce in-creased levels of the K99 adhesin upon induction. A repro-ducibly high yield of pilus-bearing cells was found to requirethe presence of other downstream accessory genes in theK99 operon.

* Corresponding author.t Present address: Escagen, San Carlos, CA 94070.

MATERIALS AND METHODSBacterial strains and media. The bacterial strains used

included the field strain E. coli B41 (O101:K99) (12) and E.coli K-12 HB101, JM103, and W3110. LB liquid medium anddrug selection LB agar plates containing 50 ,ug of ampicillinper ml or 15 ,ug of kanamycin per ml were prepared asdescribed (22). M9 medium was prepared as previouslydescribed (22), except for the addition of 2.5 g of CasaminoAcids (Difco Laboratories, Detroit, Mich.) per liter and 20 gof glucose per liter. Stock solutions (10 mg/ml) of 3-,-indoleacrylic acid (IAA; Sigma Chemical Co., St. Louis,Mo.) in ethanol were prepared on the day of the experiment.Stock solutions of 10 mg of tryptophan per ml were preparedin 10 mM NaOH and kept at 40C for up to 1 week.

Plasmids. Plasmids pBR322, pDR540, pUC8, and pUC9have been described before (4, 30, 33). The source of the trppromoter DNA fragment was plasmid pNO4, which containsa 0.45-kilobase (kb) fragment of the E. coli tryptophanoperon DNA (34) from the closest PvuII site upstream of thetrp promoter to the first Hinfl site in the trpE gene. In thisplasmid, the PvuII site was ligated to a HindIII linker and theHinfl site was filled in and ligated to a PstI linker (8-mer)before insertion between the HindIII and PstI sites of apBR322 derivative. This plasmid was a gift from Neil Oshe-roff, Biochemistry Department, Vanderbilt University Med-ical School, Nashville, Tenn.Enzymes. Restriction endonucleases AccI, AvaI, BglII,

EcoRI, HincII, HpaI, PvuII, SmaI, and TaqI, T4 DNAligase, and E. coli DNA polymerase I (Klenow fragment)were purchased from Bethesda Research Laboratories, Inc.,Gaithersburg, Md. BssHII and XbaI were purchased fromNew England BioLabs, Inc., Beverly, Mass. T4 polynucle-otide kinase was obtained from P-L Biochemicals, Inc.,Milwaukee, Wis. Enzymatic reactions were performed un-der conditions recommended by the suppliers.

Isolation and manipulation of DNA. Plasmid DNA waspurified from cleared lysates (8) by cesium chloride-ethidiumbromide density gradient centrifugation as previously de-

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2318 BAECKER ET AL.

scribed (3). An alkaline extraction procedure was used toprepare DNA for rapid characterization (6). Transformationwas performed by using a standard CaC12 technique (9).Electrophoresis of DNA was performed on agarose or poly-acrylamide gels in Tris-borate buffer (90 mM Tris, 90 mMboric acid, 2.5 mM EDTA, pH 8.3) plus 0.5 ,ug of ethidiumbromide per ml.

Immunological assays. A cellular agglutination assay (15)using a polyclonal anti-K99 serum was used as a semiquan-titative assay for the amount of cell-borne K99 pili (0 to +4scale). An enzyme-linked immunosorbent assay (ELISA)based on a monoclonal antibody (23) was developed toquantitate the pilus protein present in bacterial cell lysates.The final procedure is similar to that described to quantitatethe K88 pilus antigen (24). Briefly, cells were pelleted fromthe culture medium, washed, and suspended at 1 A550 U/mlin 50 mM Tris hydrochloride (pH 7.4)-0.1 mM EDTA-1 mMphenylmethylsulfonyl fluoride before sonic disruption.Costar (Cambridge, Mass.) 96-well vinyl microtiter plateswere coated at room temperature for 16 h with rabbitanti-K99 pilus immunoglobulin G diluted 1/1,000 in 140 mMNaCl. The wells were washed twice with TN buffer (10 mMTris hydrochloride [pH 7.4], 144 mM NaCI) containing0.01% Tween 20 before incubation at 37°C for 1.5 h withduplicate twofold dilutions of the sample in NBT buffer (140mM NaCl, 0.2% bovine serum albumin, 0.02% Tween 80).Dilutions of a standard preparation of pilus antigen purifiedby repeated cycles of polymerization-depolymerization were

included on each plate. The wells were then washed twicewith TN buffer plus 0.01% Tween 20 and twice with TNbuffer alone before incubation for 1.5 h at 37°C with a 1/40,000 dilution in NBT buffer of the mouse monoclonalanti-K99 sera (The E. coli Center, Pennsylvania State Uni-versity, University Park). The plates were then incubated for1.5 h at 37°C with a 1/2,000 dilution of horseradish peroxi-dase-conjugated goat anti-mouse immunoglobulin G (ZymedLaboratories) in NBT. After washing, the enzyme-substratebuffer [0.1 M citrate-phosphate buffer (pH 4.0), 0.25 mg/ml2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid), 0.03% hy-drogen peroxide] was added and the color was quantitatedafter 15 min with a model MR600 microplate reader (Dyna-tech Laboratories, Inc., Alexandria, Va.) at 415 nm. Theaverage of duplicate readings was used to estimate theamount of pilus antigen based on a standard curve run on

each plate. Generally, a linear response from 0.1 to 1 ng ofantigen per well was observed. Each value reported is an

average from at least three sample dilutions.Immunogenicity test. To obtain a recombinant K99 pilus

preparation for immunogenicity testing, E. coli containingplasmid pKO-TrpK99 was grown in 9 liters of LB mediumand induced by IAA addition. Pili were prepared essentiallyas previously described (10). Briefly, pili were released fromthe pelleted cells by heat shock at 60°C and concentrated by60% ammonium sulfate precipitation, and the precipitatewas dialyzed against TN buffer. The concentration of piliwas estimated by a high-pressure liquid chromatographywith a K99 pilus preparation obtained from the Animal andPlant Health Inspection Service of the U.S. Department ofAgriculture as the standard. Pilus protein was formulatedinto vaccines at 150,ug/ml. Two vaccines were prepared,with aluminum hydroxide added as an adjuvant, one con-taining only the K99 antigen and the other containing acombination of the K99 component plus K88ab, K88ac, and987p (17, 24, 31). The reference vaccine, which was ap-proved by the Animal and Plant Health Inspection Service of

PLASMID

B-41

pK99-6F

K99 DNA PHYSICAL MAP

101 151 1 110 115 Kb1

86 Kb

Hind III Hpa I Hpa I Hpa I Hpa I Hind IIII I.,I.l, | , . IBamH I BamH I Xba I Kpn I BamH I BamH I

p, P2pK99-64 1

Xba Bgl IIPT2p P2

pKO-TrpK99 - ----and Mae I

pKON-TrpK99 Xba Bgl II

16 76 17 23 29 16.5

FIG. 1. Genetic maps of cloned K99 DNAs. The horizontal linesrepresent K99-derived insert DNAs in various clones. The locationsof various structural genes are indicated by boxes below the map ofpKO-TrpK99. The numbers above the boxes refer to the molecularweights (103) of the corresponding protein products. Horizontalarrows indicate the approximate locations of promoters and theirdirection of transcription. P1 and P2 are endogenous K99 promoters,and PT,P is theE. coli tryptophan operon promoter (represented bya vertically hatched box at the end of pKO-TrpK99). The relativepositions of selected restriction endonuclease cleavage sites areindicated.

the U.S. Department of Agriculture as a reference standardfor K99 potency, contained similar amounts of the antigen.The relative potencies of the vaccines were compared in a

guinea pig assay. Guinea pigs (300 to 500 g, female, Hartleystrain) were obtained from Charles River Breeding Labora-tories, Inc., Wilmington, Mass., and inoculated with threedilutions of each vaccine. Groups of eight guinea pigs werevaccinated subcutaneously with 1 ml of a 1/5, 1/125, or 1/3,125 dilution. A second injection was administered 14 to 16days after the first vaccination. At 10 to 12 days after thesecond vaccination, serial dilutions of serum from eachanimal were prepared and analyzed by the cellular aggluti-nation assay (15). The potency of the vaccine was deter-mined by calculating the geometric mean titer of sera for thethree dilutions. Relative potency was determined by com-parison of the geometric mean titer of the test vaccine withthat of the reference vaccine.

RESULTS

In nature, the K99 adhesin is encoded by the 78-kbconjugative plasmid pRI9901. We and others (32) havesubcloned from pRI9901 a 7.0-kb BamHI fragment whichappears to contain the entire genetic determinant necessaryfor expression of the K99 antigen. One such subclone, whichcontains the 7.0-kb BarnHI fragment in the BamHI site ofpBR322, is known as pK99-64. In the presence of anti-K99serum, cells harboring plasmid pK99-64 are positive forcellular agglutination. By using a combination of deletion orinsertion mutations introduced at specific restriction enzymesites or by random insertions of transposable element TnS,we generated a genetic map which was in reasonable agree-ment with that described by de Graaf et al. (11) (Fig. 1). Ourdata, however, suggested that the essential genes wereorganized in two separate operons. One can divide andsubclone the 7.0-kb BamHI fragment in two compatibleplasmids and observe full complementary K99 expression(H.W.C., unpublished data).DNA sequence analysis of the K99 pilus structural gene. To

identify the K99 pilus subunit gene positively and to facili-

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HIGH-YIELD EXPRESSION OF K99 ANTIGEN 2319

A mRNA: 5'

Proteins: Leadr Trp E'16K PliusSubunit

B1 10 20 30 40 50

AAGTTCACGT AAAAAGGGTA TCGACA ATG AAA GCA ATT TTC GTA CTG AAA GGT TGG TGG5' (mRNA start) Met Lys Ale lie Phe Val Leu Lys Gly Trp Trp

Leadr Peptide

60 70 80 90 100 110 120

CGC ACT TCC TGA AACGGGCAG TGTATTCACC ATGCGTAAAG CAATCAGATA CCCAGCCCGCArg Thr Ser -

130 140 150 160 170 180

CTAATGAGCG GGCTTTTTTT TGAACAAAAT TAGAGAATAA CA ATG CAA ACA CAA AAA CCGMet Gin Thr Gln Lys ProTrp E' Peptide

190 200 S.D. Xbs I

1) Hind III; Pst I i1) Hind IlIl; Pat1 2) Isolate 0.5Kb FrgwmentI Ugate

Nru IHind Ill

-<../-Trp16K

FIG. 2. Construction of pUC9-Trp16K. The thin circle of thepK99-64 diagram indicates pBR322 DNA, and the double semicirclerepresents K99-derived DNA. The hatched region between HpaIrestriction sites contains the structural gene for the K99 pilussubunit (16K gene). Directions of transcription for the 3-lactamasesgene (Apr) and the K99 pilus subunit gene (16K) are indicated. Thelarge semicircular arrow in diagrams of pUC8 and pUC9, labeledLAC, represents the promoter and the N-terminal coding region ofE. coli P-galactosidase and indicates the direction of transcription.The E. coli tryptophan operon promoter in plasmids pNO4 andpUC9-Trp16K (PTrp) and the direction of transcription from thispromoter are indicated. The relative positions of selected restrictionendonuclease cleavage sites are shown. The plasmid diagrams arenot drawn to scale.

tate overexpression with an exogenous promoter, the DNAsequence encoding the entire 16-kilodalton pilus protein wasdetermined. This was accomplished by subcloning a 1.7-kbHpaI fragment of pK99-64 into the HincIl site of pUC8 toproduce plasmid pUC8-16K. Application of the Maxam-Gilbert technique (21) led to a nucleotide and deduced aminoacid sequence in complete agreement with that alreadypublished (29).

Construction of K99 expression plasmids. Figure 2 depictsthe scheme by which we incorporated the strong, inducibletryptophan (trp) promoter immediately upstream from theK99 pilus subunit. Plasmid pUC8-16K was cleaved withTaqI, an enzyme which cuts about 20 base pairs in front ofthe translational initiation codon (ATG) for the K99 subunitgene, and filled in with Klenow fragment; secondary cleav-age with EcoRI released a 1.1-kb DNA fragment. Ligation ofthis 1.1-kb fragment with pUC9 DNA which had beenpreviously cleaved with AccI, filled with Klenow fragment,and secondarily cleaved with EcoRI yielded plasmid pUC9-16K after transformation. Blunt-end ligation between filled-in TaqI and AccI restriction enzyme termini created a newNruI site which was used to screen for correct plasmidconstructs. To introduce the exogenous trp promoter intopUC9-16K, a HindIII-to-PstI DNA fragment containing the

ACT GCT GCA GGT CGC GAA CAA TGG AGA ATC TAG ATG AAA AThr Ala Ala Gly Arg Glu Gin Trp Arg lie - Met Lye

16K PILUSSUBUNiT

FIG. 3. DNA and amino acid sequence of pUC9-Trpl6K trans-lational initiation region. (A) Linear representation of the beginningof the polycistronic mRNA encoding the tryptophan operon leaderpeptide (Leader), the beginning of the trpE gene product (Trp E'),and the K99 pilus subunit (16K PILUS SUBUNIT). (B) DNAsequence and encoded amino acid sequence of the region dia-grammed above. Only the sense strand of DNA is shown. Number-ing of the DNA sequence starts with the nucleotide analogous to thestart of the polycistronic mRNA. S.D., Shine-Dalgarno sequence.

desired region was isolated from plasmid pNO4 and ligatedto pUC9-16K DNA previously cleaved with the same twoenzymes. The resultant plasmid was designated pUC9-Trpl6K.Upon induction, pUC9-Trpl6K should produce a polycis-

tronic mRNA molecule that codes for three proteins: the trpleader peptide, a short stretch of the trpE protein, and theK99 pilus subunit (Fig. 3). The cloning strategy avoidschanging the distance between the ATG starting codon of thepilus subunit and its proximal ribosomal binding site.When cells containing plasmid pUC9-Trp16K were incu-

bated with IAA, an inducer for the tryptophan promoter,there was no evidence of K99 pilus antigen expression basedon a cell agglutination assay, although low levels of antigenwere detected in cell sonicates by the ELISA. To restoreK99 pilus expression, the remainder of the K99 geneticdeterminant was reintroduced into plasmid pUC9-Trpl6Kby incorporating a BglII-to-EcoRI DNA fragment derivedfrom plasmid pK99-64 into pUC9-Trp16K via the same tworestriction enzyme cleavage sites. The resultant plasmid wasdesignated pUC9-TrpK99 (Fig. 4).

Expression of the K99 pilus antigen from pUC9-TrpK99,as assayed by cellular agglutination, was routinely achieved(data not shown). The absolute amount of K99 pilus antigenproduced, however, fluctuated widely among experiments.This fluctuation was also found with a similar clone in whichthe Tac promoter (30) replaced the trp promoter (range, 0.37to 10.66% of total protein).We attribute this variation to the high copy number of

plasmid pUC9, which out-titrated the available repressormolecules, rendering suppression of the trp or Tac promoterdifficult. To circumvent this problem, new recombinantmolecules were constructed in which determinants respon-sible for DNA replication and copy number control areprovided by plasmid pBR322 or pDR540 instead of pUC9.This was achieved by transferring a BamHI-to-HindIII DNA

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2320 BAECKER ET AL.

NruXba Pat I

EcoR

pUC9-TrplSK |

1) Bgl 11; EcoR

Hind III SamH I

pDR54O Gal K PVU

Pvul

1) eamH I;

Hind III2) Phosphatse

Hind III Nru I

|, pKffiTrpK99 l

K 1

EcoR Hind IIIkB mH

pK99-64

BamHl

og1 11 1) Bgl 11; EcoR 1

I Sol IrLigate Nru

1< Xba \pt Hind IlI

BwmH l;Hlnd 111;

Ap/~

Pvul LA

BamHI

Hind 11 Bgl 1EcoR I

L BROTH

10

0

49

0.1

z0FizF

IClI--J

0.z

Ligate

+5

+3

+1

6

4

2

5 10 15 24TIME (hr)

L BROTH + IAA

5 10 15 24

FIG. 4. Construction of pKO-TrpK99. The hatched area ofpUC9-Trpl6K represents the structural gene for the K99 pilussubunit (16K gene) with the direction of transcription indicated. Therelative locations and directions of transcription are also shown forthe E. coli tryptophan operon promoter (PTrP) and the ,-lactamasegene (Apr). The stippled area of pK99-64 represents K99-derivedinsert DNA, and the remaining thin semicircular line representspBR322 DNA. The relative position and direction of transcription ofthe galactokinase gene (Gal K) are shown in diagrams of pDR540and pKO-TrpK99. The Tac promoter (PTac) in plasmid pDR540 iseliminated in the process of forming pKO-TrpK99. The relativepositions of selected restriction endonuclease cleavage sites areshown. The plasmid diagrams are not to scale.

fragment from pUC9-TrpK99 into pDR540 or pBR322 via thesame two restriction enzyme cleavage sites. The resultantplasmids were designated pKO-TrpK99 (Fig. 4) and pBR-TrpK99, respectively. Both of these plasmids produced highlevels of K99 pilus antigen in a consistent manner. Forunknown reasons, the pKO-TrpK99 derivative generallyoutproduced the pBR-TrpK99 clone.

Control of K99 pilus antigen expression by the tryptophanpromoter. To ascertain whether E. coli cells harboringplasmid pKO-TrpK99 produce the K99 pilus, cells werepropagated in LB medium with or without addition of IAA asan inducer. Whereas uninduced cells produced a low basallevel of the K99 pilus, cells grown in the presence of IAAproduced fourfold higher amounts of the antigen whenquantitated by ELISA (Fig. 5). This represents about 10-foldmore antigen than that which accumulates with plasmidsbearing the natural K99 promoter (i.e., pK99-64). One cansuppress the basal level of K99 antigen production bypKO-TrpK99 by adding tryptophan to the growth medium.

Further evidence for control of pilus antigen gene expres-sion by the trp operon was obtained by growing cells in M-9medium containing 2% glucose and various amounts oftryptophan. As expected, the results of agglutination assays(Fig. 6; Table 1) clearly show that inclusion of 25 to 100 ,ug

FIG. 5. Induction of K99 pilus expression in E. coli bearingplasmid pKO-TrpK99. (A) Growth curves of E. coli bearing plasmidpKO-TrpK99 when grown in L broth alone (left) or L broth plus 40pLg of IAA per ml (right). Growth curves are shown as semilogarith-mic plots of A550 versus time. (B) K99 pilus expression, as deter-mined by a cellular agglutination assay. Samples were taken at timepoints indicated in the upper panels and subjected to an agglutina-tion assay with polyclonal anti-K99 pilus antibody. The results areplotted versus the times at which the samples were taken. (C) K99pilus expression of E. coli cellular sonicates as determined byELISA. Samples were taken at the time points indicated in theupper panels and subjected to an ELISA. The results, expressed asnanograms of pilus protein per milliliter of culture (103), are plottedversus the times at which the samples were taken.

of tryptophan per ml repressed antigen expression. Degra-dation of tryptophan by tryptophanase, a catabolite-repress-ible enzyme (5), was minimized in this experiment by thehigh glucose concentration in the medium. Lower concen-trations of tryptophan (1 to 10 ,ug/ml) initially repressedantigen expression but later, as tryptophan was depleted bygrowth, led to positive cellular agglutination. These dere-pressed levels of protein expression produced some celllysis, as manifested by a dip in the growth curve.

Overproduction of the antigen created a selective pressureagainst cells harboring the pKO-TrpK99 plasmid, such thatby 24 h, in the absence of tryptophan supplements, only 11%of the cells remained ampicillin resistant. In contrast, 100%of the cells grown in the presence of 100 ,ug of tryptophan perml were ampicillin resistant at 24 h. The instability of theplasmid under induced conditions could be partially over-come by repeated additions of ampicillin.As an alternative to increase the selective pressure for

plasmid retention, the intracellularly active neomycin-kana-mycin resistance gene from transposon TnS (20) was incor-porated as a HindIII fragment into pKO-TrpK99. Trans-formants bearing the resultant plasmid, pKON-TrpK99,when grown and induced in M9 medium supplemented with

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HIGH-YIELD EXPRESSION OF K99 ANTIGEN 2321

10 0,1

25,50,100

5,10i

1.0

0.52

0.112 24

TIME (hr)

FIG. 6. Growth and repression of K99 pilus expression of E. colibearing plasmid pKO-TrpK99 in minimal medium by tryptophanaddition. Growth curves of E. coli bearing plasmid pKO-TrpK99when grown in M9 minimal medium alone or in minimal mediumplus various concentrations (micrograms per milliliter) of L-trypto-phan. Growth curves are shown as semilogarithmic plots of A550versus time.

TABLE 1. K99 pilus expressiona

Tryptophan Agglutination titer at time (h):concn(Lg/ml) 2 3 5 6 7 10 24

0 3 3 4 4 4 4 41 0 3 4 4 4 4 45 0 0 4 4 4 4 410 0 0 4 4 4 4 425 0 0 1 1 1 0 150 0 0 1 1 3 0 1100 0 0 1 1 1 0 1

a Samples of each culture were taken at the indicated times and subjectedto a cellular agglutination assay with polyclonal anti-K99 pilus antibody.

kanamycin, led to production of antigen levels similar tothose obtained with pKO-TrpK99 under favorable condi-tions when plasmid instability was not encountered (data notshown).

Purification and antigenicity testing of the K99 pilus anti-gen. Pilus antigen can readily be released from K99-positivebacteria by heat treatment. This characteristic was unalteredin E. coli cells harboring either pK99-64 or pKO-TrpK99(Fig. 7).To ascertain its utility as an immunogen, the recombinant

K99 pilus antigen was used in a vaccine formulation. Prep-aration of pilus antigen involved release by heat treatment,concentration by ammonium sulfate precipitation, and dial-ysis before injection into guinea pigs. The results of serum

immunogenicity assays indicate that K99 pilus antigen pro-duced by pKO-TrpK99 has a relative potency comparable toor greater than that of the standard K99 preparations.

200

-ILain

z

100

0

60 120

TIME (min)

180

FIG. 7. Release of K99 pilus antigen from cells by heat treat-ment. E. coli organisms bearing plasmid pKO-TrpK99 or pK99-64were collected by centrifugation after overnight growth underinducing conditions (L broth plus 40 ,ug of IAA per ml). Aftersuspension in 1/10 volume of 50mM sodium phosphate (pH 7.2)-2 Murea, the cells were subjected to heating at 65°C for the indicatedtimes. The amount of K99 pilus protein released was quantitated byELISA.

Similarly, in the presence of the other enterotoxigenic anti-gens K88ab, K88ac, and 987p (17, 24, 31), the vaccine withrecombinant K99 antigen outperformed a vaccine with thestandard K99 antigen. Specifically, the relative potency of a

monovalent K99 vaccine prepared in E. coli (pKO-TrpK99)was 2.0 with respect to that of the Animal and Plant HealthInspection Service K99 reference standard, and that of a

similarly prepared vaccine containing K99, K88ab, K88ac,and 987p antigens was 1.5 (see Materials and Methods forcalculation of relative potencies).

DISCUSSION

A common approach to increase heterologous proteinexpression in E. coli is to incorporate a strong and induciblepromoter upstream proximal to the gene of interest. WithK99, such an approach could be complicated by separationof the genes responsible for K99 pilus expression into twotranscriptional units. Moreover, within one of these operonsthe endogenous promoter is at least 1.2 kb removed from itsmost proximal identified gene, that which encodes the K99pilus subunit protein (H.W.C., unpublished data; 11).Our results indicate that in the presence of the exogenous

promoter, the 1.2-kb intervening sequence is clearly dispens-able. Substitution of this sequence with the trp promoter inthe presence of the remainder of the operon led to overpro-duction of the K99 pilus antigen. This was true with otherstrong exogenous promoters as well. When the Tac pro-moter was used in place of the trp promoter, K99 synthesiscould be brought about by addition of isopropyl-p-D-thioga-lactopyranoside (data not shown). Thus, we feel that theupstream 1.2-kb sequence represents a regulatory regionwhich modulates the level of K99 pilus expression ratherthan a coding region.Kehoe et al. (19) had previously examined the effect of

using the trp promoter to direct K88ac pilus antigen expres-sion. Surprisingly, cells harboring the TrpK88ac hybridplasmid expressed high levels of K88 antigen only when thetrp promoter was repressed. Likewise, cell growth and pilusproduction were decreased when the trp promoter was

derepressed. This phenomenon was attributed to the delete-rious effect of excess membrane protein accumulation.

In contrast, our results clearly demonstrated overproduc-tion of the K99 pilus antigen on the surface of cells under

- ~~~~~~~pKOTrpK99

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control of the inducible trp promoter. We believe the dis-crepancy lies in the differences in genetic organizationbetween the two pilus operons. In the K88ac operon, thepilus subunit gene is located within the second cistron, distalto and independent of the exogenously introduced trp pro-moter (19). Consequently, induction of the trp promoterresulted in enhanced synthesis of other membrane proteinsin the absence of enhanced expression of the K88 pilussubunit. Within the K99 operon, the pilus subunit gene isimmediately proximal to its upstream promoter (natural orexogenously introduced). In this situation, successful coor-dination of gene expression within the K99 operon is appar-ently more easily achieved.

Overproduction of the K99 antigen nonetheless createdselective pressure against cells harboring the pKO-TrpK99plasmid. After overnight growth, a majority of the cells hadlost the recombinant plasmid. Such plasmid segregation wasavoided by substitution of kanamycin resistance for ampicil-lin resistance in plasmid pKON-TrpK99. In this case, theintracellularly active aminoglycoside 3'-phosphotransferaseapparently led to stronger selective pressure for plasmidretention than did the secreted ampicillinase.The immunogenicity assay results in guinea pigs demon-

strated that K99 pilus antigen derived from pKO-TrpK99compares favorably with the Animal and Plant Health In-spection Service standard K99 preparation. This was thecase regardless of whether our K99 pilus antigen was formu-lated either in a monovalent vaccine or in combination withother antigens. Since equal amounts of pilus protein wereincorporated into the vaccines, similar titers should havebeen obtained. We have no explanation for the apparentgreater immunogenicity of the pKO-TrpK99-derived anti-gen.

Aside from the high yield of K99 antigen produced by cellsbearing plasmid pKO-TrpK99 or its derivatives, there areother advantages to using this source of antigen. The natu-rally occurring K99 pilus subunit promoter has been re-ported to be sensitive to alanine repression and to betemperature sensitive (12). Substitution of the trp promotereliminates these influences. We have observed at low fre-quency genetic inversion events in plasmid pK99-64 deriva-tives which invert the gene coding for the pilus subunitrelative to the remainder of the plasmid (H. W. Chan et al.,manuscript in preparation). One of the junction sites for thisgenetic inversion is located between the natural promoterand the gene encoding the K99 pilus subunit. We do notunderstand whether this is a mode by which the operonregulates its own expression under different physiologicalconditions or whether this is a mechanism for evading a hostimmune response. Nonetheless, substitution of the trp pro-moter for the natural promoter deletes this junction site, andthus pKO-TrpK99 and its derivatives should be stabilizedagainst such genetic rearrangements. For these reasons,cells bearing plasmid pKO-TrpK99 should be a more consis-tent and predictable source of K99 antigen.Although synthesized intracellularly, the K99 pilus sub-

unit is eventually transported through the inner and outermembranes and assembled into the mature extracellularpilus. Genetic and biochemical analyses of several operonsresponsible for pilus assembly in E. coli have led to modelsfor this process (2, 13, 14). It appears that numerous pro-teins, both soluble and membrane bound, are responsible fortransport, processing, anchoring, and assembly of the pilusprotein subunit into the mature extracellular structure. Inthis regard, it is noteworthy that production of the K99 pilussubunit by itself, as in plasmid pUC9-Trpl6K, was not high

and frequently led to cell lysis. A more detailed analysis ofcellular and plasmid-encoded factors will be required toelucidate which steps limit simple overproduction of thepilus subunit itself.

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