INFECTION AND IMMUNITY, Apr. 1985, p. 73-770019-9567/85/040073-05$02.00/0Copyright X3 1985, American Society for Microbiology

Nucleotide Sequence Comparison Between Heat-Labile Toxin B-Subunit Cistrons from Escherichia coli of Human and Porcine Origin

JENNY LEONG,' ANDREW C. VINAL,2 AND WALTER S. DALLAS'*The Wellcome Research Laboratories, Burroughs Wellcome Cc., Research Triangle Park, North Carolina 27709,1 and

Department of Microbiology, North Carolina State University, Raleigh, North Carolina 276072

Received 21 September 1984/Accepted 7 January 1985

The nucleotide sequence of the LT-BH cistron (eltBH) from an enterotoxigenic Escherichia coli straininfectious for humans was determined and compared with the LT-B cistron sequence from a porcine E. coliisolate. Both cistrons were shown to comprise 375 nucleotide base pairs, and discrepancies were detected ateight positions. Of the nonhomologous base pairs, six resulted in codon changes that would lead to amino acidvariations. The nucleotide sequence distal to both LT-B cistrons was also determined, and only threedifferences were detected in 197 base pairs. An HhaI site unique to eltBH was shown to be present in all theheat-labile (LT) genes from 31 human isolates surveyed, whereas the restriction enzyme recognition site wasabsent in the gene from 46 porcine E. coli isolates. The results suggest that two genetically discernable LTgroups are identifiable and that the groups are also distinguishable by the isolation source (human or porcine)of the infecting E. coli strains.

Toxins are obligatory virulence factors expressed by somestrains of Escherichia coli that cause a diarrheal disease inhumans and in the young of some farm animals (calves,lambs, and piglets) (38). Two general classes of toxin aremade by these pathogens. The more heterogeneous heat-sta-ble toxin group includes biologically and genetically distincttoxins (1, 23, 29, 34, 39). In contrast, the heat-labile toxin(LT) group is quite homogeneous with regard to genestructure, DNA sequence (4, 28, 42), and biochemical prop-erties (14, 15, 17, 19). Two dissimilar proteins constitute LT(3, 6, 21). The larger subunit is designated LT-A and hasbeen shown to catalyze the adenosine diphosphate-ribosyla-tion of a membrane protein that is intimately involved in theregulation of adenylate cyclase in eucaryotes (16, 31). Fivemonomers of the smaller B-subunit (LT-B) are present in theholotoxin and serve to bind LT to the membrane componentGM1 (18, 30). LT-B is the immunodominant component ofthe toxin.

Observations of immunological and biochemical differ-ences among LTs purified from E. coli strains infectious fordifferent hosts (human and porcine isolates) (2, 14, 19)prompted the investigation of the genes coding for twodistinguishable LTs. The organization of the two genes wasshown to be identical, but it was suggested that the LT-Bcistron from a human E. coli isolate (eltBH) might have a fewmore codons than an LT-B cistron from a porcine E. coliisolate (eltBp) (4). In this communication, we report thecomplete nucleotide sequence of eltBH and compare it withthat of eltBp. Also, we have noted the presence of a

restriction enzyme recognition site (HhaI) unique to eltBHand have screened the LT gene in 77 E. coli isolates for thepresence of this site.

MATERIALS AND METHODS

Bacterial strains and plasmids. Clinical isolates were pro-vided by R. A. Finkelstein, S. L. Moseley, and H. W. Moon(13). E. coli JM103 was used in all plasmid manipulationexperiments (27). The plasmid EWD299 carries the LTPgene (LT from a porcine isolate) and confers ampicillin

* Corresponding author.

resistance (7), whereas the plasmid pWD600 carries the LTHgene (LT from a human isolate) and confers tetracyclineresistance (4). The plasmid pWD602 is a sibling of pWD600in which the 5,450-base-pair (bp) PstI DNA fragment isinserted into pBR322 in the opposite orientation. The plasmidpUC8 confers ampicillin resistance and was the vector intowhich DNA fragments were inserted for DNA sequence

determination (40).DNA sequence determination. Isolation of plasmid has

already been described (4). All enzymes were purchasedfrom Bethesda Research Laboratories, Gaithersburg, Md.,and restriction enzyme and ligase reaction conditions wereas described by O'Farrell et al. (32). For cloning DNAfragments into pUC8, plasmids were digested with theappropriate enzyme, heat inactivated (70°C for 15 min), thenmixed together in a reaction volume of 10 ,ul (0.2 pug ofpUC8; 0.6 ,ug of EWD299 or pWD602). (Since EWD299 alsoconfers resistance to ampicillin, the ,-lactamase gene wasfirst inactivated by cutting the plasmid with PstI and treatingit with Klenow fragment in the presence of all four deoxynu-cleotides before cleavage with HindIII.) The ligation mixturewas used to transform JM103 (22), and ampicillin-resistant,white colonies were identified. Mini-preparations of plas-mids from these colonies were analyzed for inserts intopUC8, and the insert orientation was determined (20). Thesequencing strategy followed was the one proposed byRuther et al. (36). By using both 5'-(T4 polynucleotidekinase) and 3'-(Klenow fragment) end labeling techniques,we determined the entire nucleotide sequence of both DNAstrands by the method of Maxam and Gilbert (26).DNA hybridization. Crude plasmid preparations were iso-

lated from 1.5-ml cultures of clinical enterotoxigenic E. coliisolates by the procedure of Portnoy et al. (35). One-quarterof each sample was cut with HhaI, and the resulting DNAfragments were separated on 1.4% agarose gels. Southerntransfers were done as outlined by Davis et al. (9). A 770-bpHindIll DNA fragment from EWD299 was used as a hybrid-ization probe. The DNA fragment was isolated by themethod of Lizardi et al. (25) and radiolabeled by the protocoldescribed by Feinberg and Vogelstein (12).

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74 LEONG, VINAL, AND DALLAS

ASma I

pWD196

FIG. 1. Schematic diagram showing the construction of plasmidsused for DNA sequence determination. The heavy line denotes the5,450-bp eltH PstI DNA fragment inserted into pBR322. The smallbar approximates the location of eltBH. An EcoRI fragment frompWD602 was inserted into pUC8, forming pWD195. The smallerSmaI fragment from pWD195 was deleted, forming pWD196. A771-bp HindIII fragment was inserted into pUC8 to form pJL9.Abbreviations: A, AccI; B, BamHI; E, EcoRI; H, HindIll; P, PstI;S, SmaI; Ss, SstI; X, XbaI.

RESULTS

DNA sequencing. The molecular cloning of elt from an E.coli infectious for humans has already been described (4).The recombinant plasmid pWD600 was isolated and shownto comprise a 5,450-bp DNA fragment that had been insertedinto the PstI site of pBR322. The location of eltBH within thePstI DNA fragment had been determined, and we wereconfident that it mapped to the region (582 bp in size)demarcated by an EcoRI site and a HindIll site that alsoenclosed a unique SmaI site (Fig. 1).The plasmid pUC8 developed by Vieira and Messing (40)

was used to clone DNA fragments for sequence determina-tion. The strategy used is shown in Fig. 1. An EcoRIfragment from pWD602 (a sibling of pWD600 in which thePstI DNA fragment was inserted into pBR322 in the oppo-

site orientation) was cloned into pUC8 to create pWD195.With the use of this plasmid the DNA sequence was deter-mined, starting from the EcoRI site and proceeding throughthe SmaI site (240 bp). To extend the sequence, anotherplasmid was derived. This plasmid, pWD196, was isolated as

a SmaI deletion of pWD195. With the use of pWD196 theremaining portion of the eltBH sequence was determined.We were also interested in determining the DNA sequence

3' to eltBH. This was accomplished by cloning a 771-bpHindIII fragment from pWD602 into pUC8 (Fig. 1). Theresultant plasmid, pJL9, was used to determine the 3'flanking DNA sequence of eltBH. In a similar fashion, a

771-bp HindlIl fragment from EWD299 was also cloned intopUC8, forming pJL3, and this plasmid enabled us to deter-mine the DNA sequence 3' to eltBp.

Like eltBp, eltBH was found to be 375 bp in size, startingfrom the initiation codon ATG through the termination

codon TAG (Fig. 2). After reexamination of the sequencinggels for eltBp and completion of additional sequencing, wediscovered three errors in the published sequence. Codon 7was found to be GAA (instead of CAA as published), codon43 was found to be AAG (instead of ATG), and codon 89 wasfound to be AAT (instead of AAC). The only amino acidaffected is that for codon 43, which is lysine (not methio-nine). Eight nucleotide differences between eltBH and eltBpwere detected. Of the disparate bases, two would be silent,having no effect on the resulting amino acid sequence. Theseare at codons 2 (CCT and CCC are both proline codons) and61 (CAA and CAG are both glutamine codons). The other sixdifferences would be expected to result in amino acidvariations. LT-BP has been shown to be synthesized as aprecursor with an N-terminal extension of 21 amino acids (5,33). This leader peptide directs the protein through mem-branes and is cleaved from the polypeptide, releasing themature protein. Two of the predicted amino acid variationsare in the leader peptide (codons -16 and -4) and would notbe a part of the mature protein. In eltBH the phenylalaninecodon TTT is at position -16 and the cysteine codon TGT isat position -4. In the LT-BP cistron, TGT is at position -16and the tyrosine codon TAT occupies position -4. Two ofthe amino acid differences are near the amino terminus of themature protein. Codon 4 is serine (TCT) in eltBH andthreonine (ACT) in eltBp, and codon 13 is histidine (CAC) ineltBH and arginine (CGC) in eltBp. The next differenceoccurs near the middle of the coding region at codon 46,which is GCA (alanine) for eltBH and GAA (glutamate) foreltBp. The final difference occurs at the penultimate codon,102. A glutamate codon (GAA) is present in eltBH, and alysine codon (AAA) occupies this position in eltBp. Whenthe nucleotide sequences distal to eltB were compared (197bp), three nucleotide variations were found: loci 489, 552,and 577 (Fig. 2).

Restriction enzyme site polymorphisms. A consequence ofnucleotide sequence variation can be the loss or acquisitionof restriction enzyme recognition sites. Three additionalrecognition sequences were found to be present in eltBHthan were in eltBp: an HgiAI site spanning codons -4, -3,and -2, a DdeI site at codons 2 and 3, and an HhaI site atcodons 45 and 46. When the nucleotide sequence of theentire LTP gene was analyzed for restriction enzyme recog-nition sites, six DdeI sites and three HgiAI sites werelocated but no HhaI sites were present. We were interestedin determining whether an HhaI site in eltBu might becharacteristic of human LT' strains. Therefore, LT genesfrom E. coli isolates were screened for HhaI sites bySouthern blot analysis.

Plasmids were isolated from 77 LT' isolates and cut withHhaI. The DNA fragments were separated by electropho-resis through 1.4% agarose gels, and Southern transferswere prepared. A 771-bp HindlIl fragment (Fig. 1) thatincluded all of eltBp, 197 bp distal to eltBp, and the terminal69 codons of eltAp was used as a hybridization probe. Anexample of the results obtained from this analysis is shownin Fig. 3. The plasmid preparations in the lanes marked Pwere from porcine E. coli isolates and showed only one bandof hybridization. In contrast, the other preparations werefrom human E. coli isolates and each had two HhaI DNAfragments hybridizing with the probe. Two lanes (6 and 9)show an extra faint hybridizing band which was subse-quently shown to be a partial digestion product. Each of the46 porcine preparations analyzed showed one band of hy-bridization (no HhaI site within the probe hybridizing se-quence), whereats all 31 human isolates were found to have

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LT-BH CISTRON SEQUENCE 75

EcoRI GG A A T T C G G G A T G A A T T ATG AAT AAA GTA AAA TTT TAT GTT TTA TTT

Met- Asn - Lys- Val- Lys- Ph.- Tyr- Val- Lou- Ph.-20

A HgiAI C DdeI AACG GCG TTA CTA TCC TCT CTA TQT GC& r C GGA GCT CZL_SAA TCTThr - Ala- Lou - Lou- Smr- Sm - Lou-Cys - Ala- His - Gly- Ala- Pro - Gin- Sr

-10 1G

ATT ACA *GAA CTA TGT TCG GAA TAT CAC AAC ACA CAA ATA TAT ACGIIb - Thw- Glu- Lou- Cys- Ser - Glu- Tyr - His- Ase- Thr - Gln- II* - Tyr - Thr

10

ATA AAT GAC AAG ATA CTA TCA TAT ACG GAA TCG ATG GCA GGC AAAII - Asn- Asp- Lys - Ib- Lou- Ser Tyr - Thr- Glu- Ser Met- Ala- ay- Lys20 30

* HhaI AAGA GAA ATG GTT ATC ATT ACA TTT AAG AGC GGC GCA ACA TTT CAGArg- Glu- Met Vol IIb- II - Thr- Ph.- Lys- Sw - Gly- Ala- Thr Ph- Gin

40Sma I G

GTC GAA GTCCCG AGT CAA CAT ATA GAC TCC CAA AAA AAA GCCVald Gu - Val Pro - Gly- Sr- Gln- His- II Asp- Ser- Gln Lys Lys- Ala50 60

ATT GAA AGG ATG AAG GAC ACA TTA AGA ATC ACA TAT CTG ACC GAGIle- Glu - Arg- Met- Lys -Asp - Thr Lou - Arg- II- Thr - Tyr - Lou - Thr - Glu

70

ACC AAA AU GAT AAA TTA TGT GTA TGG AAT AAT AAA ACC CCC AATThr - Lys - Ile - Asp - Lys - Lou - Cys Val - Trp - Asn - Asn - Lys - Thr - Pro - Asns0 90

ATCA ATT GCG GCA ATC AGT ATG GAA AAC TAG TT T GCT T T #AAAGCAS-- Ile - Ala- Ala- II- Setr - Mt Glu Asn Amber 400

100

T GT CT AAT GCt AGG9AACCT AT AT AACAACT ACT GT ACTT AT AC420 440

GT AAT GAGCC T T AT GC T GC ATTtGAAAAGGC GTAGAGGATGCA

440 460

A T A C C G A T C C T T A A A C T G T A A C A C T A T A A C A G C T T C C A C T AC AS6o 520

C AGGGAG TGTTATAGCAAACAGAAAA AACTAAGCTAGGCTGGGG

5AO 560

G9GC AA GC T T580

FIG. 2. Nucleotide sequence of eltBH. Assignment of the initiation codon (-21) and N terminus was based on homology to eltBp (5) andLT-BP (3). Single nucleotides above the main string of nucleotides indicate positions which differ between eltBH and eltBp. The * indicatespositions in eltBp that were in error in the original publication (5). Underlined is a long inverted repeat first described by Yamamoto andYokota (42) and suggested to be a transcription termination signal.

two probe-positive bands, indicating the presence of anHhaI site within the LTH gene.

DISCUSSIONGeary et al. compared properties of two purified LTs and

discovered both biochemical and immunological differences(14). So that we might begin to understand the nature andsignificance of the differences, we have deduced the primarystructure of the immunodominant subunit of the toxin,LT-B, for both LTs. In this communication we present theprimary structure of one of the polypeptides, LT-BH, asdeduced from the nucleotide sequence of its gene andcompare it with the primary amino acid sequence of anotherB-subunit that one of us had previously reported, also byDNA sequencing (5). The eltBs were both 375 bp in size,coding for polypeptides 124 amino acids in length. Eightnucleotide differences between the two cistrons were de-tected (Fig. 2). Two of the differences are in the third orwobble codon position and do not affect the amino acidinserted into the growing polypeptide chain (codons 2 and61). Two of the changes that would alter the incorporatedamino acid occur in the leader peptide and would have noinfluence on the properties of the mature protein. Themature LT-Bs are predicted to have different amino acids at

four positions: 4, 13, 46, and 102. Each of the amino acidvariations between the two proteins would result from asingle base-pair change per codon, with both transitions andtransversions occurring. Although the compositional molec-ular weight of both proteins is nearly identical (LT-BP is 90daltons larger), the difference in the migration of the twoproteins in sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis suggested that LT-BH was larger by 1,500daltohs. Obviously, the four amino acid variations influencethe migration of the protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis to an extent that isdisproportionate to the mass of the four amino acids. Theinfluence of amino acid substitutions on protein mobility insodium dodecyl sulfate-polyacrylamide gel electrophoresishad been noted in mammalian a-crystallin A chains (10).Immunological investigations would also predict that theseamino acids are fundamental in defining the immunodomi-nant epitopes of the proteins.Our analysis of the structure and part of the nucleotide

sequence for two LT genes is consistent with the perspectivethat LTs form a homogeneous group of toxins and corre-sponding toxin genes. The work of Yamamoto and Yokotaon an LT gene from a different human E. coli isolatesupports this view (42). In contrast, the other diarrhea-in-

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76 LEONG, VINAL, AND DALLAS

TABLE 1. Comparison of the nucleotide sequences from three LT-B cistrons and flanking basesCodon no. Nucleotide no.

Source-16 -4 -2 2 4 13 46 61 102 9 489 552 577 581

Porcinea TGT TAT CAC CCC ACT CGC GAA CAG AAA G G C A GHumanb TGT TGT TAC CCC TCT CGC GCA CAA GAA A T C G XdHumanc TTT TGT CAC CCT TCT CAC GCA CAA GAA G T A G G

a P307 isolated in Great Britian.b H10407 isolated in Bangladesh.' H74-114 isolated in the United States.d Nucleotide absent at this position.

ducing toxin made by E. coli, hepolypeptides exhibiting a broader rierties and gene nucleotide sequencDNA sequences for the three LTTable 1. Nucleotide differences arecistron from H10407 (42) has thenucleotides (one in codon -2) and tas a progenitor for eltBs. The poquence differs from this at five pohuman-derived strain carries An Lthree loci. Evolutionarily, the threein the simplest manner as diverging(most similar to the LTH gene frcmulation of point mutations. The pin amino acid variations, which in lable epitopic differences.The two human isolate LT-BH

GCA (alanine) at position 46, wherethe codon GAA (glutamate) at thispart of the recognition site (GCenzyme HhaI which is present in t

(Fig. 2). We surveyed a geographichuman and porcine LT' isolates foisite within elt. All 31 human isolatean HhaI site within elt, whereasisolates had this particular recogibasis of these data, it is tempting tsite is a genetic marker for eltH. Tthe presence of the GCA codon at

I ,, B

IHP PP

2.0 Kb -- t.10

FIG. 3. Southern transfer of HhaI-rdiagram at the top depicts the HindIIhybridization orobe for elt. The relatiN

at-stable toxin, includes LT-BH cistrons but are consistent with this interpretation.ange of biochemical prop- An intriguing possibility suggested by these data is that elts.es. A comparison of the exist as two segregated classes (19). One class only occurs in7-B cistrons is shown in strains infectious for humans (has an HhaI site), whereas thehighlighted. The LT-BH other is found only in E. coli infecting piglets. That therefewest uniquely different should be two segregated classes of LT genes is a curious;herefore might be viewed situation. In both human and porcine isolates, elt is carried)rcine LT-BP cistron se- on plasmids. Indeed, there have been no reports of a)sitions, whereas another chromosomal locus for elt. Furthermore, the plasmids have.T-BH cistron varying at been demonstrated to be transmissible (37). Why then has acistrons might be viewed human Ent plasmid not been found in porcine pathogenic E.from a common ancestor coli and vice versa? There exist in the literature documented)m H10407) by the accu- cases of the transfer of plasmids in situ (reviewed in refer-)oint mutations can result ence 11) as well as from animal-commensal E. coli toturn may result in detect- human-colonizing E. coli (24). One plausible explanation

would be incompatibility between a human (porcine) Entcistrons share the codon plasmid and a porcine (human) plasmid carrying a coloniza-,as the LT-BP cistron has tion factor. Such a situation would exclude one obligatorys position. GCA is also a virulence factor, rendering the strain avirulent. AnothertGC) for the restriction possibility is that interchange between human- and piglet-in-eltBH and absent in eltBp fecting strains is a rare event and we have not exanlined a,ally diverse collection of sufficiently large population. However, our E. coli collectionr the presence of an HhaI represents both chronologically and geographically diverse6is were shown to possess strains. It could prove informative to examine both humannone of the 46 porcine and porcine LT' strains isolated in the same locale to

nition sequence. On the determine whether transfer of Ent occurs between humano speculate that the HhaI and porcine strains.hese data do not confirm Interestingly, when the sizes of the hybridizing HhaIposition 46 in all human fragments are compared among the porcine plasmids, 45 out

of 46 are, as far as we can discern, identical (2 kilobases).The lone exception is smaller by a few hundred base pairsand could therefore have resulted from a deletion eventwithin the 2-kilobase fragment. This result suggests that~~~~ ~~

H DNA immediately flanking eltp is the same and may indicatea significant homology among porcine E. coli Ent plasmids,possibly even the same incompatibility group. We do knowthat the plasmids are not identical, since different size

JiPa~ * w- 1.7 Kb classes of Ent plasmids are represented (data not shown). In

1.7 Kb contrast, a variety of sizes of hybridizing DNA fragmentsare represented in HhaI digests of the human E. coli Entplasmids. We have also found these Ent plasmids to be

* 1.-i.i Kb heterogeneous in size (data not shown). The results are__*consistent with the possibility that eltH may be a part of or

associated with a transposable element, as first suggested by--.8 Kb Yamamoto and Yokota (41). At present we have no expla-

nation of why there appear to be two segregated classes of*estricted Ent plasmids. The LT genes, but the data presented in this communication are

ve locations of eltA (A) and consistent with this speculation.*-} ....... F.-- .,J 11-9 * A .2% II IsCI Ist'v% &_ IIWsascJ*:II X k"IXwsIM

eltB (B) are shown. The arrow indicates the position of the Hhalsite. Samples in lanes marked P were of porcine isolation, andsamples in all other lanes were from E. coli of human origin.Estimated molecular weights of some hybridizing DNA fragmentsare shown. The * denotes the 5' end of elt.

LITERATURE CITED

1. Burgess, N. M., R. J. Bywater, C. M. Cowley, N. A. Mullan, andP. M. Newsome. 1978. Biological evaluation of a methanol-sol-

INFECT. IMMUN.

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om h

ttps:

//jou

rnal

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15 D

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2021

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188.

75.1

43.9

8.

LT-BH CISTRON SEQUENCE 77

uble, heat-stable Escherichia coli enterotoxin in infant mice,pigs, rabbits, and calves. Infect. Immun. 21:526-531.

2. Clements, J. D., D. C. Flint, and F. A. Klipstein. 1982. Immu-nological and physicochemical characterization of heat-labileenterotoxins isolated from two strains of Escherichia ceoli.Infect. Immun. 38:806-809.

3. Clements, J. D., R. J. Yancey, and R. A. Finkelstein. 1980.Properties of homogeneous heat-labile enterotoxin from Esch-erichija coli. Infect. Immun. 29:91-97.

4. Dallas, W. S. 1983. Conformity between heat-labile toxin genesfrom human and porcine enterotoxigenic Escherichia coli. In-fect. Immun. 40:647-652.

5. Dallas, W. S., and S. Falkow. 1980. Amino acid sequencehomology between cholera toxin and Escherichia coli heat-labile toxin. Nature (London) 288:499-501.

6. Dallas, W. S., and S. Falkow. 1979. The molecular nature ofheat-labile enterotoxin (LT) of Escheric/hia coli. Nature (Lon-don) 277:406-407.

7. Dallas, W. S., D. M. Gill, and S. Falkow. 1979. Cistronsencoding Escherichiia c oli heat-labile toxin. J. Bacteriol.139:850-858.

8. Dallas, W. S., S. Moseley, and S. Falkow. 1979. The character-ization of an Escherichia coli plasmid determinant that encodesfor the production of a heat-labile enterotoxin, p. 113-122. InK. N. Timmis and A. Puhler (ed.), Plasmids of medical, envi-ronmental, and commercial importance. Elsevier/North-Holland Publishing Co., Amsterdam.

9. Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advancedbacterial genetics: a manual for genetic engineering. p. 159-179.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

10. de Jong, W. W., A. Zweers, and L. H. Cohen. 1978. Influence ofsingle amino acid substitutions on electrophoretic mobility ofsodium dodecyl sulfate-protein complexes. Biochem. Biophys.Res. Commun. 82:532-539.

11. Falkow, S. 1975. Infectious multiple drug resistance. Pion Ltd.,London.

12. Feinberg, A. P., and B. Vogelstein. 1983. A technique forradiolabeling DNA restriction endonuclease fragments to highspecific activity. Anal. Biochem. 132:6-13.

13. Finkelstein, R. A., Z. Yang, S. L. Moseley, and H. W. Moon.1983. Rapid latex particle agglutination test for Escherichia c olistrains of porcine origin producing heat-labile enterotoxin. J.Clin. Microbiol. 18:1417-1418.

14. Geary, S. J., B. A. Marchlewicz, and R. A. Finkelstein. 1982.Comparison of heat-labile enterotoxins from porcine and humanstrains of Escherichia coli. Infect. Immun. 36:215-220.

15. Gill, D. M., J. D. Clements, D. C. Robertson, and R. A.Finkelstein. 1981. Subunit number and arrangement in Escheri-chia coli heat-labile enterotoxin. Infect. Immun. 33:677-682.

16. Gill, D. M., and S. H. Richardson. 1980. Adenosine diphos-phate-ribosylation of adenylate cyclase catalyzed by heat-labileenterotoxin of Escherichia coli: comparison with cholera toxin.J. Infect. Dis. 141:64-70.

17. Gyles, C. L., and D. A. Barnum. 1969. A heat-labile enterotoxinfrom strains of Escherichia coli enteropathogenic for pigs. J.Infect. Dis. 120:419-426.

18. Holmgren, J. 1973. Comparison of the tissue receptors forVibrio c/zolerae and Escherichia coli enterotoxins by means ofgangliosides and natural cholera toxoid. Infect. Immun.8:851-859.

19. Honda, T., T. Tsuji, Y. Takeda, and T. Miwatani. 1981. Immu-nological nonidentity of heat-labile enterotoxins from humanand porcine enterotoxigenic Escherichia co/i. Infect. Immun.34:337-340.

20. Ish-Horowicz, D., and J. F. Burke. 1981. Rapid and efficientcosmid cloning. Nucleic Acids Res. 9:2989-2998.

21. Kunkel, S. L., and D. C. Robertson. 1979. Purification andchemical characterization of heat-labile enterotoxin producedby enterotoxigenic Escheric/hia coli. Infect. Immun. 25:586-596.

22. Lederberg, E. M., and S. N. Cohen. 1974. Transformation ofSa/lnonella tvphiinnuriim by plasmid deoxyribonucleic acid. J.

Bacteriol. 119:1072-1074.23. Lee, C. H., S. L. Moseley, H. W. Moon, S. C. Whipp, C. L.

Gyles, and M. So. 1983. Characterization of the gene encodingheat-stable toxin 11 and preliminary molecular epidemiologicalstudies of enterotoxigenic Escherichia coli heat-stable toxin 11producers. Infect. Immun. 42:264-268.

24. Levy, S. B., G. B. FitzGerald, and A. B. Macone. 1976. Spreadof antibiotic-resistant plasmids from chicken to chicken andfrom chicken to man. Nature (London) 260:40-42.

25. Lizardi, P. M., R. Binder, and S. A. Short. 1984. Preparativeisolation of DNA and biologically active mRNA from DEAE-membrane. Gene Anal. Tech. 1:22-28.

26. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labeledDNA with base-specific chemical cleavages. Methods Enzymol.65:499-560.

27. Messing, J., R. Crea, and P. H. Seeburg. 1981. A system forshotgun DNA sequencing. Nucleic Acids Res. 9:309-321.

28. Moseley, S. L., P. Echeverria, J. Seriwatana, C. Tirapat, W.Chaicumpa, T. Sakuldaipeara, and S. Falkow. 1982. Identifica-tion of enterotoxigenic Esceherichia coli by colony hybridizationusing three enterotoxin gene probes. J. Infect. Dis. 145:863-869.

29. Moseley, S. L., J. W. Hardy, M. 1. Huq, P. Echeverria, and S.Falkow. 1983. Isolation and nucleotide sequence determinationof a gene encoding a heat-stable enterotoxin of Escherichia coli.Infect. Immun. 39:1167-1174.

30. Moss, J., J. C. Osborne, Jr., P. H. Fishman, S. Nakaya, andD. C. Robertson. 1981. Escherichia coli heat-labile enterotoxin.Ganglioside specificity and ADP-ribosyltransferase activity. J.Biol. Chem. 256:12861-12865.

31. Moss, J., and S. H. Richardson. 1978. Activation of adenylatecyclase by heat-labile Escherichia co/i enterotoxin. Evidencefor ADP-ribosyltransferase activity similar to that of choler-agen. J. Clin. Invest. 62:281-285.

32. O'Farrell, P. H., E. Kutter, and M. Nakanishi. 1980. A restric-tion map of the bacteriophage T4 genome. Mol. Gen. Genet.179:421-435.

33. Palva, E. T., T. R. Hirst, S. J. S. Hardy, J. Holmgren, and L.Randall. 1981. Synthesis of a precursor to the B subunit ofheat-labile enterotoxin in Escherichia coli. J. Bacteriol. 146:325-330.

34. Picken, R. N., A. J. Mazaitis, W. K. Maas, M. Rey, and H.Heyneker. 1983. Nucleotide sequence of the gene for heat-stableenterotoxin II of Escherichia coli. Infect. Immun. 42:269-275.

35. Portnoy, D. A., S. L. Moseley, and S. Falkow. 1981. Character-ization of plasmids and plasmid-associated determinants ofYersinia enterocolitica pathogenesis. Infect. Immun. 31:775-782.

36. Ruther, U., M. Koenen, K. Otto, and B. Muller-Hill. 1981.pUR222. a vector for cloning and rapid chemical sequencing ofDNA. Nucleic Acids Res. 9:4087-4098.

37. Smith, H. W., and S. Halls. 1968. The transmissible nature ofthe genetic factor in Escherichia coli that controls enterotoxinproduction. J. Gen. Microbiol. 52:319-334.

38. Smith, H. W., and M. A. Linggood. 1971. Observations on thepathogenic properties of the K88, Hly and Ent plasmids ofEscherichia coli with particular reference to porcine diarrhoea.J. Med. Microbiol. 4:467-485.

39. So, M., and B. J. McCarthy. 1980. Nucleotide sequence ofbacterial transposon Tn1681 encoding a heat-stable (ST) toxinand its identification in enterotoxigenic Escherichia coli strains.Proc. Natl. Acad. Sci. U.S.A. 77:4011-4015.

40. Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing withsynthetic universal primers. Gene 19:259-268.

41. Yamamoto, T., and T. Yokota. 1981. Escherichia coli heat-labileenterotoxin genes are flanked by repeated deoxyribonucleicacid sequences. J. Bacteriol. 145:850-860.

42. Yamamoto, T., and T. Yokota. 1983. Sequence of heat-labileenterotoxin of Escherichia coli pathogenic for humans. J. Bac-teriol. 155:728-733.

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