Vol. 169, No. 3

Evolutionary Origin of Pathogenic Determinants in EnterotoxigenicEscherichia coli and Vibrio cholerae 01

TATSUO YAMAMOTO,'* TAKASHI GOJOBORI,2 AND TAKESHI YOKOTA1

Department of Bacteriology, School of Medicine, Juntendo University, Bunkyo-ku, Tokyo, Japan,1 and National Instituteof Genetics, Mishima, Shizuoka, Japan2

Received 14 October 1986/Accepted 12 December 1986

Three families of the evolutionarily related pathogenic determinants in enterotoxigenic Escherichia coli andVibrio cholerae 01, a family of cholera enterotoxin (CT) and heat-labile enterotoxin (LT) including CT, LTh,and LTp, a family of heat-stable enterotoxin I (STI) including STIa and STIb, and a family of K88enteroadhesion fimbriae including K88ab, K88ac, and K88ad were analyzed for synonymous (silent) nucleotidesubstitutions by using the gene nucleotide sequences of earlier reports and the LTp gene nucleotide sequencepresented in this paper. The data suggested that the divergences between LT and CT and between STIa andSTIb occurred in the remote past, whereas those betwen LTh and LTp and between members of the K88 familyoccurred very recently. We concluded that the LT gene is a foreign gene that has been acquired by E. coli toform an enteropathogen. This provides evolutionary evidence of species-to-species transfer of pathogenicdeterminants in procaryotes.

Enterotoxigenic Escherichia coli causes choleralike diar-rhea in humans and food animals. It colonizes the intestinalepithelium with fimbrial adhesins and produces enterotoxinssuch as heat-labile toxin (LT) and heat-stable toxin I (STI)(46). Human-colonizing enterotoxigenic E. coli producesfimbriae named (e.g.) CFA, and swine-colonizing enterotox-igenic E. coli produces fimbriae named (e.g.) K88 (11, 30).The K88 family includes K88ab, K88ac, and K88ad havingdifferent epitopes. LT has structural and functional featuresin common with cholera enterotoxin (CT) produced fromVibrio cholerae 01, a causative agent of cholera, and issubdivided into two (LTp and LTh) having differentepitopes; LTp is found only in porcine isolates of enterotox-igenic E. coli, whereas LTh is found only in human isolates(6, 13, 14, 18, 45, 55). STI also has two members, one (STIa)being produced from both porcine and human isolates ofenterotoxigenic E. coli and another (STIb) being producedonly from human isolates (1, 3, 42, 48, 52). The virulencegenes for LT, STI, and K88 of enterotoxigenic E. coli are

carried by plasmids, whereas the gene for CT of V. cholerae01 is on the chromosome (38). Nucleotide sequence knowl-edge has been accumulated in the three multigene families:the CT-LT gene family (8, 33, 34, 39, 49, 60, 61), the STIgene family (42, 48), and the K88 gene family (10, 12).However, little is understood of their evolutionary related-ness. In this paper, we describe the corrected, completenucleotide sequence of the LTp subunit A (LTp-A) gene

and, by analyzing synonymous nucleotide substitutions ofthe gene sequences, we discuss the divergence in evolutionof the bacterial pathogenic determinants.The genes for LTp-A and LTp-B, originating from an

enterotoxin plasmid (ENT plasmid), P307 (47), and an ENT-R plasmid, pCG86 (17) which codes for antibiotic resistanceas well, were sequenced by the chemical method of Maxamand Gilbert (36) as previously described (60). In theseexperiments, recombinant plasmids EWD299 (carrying the

* Corresponding author.

LTp genes of ENT plasmid P307 [9, 47]) and pPMC2(carrying the LTp genes of ENT-R plasmid pCG86 [4]) wereused as DNA sources. The LTp-A genes on P307 and pCG86were identical in sequence (Fig. 1). The LTp-B genes on

P307 (8, 32) and pCG86 (Fig. 1) were also identical insequence. It was also clear (Fig. 1) that the differencesbetween the three subunit A gene sequences of LTp, LTh,and CT are the result of a series of single base changes. Thepresent sequence of the LTp-A gene (Fig. 1) disagrees withearlier sequence data (49) made on the LTp-A gene of P307,with which significant deletion and insertion of nucleotideshad been pointed out (34, 39, 60). The amino acid sequenceof the LTp-A gene product (precursor to LTp-A), deducedfrom the present nucleotide sequence, is also shown in Fig.1. When an amino acid sequence comparison was madebetween the LTp-A1 and LTh-A1 fragments (ADP-ribosyltransferase; a proteolytic cleavage product of subunitA which plays a key role in the toxin function [position 1 to192 in Fig. 1 A gene region]), LTp-A1 differed from LTh-A1by only one amino acid at position 4 (Arg++Lys). In addition,in terms of a local hydrophilicity profile (19) and a predictedsecondary structure (5), LTp-A1 was identical to LTh-A1which had been previously described (58, 60).A promoter region sequence of the LTp operon was also

determined (Fig. 1). This sequence was similar to the corre-

sponding sequence of the LTh operon but markedly differentfrom that of the CT operon (Fig. 1). Tandem repetitions ofthe unique sequence TTTTGAT located 77 base pairs up-stream of the start of the CT-A gene, which play a role in theexpression of the CT operon (39), were not found in thepromoter region of the LTp or LTh operon (Fig. 1).

Using the sequence data in Fig. 1, as well as data fromearlier reports, we estimated the number of synonymous

(silent) nucleotide substitutions (40) between the homolo-gous genes in the virulence gene families of CT-LT, STI, andK88 in an attempt to determine their evolutionary relation-ships (Table 1). Synonymous substitutions were the mostsignificant between the LT and CT genes and between the

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VOL. 169, 1987 NOTES 1353

LTP(P307) TTGACATCATGTTGCATATAGGTTAZATAAAACAAGTGGCGTTATCTTTTTCCGGATTGTCTTCTTGTATGATATATAAGTTTTCCTCG

JAge7ne-10 -10 -1 10 20 30

CT 000 GTA AAG ATA ATA TTT GTG TTT TTT ATT TTC TTA TCA TCA TOT TCA TAT GCA MAT GAT GAT MAG TTA TAT COG GCA GAT TCT AGA CCT CCTGOAT GMA ATA 80G CAG TCA GGT GGT CTT ATG CCA AMA GGA CAG AGT GAO TAC TTT GAC CMA GOTITh ATG A.M.A MAT ATA AC*T TTC* ATT TTT TTT ATT TTA TTA GCA TCG C*C*A TTTA TAT GCA MAT GO* MAC AM! TTA TAC CGT* OCT MAC TCT AMA CCC* CCA MAT GAA ATA AAA COT TCC GM GGT CTT ATG CCC AMA GGa CAT' MT MAG TAC TTC MAT AMG GMI

LTp(pCG8O) ATG AMA MAT ATA ACT TTC ATT TTT TTT ATT TTA TTA GCA TCG CCA TTA TAT GCA MAT GGC 'GAC AGA TTA TAC CGT OCT MAC TCT AMA CCC CCA MAT GMA ATA AMA CGT TCC GMA GOT CTT ATG CCC AMA GMG CAT MAT GAG TAC TTC OAT AMA GMALTp(P307) ATG AMA MAT ATA ACT TTC ATT TTT TTT ATT TTA TTA GCA TCG CCA TTA TAT GCA MAT GGC MAC AGA TTA TAC CGT GCT MAC TCT AMA CCC CCA MAT GMA ATA MAA CGT TCC GMA GGT CTT ATG CCC AMA 600 CAT MAT GAG TAC TTC MAT AMA GMA

LTp met Lys Asn Ile Thr Phe Ilie Phe Phe Ilie Leu Leu Ala Ger PrA Leu Tyr Ala Asn Gly Asp !Ag Lea Tyr ArA Ala Asp Ser Arg PrA Pea Asp GTu Ilie Lys Arq Ge- Gly Gly Leu Met P-a Arg Gly His Asn Glu Tyr Phe Asp Arg Gly

4G so 60 00 AGACT CAA ATG OAT ATC AAC COT TAT MAT CAT GCA AGA GMA ACT COG ACG GMA TOT GOT AGO CAC MOT MAT GGM TAT GOT 0CC ACC TCA ATT AGO TOG AMA AGO GCC* CAC TOO GTG* GGO CM* A*CT ATA TOG* OCT GOT* CAT OCT ACT OA*T TAT ATA TATACT CMA ATG MT ATT MIT CIT TAT MIGAT OCACC AMA GMA ACA CM; ACT GGt TOT GOT AGAA TAT MAT MAC GMA TAT GOT 0CC ACT OCT COT AGO TOG AGA AGO OCT CAC TOO GCA GMA CAG OCT AlA 00A OCA GGA TAT OCT ACT OAC TAT ATA TATACT CMA ATG MAT OTT MAT COT TAT MAT CAC GCG AMA GMA ACA CMA ACC MGC TOT GTC AGA TAT GAO MAC GGA TAT GOT 0CC ACT OCT COT AGO TOG AMA AGO GCT CAC TTA GCA GMA CAG OCT AlA TTA TCA GMA TAT TCC ACT TAC TAT ATA TATACT CMA ATG MOT ATT MAT COT TAT GAO CAC GCG AMA GMA ACA CMA ACC GGC TOT GTC AGA TAT MAT MAC GMA TAT GOT TCC ACT OCT COT AGO TOG AMA AGO GCT CAC TTA GCA GGA CAG OCT ATA TTA TCA GGA TAO 0CC ACT TAC TAT AlA TAT

Thr GTn Met Asn Ilie Asn Lea Tyr Asp His Ala Arg Gly The GTe Thr Gly Phe Val Arg Tye Asp Asp Gly Tye OaT Gee The Gee Leu Gee Leu Aeg Gee Ala His Leu Ala Gly GTn Gee Ile Lea Gee Gly Tye Gee The Tye Tye Ile Tye

90 100 010 120 130GOT ATA GCC ACT GCA CCC MAC ATG TOT MAC OTT MAT MAT GTA TTA MOG GCA TAC AGO CCT CAT CCA MIT GMA CMA GMA 000 OCT GCT TTA GOT GGG ATT CCA TAC 0CC CMA ATA TAO GMA TOO TAT CMA GOT CAT ITOT GGG GIG COT GAO GMA CMAGOT ATA GCG ACA GCA CCAA AATT ATG TOT AAf GOT AAT MAT GTA TTA GOT GITA TAC MGC CCT CAT CCA TAT GMA COG MGAG GOT OCT GCG TTA GOT GM; AlA CCA 000T OCT CA* T A OGAGO TAO CGI GOT M*T TOT GGI GIG AOlT GAO GMA COOAGOT ATA GCG ACA GCA CCA MAT ATG TOT MAT GOT MAT MAT GTA TTA GGC GTA TAC AGC CCT CAC CCA TAT GMA CAG MOG GOT OCT GCG TTA GOT GMA ATA CCA TAT OCT CAG ATA TAT GGA TGG TAT CGT GOT MAT TOT GGT GTG AlT OAT GMA CGAGOT ATA GCG ACA GCA CCA MIT ATG OTT AAT GOT AAT MAT 000 TTA GGC 000 TAC AGC CCT CAC CCA OAT GMA CAG MAG GOT OCT GCG TTA GOT GMA AlA CCA TAT OCT COG 000 001 GMA TGG TAO COO GOT MAT TOT GGT GIG OTT GAO GMA COOVal Ile Ala The Ala Pea Asn Met Phe Asn Aal Asn Asp Aal Lea Gly Val Tye Gee Pea His Pea Tye Gla Gln Glu Aal Gee Ala Lea Gly Gly Ile Pea Tye Gee Gln Ilie lye Gly Tep Tye Aeg Val Asn Phe Gly OaT Ile Asp Glu Aeg

140 180 TM0 170 180 19000A CAT CGT MIT AGG GGC TAC AMA GAO AMA OAT TAC AGO AAC 010 MIT 000 OCT CCA GCA GCA MAT GGT OAT GMA TOG GCA GOT TIC CCI CCG MAG CAT AGA OCT 000 AMG GMA MAG CCG TGG OTT CAT COO GCA CCG CCG GOT TGT GGG MOT GCTTOO CAT CGT MAC AGO GMA 001 000 MAC TOG TAT TAC MAG OAAT CTOG OAT ATA GCT CCa GCA GAAG MAT GOT TAC AMA TOA GCA GOT TIC CCA CCG MOT CAT TMA GCT TGO AM* GMA GAM CCC* 000 001 CAT COO GCA CC*A CM* OGG TOO GMA MAT OCAITOA CAT CGT MAC AGO GMA TAT AGO GAC COG OAT TAC AMA MAT COG MAT AlA GCT CCG GCA MOG GOT GOT TAC AGM TTA GCA GOT TTC CCA CCG MAT CAC CMA OCT 000 AMA GMA GMA CCC TGG 000 COT CAT GCA CCA CMA GGT TGT GMA OAT TCATTA CAT CGT MAC MGG MAA TAT AGO MAC CGG OAT OAt AMA MAT COG MAT 000 OCT CCG GCA MOG MAT GGT TAC AGA TOO GCA GGT TTC CCA CCG MOT CAC CMA OCT 000 AMA GMA MAA CCC TGG All CAT CAT GCA CCA CAA GGT TGT GMA OAT TCA

Lea His Aeg Asn Aeg Gla Tye Aeg Asp Aeg Tye Tyr Aeg Asn Lea Asn Ilie Ala Pea Ala Gla Asp 012 Tye Aeg Lea Ala Gly Phe Pea Pea Asp His GTn Ala Tep Arg Gla Gla Pea Tep Ilie His His Ala Pea Gln Gly Cys Gly Asn Gee

200 210 220 230 2400CCA AMA TCA TCG ATG AGO MAT ACT TGC GAO GMA AMA 0CC CMA AGO CIA GOT GTA AMA TTC COT MAC GMA TAC CMA OCT AMA GOT AMA AMA CMA 000 010 TCA GGC OAT CMA OCT 000 000 MAT ACA CAT AAT AGO 010 000 MAT MAA TTATGTCA AGO ACA VTT AC GOT MIT ACT TGO MAT MOG GMA* 0CC COO MAT COG ACOC ATA000T00 COTC AGO IM TAT00 CMA OC MAA GOT MGa AG! COG 000 000 TCA MIC TAT COG* TC AMG GOT MAC 000 000 MAC AGO OTT C*GG MIA GMA ITT...TCA AMA ACA OTT ACA GOT GAO ACT TOT MIT MOG MOG 0CC CMG MAT COG AGC ACA 000 000 CTC AMGM 000A CMA TCA AMA GOT MAG AGO COG 000 000 TCA GAC TOT CAG TCA GAG GOT MAC 000 TAO AAC AMA 000 CGG GAO GMA TTAITCA AMA ACA OTT ACA GGT MAT ACT TGT AAI MAG GAM 0CC CMG MAT COG AGC ACA 010 100 CTC AGOGM 000TA CMA TCA AMA GOT MAG AGO COG 000 TTT TCA MAC TAT COG TCA MAG GOT MAC 000 000 MAC AGO 000 COG G*AO MAA TTA TGA

Gee Aeg The Ilie The Gly 0sp The Cys Asn Gla Gla The Gin Asn Lea Gee The Ile Tye Lea Aeg Gla lye Oln Gee Lys OaT Lys Aeg GTe Ilie Phe Gee Asp Tye Gln Gee Glu Aal Asp Ile Tye Asn Aeg Ile Aeg Asp Glu Lea .aae

IB gene-21 -20 -10 -l 10 20 30

CThli&TT AMA 000 AMA TTT 001 000 000 000 ACA GOT TOO COO OCT TCA GCA TAT GCA COO 000 ACA CCI CMA MOT 001 OCT GAO ITOG TOT GCA MAA TAC CAC AAC ACA CMA ATA TAT ACG CTA OAT GAO MAG 000 000 TCG TAT ACA GMA OCT COOITh 8.A" T AM 000A AMA TGT TA*T GOT 000A 001 ACO GCG6 TOO COO ICC OCTT COOT TTGO CA TAC GAM O*CIT CCC CM8 ITO* 000 ACA GAA CTOO TGT TC6GAMA TAOT CG*C AAC ACA CMA ATA TAO ACG 000A MAT GA*C MAG 008 COO* TCA TAO ACG GMA TC*G ATG

LTp(pC-G86) AIL,& T AMA 000 AMA TGT TAT GOT TOO ITOT ACG GCG TOO COO ICC OCT COO TOO GCA *CAC GGA GCT CCC COG ACT 001 ACA GMA COO TGT TCG GMA TAO CGC MAC ACA CMA 000 TAT ACG 000 MAT MAC MAG 000 COO TCA TAT ACG GMA TCG ATG

40 hO0 00 00 00GCT GGAAAGAAM AGOMO T00 GCT ATC 000 ACT OTT MOG MAT GOT GCA 001 000 CMA 000 GMA 000 CCA GOT 000 CMA COO 010 MIT TCA CMA AMA AMA GCG OTT GMA AGO ATG MAG MAT 0CC COG AGG OTT GCA TAO COT OCT MAA GCT AMA GTC GMAGCA GOT AMA 000 000 000 GOT AlC OTT ACIA 000 000 AOC* GGC GCA ACAA TOT COO GTC MAA GTC CCG MGC 000 CMA CAT 000 GA*C 0CC CMA AMA MOA 0CC 000 MAA AGG ATG MAG MAC ACA I0N 000 OTT ACA TAT COG ACT MAG ACT AMA AT GATTGCA MGC AMA AMA GMA 000 000 ATC 000 ACA 110 MAG MGC GGC GMA ACA TOO COG GTC GMA GTC CCG MGC 001 CMA CAT 000 GAC 0CC COG AMA AMA 0CC 000 GMA 000 000 000 MAC ACA 000 AMA ATC ACA TAT COG 0CC MAG 0CC AMA 000 000

90 100 103MAG 010 000 000 100 MAT MAT AMA ACO CCI COO GCG 001 0CC GCA 000 000 010 GCA MAT TAMMAA 000 000 000 000 MIT MAT AMA OCT CCC* 001 OCA* 000 OCO GCA ATC 000 000 GMA MAC TAOAMA 000 100 000 000 MAT 000 AMA 0CC CCC MAT TCA OTT GCG GCA ATC 000 000 00 MC TAG

FIG. 1. DNA sequences of the LTp-A and LTp-B genes compared with those of the LTh and CT genes. The sequence data of the LThand CT genes are from references 60 and 61 and references 33 and 39, respectively. The two underlined nucleotides (ATGA) of the A andB genes overlap. The LTp and CT gene nucleotides that differ from the nucleotides of the LTh genes are marked with asterisks. In the A genesequences, the numbers -1 to -18 and 1 to 240 represent the coding sequence regions for a signal peptide (18 amino acids) and for subunitA (240 amino acids), respectively. The previously reported codon sequence, GAT, at position 189 of the LTh-A gene (60) was a misreading,and the corrected sequence, AAT (position 189), is indicated. The predicted amino acid sequence of the LTp-A gene product is shown belowthe LTp nucleotide sequences; underlined amino acids differ from those of LTh (60). In the B gene sequences, the numbers -1 to -21 and1 to 103 represent the coding regions for a signal peptide and for subunit B, respectively. Nucleotide sequences upstream from the A geneare shown at the top.

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E. coli 5S rRNAS. dysenteriaeS. typhimurium 5S rRNA

700 600 500 400 300 200 100Divergence time(millions of years ago)

0

FIG. 2. Divergence in evolution of the pathogenic determinants and the 5S rRNA sequences of the bacterial pathogens causing diarrhealdiseases. The evolutionary distance between two bacterial species was calculated based on the nucleotide substitutions of5S rRNA sequencesas described by Kimura and Ohta (29). The proportion of different nucleotides between the two 5S rRNA sequences compared (E. coli 5SrRNA and V. cholerae 01 5S rRNA [35]) was corrected for multiple substitutions as described in the footnote to Table 1. Evolutionary timewas estimated by using the corrected number (K') obtained and (1.8 ± 0.5) x 10-10 per nucleotide site per year as the evolutionary rate ofnucleotide substitution for 5S rRNA sequences (20, 21). The divergence between E. coli 5S rRNA and Salmonella typhimurium 5S rRNA andthat between E. coli ribosomal proteins and Shigella dysenteriae ribosomal proteins (drawn as a dotted line in the figure) are from references20 and 23 and reference 22, respectively.

STIa and STIb genes, markedly low between the LTp andLTh genes, and also very low or even rare among the K88ab,K88ac, and K88ad genes.

Since the evolutionary rate of synonymous substitutionfor different genes is known to be roughly uniform (41), weestimated the divergence time for each pair of the homolo-gous virulence genes (Fig. 2). The STIa and STIb genes, aswell as the CT and LT genes, seemed to have diverged in theremote past, -130 million years ago. In contrast, the diver-gence between the LTp and LTh genes seemed to have beenvery recent, -0.9 million years ago. During the Cretaceousperiod, to which time the divergence between the LT and CTgenes corresponds, mammalian forms with a placenta seemto have arisen (57). Moreover, the European wild pig, Susscrofa, seems to have appeared in the earlier Pleistocene(the Quaternary period), -0.011 to 3 million years ago (7);this period of time roughly overlaps the predicted time ofdivergence of the LTp and LTh genes. From this viewpoint,we speculated that the divergence of the toxin genes may

reflect the divergence of animal hosts of the toxin-producingbacterial parasites. Occurrence of divergence among mem-

bers of the K88 gene family may overlap or be much morerecent than divergence of the LTp and LTh genes (Table 1;Fig. 2). It is believed that V. cholerae 01 produces diseasesonly in humans (humans are natural reservoir hosts of V.cholerae 01). Therefore, it is extremely interesting that CT,

a major virulence factor of cholera, was already presentbefore the appearance of humans.The time of divergence of the E. coli and V. cholerae 01

5S rRNAs was estimated by using the reported sequences(35) as described by Kimura and Ohta (29); the number ofnucleotide substitutions (K' [Fig. 2, legend]) was 0.24210.0519. The estimated divergence time, -670 million yearsago, was apparently much earlier than that between the LTand CT genes (Fig. 2).We therefore concluded that the LT gene is a foreign gene

which was acquired by E. coli to form an enteropathogen inthe recent evolutionary past compared with the origin of thetwo bacterial species E. coli and V. cholerae. It presumablyoriginated in the V. cholerae CT gene ancestor. This con-clusion is consistent with the finding that, with respect tocodon usage, the correlation coefficient between the LThgenes (A and B) and the E. coli gene mixture (Table 2; 16)was only 0.0646 (this correlation may be obtained with aprobability of 0.62 by chance), whereas the correlationcoefficient between the LTh genes (A and B) and the CTgenes (A and B) was 0.6313 (this correlation may be obtainedwith a probability of less than 10-6 by chance alone; seeTable 2 for each subunit gene). In agreement with this, thefrequency of usage of E. coli optimal codons (24, 25) wasvery low (Table 2) compared with that of other E. coli genes(24, 25), indicating that, as far as codon usage is concerned,

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NOTES 1355

TABLE 1. The number of synonymous substitutions betweenevolutionarily related virulence genes'

Gene Homologous Synonymous substitutionfamily virulence genes number (K)

CT-LT CT-A, LTh-A 1.1604 + 0.1728CT-A, LTp-A 1.1578 ± 0.1721LTh-A, LTp-A 0.0055 ± 0.0055CT-B, LTh-B 1.3246 ± 0.3116CT-B, LTp-B 1.4243 ± 0.3539LTh-B, LTp-B 0.0122 ± 0.0122

STI STIa, STIb 1.3504 ± 0.4437

K88 K88ab, K88ad 0.039 ± 0.015K88ab, K88ad 0.000 ± 0.000K88ab, K88ac 0.000 ± 0.000K88ac, K88ad 0.009 ± 0.007

a The nucleotide sequence data of the LTp, LTh, and CT genes are those inFig. 1. The sequence data of the STIa and STIb genes (each 216 base pairslong) are from references 48 and 42, respectively. See reference 12 for the firstK88ab-K88ad comparison listed and reference 10 for the remaining K88 pairs;the K88ab and K88ad gene sequences are 855 base pairs long, and thereported K88ac gene sequence is 849 base pairs long. The number ofsynonymous (silent) nucleotide substitutions was calculated by the method ofMiyata and Yasunaga (40) as follows. First, the proportion of synonymoussubstitution (P) was obtained by the formula P = number of synonymoussubstitutions observed (n,)/number of synonymous sites in the gene (N,).Then, we corrected P for multiple substitutions by the formula (26, 28) K(average number of synonymous substitutions per site) = -3/4 In (1-4/3P),assuming that the rates of nucleotide substitutions among nucleotides areequal; we also used other methods (15, 27, 51), in which the rates of nucleotidesubstitutions among nucleotides were unequal, but the values of evolutionarydistances did not change much. Finally, we estimated divergence time (7)from the formula T = K12v, where v is the evolutionary rate of synonymoussubstitution. Here, we used (5.1 ± 0.3) x 10-9 per nucleotide site per year asv (41). The data obtained are shown in Fig. 2.

the LT gene is not fully efficient in the E. coli translationalsystem. These data on the LT and CT genes provide strongsupport for a species-to-species transfer event in the evolu-tion of bacterial pathogenic determinants. It has been shownthat the LTp gene can be expressed in V. cholerae 01 (43).The concept of species-to-species transfer of genes in evo-lution has been described as a cross-species gene transfer(50), a horizontal gene transfer (2), or a nondivergencetheory of evolution (56).

Similarly, based on the codon usage data summarized inTable 2, we concluded that the STI (a or b) gene, as well as

the STII gene (a virulence gene of porcine isolates) ofenterotoxigenic E. coli, is a foreign gene which was acquiredin the past. The STI gene may have originated in bacterialspecies such as Yersinia enterocolitica or V. cholerae non-01 which produce an STI-like enterotoxin (53, 54); the genesequences have not been determined. More interestingly, ithas been demonstrated that STI has extensive amino acidsequence homology with conotoxins of the marine snailConus geographus (42).

In marked contrast, K88 genes could be more efficient inthe E. coli translational system, because the frequency ofusage of E. coli optimal codons, as well as the correlationcoefficient of codon usage, compared with the E. coli genemixture was extremely high (Table 2). It was concluded thatthe K88 genes are evolutionarily closer to E. coli than are theenterotoxin genes.The ENT plasmid P307 and the ENT-R plasmid pCG86

have been shown to share a common enterotoxin-codingregion as evidenced by heteroduplex analysis by electronmicroscope (37). The present study showed that the LTpgenes on P307 and pCG86 are identical in sequence (thereare no single base changes), suggesting that occurrence of

TABLE 2. Summary of genetic characteristics of the virulence genes

Correlation coefficient (probability bychance) of codon usage when compared

Gene family G + C content Frequency of usage of with: Sourceand gene (mol%) E. coli optimal codonsa E. coli (reference) of

mixtureb Othergenesgene sequences

CT-LTLTh A 38.2 0.38c 0.034 (0.790) 0.724 (<10-5)d 60; this studyLTp A 38.6 0.38 0.046 (0.724) This studyCT A 38.5 0.41c 0.020 (0.880) 39LTh B 37.1 0.44c 0.209 (0.105) 0.701 (<10-5)e 61LTp B 36.8 0.44 0.219 (0.090) 8, 32CT B 32.8 0.40c 0.178 (0.170) 33

STISTIa 31.0 0.50' 0.131 (0.315) 0.809 (<10-5)g 48STIb 31.9 0.50 0.105 (0.421) 42

STIISTII 36.2 0.46 0.223 (0.085) 0.489 (<10-4)g 31, 44

K88K88ab 45.1 0.59 0.348 (0.006) 12K88ab 45.1 0.61 0.355 (0.005) 10K88ac 45.1 0.59 0.334 (0.008) 10K88ad 46.4 0.57 0.379 (0.003) 12K88ad 44.6 0.60 0.373 (0.003) 10

a See references 24 and 25.b Genes for lacI, lac Y, trpA, recA, lipoprotein, and a part of RNA polymerase (16).c Taken from reference 59.d CT-A.' CT-B.f Taken from reference 24.g STIb.

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the ENT-R plasmid (from the ENT plasmid) is a very recentevent-apparently in the more recent evolutionary pastcompared with the divergence of the LTp and LTh genes andprobably since clinical use of antibiotics by humans, whichmust have acted as a very strong selective agent.

We thank Werner K. Maas for the LTp coding plasmids andhelpful suggestions; Takaaki Tamura for partial LTp gene sequenc-ing; Etsuko Nakamura for synonymous substitution analysis in part;Hiroshi Hori, Takeru Akazawa, and Nobuo Tamiya for stimulatingdiscussions; and Yoshifumi Takeda for continuous support andinterest.

This work was supported by a grant-in-aid for special projectresearch and a grant-in-aid for scientific research from the Ministryof Education, Science, and Culture of Japan.

LITERATURE CITED1. Aimoto, S., T. Takao, Y. Shimonishi, S. Hara, T. Takeda, Y.

Takeda, and T. Miwatani. 1982. Amino-acid sequence of aheat-stable enterotoxin produced by human enterotoxigenicEscherichia coli. Eur. J. Biochem. 129:257-263.

2. Busslinger, M., S. Rusconi, and M. L. Birnstiel. 1982. Anunusual evolutionary behaviour of a sea urchin histone genecluster. EMBO J. 1:27-33.

3. Chan, S.-K., and R. A. Giannelia. 1981. Amino acid sequence ofheat-stable enterotoxin produced by Escherichia coli patho-genic for man. J. Biol. Chem. 256:7744-7746.

4. Chen, T.-M., A. J. Mazaitis, and W. K. Maas. 1985. Construc-tion of a conjugative plasmid with potential use in vaccinesagainst heat-labile enterotoxin. Infect. Immun. 47:5-10.

5. Chou, P. Y., and G. D. Fasman. 1978. Empirical predictions ofprotein conformation. Annu. Rev. Biochem. 47:251-276.

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