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Isolation and nucleotide sequence determination of a gene encoding a heat-stable enterotoxin of Escherichia coli
0041-0101/90 ß .00+ .00 ~ 1990 Peraemon Pnxs pk
Tor(ron Vol. 28, No. 4, pp. 453-456, 1990. Printed in Great Britain .
ISOLATION AND NUCLEOTIDE SEQUENCE DETERMINATION OF A GENE ENCODING A HEAT-STABLE ENTEROTOXIN OF
ESCHERICHIA COLT
Xirr
Zxou, LI-Plxc SxErr and CxErro-Wu CxI
Shanghai Institute of Biochemistry, Academia Sinica, Yue-Yang Road 320, Shanghai 200031, China (Accepted for publication 1 l October 1989)
Xnv Zxou, LI-Plxc SHEN and CxExc-Wu Cxi. Isolation and nucleotide sequence determination of a gene encoding a heat-stable enterotoxin of Escherichia coli. Toxicon 28, 453-456, 1990.-An enterotoxin-producing strain of E. coli has been isolated from an infant patient in Shanghai Children Hospital and the gene of its heat-stable enterotoxin has been cloned and sequenced. The pre-pro-STI was composed of 72 amino acid residues corresponding to the encoding of 216 base pairs. There was only one nucleotide difference in the pro-part between this STI gene and the STIb gene reported in the literature . A guanosine base in our STI gene was substituted for a cytosine base in STIb gene resulting in a replacement of proline by alanine. Hence, the STI genes from different human sources are highly conserved though mutagenesis still occurs .
AT LEAST two classes of enterotoxin produced by enterotoxigenic E. coli are responsible for diarrhea in men and in various domestic animals. One is heat labile and thus designated as LT. The other is heat stable and designated as ST which can be further divided into STI and STII. STI is methanol-soluble and active in suckling mice and neonatal piglets, but inactive in weaned pigs . Whereas, STTI is methanol-insoluble, active in weaned and inactive in suckling mice. Each year about 700 million episodes of acute diarrhea occur in the world, resulting in five million deaths of children which account for one-third to one-half of infant mortality (DALLAS et al., 1980). STI from different strains of E. coli, composed of 18 or 19 amino acid residues encoded by bacterial transposons (So et al., 1979), acts through the activation of guanyl cyclase (DREYFUS et al., 1984). The sequence of several STI toxins from different E. coli strains have either been determined by a chemical approach (LALLIER et al., 1982 ; AntOTO et al., 1984) or deduced from nucleotide sequence (So et al., 1980 ; MOSELEY et al., 1983). The nucleotide sequence analysis showed that STI came from the precursor pre-pro-STI; the pre-part functions as a signal peptide leading to excretion of the toxin from E. coli, while the pro-part is released during processing of the toxin and its function remains unclear. Though a rather apparent difference was found in the pro-part of STI obtained from different animal sources of 453
45 4
Short Communications
CTAG Corresponding Sequence of STIb
C C T T
C C T T
r c
r c
c r
c r
F~a. 1. GEL MAP oF Tf~ sEQuEwvcE oF STI cEivE nv pST19 .
E. coli strains, the STI itself has a remarkable similarity . Our previous work (Zxno Buo et al., 1986) reported that an enterotoxigenic strain of E. coli was isolated from feces of a
diarrheal infant in Shanghai Children Hospital . This strain produced only a heat stable enterotoxin (STI) of 19 amino acid residues . Our present study was undertaken in order to observe whether the apparent difference in the pre- or pro-part of the toxin also exists in E. coli strains from the same human source . The gene encoding STI is located in the E. coli plasmid p71 which is 82 kilobases in length . This plasmid was purified by alkaline lysis and ultra-centrifugation to equilibrium in cesium chloride-ethidium bromide gradients (35,000 x g, 48 hr at 20°C) by the method of MAx~.Trs et al. (1982) . The purified plasmid was digested with restriction endonuclease PstI followed by Southern blot hybridization analysis with a synthetic STI probe. Only a 5.2 kilobare fragment could hybridize with the probe. The above fragment was prepared and inserted into PstI-cleaved pBR322 DNA. The recombinant plasmid with the fragment inserted was designated as pST52 . pST52 was further digested with EcoRI-PstI, HindIIIPstI and BamHI-PstI respectively, and then hybridized to the radiolabeled probes. Southern blot showed that a 1 .9 kilobare fragment positive to the probe could be obtained by digesting pST52 with BamHI-PstI . The BamHI-PstI~leaved pST52 DNA was then ligated to BamHI-PstI-cleaved pWR13 vector . The ligation solution was used to trans-
Short Communications (A) (H) (a) (b)
45 5
SD 1 TGCCATGTCC r~AGrTMT ATG AAG MA TCA ATA TTA Ttt ATT TTT CTT ..... . .... . . ... . ... .. . .. . . .. . . . .. . ... . . . . . . ... ... Net Lys Lys Ser Ile Leu Phe Ile Phe Leu 55 ?CT GTA T?G ?CT T?? ?CA CCT TTC GCT CAG GAT GCT MA CCA GTA C~ Ser Val Leu Ser Phe Ser Pro Phe Ala Gln Asp Ala Lys Pro Val Pro 115 GAG TCT TCA AM GM MA ATC ACA CTA GM TCA MA MA(iGT MC Glu Ser Ser Lys Glu Lys Ile Thr Leu Glu Ser Lys Lys Cys Asn 132 ATT GCA MA MA>AG? MT MA AGT GG? CCT GM AGC ATG MT AGT Ile Ala Lys Lys Ser Asn Lys Ser Gly Pro Glu Ser Met~Asn Ser lez 20~ AGC MT TAC TGA TGi GM ?TG(TG? TGT Mi CCT GCT TGi)ACC GGG Ser Asn ?yr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly TGC TAT TM TMTATAAAGGGMCiMACAGTTCCCTT?A?ATT?GTTCTGAT?CT Cys ?y
FIO . 2 . COMPARISON OF NUCLEOTIDE AND AMINO ACll) SEQUENCES OF STIb STRAIN P71 . (A) Nucleotide sequence of STI of E. coli strain P71 . (B) The STIb nucleotide MOSELEY (1983) . (a)(b) The amino acid sequences corresponding to (A) and
and STI,
OF
E.
Coli
sequence reported by (B) . Boxed region in position 55 indicates the divergence between STIb and STI of E. coli strain P~1 . The bottom boxed region indicates the 19 amino-acid peptides with STI activity . Two primers for sequencing were synthesized according to the sequences of positions 115-132 and 187-204.
form competent JM83 cell . In situ colony hybridization technique was used to screen the recombinant plasmids . The infant mouse assay showed that STI activity could be detected in those transformants positive to the probe ; the restriction enzyme digestion analysis of these subclones further verified that a 1 .9 kilobase fragment was inserted in pWR13 . The shortened STI gene clone was designated as pST19 . Two oligonucleotides each of 18 bases in length were synthesized and used as sequencing primers according to the nucleotide sequence of STIb reported by M06ELEY et al. (1983) . Plasmid pST19 was subjected to nucleotide sequence analysis by the dideoxynucleotide chain termination method (SANDER et al., 1981) . The nucleotide sequence of pST19 showed that among 216 base pairs encoding pre-proSTI there was only one nucleotide difference between this STI gene and the gene reported by MOSELEY et al. (1983) . A guanosine base in our STI was substituted for a cytosine base in STIb gene (Fig. 1) . As a result, the corresponding amino acid encoded was changed from proline to alanine . This single base mismatch occurred at the pro-part (Fig. 2) . The
456
Short Communications
possible functions of this region in post-processing of STI precursor, correct pairing of disulfide bonds or folding of STI peptide conformation, remain to be elucidated. The slight difference between these two STI genes of E. coli, from different human sources, seems to be a common random mutation and would not function as the codon at this position as the STIa gene of E. coli from a bovine source was also mutated, leading to a replacement of proline by serine (So et al., 1980). STI toxins are composed of 18-19 amino acid residues with high homology . The STI obtained from E. coli p71 and STIb (Mosa1 .EY et al., 1983) are both from human sources. There is only one single base substitution between these two genes. In contrast, the difference in nucleotide sequence between STIa from a bovine source and STIb from a human source is up to 31 % (So et al., 1980). Our result demonstrated that STI genes from the same source are highly conserved. Therefore an STI gene probe can be used to detect enterotoxigenic E. coli strains. When comparison of the structures of the signal was made, the pro- and active part of STI from different sources revealed that the conservation of the pro-part is less than the other two parts. However, the exact function of the pro-part and the importance of its individual residues remains to be clarified. REFERENCES Auto~ro, S ., T~uo, T ., Sl~totVISH, Y ., H~u, S ., TAKEDA, T ., TAICPEDA, Y. and MnvATern, T . (1982) Amino-acid sequence of a heat stable enterotoxin produced by human enterotoxigenic E . coli. Ew . J. Biachem. 129, 257-263 . Det.t.ns, W. S . and Fet.xow, S . (1980) Amino acid sequence homology betweèn cholera toxin and Escherichia coli heat labile toxin . Nature 288, 499-501 . Dx~us, L . A., J~so-Fx>EOtN~rtrt, L . and Ronax~rsotv, D . C . (1984) Characterization of the mechanism of action of E. coli heat-stable enterotoxin . Inject. Immun. 44, 493-501 . LALtaatt, R ., Baxruxo, F ., GENDREAU, M ., Lezutea, C ., Sato~, N . G ., Cttxanau, M . and Sr-P~xxa, S . A. (1982) Isolation and purification of E. coli heat stable enterotoxin of porcine origin. Analyt. biochem . 127, 267-275 . Metvt~Tts, T ., Ftursctt, E . F. and Snstexoox, J . (1982) Molecular Cloning, A laboratory Manual, pp .92-94 . Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Mosat .av, S. L ., H~xnY, J . W., HuQ, M . L, Ect>EVatexte, P. and Fet.xow, S . (1983) Isolation and nucleotide sequence determination of a gene encoding a heat stable enterotoxin of Escherichia coli. Infect . Immw . 39, 1167-1174 . SANGIat, F . (1981) Determination of nucleotide sequences in DNA. Science 214, 1305-1312 . So, M ., Hi~xort, F . and MCCARi'HY, B. J . (1979) The F,scherichia coli gene encoding heat stable toxin is a bacterial transposon $anked by inverted repeats of ISI . Nature 284, 47374. So, M. and McCnxrxv, H . J . (1980) Nucleotide sequence of the bacterial transposon TN1681 encoding a heat stable (ST) toxin and its identification in enterotoxigenic Escherichia coli strains . Proc. Natn . Acad. Sci. U.S.A . 77, 4011015 . Zfuo Buo, Ct-n C~tvo-Wu, C~tv YAN, X~ Mat-WEx and Sut~ Hut-L~x (1986) Purification and sequence determination of a heat stable peptide enterotoxin of E . coJi. Chinese J. Physiol. Sci. 2, 101-107 .
Tor(ron Vol. 28, No. 4, pp. 453-456, 1990. Printed in Great Britain .
ISOLATION AND NUCLEOTIDE SEQUENCE DETERMINATION OF A GENE ENCODING A HEAT-STABLE ENTEROTOXIN OF
ESCHERICHIA COLT
Xirr
Zxou, LI-Plxc SxErr and CxErro-Wu CxI
Shanghai Institute of Biochemistry, Academia Sinica, Yue-Yang Road 320, Shanghai 200031, China (Accepted for publication 1 l October 1989)
Xnv Zxou, LI-Plxc SHEN and CxExc-Wu Cxi. Isolation and nucleotide sequence determination of a gene encoding a heat-stable enterotoxin of Escherichia coli. Toxicon 28, 453-456, 1990.-An enterotoxin-producing strain of E. coli has been isolated from an infant patient in Shanghai Children Hospital and the gene of its heat-stable enterotoxin has been cloned and sequenced. The pre-pro-STI was composed of 72 amino acid residues corresponding to the encoding of 216 base pairs. There was only one nucleotide difference in the pro-part between this STI gene and the STIb gene reported in the literature . A guanosine base in our STI gene was substituted for a cytosine base in STIb gene resulting in a replacement of proline by alanine. Hence, the STI genes from different human sources are highly conserved though mutagenesis still occurs .
AT LEAST two classes of enterotoxin produced by enterotoxigenic E. coli are responsible for diarrhea in men and in various domestic animals. One is heat labile and thus designated as LT. The other is heat stable and designated as ST which can be further divided into STI and STII. STI is methanol-soluble and active in suckling mice and neonatal piglets, but inactive in weaned pigs . Whereas, STTI is methanol-insoluble, active in weaned and inactive in suckling mice. Each year about 700 million episodes of acute diarrhea occur in the world, resulting in five million deaths of children which account for one-third to one-half of infant mortality (DALLAS et al., 1980). STI from different strains of E. coli, composed of 18 or 19 amino acid residues encoded by bacterial transposons (So et al., 1979), acts through the activation of guanyl cyclase (DREYFUS et al., 1984). The sequence of several STI toxins from different E. coli strains have either been determined by a chemical approach (LALLIER et al., 1982 ; AntOTO et al., 1984) or deduced from nucleotide sequence (So et al., 1980 ; MOSELEY et al., 1983). The nucleotide sequence analysis showed that STI came from the precursor pre-pro-STI; the pre-part functions as a signal peptide leading to excretion of the toxin from E. coli, while the pro-part is released during processing of the toxin and its function remains unclear. Though a rather apparent difference was found in the pro-part of STI obtained from different animal sources of 453
45 4
Short Communications
CTAG Corresponding Sequence of STIb
C C T T
C C T T
r c
r c
c r
c r
F~a. 1. GEL MAP oF Tf~ sEQuEwvcE oF STI cEivE nv pST19 .
E. coli strains, the STI itself has a remarkable similarity . Our previous work (Zxno Buo et al., 1986) reported that an enterotoxigenic strain of E. coli was isolated from feces of a
diarrheal infant in Shanghai Children Hospital . This strain produced only a heat stable enterotoxin (STI) of 19 amino acid residues . Our present study was undertaken in order to observe whether the apparent difference in the pre- or pro-part of the toxin also exists in E. coli strains from the same human source . The gene encoding STI is located in the E. coli plasmid p71 which is 82 kilobases in length . This plasmid was purified by alkaline lysis and ultra-centrifugation to equilibrium in cesium chloride-ethidium bromide gradients (35,000 x g, 48 hr at 20°C) by the method of MAx~.Trs et al. (1982) . The purified plasmid was digested with restriction endonuclease PstI followed by Southern blot hybridization analysis with a synthetic STI probe. Only a 5.2 kilobare fragment could hybridize with the probe. The above fragment was prepared and inserted into PstI-cleaved pBR322 DNA. The recombinant plasmid with the fragment inserted was designated as pST52 . pST52 was further digested with EcoRI-PstI, HindIIIPstI and BamHI-PstI respectively, and then hybridized to the radiolabeled probes. Southern blot showed that a 1 .9 kilobare fragment positive to the probe could be obtained by digesting pST52 with BamHI-PstI . The BamHI-PstI~leaved pST52 DNA was then ligated to BamHI-PstI-cleaved pWR13 vector . The ligation solution was used to trans-
Short Communications (A) (H) (a) (b)
45 5
SD 1 TGCCATGTCC r~AGrTMT ATG AAG MA TCA ATA TTA Ttt ATT TTT CTT ..... . .... . . ... . ... .. . .. . . .. . . . .. . ... . . . . . . ... ... Net Lys Lys Ser Ile Leu Phe Ile Phe Leu 55 ?CT GTA T?G ?CT T?? ?CA CCT TTC GCT CAG GAT GCT MA CCA GTA C~ Ser Val Leu Ser Phe Ser Pro Phe Ala Gln Asp Ala Lys Pro Val Pro 115 GAG TCT TCA AM GM MA ATC ACA CTA GM TCA MA MA(iGT MC Glu Ser Ser Lys Glu Lys Ile Thr Leu Glu Ser Lys Lys Cys Asn 132 ATT GCA MA MA>AG? MT MA AGT GG? CCT GM AGC ATG MT AGT Ile Ala Lys Lys Ser Asn Lys Ser Gly Pro Glu Ser Met~Asn Ser lez 20~ AGC MT TAC TGA TGi GM ?TG(TG? TGT Mi CCT GCT TGi)ACC GGG Ser Asn ?yr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly TGC TAT TM TMTATAAAGGGMCiMACAGTTCCCTT?A?ATT?GTTCTGAT?CT Cys ?y
FIO . 2 . COMPARISON OF NUCLEOTIDE AND AMINO ACll) SEQUENCES OF STIb STRAIN P71 . (A) Nucleotide sequence of STI of E. coli strain P71 . (B) The STIb nucleotide MOSELEY (1983) . (a)(b) The amino acid sequences corresponding to (A) and
and STI,
OF
E.
Coli
sequence reported by (B) . Boxed region in position 55 indicates the divergence between STIb and STI of E. coli strain P~1 . The bottom boxed region indicates the 19 amino-acid peptides with STI activity . Two primers for sequencing were synthesized according to the sequences of positions 115-132 and 187-204.
form competent JM83 cell . In situ colony hybridization technique was used to screen the recombinant plasmids . The infant mouse assay showed that STI activity could be detected in those transformants positive to the probe ; the restriction enzyme digestion analysis of these subclones further verified that a 1 .9 kilobase fragment was inserted in pWR13 . The shortened STI gene clone was designated as pST19 . Two oligonucleotides each of 18 bases in length were synthesized and used as sequencing primers according to the nucleotide sequence of STIb reported by M06ELEY et al. (1983) . Plasmid pST19 was subjected to nucleotide sequence analysis by the dideoxynucleotide chain termination method (SANDER et al., 1981) . The nucleotide sequence of pST19 showed that among 216 base pairs encoding pre-proSTI there was only one nucleotide difference between this STI gene and the gene reported by MOSELEY et al. (1983) . A guanosine base in our STI was substituted for a cytosine base in STIb gene (Fig. 1) . As a result, the corresponding amino acid encoded was changed from proline to alanine . This single base mismatch occurred at the pro-part (Fig. 2) . The
456
Short Communications
possible functions of this region in post-processing of STI precursor, correct pairing of disulfide bonds or folding of STI peptide conformation, remain to be elucidated. The slight difference between these two STI genes of E. coli, from different human sources, seems to be a common random mutation and would not function as the codon at this position as the STIa gene of E. coli from a bovine source was also mutated, leading to a replacement of proline by serine (So et al., 1980). STI toxins are composed of 18-19 amino acid residues with high homology . The STI obtained from E. coli p71 and STIb (Mosa1 .EY et al., 1983) are both from human sources. There is only one single base substitution between these two genes. In contrast, the difference in nucleotide sequence between STIa from a bovine source and STIb from a human source is up to 31 % (So et al., 1980). Our result demonstrated that STI genes from the same source are highly conserved. Therefore an STI gene probe can be used to detect enterotoxigenic E. coli strains. When comparison of the structures of the signal was made, the pro- and active part of STI from different sources revealed that the conservation of the pro-part is less than the other two parts. However, the exact function of the pro-part and the importance of its individual residues remains to be clarified. REFERENCES Auto~ro, S ., T~uo, T ., Sl~totVISH, Y ., H~u, S ., TAKEDA, T ., TAICPEDA, Y. and MnvATern, T . (1982) Amino-acid sequence of a heat stable enterotoxin produced by human enterotoxigenic E . coli. Ew . J. Biachem. 129, 257-263 . Det.t.ns, W. S . and Fet.xow, S . (1980) Amino acid sequence homology betweèn cholera toxin and Escherichia coli heat labile toxin . Nature 288, 499-501 . Dx~us, L . A., J~so-Fx>EOtN~rtrt, L . and Ronax~rsotv, D . C . (1984) Characterization of the mechanism of action of E. coli heat-stable enterotoxin . Inject. Immun. 44, 493-501 . LALtaatt, R ., Baxruxo, F ., GENDREAU, M ., Lezutea, C ., Sato~, N . G ., Cttxanau, M . and Sr-P~xxa, S . A. (1982) Isolation and purification of E. coli heat stable enterotoxin of porcine origin. Analyt. biochem . 127, 267-275 . Metvt~Tts, T ., Ftursctt, E . F. and Snstexoox, J . (1982) Molecular Cloning, A laboratory Manual, pp .92-94 . Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Mosat .av, S. L ., H~xnY, J . W., HuQ, M . L, Ect>EVatexte, P. and Fet.xow, S . (1983) Isolation and nucleotide sequence determination of a gene encoding a heat stable enterotoxin of Escherichia coli. Infect . Immw . 39, 1167-1174 . SANGIat, F . (1981) Determination of nucleotide sequences in DNA. Science 214, 1305-1312 . So, M ., Hi~xort, F . and MCCARi'HY, B. J . (1979) The F,scherichia coli gene encoding heat stable toxin is a bacterial transposon $anked by inverted repeats of ISI . Nature 284, 47374. So, M. and McCnxrxv, H . J . (1980) Nucleotide sequence of the bacterial transposon TN1681 encoding a heat stable (ST) toxin and its identification in enterotoxigenic Escherichia coli strains . Proc. Natn . Acad. Sci. U.S.A . 77, 4011015 . Zfuo Buo, Ct-n C~tvo-Wu, C~tv YAN, X~ Mat-WEx and Sut~ Hut-L~x (1986) Purification and sequence determination of a heat stable peptide enterotoxin of E . coJi. Chinese J. Physiol. Sci. 2, 101-107 .