FEMS Microbiology Letters 90(19921 141-146 ~i'! 1992 Federation of European Microbiological Societies 11378-1tJ97/92/St15.tltl i'ublished by Elsevier
FEMSLE 04728
Analysis of K99 plasmids from enterotoxigenic Escherichia colt R i c h a r d E. lsaacson and Gall L. Start Department of l,~,terim~r)' Pathohh~h~.~v. Dil'ixion ~f .~licrohhJh~!~' aml hntntmohJk'y, Unit-t'r~ity of Illinoi.~. Urbana, lllinoi.~, U.S.A. Received 18 July 1991 Revision received 4 October It;91 Accepted 8 October 1991
Key words: Pill; Adhesion: Southern hybridization: Rcstrictio,1 fragmcnts; Enterotoxigenic; Epidemiology; Escherichia colt 1. S U M M A R Y Evaluation of 9 wild-type K99 positive strains of Escherichia colt showed that each had a plasmid of approximately 87.8 kb that hybridized with two D N A probes specific for K99 genes. The K99 reference plasmid from E. colt also is 87.8 kb. Each of these strains had a conserved 7.15-kb B a m H l fragment that also l,ybridized to these probes. Several K99 negative mutants and three 3 P - strains also contained K99 plasmids as well as the 7.15-kb B a m H l fragment. These results suggest that there is a conservation in size of the K99 plasmids of diverse strains.
2. I N T R O D U C T I O N The K99 pilus [1] is frequently produced by entcrotoxigenic Escherichia colt (ETEC) that cause severe, fatal diarrhoea in calves, lambs, and pigs [2,3]. K99 functions as an adhesin conferring
('on'espondt'nct' to; R.E. Isaacson, Department of VelerinaD' Pathobiology, Divisionof Microbiologyand Immunology,University of Illinois, Urbana, IL 61802, U.S.A.
on K99" E T E C the ability to attach to small intestinal epithelium and thus facilitate the colonization of small intestines. K99 is encoded on plasmid of 87.~ kb. The K99 genes in the type strain B41 have been cloned in several laboratories [4-6] and found to reside on a 7.15-kb B a m H ! fragment. Like other E. colt pill, the synthesis of K99 requires the expression of 7 - 8 unique proteins [5,6]. One of the proteins has a molecular mass of 18.2 kDa and is called pilin [7]. Pilin is the major constituent of mature K99 pill. Genetic evidence has demonstrated that, unlike other pilus-adhesins of E. colt, the K99 pilin protein contains the specificity for binding to host receptors [8]. K99 is widely distributed in nature and the serologic detection of K99 has been used as a diagnostic indicator of etiology. However, since growth in the laboratory does not favor expression of K99, serologic detection is often ambiguous. Moseley et al. [9] have used D N A - D N A hybridization to detect K99 in a number of naturally occurring E. colt strains, Using a K99specific DNA probe, they were able to demonstrate the presence of K99-related D N A sequences in 3 of 3 E T E C designated K99 ~- by serologic techniques. They also detected K99-re-
142 Table 1 E. coil strains used
lated sequences in 4 of 5 serologically K99ETEC. Of these strains the one probe-negative strain produced K88 while the 4 probe-positive strains produced F41. Three of the probe-positive strains we,re previously designated 3 P - and the fourth strain was a mutant of the K99 type strain, B41, that no longer produced K99 (B41M). Since there is no homology between K99 and F41 [9] it was concluded that indeed these strains contained K99-related sequences. However, since these strains were not extensively analyzed it was not possible to determine how much of the K99 sequence resided in these probe-positive, serologically negative strains. The objectives of the experiments described in this manuscript were: (1) to use a more diverse set of ETEC strains to determine whether there was conservation in the size of the K99 plasmids as well as the restriction fragment encoding K99 in this group of strains; and (2) to determine the size of the K99 plasmids and the K99 coding region in the E. coil strains that were serologically K99 negative but DNA probe-positive.
Strain 1536 1537 1538 1539 1593 1594 1596 1597 1598 BI 17 B117B41M 1476 987 VAC-1676 KATI-1706 VC-1751 1297
Genotype K99, ST (biotype ID) K99. ST (biotype2A) K99. ST (biotype4A-C) K99. ST (biotype 4G-H) 09: K35, 99, ST OI0l :K30. 90. ST O20:K +, 99, ST O8 : K25, 99, ST OIOI :K28, 99, ST 08 : K85, 99, ST I4,99- mutant of B117 K99- mutant of B41 (OI01 :K?) E. coli KI2 strain 711 containingpK88ac 09: KI03, 987P, ST Ol0l :K?, F41, ST Ol0l :K?, F41, ST OI01 :K?, 1741,ST E. coli KI2 strain 711 containingplX12
plX12 and the 7.15-kb BamHl fragment encoding K99. The 9 field strains were from calves with diarrhoea. They were obtained from geographically diverse farms over a ten year period and were of various serotypes. As well, the four strains 1536-1539 represented four different biotypes. Biotype data on the other strains were not available.
3. MATERIALS AND M E T H O D S
3.1. E. coil strains used The E. coil strains used (see Table 1) included 9 wild-type K99 ÷ ETEC strains, 2 K99- mutants derived from the virulent, K99 ~ ETEC strains B41 and BI17, 3 virulent ETEC strains that were designated 3 P - by Moon et al. [10] and produce F41. As controls, plasmid DNA from K88 + and 987P ÷ ETEC strains were used as was purified
3.2. Preparation of plasmid DNAs and DNA probes The 7.15-kb BamHl fragment encoding K99 was cloned into the vector pBR322 [11] generating plX12 [5]. The physical map of plX12 (Fig. 1) is identical to pFK99 which also encodes K99 and
pIX12 (7.15 kb Barn HI insert) 8.
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Fig. I. Restriction map of plXI2 and locationsof the two DNA probes used for hybridization experiments.Coding locationsand sizesof the various K99 proteins are shown.
l'J.3
was constructed by de G r a a f ct al. [12]. The K99 pilin gene has been located in the region of the single Bglll site using site directed mutagcncsis, transposon mediated mutagenesis, and D N A sequencing (Fig. 1). For the purpose of analyzing a series of field isolates for the presence of K99-related D N A sequences, we have employed two D N A probes in hybridization experiments. The two probes are: (1) the entire 7.15-kb BamH! fragment that will detect the presence of any of the K99-related gene sequences; and (2) an 810-bp H i n f l probe that is located within the K99 pilin gene and 200-bp of the adjacent 76-kDa protein gene (Fig. 1) and will detect the presence of these two genes only. Plasmid D N A was isolated from the E. colt strains listed in Table ! after growth in LuriaBertaini broth [13] using the mini lysis procedure of lsh-Horowicz and Burke [14]. To prepare D N A probes, the plasmid piX12 was purified from strain 1297 by equilibrium cehtrifugation in cesium chloride-ethidium bromide [15] followed by extraction with butanol to remove the bound ethldlum bromide. Purified p l X l 2 was digested with the restriction endonuclease B a m H i or Hinfl and the digests were separated by electropl-_,oresis in an agarose gel (0.7%). The desired fragment was obtained by electrophoresis onto a piece of dialysis tubing. After elution from the
dialysis tubing, phenol extraction, and ethanol precipitation the probes were labelled with [3Zp] by nick translation (Amersham) using a-[3ZP]-deoxyadcnosine triphosphate (Amersham).
3.3. Southern blot hybridization Mini lysatcs of various E. colt strains were separated by clectrophoresis through an agarose gel (0.7%). When indicated, the plasmids were digested with the restriction enzyme BamHl prior to electrophoresis. Separated D N A was transferred to nylon filters (Hybond N, Amersham) by the procedure of Southern [16]. The D N A was cross-linked to the filter by exposure to ultraviolet light. Hybridization was for 16 h at 68°C in 2 × saline-sodium citrate (SSC). The final stringency wash was II.I × SSC at 50°C. To hybridize with another probe, it was first necessary to strip the labe[!ed probe from the immobilized hybrid. To do so the filters were immersed in a hot (100°C) solution of 0.1 × SSC containing 0.5% sodium dodccyl sulfate and agitated for 15 min.
4. RESULTS Electrophoresis of the plasmids from each of the strains showed that each contained several plasmids (Fig. 2A). None of the wild-type strains
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Fig. 2. Agarose gel of plasmids from various E. coil strains stained with ethidium bromide (A) and an autoradiograph of hybridizationwith the Hinfl probe (B). Lanes: a. 1536:b. 1537;c, 1538;d. 1539: e, 1593: f, 1594;g, 1596; h, 1597- i, 1598:j, B! 17: k. Bll7-; I, B41M; m, 1476: n, VAC-1676- o, KATI-1706: p, VC-1751- q. ~87: r. pIXl2: and s, 7.15-kh BamHl fragment of piXI2. CCC, covalentlyclosed circular DNA: PC. open circular DNA.
144 appeared to have the same plasmid profile. The separated plasmids were transferred to a nylon filter and sequentially probed with the labelled Hinfl fragment and after stripping the filter it was re-probed with the BamHl p~obe. Regardless of the probe used, the results in Fig. 2B were obtained. Each of the K99-positive isolates contained a plasmid in the size range of 87.8-kb that hybridized to the probes (Fig. 2B, tracks a-j). The K99- mutants B41M and B l 1 7 - also contained plasmids of approximately 87.8-kb as did the three 3 P - strains (Fig. 2B, tracks k, I, n, o, p). On the other hand, plasmid D N A from the K88 + and 987P + strains showed no homology to either of the probes (Fig. 2B, tracks m, q). These results demonstrated the presence of K99-related sequences in each of the wild-type strains, the two K99- mutants and the 3P-strains, and that these sequences were always associated with a plasmid of approximately 87.8-kb. Since there was such a close similarity in size of the K99 plasmids from each of the strains tested, we wanted to know if there was physical conservation of the K99 coding region. Therefore, each of the plasmid preparations from these E. coli strains was subsequently digested with BamHl, separated clectrophoretically, blotted to a filter, and probed sequentially with the BamHl
a
b
c
de
f
g
h
i
and Hinfl probes. Each of the strains containing a K99 plasmid (including B41M, B I I 7 - , and the 3 P - strains) contained a single BamHl fragment that hybridized to both probes (Fig. 3). With the exception of strain B I 1 7 - , (Fig. 3, track K), the size of these BamHl fragments were all the same: 7.15-kb. In strain B i I 7 - , the BamH! fragment was slightly larger possibly due to an insertion in the 7.15 BamHl fragment. As expected, the digested plasmid D N A from the K88 + and 987P ÷ strains did not hybridize with either of the probes, Digested chromosomal D N A from the 987P ÷ strain also failed to hybridize with either of the K99-specific D N A probes.
5. DISCUSSION K99 is a widely distributed pilus-adhesin that facilitates the colonization of calf, lamb, and piglet small intestines. The results from hybridization experiments with the K99 ÷ field isolates demonstrated that K99 was plasmid-encoded in each of the field isolates and that in each strain the size of the K99 plasmld was conserved at approximately 87.8 kb. These results suggest that DNAD N A hybridization may be a useful technique to detect K99 + strains especially since the technique
j
k
I
m
n
o
p
q
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s
Fig. 3. Auloradiograph of plasmids digested with BamHI and hyb~!dizedwith the BamH! probe. Lanes: a, 1536;b, 1537"c, 1538:d, 1539; e, 1593:f, 1594:g, 1596: h, 1597: i, 1598:j, BI17: k, B117-: I, B41M: m, 1476. n, VAC-1676"o, KATI-1706"p, VC-1751"q, 987 chromosomal DNA" r, 987 plasmid DNA: and s, plXI2.
145 does not require expression of K99. However, as discussed below, this technique will also detect E. coil strains that are phenotypically K99- but remain genotypically K99 + such as the 3 P strains. Analysis of restriction digests of the plasmids from these strains by D N A - D N A hybridization showed that each strain contained a 7.15-kb BamHl fragment that hybridized with the cloned 7.15-kb fragment that originated in strain B41. Thus, not only is there a conservation in the size of the K99 plasmids but as well there is conservation in the size of the BamH! fragment on which it resides. Since the field strains were obtained over a tO year period and represented different serotypes and biotypes, it is interesting that this conservation in size is so pronounced. The plasmids encoding K88 do vary in size. As well there are antigenic variants of the K88 pilin molecules. The different antigenic variants of K88 have been associated with differences in the restriction maps of the K88 genes. Although the n u m b e r of K99 ÷ strains analyzed was not extensive, the conservation of the size of the K99 plasmids and K99 coding region is consistent with the tact that there have been no reports of antigenic variants of K99. The coding region of K99 is approximately 7.15-kb. Allowing for essential functions such as plasmid replication, segregation and incompatibility there is sufficient coding capacity available /,approximately 4 0 - 6 0 kb) to encode additional proteins of unspecified function in each of the K99 plasmlds. The maintenance of this large excess of coding capacity leads to speculation that these sequences may encode important products that enhance survival in the their natural habitat and may represent other yet-undlscovered virulence factors. Moseley et al. [9] previously reported the presence of K99-related sequences in the plasmid fraction of the three 3 P - strains and B41M. We have confirmed this observation and extended it by demonstrating that the plasmids containing the sequences homologous to K99 also contain a 7.15-kb BamHl fragment that hybridized to the K99 probes. As well, these plasmids have the same molecular mass as the wild-type K99 plas-
raids. The 3P- strains were collected in 1977 and stored at room temperature in the dark for two years prior to analysis of pilus production [10]. B41M and B117- are spontaneous variants that no longer produce Kqg. it is likely that the 3 P strains 6riginated from previously K99-poshive isolates and that on storage have, for unknown reasons, lost the ability to producc K99. The same can be said for B41M. The size of the K09 coding region in B I l 7 - i s somewhat larger than 7.15-kb and ma~ have resulted from an inscrtional event. K99 plays an essential role in the pathogenesis of E T E C induced diarrhoeal disease. As a result, K99 has been effectively used in vaccines to prevent disease. With increased use of such vaccines, there is concern that there will be selection for new or different adhesins that are antigenically unrelated to K99. The fact that the 3 P - strains, B41M and B117- are virulent demonstrates this potential, in these particular cases, the resident F41 adhesin substituted for K99. In the field it is likely that similar changes will occur and that these variants will be responsible for disease in vaccinated herds.
ACKNOWLEDGEMENT This matcrial is based upon work supported by the Cooperative State Research Service, U.S. Department of Agriculture under agreement No. 9001678.
REFERENCES [ll Isaacson. R.E. (1977) Infect. Immun. 15. 272-279. [2] Moon. H.W.. Nagy. B.. lsaacson. R.E.. and Orskov. I. (1977) Infect. Immun. 15, 614-620. [3] Smith, H.W. and Lingg~md, M.A. (1971)J. Mcd. Microbiol. 4, 467-485. [4] Baecker, P.A., Shehon. E.R., Bursztyn-Pettegrew. H., Salazar. F.II., Osen. E.C.. Stoufer. S.D.. Lee. S.W.. and Chan. H.W. (1q88) Infect. Immun. 56. 2317-2323. [5] isaacson, R.E. (1985)Avian Dis. 30. 28-36. [6] van Embden. J.D.A.. de Graal. F.K., Schouls, L.M., and Teppema, J.S. (198(I) Infect. Immun. 26, 1125-1133. [7] lsaacson. R.E., Colmenero, J., and Richter. P. (1981) FEMS Microbiol. Lett. 12, 229-232.
14h
[8] Jacobs. A.A.C., Simons. B.It., and de Graal~ F.K. (1987) EMBO J. 6, 1805-1808. [9] Moseley, S.L., Dougan. G.. Schnieder, R. A.. and Moon, H.W. (1986) !. Bacteriol. 167. 799-8114. [1(}] Moon, H.W., Kohler, E.M., Schnieder, R.A.. and Whipp, S.C. (1980) Infect. Immun. 27, 222-230. [I I] Bolivar, F., Rodriquez, R.L., Greene, P.J.. Betlack. M.C.. Crosa, J.H., and Falkow. S. (1977) Gene 2.95-113. [12] de Graaf, F.K.. Krenn, B.E.. and Klaasen, P. (1984) Infect. Immun. 43, 5(18-514.
[13] Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring, New York. [14] Ish-Horowicz, D. and Burke. J.F. {1981) Nucleic Acids Res. 9, 2989-2998. [15] Clewell, D.B. and HelinskL D.R. (1970) Biochem. 9, 4428-444O. [16] Southern. E.M. 11975) J. Mol. Biol. 98, 5113-517.
FEMSLE 04728
Analysis of K99 plasmids from enterotoxigenic Escherichia colt R i c h a r d E. lsaacson and Gall L. Start Department of l,~,terim~r)' Pathohh~h~.~v. Dil'ixion ~f .~licrohhJh~!~' aml hntntmohJk'y, Unit-t'r~ity of Illinoi.~. Urbana, lllinoi.~, U.S.A. Received 18 July 1991 Revision received 4 October It;91 Accepted 8 October 1991
Key words: Pill; Adhesion: Southern hybridization: Rcstrictio,1 fragmcnts; Enterotoxigenic; Epidemiology; Escherichia colt 1. S U M M A R Y Evaluation of 9 wild-type K99 positive strains of Escherichia colt showed that each had a plasmid of approximately 87.8 kb that hybridized with two D N A probes specific for K99 genes. The K99 reference plasmid from E. colt also is 87.8 kb. Each of these strains had a conserved 7.15-kb B a m H l fragment that also l,ybridized to these probes. Several K99 negative mutants and three 3 P - strains also contained K99 plasmids as well as the 7.15-kb B a m H l fragment. These results suggest that there is a conservation in size of the K99 plasmids of diverse strains.
2. I N T R O D U C T I O N The K99 pilus [1] is frequently produced by entcrotoxigenic Escherichia colt (ETEC) that cause severe, fatal diarrhoea in calves, lambs, and pigs [2,3]. K99 functions as an adhesin conferring
('on'espondt'nct' to; R.E. Isaacson, Department of VelerinaD' Pathobiology, Divisionof Microbiologyand Immunology,University of Illinois, Urbana, IL 61802, U.S.A.
on K99" E T E C the ability to attach to small intestinal epithelium and thus facilitate the colonization of small intestines. K99 is encoded on plasmid of 87.~ kb. The K99 genes in the type strain B41 have been cloned in several laboratories [4-6] and found to reside on a 7.15-kb B a m H ! fragment. Like other E. colt pill, the synthesis of K99 requires the expression of 7 - 8 unique proteins [5,6]. One of the proteins has a molecular mass of 18.2 kDa and is called pilin [7]. Pilin is the major constituent of mature K99 pill. Genetic evidence has demonstrated that, unlike other pilus-adhesins of E. colt, the K99 pilin protein contains the specificity for binding to host receptors [8]. K99 is widely distributed in nature and the serologic detection of K99 has been used as a diagnostic indicator of etiology. However, since growth in the laboratory does not favor expression of K99, serologic detection is often ambiguous. Moseley et al. [9] have used D N A - D N A hybridization to detect K99 in a number of naturally occurring E. colt strains, Using a K99specific DNA probe, they were able to demonstrate the presence of K99-related D N A sequences in 3 of 3 E T E C designated K99 ~- by serologic techniques. They also detected K99-re-
142 Table 1 E. coil strains used
lated sequences in 4 of 5 serologically K99ETEC. Of these strains the one probe-negative strain produced K88 while the 4 probe-positive strains produced F41. Three of the probe-positive strains we,re previously designated 3 P - and the fourth strain was a mutant of the K99 type strain, B41, that no longer produced K99 (B41M). Since there is no homology between K99 and F41 [9] it was concluded that indeed these strains contained K99-related sequences. However, since these strains were not extensively analyzed it was not possible to determine how much of the K99 sequence resided in these probe-positive, serologically negative strains. The objectives of the experiments described in this manuscript were: (1) to use a more diverse set of ETEC strains to determine whether there was conservation in the size of the K99 plasmids as well as the restriction fragment encoding K99 in this group of strains; and (2) to determine the size of the K99 plasmids and the K99 coding region in the E. coil strains that were serologically K99 negative but DNA probe-positive.
Strain 1536 1537 1538 1539 1593 1594 1596 1597 1598 BI 17 B117B41M 1476 987 VAC-1676 KATI-1706 VC-1751 1297
Genotype K99, ST (biotype ID) K99. ST (biotype2A) K99. ST (biotype4A-C) K99. ST (biotype 4G-H) 09: K35, 99, ST OI0l :K30. 90. ST O20:K +, 99, ST O8 : K25, 99, ST OIOI :K28, 99, ST 08 : K85, 99, ST I4,99- mutant of B117 K99- mutant of B41 (OI01 :K?) E. coli KI2 strain 711 containingpK88ac 09: KI03, 987P, ST Ol0l :K?, F41, ST Ol0l :K?, F41, ST OI01 :K?, 1741,ST E. coli KI2 strain 711 containingplX12
plX12 and the 7.15-kb BamHl fragment encoding K99. The 9 field strains were from calves with diarrhoea. They were obtained from geographically diverse farms over a ten year period and were of various serotypes. As well, the four strains 1536-1539 represented four different biotypes. Biotype data on the other strains were not available.
3. MATERIALS AND M E T H O D S
3.1. E. coil strains used The E. coil strains used (see Table 1) included 9 wild-type K99 ÷ ETEC strains, 2 K99- mutants derived from the virulent, K99 ~ ETEC strains B41 and BI17, 3 virulent ETEC strains that were designated 3 P - by Moon et al. [10] and produce F41. As controls, plasmid DNA from K88 + and 987P ÷ ETEC strains were used as was purified
3.2. Preparation of plasmid DNAs and DNA probes The 7.15-kb BamHl fragment encoding K99 was cloned into the vector pBR322 [11] generating plX12 [5]. The physical map of plX12 (Fig. 1) is identical to pFK99 which also encodes K99 and
pIX12 (7.15 kb Barn HI insert) 8.
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Fig. I. Restriction map of plXI2 and locationsof the two DNA probes used for hybridization experiments.Coding locationsand sizesof the various K99 proteins are shown.
l'J.3
was constructed by de G r a a f ct al. [12]. The K99 pilin gene has been located in the region of the single Bglll site using site directed mutagcncsis, transposon mediated mutagenesis, and D N A sequencing (Fig. 1). For the purpose of analyzing a series of field isolates for the presence of K99-related D N A sequences, we have employed two D N A probes in hybridization experiments. The two probes are: (1) the entire 7.15-kb BamH! fragment that will detect the presence of any of the K99-related gene sequences; and (2) an 810-bp H i n f l probe that is located within the K99 pilin gene and 200-bp of the adjacent 76-kDa protein gene (Fig. 1) and will detect the presence of these two genes only. Plasmid D N A was isolated from the E. colt strains listed in Table ! after growth in LuriaBertaini broth [13] using the mini lysis procedure of lsh-Horowicz and Burke [14]. To prepare D N A probes, the plasmid piX12 was purified from strain 1297 by equilibrium cehtrifugation in cesium chloride-ethidium bromide [15] followed by extraction with butanol to remove the bound ethldlum bromide. Purified p l X l 2 was digested with the restriction endonuclease B a m H i or Hinfl and the digests were separated by electropl-_,oresis in an agarose gel (0.7%). The desired fragment was obtained by electrophoresis onto a piece of dialysis tubing. After elution from the
dialysis tubing, phenol extraction, and ethanol precipitation the probes were labelled with [3Zp] by nick translation (Amersham) using a-[3ZP]-deoxyadcnosine triphosphate (Amersham).
3.3. Southern blot hybridization Mini lysatcs of various E. colt strains were separated by clectrophoresis through an agarose gel (0.7%). When indicated, the plasmids were digested with the restriction enzyme BamHl prior to electrophoresis. Separated D N A was transferred to nylon filters (Hybond N, Amersham) by the procedure of Southern [16]. The D N A was cross-linked to the filter by exposure to ultraviolet light. Hybridization was for 16 h at 68°C in 2 × saline-sodium citrate (SSC). The final stringency wash was II.I × SSC at 50°C. To hybridize with another probe, it was first necessary to strip the labe[!ed probe from the immobilized hybrid. To do so the filters were immersed in a hot (100°C) solution of 0.1 × SSC containing 0.5% sodium dodccyl sulfate and agitated for 15 min.
4. RESULTS Electrophoresis of the plasmids from each of the strains showed that each contained several plasmids (Fig. 2A). None of the wild-type strains
A abedefgh
ij
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a bcde
f g hi
j klmnopqrs
~-CCC ~--oc
Fig. 2. Agarose gel of plasmids from various E. coil strains stained with ethidium bromide (A) and an autoradiograph of hybridizationwith the Hinfl probe (B). Lanes: a. 1536:b. 1537;c, 1538;d. 1539: e, 1593: f, 1594;g, 1596; h, 1597- i, 1598:j, B! 17: k. Bll7-; I, B41M; m, 1476: n, VAC-1676- o, KATI-1706: p, VC-1751- q. ~87: r. pIXl2: and s, 7.15-kh BamHl fragment of piXI2. CCC, covalentlyclosed circular DNA: PC. open circular DNA.
144 appeared to have the same plasmid profile. The separated plasmids were transferred to a nylon filter and sequentially probed with the labelled Hinfl fragment and after stripping the filter it was re-probed with the BamHl p~obe. Regardless of the probe used, the results in Fig. 2B were obtained. Each of the K99-positive isolates contained a plasmid in the size range of 87.8-kb that hybridized to the probes (Fig. 2B, tracks a-j). The K99- mutants B41M and B l 1 7 - also contained plasmids of approximately 87.8-kb as did the three 3 P - strains (Fig. 2B, tracks k, I, n, o, p). On the other hand, plasmid D N A from the K88 + and 987P + strains showed no homology to either of the probes (Fig. 2B, tracks m, q). These results demonstrated the presence of K99-related sequences in each of the wild-type strains, the two K99- mutants and the 3P-strains, and that these sequences were always associated with a plasmid of approximately 87.8-kb. Since there was such a close similarity in size of the K99 plasmids from each of the strains tested, we wanted to know if there was physical conservation of the K99 coding region. Therefore, each of the plasmid preparations from these E. coli strains was subsequently digested with BamHl, separated clectrophoretically, blotted to a filter, and probed sequentially with the BamHl
a
b
c
de
f
g
h
i
and Hinfl probes. Each of the strains containing a K99 plasmid (including B41M, B I I 7 - , and the 3 P - strains) contained a single BamHl fragment that hybridized to both probes (Fig. 3). With the exception of strain B I 1 7 - , (Fig. 3, track K), the size of these BamHl fragments were all the same: 7.15-kb. In strain B i I 7 - , the BamH! fragment was slightly larger possibly due to an insertion in the 7.15 BamHl fragment. As expected, the digested plasmid D N A from the K88 + and 987P ÷ strains did not hybridize with either of the probes, Digested chromosomal D N A from the 987P ÷ strain also failed to hybridize with either of the K99-specific D N A probes.
5. DISCUSSION K99 is a widely distributed pilus-adhesin that facilitates the colonization of calf, lamb, and piglet small intestines. The results from hybridization experiments with the K99 ÷ field isolates demonstrated that K99 was plasmid-encoded in each of the field isolates and that in each strain the size of the K99 plasmld was conserved at approximately 87.8 kb. These results suggest that DNAD N A hybridization may be a useful technique to detect K99 + strains especially since the technique
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k
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m
n
o
p
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r
s
Fig. 3. Auloradiograph of plasmids digested with BamHI and hyb~!dizedwith the BamH! probe. Lanes: a, 1536;b, 1537"c, 1538:d, 1539; e, 1593:f, 1594:g, 1596: h, 1597: i, 1598:j, BI17: k, B117-: I, B41M: m, 1476. n, VAC-1676"o, KATI-1706"p, VC-1751"q, 987 chromosomal DNA" r, 987 plasmid DNA: and s, plXI2.
145 does not require expression of K99. However, as discussed below, this technique will also detect E. coil strains that are phenotypically K99- but remain genotypically K99 + such as the 3 P strains. Analysis of restriction digests of the plasmids from these strains by D N A - D N A hybridization showed that each strain contained a 7.15-kb BamHl fragment that hybridized with the cloned 7.15-kb fragment that originated in strain B41. Thus, not only is there a conservation in the size of the K99 plasmids but as well there is conservation in the size of the BamH! fragment on which it resides. Since the field strains were obtained over a tO year period and represented different serotypes and biotypes, it is interesting that this conservation in size is so pronounced. The plasmids encoding K88 do vary in size. As well there are antigenic variants of the K88 pilin molecules. The different antigenic variants of K88 have been associated with differences in the restriction maps of the K88 genes. Although the n u m b e r of K99 ÷ strains analyzed was not extensive, the conservation of the size of the K99 plasmids and K99 coding region is consistent with the tact that there have been no reports of antigenic variants of K99. The coding region of K99 is approximately 7.15-kb. Allowing for essential functions such as plasmid replication, segregation and incompatibility there is sufficient coding capacity available /,approximately 4 0 - 6 0 kb) to encode additional proteins of unspecified function in each of the K99 plasmlds. The maintenance of this large excess of coding capacity leads to speculation that these sequences may encode important products that enhance survival in the their natural habitat and may represent other yet-undlscovered virulence factors. Moseley et al. [9] previously reported the presence of K99-related sequences in the plasmid fraction of the three 3 P - strains and B41M. We have confirmed this observation and extended it by demonstrating that the plasmids containing the sequences homologous to K99 also contain a 7.15-kb BamHl fragment that hybridized to the K99 probes. As well, these plasmids have the same molecular mass as the wild-type K99 plas-
raids. The 3P- strains were collected in 1977 and stored at room temperature in the dark for two years prior to analysis of pilus production [10]. B41M and B117- are spontaneous variants that no longer produce Kqg. it is likely that the 3 P strains 6riginated from previously K99-poshive isolates and that on storage have, for unknown reasons, lost the ability to producc K99. The same can be said for B41M. The size of the K09 coding region in B I l 7 - i s somewhat larger than 7.15-kb and ma~ have resulted from an inscrtional event. K99 plays an essential role in the pathogenesis of E T E C induced diarrhoeal disease. As a result, K99 has been effectively used in vaccines to prevent disease. With increased use of such vaccines, there is concern that there will be selection for new or different adhesins that are antigenically unrelated to K99. The fact that the 3 P - strains, B41M and B117- are virulent demonstrates this potential, in these particular cases, the resident F41 adhesin substituted for K99. In the field it is likely that similar changes will occur and that these variants will be responsible for disease in vaccinated herds.
ACKNOWLEDGEMENT This matcrial is based upon work supported by the Cooperative State Research Service, U.S. Department of Agriculture under agreement No. 9001678.
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[8] Jacobs. A.A.C., Simons. B.It., and de Graal~ F.K. (1987) EMBO J. 6, 1805-1808. [9] Moseley, S.L., Dougan. G.. Schnieder, R. A.. and Moon, H.W. (1986) !. Bacteriol. 167. 799-8114. [1(}] Moon, H.W., Kohler, E.M., Schnieder, R.A.. and Whipp, S.C. (1980) Infect. Immun. 27, 222-230. [I I] Bolivar, F., Rodriquez, R.L., Greene, P.J.. Betlack. M.C.. Crosa, J.H., and Falkow. S. (1977) Gene 2.95-113. [12] de Graaf, F.K.. Krenn, B.E.. and Klaasen, P. (1984) Infect. Immun. 43, 5(18-514.
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