- Email: [email protected]
The characterization of a conjugative R-plasmid isolated from Aeromonas salmonicida
PLASMID
16,213-218
(1986)
NOTES The Characterization of a Conjugative R-Plasmid Isolated from Aeromonas salmonicida TAKASHIAOKI,*' YASUTAMIMITOMA,*~~ ANDJORGEH.CROSA~ “Department of Fisheries, Faculty of Agriculture. Miyazaki University. Miyazaki 889-21. Japan and tDepar?ment of A4icrobioIogy and Immunology, School of Medicine, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201 Received August 6, 1985; revised July 9, 1986 We have analyzed two related R-plasm& isolated from Aeromonas salmonicida: pAr32 and pJA8102-1, which encode resistance. to chloramphenicol, streptomycin, and sulfonamides (Su). The genetic map of pJA8 102- 1 indicated that this plasmid consists of at least three functionally different regions. One is the region conferring ability to transfer (tr), one is the region coding for drug resistance genes (r-det), and one is the region encoding the replication functions (rep). Roth plasmids contain repeated sequences (RS); pJA8 102-l has four copies of this RS, while pAr-32 has three copies. The RS sequence of pJA8 102- 1 ax located adjacent to each of the drug-resistance genes. These sequences might have been responsible for the duplication of the Su-resistance gene on this plasmid. Q 1986AcademicKerr, IIK.
Aeromonas salmonicida is the causative agent of furunculosis in salmonid fish. This disease has been one of the most important causes of mortalities in salmonid culturing, aggravated by the recent appearance of strains resistant to the action of a large number of antibiotics (I). In most cases drug resistance was ascribed to the presence of an R-plasmid. The R-plasm& found in A. salmonicida belong to two incompatibility (inc) groups. One is the inc A group and the other is the inc U group (2). The inc A group R-plasmids are frequently found in fish pathogens such as A. hydrophila and Edwardsiella tarda, while inc U group R-plasmids appear to be specific for A. salmonicida and A. hydrophila (2-S). However, recently, inc U group R-plasmids were also found in enteric bacteria (6). Recent work has demonstrated the existence of a 29megadalton (MDa) conjugative R-plasmid from the inc U group in a large number of A. salmonicida strains, which conferred resistance to chloramphenicol (Cm?, streptomycin (Sm”), and sulfonamide (Sur) (3). We report in this paper the molecular and functional characterization of this R-p&mid as well as
comparisons with pAr32 and one other Rplasmid isolated from A. salmonicida. The A. salmonicida. Escherichia coli strains, and plasmids used in this work are described in Table 1. The transferable R-plasm& pAr32 (7) and pJA8 102- 1 (8), encoding resistance to Cm’, Sm’, and Su*, were detected in A. salmonicida. Plasmid DNA was isolated by a modified Triton X- 100 method (9), purified by ethidium bromide-c&urn chloride density gradient centrifugation, and subsequently digested with the appropriate restriction endonuclease under the conditions described by the suppliers (Bethesda Research Laboratories). The restriction endonuclease digestion patterns of pAr32 and pJA8102-1 DNA are shown in Fig. 1. The molecular weight of pAr32 was 47-kilobase pairs (kb) and of pJA8 102- 149 kb, the plasmids are related, and all subsequent experiments were carried out using pJA8 102- 1. The plasmids pBR322 (IO), pACYC177 (II), pKY2662 (12), and pKY2700 (13) were used as vectors for cloning the drug-resistance genes. To construct a restriction enzyme map of pJA8 102-l) three approaches were used. The first one was carried out by EcoRI partial cloning. The second approach was performed
’ To whom all correspondence should be addressed. 213
014%619X/86
$3.00
cowlight 0 1986 by AcadrrniC Rcrs, 1°C. All rights of reproduction in any form reserved.
214
NOTES TABLE 1 BACTERIALSTRAINSAND PLASMIDS Sample
Bacterial strains Aeromonas salmonicida MZ8 102 Ar-32 Escherichia coli C600 W1485-1 HBlOl KS2120 P3478 Plasmids pJA8102-1 pAr32 pBR322 pACYC177 pKY2662 pKY2700 pJHC-YE6
Relevant characteristics
Wild type Wild type
References
8 7
F thi thr 1euBlacy ton4 SupE str’ nal’ thyrecA- r;; rn, ara proA lacy galK str’ mtl SupE recA endA sup’ (supplied by K. Shimada) poti- thy- lac-
14 15, 16 14 This study 17
Cm’ Sm’ Su’ Cm’ Sm’ Su’ Tc’ Ap’ Cm’ Ap’ AP’ AP’ pKY2700 with EcoRI fragment 6 of pJA8102-1:Su’
8 7 10 II I2 13 This study
by double digestion with different restriction endonucleases. This technique was mainly applied to determine the restriction endonuclease map for enzymes with a few cleavage sites on pJA8 102-l DNA such as BamHI, KpnI, SalI, and XhoI. The third approach was carried out by cloning and recutting with other enzymes. BumHI fragment 1 was cloned into pKY2662 which is a cosmid vector, by using an in vitro packaging system.BumHI fragment 2 was cloned into pBR322 by standard methods (14). Clones containing EcoRI partial digests were also used. A restriction map of pJA8 102-l was developed for BumHI, BgnI, EcoRI, &MI, SalI, and X/z01(Fig. 2). To construct a genetic map of pJA8 102-1, we examined the phenotype of strains carrying the EcoRI partial digests or clones containing other restriction fragments. The antibiotic-resistance patterns of strains carrying recombinant plasmids wasalso determined. The results shown in Table 2 indicated that EcoRI fragment 2 (E2) carries determinants for resistance to chloramphenicol and that both fragments E5 and E6 could confer resistance to sulfonamide. Fragments E5 and E4 contain the gene for streptomycin resistance.
To determine the location of the replication region of pJA8 102-l we generated a miniplasmid from pJA8 102-1. Plasmid DNA was cleaved by either EcoRI or Bg/II, ligated without adding any vector DNA, and then transformed into HB 101 with selection for Cm’. By using this procedure several Cm’ transformants were obtained. In the caseof the DNA cleavedwith EcoRI, the miniplasmid consisted of fragmentsE 1, E2, and E9. This miniplasmid was designated pJA8 102-1E. In the case of BgfiI-cleaved DNA, we obtained a Cm’ miniplasmid which is composedof BglII fragments Bg2, Bg7, and Bg9. This miniplasmid was named minLpJA8 102-1B. The consensus region between these two miniplasmids was called the rep region. The location of the incompatibility genes of pJA8 102-1 was assessedby monitoring whether two plasmids (i.e., pJA8 102-l and somederivative plasmids) could coexist stably in the absenceof selection for resistance. The results, shown in Table 2, indicate that the plasmid containing either EcoRI fragment E 1 or E9 was incompatible with pJA8 102-1; plasmids carrying the other fragments could coexist stably with pJA8 102-1. From these data
215
NOTES ABC
DE
F
G
H
I
JKLMNO
FIG. 1. Restriction endonucleasecleavage patterns of pJA8 102-1 and pAr32,0.7% agarosegels. Channels A-G: pJA8 102-l; channels I-O: pAr32 DNA; channel I-L fragments of lambda DNA cleaved with HirrdIII + pBR322 DNA cleaved with HiniT. A, I: BarnHI; B, J: BgfiI; C, K: .&RI; D, L:HindIII; E, M: PstI; F, N: PvuII; G, 0: XhoI. Plasmid DNA was digestedwith the appropriate endonucleaseand the resulting fragments were subjected to electrophoresis in horizontal O.S%(w/v)agarosegels at 25 mA for about 15 h. The DNA was stained with ethidium bromide and visualized under uv light.
we conclude that the incompatibility region overlaps the replication region and that there at least two regions involved in incompatibility, contained in fragments El and E9, respectively. Several clones were checked to determine whether the plasmids were self-transmissible. The only conjugative derivative was that containing BumHI fragment B 1. A combination of the data in this paper permitted the assignment of functions to the physical map of pJA8 102-1, resulting in the genetic map shown in Fig. 2. In previous experiments, when the cloned E2 fragment was used as a probe in hybridization experiments (18-20), hybridization FIG. 2. Genetic and physical map of pJA8102-1. Tra, with severalother EcoRI fragments was noted. transfer regions;rep, replication; inc, incompatibility; Cm’, In addition to E2, the fragments ES and E6 chloramphenicol resistance;Sm’, streptomycin resistance; Su’, sulfonamide resistance.The following restriction en- showed strong homology, indicating that certain sequences,designated RS, present in E2 donucleases were used: EC&I, BarnHI, BgiII, X/z&I, may be repeated on the other EcoRI fragKpnI, and SalI.
216
NOTES TABLE 2 THE F’HENOTYP~ OF Escherichiu coli STRAINS CARRYING Vmous
DERIVATIVES OF pJA8 102-l
Phenotype of strain carrying clones Plasmid
Cloned fragments
pJHC-YEPl pJHC-YEP2 pJHC-YEP7 pJHC-YEP17 pJHC-YEP32 pJHC-YEP46 pJHC-YEP49 pJHC-YEPSO pJHCYEP60 pJHC-YE2 pJHC-YBl pJHC-YB2 pKY2700
E4, ES E5, E6 I3 E4, E5, E6 El, E4, E9 E4, E9 E6 E5 El E2 Bl B2 -
Cm
Sm
SU
S
r
S
s
S
r
S
S
S
S
r
S
S
S
S
S
S
S
S
S
s
S
r r
S
s
S
r
S
S
S
S
S
r s
r
r
S
S
Tm
Inc
-
+ + + + -
+ ND -
Note. Tra, Self-transmissibiIity;inc, incompatibility against pJA8 102-1; s, sensitive;r, &stance; E, EcoRI, B, BumHI; ND, not detetmined. To test tra functions of recombinant plasmids, E. coli KS2 120 strain containing the derivative was mixed with E. coli W 1485-1. In order to examine whether cloned fragments contained inc regions, KS2 120 strain containing pJA8lOZ1 was transferred with several pairs of cloned derivatives. The transconjugants catrying both plasmids were grown in L broth overnight at 37°C without drug selection for 100 generations and cells were spread on L agar plates. The colonies were examined for ampicillin and chloramphenicol, or streptomycin sensitivity. In those casesin which significant curing of either plasmid was observed, the pair was designated inc positive.
ments. Comparison of the restriction endonucleasepatterns of pJA8 102-1 and pAr32 revealed that both plasmids were almost identical, with pJA8 102-1 possessingE6 asan extra EcuRI fragment. Genetic mapping indicated that both E5 and E6 coded for sulfonamide resistance, suggesting a duplication of this gene. In order to determine the location and copy number of the repeated sequence (RS) elements on pJA8 102-1 and pAr32, we carried out Southern blot hybridization experiments (18) using the E6 fragment clone as a probe against doubly digested pJA8 102-l or pAr32 plasmid DNA. The results, shown in Fig. 3, indicate that pJA8 102-1 had four copies of this sequencewhile pAr32 has three copies. To determine the location of the RS element we constructed a restriction map of E5 and E6 (Fig. 4) and compared it to the map for the E2 fragment found in the previous section. It appears that the RS has single Hind111 and
Pm11 sites on fragment E6, although it may contain two HinIII sitesinstead of HMIII and PvuII siteson fragment E5. Thus, the RS could be positioned adjacent to each drug-resistance gene. The duplication of the sulfonamide-resistance gene on pJA8 102-l may have been mediated by this RS. The inc U group R-plasm& were originally classified among the inc W group (21). Subsequently, it was reported that these R-plasmids belonged to a new incompatibility group (3,4). pAr32 was proposed as the prototype inc U group R-plasmid (4), and we included this plasmid, as well as a more recent Japanese isolate, with the same resistance markers, pJA8 102-l. Inc U group plasmids are also found in A. hydrophila (5) and RA3 is the prototype inc U plasmid found in this bacterium (4). Comparison of the restriction endonucleasepattern of pAr32 shown in Fig. 1 with the pattern published by TschQe et al. (6) for RA3 indicates that the two plasmids are identical.
217
NOTES
(A) ABCDEFGHI
(W ABCDEFGHI
FIG. 3. Copy number of RS elements in pJA8 102-I and pAr32. Channels B-E: pJA8 102-I DNA, channels F-I: pAr32 DNA; A, lambda DNA cleaved with HindI& B, F: EC&I; C, G: EcoRl + BumHI; D, H: EcoRI + &!I; E, I: E&U + XhoI. The probe was ‘*P-labeled DNA from pJHC-yE6. Panel A showselectrophoretic profiles of R-plasmid DNAs digested by endonuclease.Panel B is an autoradiogram of a nitrccellulose filter blotted with the DNA in panel A (18) and probed with ‘*P-labeled pJHC-YE6, which was prepared by nick translation (19). Hybridization was carried out under the conditions described previously (14,20).
These results suggest that there might have been transfer between A. hydrophifa and A. salmonicida. Since A. salmonicida is psychophilic it is difficult to envisage that the inc U plasmids in this bacterium could have been transferred to A. salmonicida from enteric bacteria of human and animal sourcesknown to possessinc U plasmids. However, the existence of a potential intermediate host, such as A. hydrophila, could explain the rapid spread of R-plasm& in these bacteria. It is of interest that the restriction map of the r-det region of pJA8 102-l is very similar to that of RSa described by Tait et al. (22).
The Cm’ region (fragment E2) and the Su’ region (fragment ES) are strongly conserved. However, the other regions of pJA8 102-1 are completely different from those of RSa (manuscript in preparation). Hedgesand Datta (22) have reported the spontaneous appearance of Cm” mutants in cells containing either Rsa or RA3 (identical to pAr32). These Cm” mutants have also been generated by deletion events mediated by the RS, suggestingthat the RS may be an insertion sequence.In the case of pJA8 102-1 the Cm’ and the Su’ genes are flanked by repeated sequences.If these RS are insertion sequencesit is possible that the Cm’
218
NOTES
pKY 2700
pJHC-YE5
pJHC-YE6
m
f.
m”
1
I
1
,
IKb
2: I,
-1 +---
cm’-___)
pBR322
pJHC-YII
,
FIG. 4. Location of RS elements on the R-determinant region of pJA8 102-l. RS, repeated sequence. Symbols are the same as in Fig. 2.
and/or Su’ genes may be components of a transposon, although, so far, no transposition has been detected. We are presently analyzing the molecular nature of these repeated sequences and their possible role in the epidemiological spread of their associateddrug-resistance determinants.
6. TSCHAPE,H., TIETZE,E., AND KOCH, F., J. Gen. Microbiol. 127, 155-160 (1981). 7. AOKI, T., E&JSA, S., KIMURA, T., AND WATANABE, T., Appl. Micrbiol. 22,716-717 (1971). 8. Aow, T., KJTAO,T., IEMURA,N., MITOMA, Y., AND NOMURA,T., Bull. Japan. Sot. Sci. Fish. 49, 1722 (1983). 9. CLEWELL,D. B., AND HELINSKI, D. H., Proc. Natl. Acad. Sci. USA 62, 1159- 1166 (1969). 10. BOLIVAR,F., Gene4, 121-136 (1978). 11. CHANG, A. C. Y., AND COHEN,S. N., J. Bacterial. ACKNOWLEDGMENTS 134, 1141-l 156 (1978). We thank Professor T. Kitao for valuable discussions 12. FIJJIYOSHI,T., SASAKI,M., ONO, K., NAKAMURA, and encouragement. This research was founded by the T., SHIMADA,K., AND TAKAGI, Y., J. Biochem. 94,443-450 (1983). Japan Society for the Promotion of Scienceunder the Ja13. OZAKI, L. S., KIMURA, A., SHIMADA,K., AND TAKpan-U.S. Cooperative ScienceResearchand by SeaGrant 047- 158-4402l-RA20 from NOAA (to J.H.C). Y.M. acAGI, Y., J. Biochem. 91, 1155-l 162 (1982). knowledges an exchange training scholarship from the 14. MANIATIS, T., FRITSCH,E. F., AND SAMBROOK,J., “Molecular Cloning: A Laboratory Manual.” Cold Ministry of Education, Science,and Culture of Japan. Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982. REFERENCES 15. CROSA,J. H., OLARTE,J., MATA, L. J., LUTTROPP, L. K., AND PENARANDA,M. E., Antimicrob. Agents 1. AOKI, T., EGUSA,S., YADA, C., AND WATANABE,T., Chemother. l&553-558 (1977). Japan. J. Microbial. 16,233-238 (1972). 16. MITOMA, Y., Aoru, T., AND CROSA,J. H., Plasmid 2. AOKI, T., KITAO, T., AND ARAI, T., In “Plasmids, 12, 143-148 (1984). Medical and Theoretical Aspects” (S. Mitsuhashi, 17. CROSA,J. H., LUTTROPP,L. K., AND FALKOW,S., J. L. Rosvival, and V. KrEmery, eds.), pp. 39-45. Mol. Biol. 124443-460 (1978). Czechoslovak Medical Press,Prague, 1977. 18. SOUTHERN,E., J. Mol. Biol. 98,503-517 (1975). 3. AOKI, T., KITAO, T., ANDO, T., AND ARAI, T., In 19. RIGBY,P., DIM, M., RHODES,C., AND BERG, “Microbial Drug Resistance” (S. Mitsuhashi, ed.), P., J. Mol. Biol. 98, 503-517 (1977). Vol. 2, pp. 2 19-222.Japan Scientific SocietiesPress, 20. DENHARDT,D. T., B&hem. Biophys. Res. Commun. Tokyo, 1979. 23,64 l-646 ( 1966). 4. BRADLEY,D. E., AOKI, T., KITAO, T., AUI, T., AND 21. HEDGES,R. W., AND DANA, N., Nature (London) T%xIAPE, H., Plasmid 889-93 (1982). 234,220-22 1 (197 1). 5. AKAsHl A., AND AOKI, T., Bull. Sot. Sci. Fish. 52, 22. TAIT, R. C., LIJNDQUIST,R. C., AND KADo, C. I., 649-655 (1986). Mol. Gen. Genet. 186, lo-15 (1982).
16,213-218
(1986)
NOTES The Characterization of a Conjugative R-Plasmid Isolated from Aeromonas salmonicida TAKASHIAOKI,*' YASUTAMIMITOMA,*~~ ANDJORGEH.CROSA~ “Department of Fisheries, Faculty of Agriculture. Miyazaki University. Miyazaki 889-21. Japan and tDepar?ment of A4icrobioIogy and Immunology, School of Medicine, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201 Received August 6, 1985; revised July 9, 1986 We have analyzed two related R-plasm& isolated from Aeromonas salmonicida: pAr32 and pJA8102-1, which encode resistance. to chloramphenicol, streptomycin, and sulfonamides (Su). The genetic map of pJA8 102- 1 indicated that this plasmid consists of at least three functionally different regions. One is the region conferring ability to transfer (tr), one is the region coding for drug resistance genes (r-det), and one is the region encoding the replication functions (rep). Roth plasmids contain repeated sequences (RS); pJA8 102-l has four copies of this RS, while pAr-32 has three copies. The RS sequence of pJA8 102- 1 ax located adjacent to each of the drug-resistance genes. These sequences might have been responsible for the duplication of the Su-resistance gene on this plasmid. Q 1986AcademicKerr, IIK.
Aeromonas salmonicida is the causative agent of furunculosis in salmonid fish. This disease has been one of the most important causes of mortalities in salmonid culturing, aggravated by the recent appearance of strains resistant to the action of a large number of antibiotics (I). In most cases drug resistance was ascribed to the presence of an R-plasmid. The R-plasm& found in A. salmonicida belong to two incompatibility (inc) groups. One is the inc A group and the other is the inc U group (2). The inc A group R-plasmids are frequently found in fish pathogens such as A. hydrophila and Edwardsiella tarda, while inc U group R-plasmids appear to be specific for A. salmonicida and A. hydrophila (2-S). However, recently, inc U group R-plasmids were also found in enteric bacteria (6). Recent work has demonstrated the existence of a 29megadalton (MDa) conjugative R-plasmid from the inc U group in a large number of A. salmonicida strains, which conferred resistance to chloramphenicol (Cm?, streptomycin (Sm”), and sulfonamide (Sur) (3). We report in this paper the molecular and functional characterization of this R-p&mid as well as
comparisons with pAr32 and one other Rplasmid isolated from A. salmonicida. The A. salmonicida. Escherichia coli strains, and plasmids used in this work are described in Table 1. The transferable R-plasm& pAr32 (7) and pJA8 102- 1 (8), encoding resistance to Cm’, Sm’, and Su*, were detected in A. salmonicida. Plasmid DNA was isolated by a modified Triton X- 100 method (9), purified by ethidium bromide-c&urn chloride density gradient centrifugation, and subsequently digested with the appropriate restriction endonuclease under the conditions described by the suppliers (Bethesda Research Laboratories). The restriction endonuclease digestion patterns of pAr32 and pJA8102-1 DNA are shown in Fig. 1. The molecular weight of pAr32 was 47-kilobase pairs (kb) and of pJA8 102- 149 kb, the plasmids are related, and all subsequent experiments were carried out using pJA8 102- 1. The plasmids pBR322 (IO), pACYC177 (II), pKY2662 (12), and pKY2700 (13) were used as vectors for cloning the drug-resistance genes. To construct a restriction enzyme map of pJA8 102-l) three approaches were used. The first one was carried out by EcoRI partial cloning. The second approach was performed
’ To whom all correspondence should be addressed. 213
014%619X/86
$3.00
cowlight 0 1986 by AcadrrniC Rcrs, 1°C. All rights of reproduction in any form reserved.
214
NOTES TABLE 1 BACTERIALSTRAINSAND PLASMIDS Sample
Bacterial strains Aeromonas salmonicida MZ8 102 Ar-32 Escherichia coli C600 W1485-1 HBlOl KS2120 P3478 Plasmids pJA8102-1 pAr32 pBR322 pACYC177 pKY2662 pKY2700 pJHC-YE6
Relevant characteristics
Wild type Wild type
References
8 7
F thi thr 1euBlacy ton4 SupE str’ nal’ thyrecA- r;; rn, ara proA lacy galK str’ mtl SupE recA endA sup’ (supplied by K. Shimada) poti- thy- lac-
14 15, 16 14 This study 17
Cm’ Sm’ Su’ Cm’ Sm’ Su’ Tc’ Ap’ Cm’ Ap’ AP’ AP’ pKY2700 with EcoRI fragment 6 of pJA8102-1:Su’
8 7 10 II I2 13 This study
by double digestion with different restriction endonucleases. This technique was mainly applied to determine the restriction endonuclease map for enzymes with a few cleavage sites on pJA8 102-l DNA such as BamHI, KpnI, SalI, and XhoI. The third approach was carried out by cloning and recutting with other enzymes. BumHI fragment 1 was cloned into pKY2662 which is a cosmid vector, by using an in vitro packaging system.BumHI fragment 2 was cloned into pBR322 by standard methods (14). Clones containing EcoRI partial digests were also used. A restriction map of pJA8 102-l was developed for BumHI, BgnI, EcoRI, &MI, SalI, and X/z01(Fig. 2). To construct a genetic map of pJA8 102-1, we examined the phenotype of strains carrying the EcoRI partial digests or clones containing other restriction fragments. The antibiotic-resistance patterns of strains carrying recombinant plasmids wasalso determined. The results shown in Table 2 indicated that EcoRI fragment 2 (E2) carries determinants for resistance to chloramphenicol and that both fragments E5 and E6 could confer resistance to sulfonamide. Fragments E5 and E4 contain the gene for streptomycin resistance.
To determine the location of the replication region of pJA8 102-l we generated a miniplasmid from pJA8 102-1. Plasmid DNA was cleaved by either EcoRI or Bg/II, ligated without adding any vector DNA, and then transformed into HB 101 with selection for Cm’. By using this procedure several Cm’ transformants were obtained. In the caseof the DNA cleavedwith EcoRI, the miniplasmid consisted of fragmentsE 1, E2, and E9. This miniplasmid was designated pJA8 102-1E. In the case of BgfiI-cleaved DNA, we obtained a Cm’ miniplasmid which is composedof BglII fragments Bg2, Bg7, and Bg9. This miniplasmid was named minLpJA8 102-1B. The consensus region between these two miniplasmids was called the rep region. The location of the incompatibility genes of pJA8 102-1 was assessedby monitoring whether two plasmids (i.e., pJA8 102-l and somederivative plasmids) could coexist stably in the absenceof selection for resistance. The results, shown in Table 2, indicate that the plasmid containing either EcoRI fragment E 1 or E9 was incompatible with pJA8 102-1; plasmids carrying the other fragments could coexist stably with pJA8 102-1. From these data
215
NOTES ABC
DE
F
G
H
I
JKLMNO
FIG. 1. Restriction endonucleasecleavage patterns of pJA8 102-1 and pAr32,0.7% agarosegels. Channels A-G: pJA8 102-l; channels I-O: pAr32 DNA; channel I-L fragments of lambda DNA cleaved with HirrdIII + pBR322 DNA cleaved with HiniT. A, I: BarnHI; B, J: BgfiI; C, K: .&RI; D, L:HindIII; E, M: PstI; F, N: PvuII; G, 0: XhoI. Plasmid DNA was digestedwith the appropriate endonucleaseand the resulting fragments were subjected to electrophoresis in horizontal O.S%(w/v)agarosegels at 25 mA for about 15 h. The DNA was stained with ethidium bromide and visualized under uv light.
we conclude that the incompatibility region overlaps the replication region and that there at least two regions involved in incompatibility, contained in fragments El and E9, respectively. Several clones were checked to determine whether the plasmids were self-transmissible. The only conjugative derivative was that containing BumHI fragment B 1. A combination of the data in this paper permitted the assignment of functions to the physical map of pJA8 102-1, resulting in the genetic map shown in Fig. 2. In previous experiments, when the cloned E2 fragment was used as a probe in hybridization experiments (18-20), hybridization FIG. 2. Genetic and physical map of pJA8102-1. Tra, with severalother EcoRI fragments was noted. transfer regions;rep, replication; inc, incompatibility; Cm’, In addition to E2, the fragments ES and E6 chloramphenicol resistance;Sm’, streptomycin resistance; Su’, sulfonamide resistance.The following restriction en- showed strong homology, indicating that certain sequences,designated RS, present in E2 donucleases were used: EC&I, BarnHI, BgiII, X/z&I, may be repeated on the other EcoRI fragKpnI, and SalI.
216
NOTES TABLE 2 THE F’HENOTYP~ OF Escherichiu coli STRAINS CARRYING Vmous
DERIVATIVES OF pJA8 102-l
Phenotype of strain carrying clones Plasmid
Cloned fragments
pJHC-YEPl pJHC-YEP2 pJHC-YEP7 pJHC-YEP17 pJHC-YEP32 pJHC-YEP46 pJHC-YEP49 pJHC-YEPSO pJHCYEP60 pJHC-YE2 pJHC-YBl pJHC-YB2 pKY2700
E4, ES E5, E6 I3 E4, E5, E6 El, E4, E9 E4, E9 E6 E5 El E2 Bl B2 -
Cm
Sm
SU
S
r
S
s
S
r
S
S
S
S
r
S
S
S
S
S
S
S
S
S
s
S
r r
S
s
S
r
S
S
S
S
S
r s
r
r
S
S
Tm
Inc
-
+ + + + -
+ ND -
Note. Tra, Self-transmissibiIity;inc, incompatibility against pJA8 102-1; s, sensitive;r, &stance; E, EcoRI, B, BumHI; ND, not detetmined. To test tra functions of recombinant plasmids, E. coli KS2 120 strain containing the derivative was mixed with E. coli W 1485-1. In order to examine whether cloned fragments contained inc regions, KS2 120 strain containing pJA8lOZ1 was transferred with several pairs of cloned derivatives. The transconjugants catrying both plasmids were grown in L broth overnight at 37°C without drug selection for 100 generations and cells were spread on L agar plates. The colonies were examined for ampicillin and chloramphenicol, or streptomycin sensitivity. In those casesin which significant curing of either plasmid was observed, the pair was designated inc positive.
ments. Comparison of the restriction endonucleasepatterns of pJA8 102-1 and pAr32 revealed that both plasmids were almost identical, with pJA8 102-1 possessingE6 asan extra EcuRI fragment. Genetic mapping indicated that both E5 and E6 coded for sulfonamide resistance, suggesting a duplication of this gene. In order to determine the location and copy number of the repeated sequence (RS) elements on pJA8 102-1 and pAr32, we carried out Southern blot hybridization experiments (18) using the E6 fragment clone as a probe against doubly digested pJA8 102-l or pAr32 plasmid DNA. The results, shown in Fig. 3, indicate that pJA8 102-1 had four copies of this sequencewhile pAr32 has three copies. To determine the location of the RS element we constructed a restriction map of E5 and E6 (Fig. 4) and compared it to the map for the E2 fragment found in the previous section. It appears that the RS has single Hind111 and
Pm11 sites on fragment E6, although it may contain two HinIII sitesinstead of HMIII and PvuII siteson fragment E5. Thus, the RS could be positioned adjacent to each drug-resistance gene. The duplication of the sulfonamide-resistance gene on pJA8 102-l may have been mediated by this RS. The inc U group R-plasm& were originally classified among the inc W group (21). Subsequently, it was reported that these R-plasmids belonged to a new incompatibility group (3,4). pAr32 was proposed as the prototype inc U group R-plasmid (4), and we included this plasmid, as well as a more recent Japanese isolate, with the same resistance markers, pJA8 102-l. Inc U group plasmids are also found in A. hydrophila (5) and RA3 is the prototype inc U plasmid found in this bacterium (4). Comparison of the restriction endonucleasepattern of pAr32 shown in Fig. 1 with the pattern published by TschQe et al. (6) for RA3 indicates that the two plasmids are identical.
217
NOTES
(A) ABCDEFGHI
(W ABCDEFGHI
FIG. 3. Copy number of RS elements in pJA8 102-I and pAr32. Channels B-E: pJA8 102-I DNA, channels F-I: pAr32 DNA; A, lambda DNA cleaved with HindI& B, F: EC&I; C, G: EcoRl + BumHI; D, H: EcoRI + &!I; E, I: E&U + XhoI. The probe was ‘*P-labeled DNA from pJHC-yE6. Panel A showselectrophoretic profiles of R-plasmid DNAs digested by endonuclease.Panel B is an autoradiogram of a nitrccellulose filter blotted with the DNA in panel A (18) and probed with ‘*P-labeled pJHC-YE6, which was prepared by nick translation (19). Hybridization was carried out under the conditions described previously (14,20).
These results suggest that there might have been transfer between A. hydrophifa and A. salmonicida. Since A. salmonicida is psychophilic it is difficult to envisage that the inc U plasmids in this bacterium could have been transferred to A. salmonicida from enteric bacteria of human and animal sourcesknown to possessinc U plasmids. However, the existence of a potential intermediate host, such as A. hydrophila, could explain the rapid spread of R-plasm& in these bacteria. It is of interest that the restriction map of the r-det region of pJA8 102-l is very similar to that of RSa described by Tait et al. (22).
The Cm’ region (fragment E2) and the Su’ region (fragment ES) are strongly conserved. However, the other regions of pJA8 102-1 are completely different from those of RSa (manuscript in preparation). Hedgesand Datta (22) have reported the spontaneous appearance of Cm” mutants in cells containing either Rsa or RA3 (identical to pAr32). These Cm” mutants have also been generated by deletion events mediated by the RS, suggestingthat the RS may be an insertion sequence.In the case of pJA8 102-1 the Cm’ and the Su’ genes are flanked by repeated sequences.If these RS are insertion sequencesit is possible that the Cm’
218
NOTES
pKY 2700
pJHC-YE5
pJHC-YE6
m
f.
m”
1
I
1
,
IKb
2: I,
-1 +---
cm’-___)
pBR322
pJHC-YII
,
FIG. 4. Location of RS elements on the R-determinant region of pJA8 102-l. RS, repeated sequence. Symbols are the same as in Fig. 2.
and/or Su’ genes may be components of a transposon, although, so far, no transposition has been detected. We are presently analyzing the molecular nature of these repeated sequences and their possible role in the epidemiological spread of their associateddrug-resistance determinants.
6. TSCHAPE,H., TIETZE,E., AND KOCH, F., J. Gen. Microbiol. 127, 155-160 (1981). 7. AOKI, T., E&JSA, S., KIMURA, T., AND WATANABE, T., Appl. Micrbiol. 22,716-717 (1971). 8. Aow, T., KJTAO,T., IEMURA,N., MITOMA, Y., AND NOMURA,T., Bull. Japan. Sot. Sci. Fish. 49, 1722 (1983). 9. CLEWELL,D. B., AND HELINSKI, D. H., Proc. Natl. Acad. Sci. USA 62, 1159- 1166 (1969). 10. BOLIVAR,F., Gene4, 121-136 (1978). 11. CHANG, A. C. Y., AND COHEN,S. N., J. Bacterial. ACKNOWLEDGMENTS 134, 1141-l 156 (1978). We thank Professor T. Kitao for valuable discussions 12. FIJJIYOSHI,T., SASAKI,M., ONO, K., NAKAMURA, and encouragement. This research was founded by the T., SHIMADA,K., AND TAKAGI, Y., J. Biochem. 94,443-450 (1983). Japan Society for the Promotion of Scienceunder the Ja13. OZAKI, L. S., KIMURA, A., SHIMADA,K., AND TAKpan-U.S. Cooperative ScienceResearchand by SeaGrant 047- 158-4402l-RA20 from NOAA (to J.H.C). Y.M. acAGI, Y., J. Biochem. 91, 1155-l 162 (1982). knowledges an exchange training scholarship from the 14. MANIATIS, T., FRITSCH,E. F., AND SAMBROOK,J., “Molecular Cloning: A Laboratory Manual.” Cold Ministry of Education, Science,and Culture of Japan. Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982. REFERENCES 15. CROSA,J. H., OLARTE,J., MATA, L. J., LUTTROPP, L. K., AND PENARANDA,M. E., Antimicrob. Agents 1. AOKI, T., EGUSA,S., YADA, C., AND WATANABE,T., Chemother. l&553-558 (1977). Japan. J. Microbial. 16,233-238 (1972). 16. MITOMA, Y., Aoru, T., AND CROSA,J. H., Plasmid 2. AOKI, T., KITAO, T., AND ARAI, T., In “Plasmids, 12, 143-148 (1984). Medical and Theoretical Aspects” (S. Mitsuhashi, 17. CROSA,J. H., LUTTROPP,L. K., AND FALKOW,S., J. L. Rosvival, and V. KrEmery, eds.), pp. 39-45. Mol. Biol. 124443-460 (1978). Czechoslovak Medical Press,Prague, 1977. 18. SOUTHERN,E., J. Mol. Biol. 98,503-517 (1975). 3. AOKI, T., KITAO, T., ANDO, T., AND ARAI, T., In 19. RIGBY,P., DIM, M., RHODES,C., AND BERG, “Microbial Drug Resistance” (S. Mitsuhashi, ed.), P., J. Mol. Biol. 98, 503-517 (1977). Vol. 2, pp. 2 19-222.Japan Scientific SocietiesPress, 20. DENHARDT,D. T., B&hem. Biophys. Res. Commun. Tokyo, 1979. 23,64 l-646 ( 1966). 4. BRADLEY,D. E., AOKI, T., KITAO, T., AUI, T., AND 21. HEDGES,R. W., AND DANA, N., Nature (London) T%xIAPE, H., Plasmid 889-93 (1982). 234,220-22 1 (197 1). 5. AKAsHl A., AND AOKI, T., Bull. Sot. Sci. Fish. 52, 22. TAIT, R. C., LIJNDQUIST,R. C., AND KADo, C. I., 649-655 (1986). Mol. Gen. Genet. 186, lo-15 (1982).