- Email: [email protected]
Modular evolution of disseminated Inc 7-M plasmids encoding gentamicin resistance
(1982)
PLASMlD8,215-231
Modular Evolution of Disseminated Inc 7-M Plasmids Encoding Gentamicin Resistance’ AGNES LABIGNE-ROUSSEL,’ Laboratoire
de Biochimie,
JANINE WITCHITZ,
AND PATRICE COURVALIN~
L.A. CNRS 271, Unit6 de Bact&iologie Mdicale, 25, rue du Dr. Roux, F-75724 Paris, France
Institut Pasteur,
Received January 27, 1982 We have examined the relationship between Inc 7-M plasmids conferring resistance to gentamicin by synthesis of an acetylating enzyme (AAC(3)) and other plasmids of the same incompatibility group by agarose gel electrophoresis following digestion with restriction endonucleasesand by nucleic acid hybridization. Although isolated from different bacterial hosts over a 6-year period of time the three plasmids mediating acetylation of gentamicin are very similar. They are related to, but distinct from the other Inc 7-M plasmids. The in vivo evolution of these plasmids appeared modular, involving sequential deletions and stepwise acquisition of transposons (Tn6, Tn7, and a Tn9-like element). These results are in favor of a plasmid epidemic and indicate a great stability of the transfer factor part of these plasmids under natural conditions.
Plasmid-mediated resistance to gentamitin not associated with kanamycin-tobramycin resistance in enterobacteria wag detected in France in 1971 (Witchitz, 1972). The original resistance (R)4 plasmid (pIP135) encodes an aminocyclitol acetyltransferase 3(AAC(3)) (Umezawa et al., 1973), confers resistance to sulfonamide, tetracycline, and mercury, and belongs to incompatibility (Inc) group 7-M (Witchitz and Gerbaud, 1972). Plasmids conferring an identical resistance phenotype isolated later also belong to incompatibility group 7-M and, when compared by DNA annealing (Roussel and
Chabbert, 1978), exhibit a degree of nucleotide sequence homology (97-100%) significantly higher than that (80%) of the other members of the group. The stable association of a resistance gene with a type of plasmid led to the suggestion (Chabbert et al., 1974, 1979) that dissemination of this apparently new antibiotic resistance determinant (R determinant) in France was caused by the spread of an epidemic plasmid. Recently, Casewell et al. (1977) reported an outbreak in London caused by strains of Klebsiella aerogenes serotype 16 which, in addition to being resistant to gentamicin, sulfonamide, tetracycline, and mercury, were also resistant to ampicillin, chloramphenicol, ’ This work is dedicated to the memory of Janine kanamycin-neomycin, and trimethoprim. Witchitz. * Present address: Department of Medical MicrobiInvestigation of the plasmid content of these ology, Stanford University School of Medicine, Stanstrains (Datta et al., 1979) showed that a sinford, Ca., 94305. gle Inc 7-M plasmid was responsible for all 3To whom all correspondence should be addressed. 4 Abbreviations used: ColE 1, colicinogenic factor E 1; resistancesand that resistance to gentamicin and structurally related compounds was Xbb, Xb5 156519 ~1857 Sam7; kb, kilobase, R plasmid, antibiotic resistance plasmid; R determinant, antibiotic probably due to the presence of an AAC(3). resistance mediating gene; SlOO,supematant (lOO,OOOg) In order to assessthe extent of the Inc 7of a bacterial extract after sonication; genotypes include M plasmid epidemic we have compared, by sac(3), aminocyclitol acetyltransferase 3; aad(2”), amiagarose gel electrophoresis following digesnocyclitol adenylyltransferase 2”; aad(3”)(9), aminocytion with restriction endonucleases and by clitol adenylyltransferase 3”, 9; aph(3’)(5”), aminocyclitol phosphotransferase 3:5”. nucleic acid hybridization, plasmids pIPl35, 215
0 147-619X/82/0602 15-l 7$02.00/O
216
LABIGNE-ROUSSEL,
WITCHITZ,
AND COURVALIN
pIPl5 1 isolated a year later in a different hospital with pTH 1, a plasmid representative of the London outbreak. Inc 7-M plasmids pIP69 and pIP179 which do not code for an AAC(3) were also included in this study. (A preliminary report of part of this work was presented at the Symposium on Plasmids of Medical, Environmental and Commercial Importance, 26-28 April 1979, Spitzingsee, FRG.) The mechanisms of resistance toward aminocyclitols are described and the plasmid genesencoding antiboitic resistance are compared with those of known transposons.
(Courvalin et al., 1977) and the enzymes assayed (Haas and Dowding, 1975) as described. Preparation ofplasmid DNA. Transferable plasmid DNA was purified after pelleting from sarkosyl lysates on glycerol cushions as described (Labigne-Roussel et al., 198 1). Amplifiable DNA from ColEl factor and derivative plasmids was purified by two successive bandings in cesium chloride-ethidium bromide as described (Tanaka and Weisblum, 1975). DNA from bacteriophage Xc1857 and derivatives was prepared as described (Courvalin and Fiandt, 1980). Bacteriophages Xb515 b519 ~1857 Sam7::Tn6 MATERIALS AND METHODS and Xl, dgal ~1857 St68-OP306::ISl DNAs Plasmids. The sources and properties of were kindly provided by R. Jorgensen and the plasmids are listed in Table 1. Each plas- P. Starlinger, respectively. mid was introduced into Escherichia coli KAgarose gel electrophoresis. Plasmid DNA 12 strain BM2 1 (F-, prototroph, nalA) by was digested with restriction endonucleases conjugation as described before (Witchitz in reaction mixtures of 70 ~1 for analytical and Chabbert, 1971). purposes or of 350 ~1 for preparative gels, Media. Brain heart infusion broth and containing 100 mM Tris-HCl (pH 7.8), 100 agar (Difco) were used. Disc sensitivity tests mM MgClz for EcoRI, 6 mM Tris-HCl (pH were done on Mueller-Hinton agar medium. 7.0), 6 mM MgC 12,50 mM NaCl for ZZindIII, All incubations were at 37°C. and 15 mM Tris-HCl (pH 9.0), 6 mM MgC&, Testsfor incompatibility. Tests for incom- 15 mM KC1 for SmaI. The reactions were patibility were performed as described before allowed to proceed for 1 h at 37°C (except (Chabbert et al., 1972). for SmaI which was incubated at 25°C) then Assay for aminocyclitol-modifying en- heated at 68°C for 10 min and chilled at 0°C. zymes. The E. coli extracts were prepared The resulting DNA fragments were separated TABLE
1
PROPERTIES OF THE Inc 7-M PLASMIDS Plasmid No.
Phenotypic characters“
Genotypeb
Donor strain
PIP135
Gm Sm Su Tc Hg
sac(3)-I aad(3”)(9) tel(class C)
pIP135-I
Tc Hg
ter(class C)
PIP151
Gm Sm Su Tc Hg
sac(3)-I aad(3”X9) lef(chss C)
K. pneumoniae
pTH I
Ap Cm Gm Km Nm Sm Su Tc Tp Hg
sac(3)-I aad(3”)(9) aph(3’)(5”)-I b/a(Tem-1) tel(c1as.sC) ap/t(3’)(5”)-1 b/a(Tem-I) lef(class B)
K. aerogenes
aad(2”) bla(Tem- I)
K. pneumoniae
pIP69 PIP179
Ap Km Nm Tc Hg Ap Gm Km Tm
E. cloacae
68M K-16 R13 .S. paratyphi
B
source or reference
Origin
Hop&al Foch, Suresnes Witchitz and Gerbaud, I972 (1971) Witchitz and Spontaneous derivative Gerbaud, 1972 of PIP135 HopitaI Claude-Bernard, Roussel and Chabbert, 1978 Paris (1972) Datta et al., St. Thomas’s hospital, 1979 London ( 1977) Creteil ( 1969)
Chabbert et al.. 1972
New York (1973)
’ Nomenclature of phenotypic characters of plasmids is according to Novick et al. (1976). ’ Genetic symbols are according to Novick ef al. ( 1976) Davies and Smith ( 1978), and Mendez m al. ( 1980).
S. Schaeffler
IN VIVO EVOLUTION OF GENTAMICIN
by electrophoresis in horizontal slab gels containing 0.7, 0.8, or 1% agarose. Electrophoresis and staining were as described by Shinnick et al. (1975). Hybridization. 32P-Labeled complementary RNA (cRNA) (Courvalin and Fiandt, 1980) and nick-translated DNA (Maniatis et al., 1975) probes were prepared as described. Restriction endonuclease-generated fragments of plasmid DNA fractionated by agarose gel electrophoresis were transferred to cellulose nitrate sheets (Sartorius) as described by Southern (1975). Hybridization of the probes to the DNA immobilized on the membrane filters was as described by Denhardt (1966). Hybridization was revealed by autoradiography using XR 1-Omat Kodak film. Enzymes.Restriction endonucleasesEcoRI (Stimegi et al., 1977) and SmaI (Tanaka and Weisblum, 1975) were purified as described. Restriction endonuclease Hind111was kindly provided by J. Gardner and R. Jorgensen. Endonucleases BamHI, Sal I, and PstI, from Biolabs, were used according to the manufacturer’s recommendations. Lysozyme was from Sigma. Ribonuclease A (bovine pancreas) was from Calbiochem. Proteinase K was from Merck. E. coli K- 12 RNA polymerase holoenzyme (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) was from Miles. Chemicals. [ l-14C]Acetyl-coenzyme A ([ 14C]CoASAc), adenosine 5’-[a-32P]triphosphate, triethylammonium salt ([c~-~~P]ATP), adenosine 5’-[y-32P]triphosphate, triethylammonium salt ([Y-~~P]ATP), and [U14C]adenosine 5’-triphosphate, ammonium salt ([U-‘4C]ATP) were obtained from the Radiochemical Centre Amersham. The antibiotics were provided by the following laboratories: gentamicins (Gen) C 1a, C 1, C2, A, B, and Gen complex (26.3% Cla, 40.8% Cl, 32.9% C2), sisomicin (Sis), 2”-deoxy-sisomitin (2”-DSis), and netilmicin (Net), Schering; kanamycins (Kan) A, B, C, and amikacin (Ami), Bristol; neamine (NeoA), neomycin B (NeoB), spectinomycin (Spc), Upjohn; paromomycin (Par) and butirosin (But), ParkeDavis; tobramycin (Tob) and apramycin
R PLASMIDS
217
(Apr), Lilly; fortimycin (For), Kyowa; lividomycin (Liv) A, Kowa; ribostamycin (Rib), Meiji; streptomycin (Str), Pfizer; nalidixic acid, Winthrop. Sarkosyl (sodium lauryl sarcosinate) was provided by Colgate-Palmolive. RESULTS Classification
by Incompatibility
Plasmids pIP135 (later designated JR88; Kagan and Davies, 1980), PIP 135-1 (Witchitz and Gerbaud, 1972), pIP69 (Chabbert et al., 1972), and pTH1 (Datta et al., 1979) belong to the incompatibility group 7-M (Chabbert et al., 1972; Datta et al., 1975, 1979). Plasmids pIPl5 1 and pIP179 were also found to belong to the Inc 7-M group on the following observations. E. coli strains harboring PIP 151 or pIP69 were mixed and transfer in each direction was selected. The resident plasmid was eliminated from all transconjugants examined. Plasmids pIP179 and PIP 135-1 were shown to be incompatible in the same way. The properties of the Inc 7-M plasmids studied are summarized in Table 1 and the suspected structural interrelationships between some of them are diagrammed in Fig. 1. Mechanisms of Plasmid-Mediated Resistance to Aminocyclitol Antibiotics
E. coli strain BM21 and transconjugants were examined for aminocyclitol-modifying activities. No modifying activity was detected in the aminocyclitol-susceptible strains BM2 1 and BM2 1 harboring plasmid PIP 135-1. The aminocyclitol substrate profiles of the enzymes extracted from transconjugants are shown in Fig. 2 and Table 2. Strains harboring plasmids PIP 135, PIP 151, and pTH 1 were found to contain aminocyclitol acetyland adenylyltransferases (Table 1). Gentamicin C antibiotics and sisomicin were excellent substrates for the acetyltransferase, whereas gentamicin A and B, netilmicin, the kanamycin compounds, amikacin, and tobramycin were either poor substrates or not acetylated. Based on these substrate profiles
218
LABIGNE-ROUSSEL, WITCHITZ, AND COURVALIN G
“g A
C
plP135
co’
(79.3)
)
-DELETION
I
IN E
- 2 INSERTIONS
FIG. 1. In vivo modular evolution of Inc 7-M gentamicin R plasmids. (--), plasmid pIPI DNA; inserted DNA; (+ ), IS15; (), ISl; (-), transposon; (I), EcoRI recognition site. The size ( v), (in kilobases) of the plasmids is indicated in parentheses. EcoRI-generated DNA fragments 4 and B were arbitrarily located in the graph. The position of pIPl5 1 EcoRI fragments F and G relative to E is not known. Cm, chloramphenicol; Km, kanamycin; Gm, gentamicin; Sm, streptomycin; Su, sulfonamide; Tc, tetracycline.
and hybridization experiments (see below) the enzymes are AAC(3). The fact that fortimycin is a substrate indicates that the enzymes are of type I (AAC(3>1) (Davies and Smith, 1978). The adenylylating enzymes are active on streptomycin and spectinomycin (AAD(3”)(9)). The substrate profiles of enzymes of the same type, extracted from E. coli BM2 1 harboring pIPl35, pIPl5 1, or pTH1, are similar. Plasmids pTH 1 and PIP69 encode a phosphotransferase (Table 2). Since kanamycin B is phosphorylated and tobramycin is not,
this indicates that the 3’-hydroxyl group is the site of modification (APH(3’)); the fact that lividomycin A is modified but butirosin is not indicates that the enzymes are of type I (APH (3’)(Y)-1 (Davies and Smith, 1978). Strain BM2 1 harboring plasmid pIP179 was found to contain aminocyclitol-adenylyltransferase but no phospho- or acetyltransferase (Table 2). The fact that sisomicin is adenylylated and 2”-deoxy-sisomicin is not, indicates that the 2”-hydroxyl group is the site of adenylylation (AAD(2”)). In all these strains, the modifying enzymes
IN VZVO EVOLUTION
OF GENTAMICIN
219
R PLASMIDS
ments plus fragments B and D. Plasmid pIPl5 1 had a size of 82.4 kb and consisted
of the 16 common fragmentsplus fragments
ll
B, E, .F, and G. Plasmid pTH 1 had a size of 110.1 kb and consisted of the 16 common fragments plus fragments H to N. Plasmid pIP69 had a size of 78.5 kb and 13 EcoRI-generated DNA fragments designated 1’ to 13’; pIP179 had a size of 78.8 kb and 14 fragments (1” to 14”).
n --E.coli BM21/pIP151 :: I
Analysis of Plasmid DNA by Hybridization
Plasmid DNA was cleaved with EcoRI, HindIII, BumHI, and/or PstI restriction enTABLE FIG.
2. fhhStK3te
profiles
Of enzymes extracted
from
E. coli strain BM2 1 harboring plasmids pIPl35, pIPl5 1, or pTHl. Aeetylation (solid line) or adenylylation (dashed line) are expressed relative to gentamicin Cla or streptomycin as 10046, respectively.
were expressed constitutively as they were detected in cells grown in the absence of antibiotic. Restriction
Endonuclease Analysis of Plasmid DNA
The plasmid DNA from each transconjugant was purified by ultracentrifugation in cesium chloride-ethidium bromide, digested with EC&I restriction endonuclease, and analyzed by acrylamide (data not shown) or agarose gel electrophoresis. The results are shown in Fig. 3 and are summarized in Table 3. Comparative analysis of the EcoRI-generated fragment patterns of plasmid DNAs showed that PIP 135, PIP 135-1, PIP 151, and pTH 1 shared 16 fragments with indistinguishable size which were numbered in order of decreasing molecular weight. Fragments which were not common (extra) were designated with letters A to N. Plasmid pIP135 had a size of 79.3 kilobases (kb) and consisted of the 16 common fragments plus fragments A to C. Plasmid pIP135-1 had a size of 70.4 kb and consisted of the 16 common fmg-
2
SUFNRATEPROFXLESOFENZYMESEXTRATEDFROM E. coliS~R~r~BM21 HARBORINGPLASMIDS pTH1, pIP69, OR pIP179 Percentageof antibiotic Phosphorylat& Adenylylatcd’ pIPI
Aminocyclitol
pTH 1
pIP69
Ckntamicin C 1a Gentamicin Cl Gfmtamicin C2 Gentamicin A Gentamicin B Sisomicin 2’-Deoxy-sisomicin Netilmicin Kanamycin A Kanamycin B Kanamycin C Amikacin Tobmmycin Neomycin A Neomycin B Paromomycin Lividomycin A Butirosio Ribostamycin Stnptomycin Spectinomycin
0 0 0 44 30 1 -
-b 40 56 0 -
34 47 43 5 2 53
62 63 61 1 0 94
100
100
-
35
42
-
141
44
-
28 64
10 13
-
0 0
-
100 129 109 98 105 137 7 78 80 91 86 0 72 -
-
o Phosphorylation or adenylylation are expressed relative to neomycin B or gentamicin Cla as LOO%,respectively. No adenylylation of gentamicin complex was detected in E. coli strain BM21 harboring plaamids pIP135, pIP151, or pTH1. No phosphorylation of gentamicin complex, streptomycin, or spcctinomycin was detected in strain BM21 harboring pIP135 or pIPI and no adenylylation or acetylation of neomycin B was detccted in strain BM21 harboring pIP69. ’ Not determined.
PIP 135-1
PIP 135
PTHl
PIP 69 PIP 179 1’
1’
-
G --_--,-g-czz*-
--6-
p ____-
-a-5-
-
PIP 151
10-
---D
I -
PIP 135-1
-,-
-65
-5-
_
-
PIP 135 PIP 69 PIP 179
,
----M
XL
--.K
-,I’ 9’
-96’
-7’
-6’
-5’
--r .____ H - I, I -2’ -2’
pTH1
-33
-,2
-52 -49
-24, -2,.
x’
-62
-61
-” -211
X’
FIG. 3. Analysis by agarose gel electrophoresis of DNA from Inc 7-M plasmids. (A) Two to three micrograms of plasmid DNA were digested with EcoRI. Fragments obtained by digestion of Xc1857 DNA, 48 kb (Fiandt et al., 1977) with restriction endonucleases Hind111 (Xa) and EcoRI + Hind111 (Xb-Thomas and Davis, 1975; Robinson and Landy, 1977) were used as molecular size standard. Electrophoresis was carried out in a 1% agarose horizontal slab gel (18 X 13 X 0.4 cm) for 12 h at 3 V/cm. (B) Schematic representation of A. Number and letter designations are as explained in the text (cf. Results: Restriction endonuclease analysis of plasmid DNA). (- - -), EcoRI fragments not common to plasmids pIPl35, PIP 135 1, PIP 15 1, and pTH 1. The size of the reference hcI857 DNA fragments is expressed in kilobases. Plasmids pIP15 1, pIPl35-1, pIP135, and pTH1 EcoRI fragments 14, 15, and 16, plasmid pIP69 EcoRI fragments 1 I’, 12, and 13, and plasmid pIP179 EcoRI fragments 12”, 13”, and 14” were detected on 1.4% agarose gels only and are arbitrarily positioned in the graph.
PIP 151
IN VIVO EVOLUTION OF GENTAMICIN
221
R PLASMIDS
TABLE 3 EcoRI DIGESTIONOF Inc 7-M PLASMIDDNA pIP69
pIP135,” pIP135-1,b pIPl51,c pTHld Common fragment No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Added size
Sizee 11.6 (14) 9.4 (14) 8.5 (14) 5.6 (14) 4.9 (14) 3.2 (14) 2.7 (14) 2.4 (14) 2.0 (14) 1.6 (11) 1.3 (12) 1.3 (12) 1.2 (8) 0.75 (4) 0.6 (4) 0.55 (4)
Extra fragment letter
Size’
A B C D E F G H I J K L M N
10.7 (2) 8.5 (8) 2.5 (3) 4.3 (3) 9.9 (3) 3.6 (3) 2.8 (3) 16.8 (4) 9.3 (4) 8.8 (4) 6.3 (4) 5.9 (4) 4.2 (4) 1.2 (4)
57.6
PIP179
Fragment No.
Size’
1’ 2 3 4 5 6 7’ 8 9 10 11’ 12’ 13
22.6 (3) 11.6 (3) 9.7 (3) 8.8 (3) 7.7 (3) 6.2 (3) 3.7 (3) 3.4 (3) 1.6 (3) 1.3 (3) 0.75 (1) 0.6 (1) 0.55 (1)
Added size
78.5
Fragment No.
Size’
1” 2 3” 4 5” 6” 7” 8” 9” lo” 1I” 12 13 14”
30.5 (3) 11.6 (3) 9.0 (3) 8.6 (4) 4.9 (4) 3.7 (4) 2.4 (4) 2.0 (4) 1.6 (4) 1.3 (4) 1.3 (3) 0.75 (1) 0.6 (1) 0.55 (1)
Added size
78.8
Note. Number and letter designations are as explained in the text (cf., under Results: Restriction endonuclease analysis of plasmid DNA). * pIP135:57.6 + A + B + C = 79.3 kb. b plP135-1:57.6 + B + D = 70.4 kb. ‘pIP151:57.6 + B + E + F + G = 82.4 kb. dpTHl:57.6 + H + I + J + K + L + M + N = 110.1 kb. eThe size of the fragments (in kilobases) was estimated after agarose gel electrophoresis using Xc1857 DNA digested by HindIII, EcoRI + Hind111(Thomas and Davis, 1975; Robinson and Landy, 1977),and SmaI (Rosenvold and Honigman, 1977) as molecular size standard. The values are means from the number of determinations indicated in parentheses.
donucleasesand the resulting fragments were separated by agarose gel electrophoresis, denatured in situ, transferred to a nitrocellulose filter (driver), and hybridized to in vitro 32P-labeledplasmid or bacteriophage DNA or complementary RNA (cRNA) probes. The distribution of homologous sequences among the different fragments of plasmid DNA was then determined by autoradiography. The properties of the template DNAs for probes used to localize the R determinants and search for the presence of insertion sequences (IS) are summarized in Table 4. In each experiment, the reaction with PIP 135 cRNA probe which hybridizes with a majority of DNA fragments was used as an internal
standard. The results of the hybridization experiments are shown in Figs. 4 and 5, and summarized in Table 5.
(1) Comparison of the Inc 7-M Plasmids Plasmid PIP 135-1 has the smallest size and encodes the fewest resistances (Tc and Hg only). The probe made from this plasmid was therefore used for the comparison of the Inc 7-M transfer factors. Comparison of the electrophoretic mobilities of the EcoRI-generated plasmid DNA fragments (Fig. 3) with those of fragments which hybridized to 32P-labeled pIP135-1 cRNA (Fig. 4) showed that pIP135-1 shared
222
LABIGNE-ROUSSEL, WITCHITZ, AND COURVALIN TABLE 4 F’LAWIDS AND BACFERIOPHAGES USED AS PROBES FOR HYBRIDIZATION
Plasmid or bacteriophage
Relevant genotype”
Source or reference
ColE I pSF2 124 pCHl3’ ColEl::Tn’l pLHIC psc101 PRT~~ pBR325 Xe A: :Tn6’ X: :Tn601g X::ISlh PlCm PIP111
b&Tern- I ) aad(3@)(9),sul aad(3?9), dfr1 aad(3”)(9), sac(3)-I, sul tet(c1as.sC) tef(class B) bla(Tem- 1), cat, tet(class C) aph(3’)(5”)-I aph(Y)(S”)-I cat IS15
Bamral and Helinski ( 1972) So et al. (1975) J. Davies N. Datta Kagan and Davies ( 1980) Cohen and Chang ( 1973) Jorgensen and ReznikofF (1979) Bolivar (1978) Fiandt et al. (1977) Berg et al. (1975) Davies et al. ( 1977) P. Starlinger Kondo et al. (1962) Rouse1 et al. ( i 979) Labigne-Roussel and Courvalin (1982)
’ Genetic symbols are according to Novick et al. (1976), Davies and Smith (1978), and Mendez et al. (1980). bpCH13, ColElQ {aad(3”)(9), A}. ’ Plasmid pLH1 consists in pIPl35 EcoRI fragment A cloned in ColE I [ColElQ { aad(3”)(9), aad3)-I, sul}]. d pRT2, pVH5 1fl { tef(class B)}. e A, hcz857. /X::Tn6, Xb5156519 ~I857 Sam7::Tn6. gX::Tn601, Xb515b519 ~1857Sam7::Tn601. h X::ISl, Xl, Adgal cI857 St68-OP306::ISl.
DNA sequenceswith the 16 common fragments of pIP135, as expected, but also with those of pIP 151 and pTH 1. Plasmid PIP 135 1 labeled cRNA also hybridized to pIP135 &RI-generated DNA fragments B and C, to pIPl5 1 fragments B, F, and G and to pTH1 fragments H, K, L, and, possibly, I and N. The two latter fragments corn&rate with common EcoRI fragments 2 and 13, respectively. Plasmid pIPl35-1 cRNA hybridized to 9 pIP69 and 11 pIP179 EcoRI fragments. (2) Localization of the R Determinants and the Insertion Sequences Tetracycline R determinant. Plasmid pSClO1 (Tc) probe hybridized to the common EcoRI fragment four. The tetracycline determinant in pIP135, pIP135-1, pIP151, and pTH 1 therefore belongs to classC (Mendez et al., 1980).
Streptomycin, sulfonamide, and gentamitin R determinants. Probes made from pCHl3 (SmSu), pLH 1 (GmSmSu), and ColE 1 allowed us to assign the streptomycin, sulfonamide, and gentamicin R determinants to EcoRI fragment A of PIP 135, E of pIP151, and I of pTH1. Trimethoprim and streptomycin R determinants. The trimethoprim R determinant was localized with ColE 1::Tn7 (SmTp) cRNA probe on pTH1 EcoRI fragments H and L. The transposable element Tn7 (Barth et al., 1976) confers resistance to trimethoprim by synthesis of a dihydrofolate reductase type I (Pattishall et al., 1977) and to streptomycin by synthesis of an aad(3”)(9). There is an EcoRI site in Tn7 (Richards and Nugent, 1979) and HindIII generates two DNA fragmerits of 2.6 and 2.1 kb (Datta et al., 1981, Fig. 5E). Plasmid pTH 1 digested with Hind111 also gave rise to two DNA fragments of 2.6
IN VIVO EVOLUTION OF GENTAMICIN
R PLASMIDS
223
activities in plasmid pTH1 are structurally related and that, under our conditions, the gene encoding streptomycin resistance in Tn7 is not transcribed in vitro. Ampicillin R determinant. Plasmid pTH 1 encodes a TEM- 1 P-lactamase (Table 1). Datta et al. (1979) found that at least part of the ampicillin R determinant in pTH1 is located in EcoRI fragment K (fragment number five in their nomenclature) and in our experiments the pSF2 124 (Ap) probe hybridized to fragments J and K of pTH1. The widespread Tn3 element contains a BamHI site, but no EcoRI site (Heffron and McCarthy, 1979). In order to discriminate between the presence in pTH1 of two ampiPIP PIP DRIVERS PIP PIP PIP pTH1 69 179 135 135-1 151 (EcoRI) cillin R determinants as in the case of streptomycin resistance, or the occurrence of an FIG. 4. Analysis of plasmid DNA by hybridization. Ten micrograms of the driver plasmid DNAs indicated EcoRI site in the region associated with the on the bottom line were digested with EcoRI restriction P-lactamase, pSF2 124 (Ap) cRNA was hyendonuclease. The resulting DNA fragments were frac- bridized with pTH1 DNA digested with tionated by electrophoresis through horizontal slab gels BamHI or BamHI plus EcoRI (Fig. 5E). The (0.7% agarose, 18 X 13 X 0.4 cm) for 12 h at 3 V/cm, transferred to a nitrocellulose sheet, and hybridized to pSF2 124 (Ap) probe hybridized to two fragin vitro 32P-labeledplasmid PIP 135-1 cRNA as indicated ments of pTH1 digested with BamHI and on the top line (probe). The autoradiogram was exposed three fragments after digestion with BamHI for a period of 12 h. Letter and number designations are plus EcoRI, whereas the presence of two amas explained in the text (cf., Results: Restriction endopicillin R determinants should result in the nuclease analysis of plasmid DNA). hybridization with four pTH 1 BamHI-generated DNA fragments. The EcoRI site in, or close to, the gene specifying the P-lactaand 2.1 kb which shared homology with Tn7 mase could be due to the insertion, in the (Fig. 5E). This observation (Datta et al., Tn3 element, of a DNA fragment possessing 1981) confirms that, as suggested (Datta et a recognition site for this restriction endoal., 1979) on the basis of molecular weight nuclease. and restriction enzyme analysis of plasmid Chloramphenicol R determinant and ISI. DNA, the entire Tn7 element is present in Bacteriophages PlCm (Cm ISl) and h::ISl plasmid pTH 1. Plasmid pTH 1 therefore en- cRNAs hybridized to pTH1 EcoRI DNA codestwo a&( 3”)(9) activities. Hybridization fragments J and M (Fig. 5D) indicating the of pCH 13 (SmSu) and, probably, pLH1 presence in this plasmid of two copies of a (GmSmSu) probes to pTH 1 EcoRI fragment sequence homologous to IS1 separated by a L is due to the presence, in this fragment, of minimum of one EcoRI recognition site. The the portion of Tn7 coding for the adenyly- pBR325 (ApCmTc) probe hybridized to lating enzyme. Conversely, because of the pTH1 EcoRI fragments 4 (because of the presence in these fragments of the strepto- presence in this fragment of the tetracycline mycin R determinant, plasmid ColEl ::Tn7 resistance gene), J and K (because of honick-translated DNA also hybridized to mology with the ampicillin determinant) and EcoRI fragments A of pIPl35, E of pIPl5 1, M showing that at least part of the chlorand, most likely, I of pTH1 whereas the amphenicol determinant is located in this ColEl::Tn7 cRNA probe did not. These ob- fragment (data not shown). servations indicate that the two adenylylating DNA from plasmids pTH1 and ColEl:: PROBE 0
plP135-1
224
LABIGNE-ROUSSEL, WITCHITZ, AND COURVALIN
PROBES
ORIVERS FIG. 5. AP&& of p&,Mid DNA by hybridization. Ten micrograms of the driver plasmid DNAs were digested with restriction eedonucleases as indicated on the bottom line. The resulting DNA fragments were fractionated by electrophoresis (same conditions as in Fig. 31,transferred to a nitiocellulose sheet, and hybridized to in vitro 32P-laheIedplasmid cRNA as indicated on the top line (probes). The autora-
IN VW0 EVOLUTION OF GENTAMICIN
Tn9 (Cm ISl) was digested with PstI restriction endonuclease, compared by agarosegel electrophoresis, and hybridized, following transfer on a nitrocellulose sheet, to X::ISl, pBR325 (ApCmTc), and pSF2124 (Ap) probes. Transposable element Tn9 (Rosner and Gottesman, 1977) consists of a chloramphenicol determinant flanked by two copies of IS1 in direct orientation. Insofar as there is a single EcoRI recognition site in the structural gene for the chloramphenicol acetyltransferase and IS 1 contains a unique P.stI restriction site, Tn9 DNA generates upon digestion a 1.75kb PstI fragment and two EcoRI/PstI fragments 1.2 and 0.55 kb in size. Plasmid pTH1 restricted with PstI did not give rise to the characteristic Tn9 fragment but to a slightly larger one ( 1.95 kb) identified on the basis of homology with X::ISl and pBR325 (ApCmTc) but not with pSF2 124 (Ap) (data not shown). Plasmid pTH1 DNA was further digested by PstI plus EcoRI and hybridized, after electrophoresis in an agarose gel, with the same three probes. Since they each contain a copy of IS 1, and therefore a PstI site, both EcoRI fragments J and M disappeared in this analysis. Two new smaller fragments hybridized with X::ISl and pBR325 (ApCmTc) but not with the pSF2 124 (Ap) probe demonstrating that the structural gene for chloramphenicol resistance, as in Tn9 (Rosner and Gottesman, 1977), has one EcoRI recognition site and is flanked by two copies of IS 1. In conclusion, plasmid pTH1 contains a transposon closely related to but distinct from Tn9. The function, if any, coded for by the additional 200 bp in the central portion of this element remains unknown. Kanamycin R determinant and IS1.5. The kanamycin determinant was localized with X::Tn60 1 and XcRNA probes on EcoRI fragment J of pTH 1. Transposable elements Tn6 (Berg et al., 1975) and Tn601 (Davies et al.,
R PLASMIDS
225
1977), later designated Tn903 (Nomura et
al., 1978) (Table 4), confer resistance to kanamycin by synthesis of an amino glycoside phosphotransferase (3’)( 5”) type I (aph(3’)(5”) I) (Haas and Dowding, 1975), similar to that encoded by pTH1 (Table 2). The structural gene for the phosphotransferase in the 3.15-kb Tn60 1 element is flanked by two inverted copies of IS903, 1.05 kb in length (Kopecko et al., 1976; Courvalin et al., 1979). The 4. I-kb Tn6 element contains two terminal directly repeated copies of IS 15 (Labigne-Roussel et al. (1981) and (1982). Analysis of heteroduplexes by electron microscopy and hybridization experiments (Courvalin et al., 1979) indicated that the 1.05-kb structural genes for the phosphotransferases in Tn601 and Tn6 are homologous. As expected, the X::Tn6 probe hybridized to the kanamycin R determinant of pTH1, but, as opposed to X::Tn60 1 cRNA also hybridized to EcoRI fragments K of pTH1, C of pIP135, D of pIP135-1, and F and G of PIP 151. The existence of IS 15 in EcoRI-generated DNA fragments D of pIPl35-1 and J and K of pTH 1 was confirmed with a probe made from PIP 111. This IncIl plasmid does not confer resistance to kanamycin (Chabbert and Gerbaud, 1974) and contains one copy of IS15 (LabigneRoussel and Courvalin, 1982). There is a Hind111recognition site in the kanamycin R determinant of Tn6 (Berg et al., 1975). Plasmid pTH1 DNA was digested with EcoRI + Hind111and hybridized with X::Tn60 1 and pIPll1 probes (data not shown). The two pTH1 fragments which shared homology with Tn60 1 also hybridized with PIP 111 indicating the presence of two copies of IS 15 and therefore of an entire Tn6 element in pTH1 EcoRI fragment J. Bacteriophage Xc1857and Colicin E 1 factor cRNAs, used as controls, and probe from pRT2 did not hybridize to any of the plasmids studied.
diograms were exposed for a period of 12 h. Letter and number designations are as explained in the text (cf., Results: Restriction endonuclease analysis of plasmid DNA). EcoRI-generated DNA fragments 14 to 16, 11’ to 13’, and 12” to 14” are not seen in this particular analysis.
+ ? + + ?
H I J K L M N
pTH 1
+ + + -
+ +
-
-
-
+ + -
+ + -
,,-.,-
-
+ + -
+ +
+
+
A: :Tn6 (KmIS1522)
+ + -
ND ND ND ND
+
ND ND ND
PIP111 (IS15)
---
---
), indicates a weak hybridization relative
+ -
-
-
-
-
X::Tn601 (Km)
X::ISl
’ f, Positive result; -, negative result; ?, indicates comigration with an EcoRI fragment sharing homology with PIP 135-1; ( to the others; ND, not determined. b ColEl, pRT2, and Xc1857cRNA probes gave negative results. ’ Result positive with the nick-translated DNA probe and negative with the cRNA probe.
(+) -
+ -
+ + -
+ ? + + ?
+ + + +
B E F G
pIP151
($ + -
+c -
+ -
+ -
+ + +
+ +
B D
pIP135-1 -
-
-
+
-
+ +
-
-
-
+’ -
+ -
PlCm (CmISl)
pBR325 (ApCmTc)
pSF2124 (AP)
+ -
ColEl::Tn7 (Sm’W
+ +
pLH 1 (GmSmSu)
+ + +
pCH13 (SmSu)
pIP135-1
pIP135
A B C
EcoRI fragment
pIP135
Driver
Probe (Relevant characters)h
SUMMARY OF THE RESULTSOF THE HYBRIDIZATIONS~
TABLE 5
g T F 2
5
E 3 P E 1 2 =i “N
5
5
F
IN VW0 EVOLUTION OF GENTAMICIN
(3) Evolutionary Relationships among the 7-M Plasmids Plasmid pIPl35- 1 is a spontaneous derivative of pIP135 obtained in the laboratory (Witchitz and Gerbaud, 1972). This plasmid arose from an 8.9-kb deletion which fused pIP135 EcoRI fragments A (10.7 kb) and C (2.5 kb) to generate pIP135-1 EcoRl fragment D (4.3 kb) (Fig. 2). As already mentioned, this new DNA fragment retained the pIP 135 EcoRI fragment C copy of IS 15 and similar adjacent deletions induced by this insertion sequence have already been observed (Labigne-Roussel et al., 1981). Plasmid pLH1 (GmSmSu) cRNA hybridized to pIP135-1 EcoRI fragment D, but probes from pCH 13 (SmSu) and ColEl::Tn7 (SmTp) did not. Plasmid pIPl35- 1 does not confer resistance to gentamicin (nor to streptomycin and sulfonamide) and we did not detect any acetylating activity in cells harboring this plasmid. It appears therefore that a vestige of the PIP 135 EcoRI fragment A gene encoding the AAC(3) I enzyme is present in pIP135-I EcoRI fragment D but is not expressed in vivo. The fact that pIPl35- 1 cRNA did not hybridize to pIP135 EcoRI fragment A is probably due to a similar lack of transcription in vitro. As opposed to PIP 135-1, plasmid PIP 151 (82.4 kb) is larger than pIP135 (79.3 kb). Plasmid pIPl5 1 EcoRI fragment E which contains the gentamicin, streptomycin, and sulfonamide R determinants is smaller (by 0.8 kb) than pIP135 EcoRI fragment A (Fig. 3). The filiation between these two DNA fragments is confirmed by the fact that they both contain two BamHI recognition sites flanking DNA fragments which precisely differ in size by 0.8 kb (data not shown). In pIPl51, plasmid pIP135 EcoRI fragment C is replaced by the larger fragments F and G which share homology with PIP 135. This change in the EcoRI-generated fragment pattern of plasmid DNA may be due to IS15 mediated rearrangements within fragment C associated with a duplication of this insertion sequence. Presumably, for the same reason as for
R PLASMIDS
221
PIP 135 EcoRI fragment A, plasmid pIP 1351 cRNA did not hybridize to PIP 151 EcoRI fragment E. Plasmid pTH1 is also larger, by 30.8 kb, than plasmid pIP135 and seven additional EcoRI-generated DNA fragments replace the three noncommon EcoRI fragments of pIP135 (Fig. 3). Plasmid pTH1 EcoRI fragment I is responsible for resistance to gentamicin, streptomycin, and sulfonamide and has a size reduced by 1.4 kb compared to that of pIP135 EcoRI fragment A. The two BamHI recognition sites in fragment I delimit a DNA segment with migration properties indistinguishable from those of the BamHI fragment which originates from pIPl5 1 EcoRI fragment E. This latter fragment could therefore represent an intermediate step between pIP135 EcoRI fragment A and pTH1 EcoRI fragment I. The second deletion leading from E to I must be located outside the internal BamHI fragment. Based on molecular weight determinations, the most likely candidate for integration of the transposable element Tn7 (14 kb, Table 4) which overlaps fragments H (16.8 kb) and L (5.9 kb) is PIP 135 EcoRI fragment B (8.5 kb, Table 3). Compatible with this event is the fact that fragments H and L share homology with PIP 135 cRNA. Integration of Tn7 in PIP 135 EcoRI fragment B rules out, by lack of space,the presencein pTH 1 EcoRI fragment L of genes mediating resistance to gentamicin and sulfonamide. Because plasmids pCH 13 (SmSu) and pLH 1 (GmSmSu) carry aad(3”)(9) encoding genes homologous to that of Tn7 in EcoRI fragment L we could not eliminate the presence, also in this DNA fragment, of gentamicin and sulfonamide R determinants. The mapping of the remaining four noncommon EcoRI-generated fragments of plasmid pTH 1 is shown in Fig. 1 and was based on the following observations. -Acquisition of the R determinants results from the insertion of a DNA fragment into pIP135 EcoRI fragment C, which disappears. -As already discussed, EcoRl fragments
228
LABIGNE-ROUSSEL, WITCHITZ, AND COURVALIN
A and C are contiguous in plasmid pIPl35, and pTH1 EcoRI fragment I derives from PIP 135 EcoRI fragment A. Therefore EcoRI fragment I is adjacent to the insert in plasmid pTH1. -As found in pIP135 EcoRI fragment C, plasmid pTH1 EcoRI fragment K contains a copy of IS15 and thus is next to fragment I. -Fragments K and J are adjacent because Tn3 hybridized to both DNA fragments. -Fragments J and M are also adjacent in so far as the structural gene for the chloramphenicol acetyltransferase overlaps these two fragments. Plasmid pTH1 EcoRI fmgment J contains two copies of IS 15 but, being flanked by fragments K and M, this fragment cannot be adjacent to fragment I. -Plasmid pIP135 cRNA did not hybridize to pTH1 EcoRI fragment M thus indicating that M is not a junction fragment, i.e., one of the two restricted fragments composed of DNA from the insert fused with that of the pIP135 receptor. The cryptic EcoRI fragment N, which likely shares homology with plasmid pIP135, is therefore the secondjunction fragment. In all the plasmids studied, because we have no probe to trace this R determinant, the location of the genesconferring resistance to mercury remains unknown.
known plasmid of this type (Witchitz and Gerbaud, 1972). The second plasmid, PIP 151 (Roussel and Chabbert, 1978) confers to the host an antibiotic resistance phenotype indistinguishable from that due to PIP 135 and was isolated a year later in a different hospital. The third plasmid, pTH 1, was detected more recently (Casewell et al., 1977) in Great-Britain and mediates resistance to an expanded number of antibiotics. Plasmids pIPl35, pIPl5 1, and pTH 1, encode aminocyclitol acetylating enzymes with identical site specificity and indistinguishable substrate profiles (Fig. 2). Comparison by restriction endonuclease-generated patterns followed by nucleic acid hybridizations indicated that these three plasmids were similar (Figs. 3 and 4) and that they were related (Fig. 4), but distinct (Fig. 3) from the other Inc 7-M plasmids (e.g., pIP69 and pIP179). This finding confirms an earlier observation (Roussel and Chabbert, 1978) based on quantitative DNA annealing studies and constitutes further support for the notion of plasmid epidemic (Chabbert et al., 1974, 1979). Plasmid pTH 1 could derive from plasmid pIP135 following at least four distinct events: two sequential deletions in EcoRI fragment A and stepwiseacquisition of Tn7 and a large insert responsible for the remaining resistances. Assuming that plasmid pTH 1 harbors the entire Tn6 and Tn7 transposable eleDISCUSSION ments, and also a Tn9-like element, we can We have compared plasmids conferring account for 25.7 kb out ofthe 29.4 additional resistance to gentamicin by synthesis of an kb compared to plasmid pIP135. The funcacetylating enzyme and belonging to incom- tion(s), if any, encoded by the remaining 3.9 patibility group 7-M with other plasmids of kb is unknown. In any case, whatever the the same group. We focused our attention on explanation may be for the remaining structhe study of the epidemiology of this mech- tural differences, plasmid pIP135 is not the anism of resistance to aminocyclitols because direct progenitor of pTH1 and plasmid it first appeared among enterobacteria in our pIPI 1 appears as the fossil of an intermecountry (Witchitz and Gerbaud, 1972) and diate state in this in vivo modular evolution. we were therefore able to trace its subsequent Such a modular evolution has been proposed spread. The three Inc 7-M plasmids mediat- in other systems(Sharp et al., 1973; Kopecko ing acetylation of gentamicin included in this et al., 1976; Susskind and Botstein, 1978). The transition from plasmid pIPI to study were isolated from different bacterial hosts (Table 1) and were selected on the fol- plasmid pTH 1 provides an example of struclowing criteria. Plasmid pIP135 is the first tural stability combined with genetic flexi-
IN VIVO EVOLUTION OF GENTAMICIN
bility under natural conditions. The striking similarity between pIPl35, pIPl5 1, and pTH 1 reflects a high degree of stability of the transfer factor part of PIP135 in vivo over at least 6 years. By contrast, acquisition, during the same period of time, of various genesled to resistance toward major groups of antibiotics (certain @-lactams,chloramphenicol, the remaining aminocyclitols, and trimethoprim). The new selective advantages conferred by pTH1 represent an addition to the potential of plasmid pIPl35: there was no insertion-inactivation of any preexisting R determinant nor of any transfer gene upon acquisition of the new genetic information. With the exception of transposable element Tn7, the additional R determinants are clus’ tered on the DNA molecule (Fig. 1). As op- posed to pIP135, plasmid pTH1 possesses two copies of a gene encoding a streptomytin-spectimomycin adenylyltransferase (Fig. 5E), an enzyme which confers low-level resistance toward the two antibiotics. The adenylyltransferase levels in cells harboring pTH1 are two to three times higher than those found in cells harboring pIP135. The MICs of streptomycin and spectinomycin for E. coli containing pTH1 are higher than when it contains pIP135. These variations could also be explained by differences between the copy numbers of the two plasmids. However this eventually seemsmost unlikely since the acetyltransferase levels, and subsequent aminocyclitol resistance phenotypes, mediated by pIP135 and pTH1 are identical (data not shown). The resistance phenotype conferred by pTH1, compared to that due to the presence of plasmid pIPl35, is therefore not only qualitatively but also quantitatively altered. Similar gene dosage effects have already been reported for aminocyclitol-modifying enzymes (Courvalin and Fiandt, 1980). Gentamicin resistance by acetylation of the 3-amino group of the antibiotic molecule has long been associated exclusively with Inc 7-M plasmids in our observations. This stable linkage was not in favor of the location of this resistance gene on a transposable ele-
R PLASMIDS
229
ment (Calos and Miller, 1980). In fact, we have no evidence for transposition of gentamicin resistance from the Inc 7-M plasmids studied. However, recently, four transposons conferring resistance to gentamicin by a similar mechanism have been described (Rubens et al., 1979a,b; Datta et al., 1981). It is likely that the Inc 7-M plasmids, which already contain a minimum of one copy of the ubiquitous insertion sequence IS 1522 (LabigneRoussel and Courvalin, 1982) represent the pool of origin of these transposable elements. In order to test this hypothesis it will be of interest to determine the structures of these various transposons and to compare them with that of the corresponding portion of a putative Inc 7-M ancestor. Theoretically, any structural gene can be located on a transposon, provided that selective pressures are exerted. This prerequisite is obviously fulfilled in certain ecosystemsfor gentamicin resistance and may well be at the “origin” of these presumably recently evolved transposable elements. ACKNOWLEDGMENTS We thank N. Datta and S. Schaeffler for gift of strains, C. Carlier for the assaysof the aminocyclitol-modifying enzymes, E. Collatz for isoelectric point determination, 0. Rouelland for secretarial assistance,and Y. A. Chabbert for material support. This work was supported by a grant (ATP 72.79.104) from the Institut National de la Sante et de la Recherche Medicale (INSERM).
REFERENCES BARTH, P. T., DA?TA, N., HEDGES,R. W., AND GRINTER,N. J. ( 1976).Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons. J. Bacterial. 125, 800-8 10. BAZARAL,M., AND HELINSKI, D. R. (1972). Characterization of multiple circular DNA forms of colicinogenie factor El from Proteus mirabilis. Biochemistry 7,3513-3519. BERG, D. E., DAVIES, J., ALLET, B., AND ROCHAIX, J. D. (1975). Transposition of R factor genes to bacteriophage X. Proc. Nat. Acad. Sci. USA 72, 36283632. BOLIVAR, F. (1978). Construction and characterization of new cloning vehicles. Gene 4, 12I- 136.
230
LABIGNE-ROUSSEL, WITCHITZ, AND COURVALIN
CALOS, M. P., AND MILLER, J. H. (1980). Transposable
elements. Cell 20, 519-595. CASEWELL, M. W., DALTON, M. T., WEBSTER,M., AND PHILLIPS, I. (1977). Gentamicin-resistant Klebsiella aerogenes in a urological ward. Lancet 2, 444-446. CHABBERT,Y. A., AND GERBAUD, G. R. (1974). Surveillance tpidemiologique des plasmides responsables de la resistance au chloramphenicol de Salmonella typhi. Ann. Microbial. (Inst. Pasteur) 125A, 153- 166. CHABBERT,Y. A., ROUSSEL,A., WITCHITZ, J. L., SANSON-LE PORS, M. J., AND COURVALIN, P. (1979). Restriction endonuclease generated patterns of plasmids belonging to incompatibility groups 11, C, M, and N: Application to plasmid taxonomy and epidemiology. In “Plasmids of Medical, Environmental, and Commercial Importance” (Timmis and Piihler, eds.), pp. 183-193. Elsevier/North-Holland, Amsterdam/New York/Oxford. CHABBERT,Y. A., SCAVIZZI,M. R., WITCHITZ, J. L., GERBAUD,G. R., AND BOUANCHAUD,D. H. (1972). Incompatibility groups and the classification of Firesistance factors. J. Bacterial. 112, 666-675. CHABBERT,Y. A., WITCHITZ, J. L., AND GERBAUD, G. R. (1974). Epidemics of R factors mediating gentamicin resistance and belonging to different incompatibility groups. In “Progress in Chemotherapy-I” (G. K. Daikos, ed), pp. 22-28. Hellenic Society of Chemotherapy, Athens. COHEN, S. N., AND CHANG, A. C. Y. (1973). Recircularization and autonomous replication of sheared Rfactor DNA segments in Escherichia coli transforman@. Prof. Nat. Acad. Sci. USA 70, 293-297. COURVALIN, P., AND FIANDT, M. (1980). AminogJycoside-modifying enzymes of Staphylococcus aureus: Expression in Escherichia coli. Gene 9, 247-269. COURVALIN, P., FIANDT, M., AND DAVIES, J. (1979). DNA relationships between genes coding for aminoglycoside-modifying enzymes from antibiotic-producing bacteria and R plasmids. Zn “Microbiology1978” (D. Schlessinger,ed.), pp. 262-266. Amer. Sot. for Microbial., Washington, D. C. COURVALIN,P., WEISBLIJM,B., AND DAVIES,J. (1977). Aminoglycoside-modifying enzyme of an antibioticproducing bacterium acts as a determinant of antibiotic resitance in Escherichia coli. Proc. Nat. Acad. Sci. USA 74, 999-1003. DATTA, N. (1975). Epidemiology and classification of plasmids. In “Microbiology-1974” (D. Schlessinger, ed.), pp. 9-15. Amer. Sot. for Microbial., Washington, D. C. DAT~A, N., HUGHES, V. M., NUGENT, M. E., AND RICHARDS,H. (1979). Plasmids and transposons and their stability and mutability in bacteria isolated during an outbreak of hospital infection. Plasmid 2, I82196. DATTA, N., NUGENT, M., AND RICHARDS,H. (1981). Transposons encoding trimethoprim or gentamicin resistancein medically important bacteria. Cold Spring Harbor Symp. Quant. Biol. 45, 45-5 1. DAVIES, J., BERG, D., JORGENSEN,R., FIANDT, M.,
HUANG, T. S. R., COURVALIN,P., AND SEHLOFF,J. ( 1977). Transposable neomycin phosphotransferases. In “R-factors: Their properties and possible control” (Drews and Hiigenauer, eds.), pp. 101-l 10. SpringerVerlag, Wien/New York. DAVIES,J., AND SMITH, D. I. (1978). Plasmid-mediated resistance to antimicrobial agents. Annu. Rev. Microbiol. 32, 469-5 18. DENHARDT,D. T. ( 1966). A membrane-tilter technique for the detection of complementary DNA. B&hem. Biophys. Res. Commun. 23, 641-646. FIANDT, M., HONIGMAN, A., ROSENVOLD,E. C., AND SZYBALSKI,W. (1977). Precise measurement of the b2 deletion in coliphage lambda. Gene 2, 289-293. HAAS, M. J., AND DOWDING,J. E. (1975). Aminoglycoside modifying enzymes. In “Methods in Enzymology” (J. H. Hash, ed.), Vol. 43, pp. 6 1l-628. Academic Press, New York. HEFFRON,F., AND MCCARTHY, J. M. (1979). DNA sequence analysis of the transposon Tn3: Three genes and three sites involved in transposition of Tn3. Cell 8, 1153-I 163. JORGENSEN,R. A., AND REZNIKOFF,W. S. (1979). Organization of structural and regulatory genes that mediate tetracycline resistance in transposon TnlO. J. Bacterial. 138, 705-7 14. KAGAN, S. A., AND DAVIES, J. E. (1980). Enzymatic modification of aminocychtol antibiotics: Mutations affecting the expression of aminocyclitol acetyltransferase-3. Plasmid 3, 3 12-3 18. KUNDO, E., HARADA, K., AND MITSUHASHI,S. (1962). Drug-resistance of enteric bacteria. 12. Transduction of the transmissible drug resistance factor by bacteriophage Pl kc. Japan J. Exp. Med. 32, 139-147. KOPECKO,D. J., BREVET,J., AND COHEN,S. N. (1976). Involvement of multiple translocating DNA segments and recombinational hotspots in the strnctural evolution of bacterial plasmids. J. Mol. Biol. 108, 333360.
LABIGNE-ROUSSEL,A., GERBAUD, G. R., AND COURVALIN, P. (198 1). Translocation of sequencesencoding antibiotic resistance from the chromosome to a receptor plasmid in Salmonella ordonez. Mol. Gen. Genet. 182, 390-408. LABIGNE-ROUSSEL,A., BRIAUX-GERBAUD, S., AND COURVALIN,P. (1982). Tn1525, a kanamycin R determinant flanked by two direct copies of IS 15. Mol. Gen. Genet., in press. LABIGNE-ROUSSEL, A., AND COURVALIN, P. (1982). A new insertion sequence widely spread in R plasmids of gram negative bacteria. Mol. Gen. Genet., in press. MANIATIS, T., JEFFREY,A., AND KLEID, D. G. (1975). Nucleotide sequence of the rightward operator of phage h. Proc. Nat. Acad. Sci. USA 72, 1184-I 188. MENDEZ, B., TACHIBANA,~., AND LEVY, S. B.(1980). Heterogeneity of tetracycline resistance determinants. Plasmid 3, 99- 108. NOMURA, N., YAMAGISHI, H., AND OKA, A. (1978). Isolation and characterization of transducing coli-
IN VZVO EVOLUTION OF GENTAMICIN phage fd carrying a kanamycin resistance gene. Gene 3, 39-5 1. NOVICK, R. P., CL~WES,R. C., COHEN,S. N., CURTIS% R., III, DATTA, N., AND FALKOW,S. (1976). Uniform nomenclature for bacterial plasmids: A proposal. Butteriol. Rev. 40, 168-189. PATTISHALL, K. H., ACAR, J. F., BURCHALL, J. J., GOLDSTEIN,F. W., AND HARVEY, R. J. (1977). Two distinct types of Tmp-resistant dihydrofolate reductase specified by R. plasmids of different compatibility groups. J. Biol. Chem. 252, 2319-2323. RICHARDS,H., AND NUGENT, M. (1979). The incidence and spread of transposon Tn7. In “Plasmids of Medical, Environmental and Commercial Importance” (Timmis and Puhler, eds.), pp. 195-198. Elsevier/ North-Holland, Amsterdam/New York/Oxford. ROBINSON, L. H., AND LANDY, A. (1977). HindII, Hind111 and HpaI restriction fragment maps of bacteriophage X DNA. Gene 2, l-3 1. ROSENVOLD,E. C., AND HONIGMAN, A. (1977). Map ping of AvaI and XmuI cleavage sites in bacteriophage DNA including a new technique of DNA digestion in agarose gels. Gene 2, 273-288. ROSNER,J. L., AND GO~MAN, M. M. (1977). Transposition and deletion of Tn9: A transposable element carrying the gene for chloramphenicol resistance. In “DNA Insertion Elements, Plasmids and Episomes” (A. I. Bukhari, J. A. Shapiro, and S. C. Adhya, eds.), pp. 2 13-2 18. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. ROUSSEL,A., CARLIER, C., GERBAUD,G., CHABBERT, Y. A., CROISSANT,O., AND BLANGY, D. (1979). Reversible translocation of antibiotic resistance determinants in Salmonella ordonez. Mol. Gen. Genet. 169, 13-25.
ROUSSEL,A. F., AND CHABBERT,Y. A. (1978). Taxonomy and epidemiology of Gram-negative bacterial plasmids studied by DNA-DNA filter hybridization in formamide. J. Gen. Microbial. 104, 269-276. RUBENS,G. E., MCNEILL, W. F., AND FARRAR,W. E., JR. (1979a). Transposable plasmid deoxyribonucleic acid sequence in Pseudomonas aeruginosa which mediates resistance to gentamicin and four other antimicrobial agents. J. Bacterial. 139, 877-882. RUBENS,G. E., MCNEILL, W. F., AND FARRAR,W. E., JR. (I 979b). Evolution of multiple-antibiotic-resis-
R PLASMIDS
231
tancc plasmids mediated by transposable plasmid deoxyribonucleic acid sequence. J. Bacterial. 140, 7 13719. SHARP,P. A., COHEN,S. N., AND DAVIDSON,N. (1973). Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. J. Mol. Biol. 75, 235-255. SHINNICK,T. M., LUND, E., SMITHIES,O., AND BLATTNER, F. R. (1975). Hybridization of labeled RNA to DNA in agarosegels. Nucl. Acids Res. 2, 1911-1929. So, M., GILL, R., AND FALKOW,S. (1975). The generation of a ColE I -Ap cloning vehicle which allows detection of insert DNA. Mol. Gen. Genet. 142, 239249.
SOUTHERN,E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-5 17. SUMEGI, J., BREEDVELD,D., HOSSENLOPP,P., AND CHAMBON, P. (1977). A rapid procedure for purification of EcoRl endonuclease. Biochem. Biophys. Res. Commun. 16, 78-85. SUSSKIND,M. M., AND BOTSTEIN,D. (1978). Molecular genetics of bacteriophage P22. Microb. Rev. 42, 385413. TANAKA, T., AND WEISBLUM,B. (1975). Construction of colicin El-R factor composite plasmid in vitro: Means for amplification of deoxyribonucleic acid. J. Bacterial. 121, 354-362. THOMAS,M., AND DAVIS, R. W. (1975). Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease. J. Mol. Biol. 91, 315-328. UMEZAWA,H., YAGISAWA,M., MATSUHASHI,Y., NAGANAWA,H., YAMAMOTO, H., KONDO, S., TAKEUCHI, T., AND CHABBERT,Y. A. (1973). Gentamicin acetyltransferase in Escherichia coli carrying R factor. J. Antibiot. (Tokyo) 26, 6 12-6 13. WITCHITZ, J. L. (1972). Plasmid-mediated gentamicin resistance not associated with kanamycin in Enterobacteriaceae. J. Antibiot. (Tokyo) 25, 622-624. WITCHITZ, J. L., AND CHABBERT,Y. A. (197 1). Rbistance transferable & la gentamicine. I. Expression de la resistance.Ann. Inst. Pasteur 121, 733-742. WITCHITZ, J. L., AND GERBAUD,G. R. (1972). Classification de plasmides con&ant la resistance & la gentamicine. Ann. Inst. Pasteur 123, 333-339.
PLASMlD8,215-231
Modular Evolution of Disseminated Inc 7-M Plasmids Encoding Gentamicin Resistance’ AGNES LABIGNE-ROUSSEL,’ Laboratoire
de Biochimie,
JANINE WITCHITZ,
AND PATRICE COURVALIN~
L.A. CNRS 271, Unit6 de Bact&iologie Mdicale, 25, rue du Dr. Roux, F-75724 Paris, France
Institut Pasteur,
Received January 27, 1982 We have examined the relationship between Inc 7-M plasmids conferring resistance to gentamicin by synthesis of an acetylating enzyme (AAC(3)) and other plasmids of the same incompatibility group by agarose gel electrophoresis following digestion with restriction endonucleasesand by nucleic acid hybridization. Although isolated from different bacterial hosts over a 6-year period of time the three plasmids mediating acetylation of gentamicin are very similar. They are related to, but distinct from the other Inc 7-M plasmids. The in vivo evolution of these plasmids appeared modular, involving sequential deletions and stepwise acquisition of transposons (Tn6, Tn7, and a Tn9-like element). These results are in favor of a plasmid epidemic and indicate a great stability of the transfer factor part of these plasmids under natural conditions.
Plasmid-mediated resistance to gentamitin not associated with kanamycin-tobramycin resistance in enterobacteria wag detected in France in 1971 (Witchitz, 1972). The original resistance (R)4 plasmid (pIP135) encodes an aminocyclitol acetyltransferase 3(AAC(3)) (Umezawa et al., 1973), confers resistance to sulfonamide, tetracycline, and mercury, and belongs to incompatibility (Inc) group 7-M (Witchitz and Gerbaud, 1972). Plasmids conferring an identical resistance phenotype isolated later also belong to incompatibility group 7-M and, when compared by DNA annealing (Roussel and
Chabbert, 1978), exhibit a degree of nucleotide sequence homology (97-100%) significantly higher than that (80%) of the other members of the group. The stable association of a resistance gene with a type of plasmid led to the suggestion (Chabbert et al., 1974, 1979) that dissemination of this apparently new antibiotic resistance determinant (R determinant) in France was caused by the spread of an epidemic plasmid. Recently, Casewell et al. (1977) reported an outbreak in London caused by strains of Klebsiella aerogenes serotype 16 which, in addition to being resistant to gentamicin, sulfonamide, tetracycline, and mercury, were also resistant to ampicillin, chloramphenicol, ’ This work is dedicated to the memory of Janine kanamycin-neomycin, and trimethoprim. Witchitz. * Present address: Department of Medical MicrobiInvestigation of the plasmid content of these ology, Stanford University School of Medicine, Stanstrains (Datta et al., 1979) showed that a sinford, Ca., 94305. gle Inc 7-M plasmid was responsible for all 3To whom all correspondence should be addressed. 4 Abbreviations used: ColE 1, colicinogenic factor E 1; resistancesand that resistance to gentamicin and structurally related compounds was Xbb, Xb5 156519 ~1857 Sam7; kb, kilobase, R plasmid, antibiotic resistance plasmid; R determinant, antibiotic probably due to the presence of an AAC(3). resistance mediating gene; SlOO,supematant (lOO,OOOg) In order to assessthe extent of the Inc 7of a bacterial extract after sonication; genotypes include M plasmid epidemic we have compared, by sac(3), aminocyclitol acetyltransferase 3; aad(2”), amiagarose gel electrophoresis following digesnocyclitol adenylyltransferase 2”; aad(3”)(9), aminocytion with restriction endonucleases and by clitol adenylyltransferase 3”, 9; aph(3’)(5”), aminocyclitol phosphotransferase 3:5”. nucleic acid hybridization, plasmids pIPl35, 215
0 147-619X/82/0602 15-l 7$02.00/O
216
LABIGNE-ROUSSEL,
WITCHITZ,
AND COURVALIN
pIPl5 1 isolated a year later in a different hospital with pTH 1, a plasmid representative of the London outbreak. Inc 7-M plasmids pIP69 and pIP179 which do not code for an AAC(3) were also included in this study. (A preliminary report of part of this work was presented at the Symposium on Plasmids of Medical, Environmental and Commercial Importance, 26-28 April 1979, Spitzingsee, FRG.) The mechanisms of resistance toward aminocyclitols are described and the plasmid genesencoding antiboitic resistance are compared with those of known transposons.
(Courvalin et al., 1977) and the enzymes assayed (Haas and Dowding, 1975) as described. Preparation ofplasmid DNA. Transferable plasmid DNA was purified after pelleting from sarkosyl lysates on glycerol cushions as described (Labigne-Roussel et al., 198 1). Amplifiable DNA from ColEl factor and derivative plasmids was purified by two successive bandings in cesium chloride-ethidium bromide as described (Tanaka and Weisblum, 1975). DNA from bacteriophage Xc1857 and derivatives was prepared as described (Courvalin and Fiandt, 1980). Bacteriophages Xb515 b519 ~1857 Sam7::Tn6 MATERIALS AND METHODS and Xl, dgal ~1857 St68-OP306::ISl DNAs Plasmids. The sources and properties of were kindly provided by R. Jorgensen and the plasmids are listed in Table 1. Each plas- P. Starlinger, respectively. mid was introduced into Escherichia coli KAgarose gel electrophoresis. Plasmid DNA 12 strain BM2 1 (F-, prototroph, nalA) by was digested with restriction endonucleases conjugation as described before (Witchitz in reaction mixtures of 70 ~1 for analytical and Chabbert, 1971). purposes or of 350 ~1 for preparative gels, Media. Brain heart infusion broth and containing 100 mM Tris-HCl (pH 7.8), 100 agar (Difco) were used. Disc sensitivity tests mM MgClz for EcoRI, 6 mM Tris-HCl (pH were done on Mueller-Hinton agar medium. 7.0), 6 mM MgC 12,50 mM NaCl for ZZindIII, All incubations were at 37°C. and 15 mM Tris-HCl (pH 9.0), 6 mM MgC&, Testsfor incompatibility. Tests for incom- 15 mM KC1 for SmaI. The reactions were patibility were performed as described before allowed to proceed for 1 h at 37°C (except (Chabbert et al., 1972). for SmaI which was incubated at 25°C) then Assay for aminocyclitol-modifying en- heated at 68°C for 10 min and chilled at 0°C. zymes. The E. coli extracts were prepared The resulting DNA fragments were separated TABLE
1
PROPERTIES OF THE Inc 7-M PLASMIDS Plasmid No.
Phenotypic characters“
Genotypeb
Donor strain
PIP135
Gm Sm Su Tc Hg
sac(3)-I aad(3”)(9) tel(class C)
pIP135-I
Tc Hg
ter(class C)
PIP151
Gm Sm Su Tc Hg
sac(3)-I aad(3”X9) lef(chss C)
K. pneumoniae
pTH I
Ap Cm Gm Km Nm Sm Su Tc Tp Hg
sac(3)-I aad(3”)(9) aph(3’)(5”)-I b/a(Tem-1) tel(c1as.sC) ap/t(3’)(5”)-1 b/a(Tem-I) lef(class B)
K. aerogenes
aad(2”) bla(Tem- I)
K. pneumoniae
pIP69 PIP179
Ap Km Nm Tc Hg Ap Gm Km Tm
E. cloacae
68M K-16 R13 .S. paratyphi
B
source or reference
Origin
Hop&al Foch, Suresnes Witchitz and Gerbaud, I972 (1971) Witchitz and Spontaneous derivative Gerbaud, 1972 of PIP135 HopitaI Claude-Bernard, Roussel and Chabbert, 1978 Paris (1972) Datta et al., St. Thomas’s hospital, 1979 London ( 1977) Creteil ( 1969)
Chabbert et al.. 1972
New York (1973)
’ Nomenclature of phenotypic characters of plasmids is according to Novick et al. (1976). ’ Genetic symbols are according to Novick ef al. ( 1976) Davies and Smith ( 1978), and Mendez m al. ( 1980).
S. Schaeffler
IN VIVO EVOLUTION OF GENTAMICIN
by electrophoresis in horizontal slab gels containing 0.7, 0.8, or 1% agarose. Electrophoresis and staining were as described by Shinnick et al. (1975). Hybridization. 32P-Labeled complementary RNA (cRNA) (Courvalin and Fiandt, 1980) and nick-translated DNA (Maniatis et al., 1975) probes were prepared as described. Restriction endonuclease-generated fragments of plasmid DNA fractionated by agarose gel electrophoresis were transferred to cellulose nitrate sheets (Sartorius) as described by Southern (1975). Hybridization of the probes to the DNA immobilized on the membrane filters was as described by Denhardt (1966). Hybridization was revealed by autoradiography using XR 1-Omat Kodak film. Enzymes.Restriction endonucleasesEcoRI (Stimegi et al., 1977) and SmaI (Tanaka and Weisblum, 1975) were purified as described. Restriction endonuclease Hind111was kindly provided by J. Gardner and R. Jorgensen. Endonucleases BamHI, Sal I, and PstI, from Biolabs, were used according to the manufacturer’s recommendations. Lysozyme was from Sigma. Ribonuclease A (bovine pancreas) was from Calbiochem. Proteinase K was from Merck. E. coli K- 12 RNA polymerase holoenzyme (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) was from Miles. Chemicals. [ l-14C]Acetyl-coenzyme A ([ 14C]CoASAc), adenosine 5’-[a-32P]triphosphate, triethylammonium salt ([c~-~~P]ATP), adenosine 5’-[y-32P]triphosphate, triethylammonium salt ([Y-~~P]ATP), and [U14C]adenosine 5’-triphosphate, ammonium salt ([U-‘4C]ATP) were obtained from the Radiochemical Centre Amersham. The antibiotics were provided by the following laboratories: gentamicins (Gen) C 1a, C 1, C2, A, B, and Gen complex (26.3% Cla, 40.8% Cl, 32.9% C2), sisomicin (Sis), 2”-deoxy-sisomitin (2”-DSis), and netilmicin (Net), Schering; kanamycins (Kan) A, B, C, and amikacin (Ami), Bristol; neamine (NeoA), neomycin B (NeoB), spectinomycin (Spc), Upjohn; paromomycin (Par) and butirosin (But), ParkeDavis; tobramycin (Tob) and apramycin
R PLASMIDS
217
(Apr), Lilly; fortimycin (For), Kyowa; lividomycin (Liv) A, Kowa; ribostamycin (Rib), Meiji; streptomycin (Str), Pfizer; nalidixic acid, Winthrop. Sarkosyl (sodium lauryl sarcosinate) was provided by Colgate-Palmolive. RESULTS Classification
by Incompatibility
Plasmids pIP135 (later designated JR88; Kagan and Davies, 1980), PIP 135-1 (Witchitz and Gerbaud, 1972), pIP69 (Chabbert et al., 1972), and pTH1 (Datta et al., 1979) belong to the incompatibility group 7-M (Chabbert et al., 1972; Datta et al., 1975, 1979). Plasmids pIPl5 1 and pIP179 were also found to belong to the Inc 7-M group on the following observations. E. coli strains harboring PIP 151 or pIP69 were mixed and transfer in each direction was selected. The resident plasmid was eliminated from all transconjugants examined. Plasmids pIP179 and PIP 135-1 were shown to be incompatible in the same way. The properties of the Inc 7-M plasmids studied are summarized in Table 1 and the suspected structural interrelationships between some of them are diagrammed in Fig. 1. Mechanisms of Plasmid-Mediated Resistance to Aminocyclitol Antibiotics
E. coli strain BM21 and transconjugants were examined for aminocyclitol-modifying activities. No modifying activity was detected in the aminocyclitol-susceptible strains BM2 1 and BM2 1 harboring plasmid PIP 135-1. The aminocyclitol substrate profiles of the enzymes extracted from transconjugants are shown in Fig. 2 and Table 2. Strains harboring plasmids PIP 135, PIP 151, and pTH 1 were found to contain aminocyclitol acetyland adenylyltransferases (Table 1). Gentamicin C antibiotics and sisomicin were excellent substrates for the acetyltransferase, whereas gentamicin A and B, netilmicin, the kanamycin compounds, amikacin, and tobramycin were either poor substrates or not acetylated. Based on these substrate profiles
218
LABIGNE-ROUSSEL, WITCHITZ, AND COURVALIN G
“g A
C
plP135
co’
(79.3)
)
-DELETION
I
IN E
- 2 INSERTIONS
FIG. 1. In vivo modular evolution of Inc 7-M gentamicin R plasmids. (--), plasmid pIPI DNA; inserted DNA; (+ ), IS15; (), ISl; (-), transposon; (I), EcoRI recognition site. The size ( v), (in kilobases) of the plasmids is indicated in parentheses. EcoRI-generated DNA fragments 4 and B were arbitrarily located in the graph. The position of pIPl5 1 EcoRI fragments F and G relative to E is not known. Cm, chloramphenicol; Km, kanamycin; Gm, gentamicin; Sm, streptomycin; Su, sulfonamide; Tc, tetracycline.
and hybridization experiments (see below) the enzymes are AAC(3). The fact that fortimycin is a substrate indicates that the enzymes are of type I (AAC(3>1) (Davies and Smith, 1978). The adenylylating enzymes are active on streptomycin and spectinomycin (AAD(3”)(9)). The substrate profiles of enzymes of the same type, extracted from E. coli BM2 1 harboring pIPl35, pIPl5 1, or pTH1, are similar. Plasmids pTH 1 and PIP69 encode a phosphotransferase (Table 2). Since kanamycin B is phosphorylated and tobramycin is not,
this indicates that the 3’-hydroxyl group is the site of modification (APH(3’)); the fact that lividomycin A is modified but butirosin is not indicates that the enzymes are of type I (APH (3’)(Y)-1 (Davies and Smith, 1978). Strain BM2 1 harboring plasmid pIP179 was found to contain aminocyclitol-adenylyltransferase but no phospho- or acetyltransferase (Table 2). The fact that sisomicin is adenylylated and 2”-deoxy-sisomicin is not, indicates that the 2”-hydroxyl group is the site of adenylylation (AAD(2”)). In all these strains, the modifying enzymes
IN VZVO EVOLUTION
OF GENTAMICIN
219
R PLASMIDS
ments plus fragments B and D. Plasmid pIPl5 1 had a size of 82.4 kb and consisted
of the 16 common fragmentsplus fragments
ll
B, E, .F, and G. Plasmid pTH 1 had a size of 110.1 kb and consisted of the 16 common fragments plus fragments H to N. Plasmid pIP69 had a size of 78.5 kb and 13 EcoRI-generated DNA fragments designated 1’ to 13’; pIP179 had a size of 78.8 kb and 14 fragments (1” to 14”).
n --E.coli BM21/pIP151 :: I
Analysis of Plasmid DNA by Hybridization
Plasmid DNA was cleaved with EcoRI, HindIII, BumHI, and/or PstI restriction enTABLE FIG.
2. fhhStK3te
profiles
Of enzymes extracted
from
E. coli strain BM2 1 harboring plasmids pIPl35, pIPl5 1, or pTHl. Aeetylation (solid line) or adenylylation (dashed line) are expressed relative to gentamicin Cla or streptomycin as 10046, respectively.
were expressed constitutively as they were detected in cells grown in the absence of antibiotic. Restriction
Endonuclease Analysis of Plasmid DNA
The plasmid DNA from each transconjugant was purified by ultracentrifugation in cesium chloride-ethidium bromide, digested with EC&I restriction endonuclease, and analyzed by acrylamide (data not shown) or agarose gel electrophoresis. The results are shown in Fig. 3 and are summarized in Table 3. Comparative analysis of the EcoRI-generated fragment patterns of plasmid DNAs showed that PIP 135, PIP 135-1, PIP 151, and pTH 1 shared 16 fragments with indistinguishable size which were numbered in order of decreasing molecular weight. Fragments which were not common (extra) were designated with letters A to N. Plasmid pIP135 had a size of 79.3 kilobases (kb) and consisted of the 16 common fragments plus fragments A to C. Plasmid pIP135-1 had a size of 70.4 kb and consisted of the 16 common fmg-
2
SUFNRATEPROFXLESOFENZYMESEXTRATEDFROM E. coliS~R~r~BM21 HARBORINGPLASMIDS pTH1, pIP69, OR pIP179 Percentageof antibiotic Phosphorylat& Adenylylatcd’ pIPI
Aminocyclitol
pTH 1
pIP69
Ckntamicin C 1a Gentamicin Cl Gfmtamicin C2 Gentamicin A Gentamicin B Sisomicin 2’-Deoxy-sisomicin Netilmicin Kanamycin A Kanamycin B Kanamycin C Amikacin Tobmmycin Neomycin A Neomycin B Paromomycin Lividomycin A Butirosio Ribostamycin Stnptomycin Spectinomycin
0 0 0 44 30 1 -
-b 40 56 0 -
34 47 43 5 2 53
62 63 61 1 0 94
100
100
-
35
42
-
141
44
-
28 64
10 13
-
0 0
-
100 129 109 98 105 137 7 78 80 91 86 0 72 -
-
o Phosphorylation or adenylylation are expressed relative to neomycin B or gentamicin Cla as LOO%,respectively. No adenylylation of gentamicin complex was detected in E. coli strain BM21 harboring plaamids pIP135, pIP151, or pTH1. No phosphorylation of gentamicin complex, streptomycin, or spcctinomycin was detected in strain BM21 harboring pIP135 or pIPI and no adenylylation or acetylation of neomycin B was detccted in strain BM21 harboring pIP69. ’ Not determined.
PIP 135-1
PIP 135
PTHl
PIP 69 PIP 179 1’
1’
-
G --_--,-g-czz*-
--6-
p ____-
-a-5-
-
PIP 151
10-
---D
I -
PIP 135-1
-,-
-65
-5-
_
-
PIP 135 PIP 69 PIP 179
,
----M
XL
--.K
-,I’ 9’
-96’
-7’
-6’
-5’
--r .____ H - I, I -2’ -2’
pTH1
-33
-,2
-52 -49
-24, -2,.
x’
-62
-61
-” -211
X’
FIG. 3. Analysis by agarose gel electrophoresis of DNA from Inc 7-M plasmids. (A) Two to three micrograms of plasmid DNA were digested with EcoRI. Fragments obtained by digestion of Xc1857 DNA, 48 kb (Fiandt et al., 1977) with restriction endonucleases Hind111 (Xa) and EcoRI + Hind111 (Xb-Thomas and Davis, 1975; Robinson and Landy, 1977) were used as molecular size standard. Electrophoresis was carried out in a 1% agarose horizontal slab gel (18 X 13 X 0.4 cm) for 12 h at 3 V/cm. (B) Schematic representation of A. Number and letter designations are as explained in the text (cf. Results: Restriction endonuclease analysis of plasmid DNA). (- - -), EcoRI fragments not common to plasmids pIPl35, PIP 135 1, PIP 15 1, and pTH 1. The size of the reference hcI857 DNA fragments is expressed in kilobases. Plasmids pIP15 1, pIPl35-1, pIP135, and pTH1 EcoRI fragments 14, 15, and 16, plasmid pIP69 EcoRI fragments 1 I’, 12, and 13, and plasmid pIP179 EcoRI fragments 12”, 13”, and 14” were detected on 1.4% agarose gels only and are arbitrarily positioned in the graph.
PIP 151
IN VIVO EVOLUTION OF GENTAMICIN
221
R PLASMIDS
TABLE 3 EcoRI DIGESTIONOF Inc 7-M PLASMIDDNA pIP69
pIP135,” pIP135-1,b pIPl51,c pTHld Common fragment No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Added size
Sizee 11.6 (14) 9.4 (14) 8.5 (14) 5.6 (14) 4.9 (14) 3.2 (14) 2.7 (14) 2.4 (14) 2.0 (14) 1.6 (11) 1.3 (12) 1.3 (12) 1.2 (8) 0.75 (4) 0.6 (4) 0.55 (4)
Extra fragment letter
Size’
A B C D E F G H I J K L M N
10.7 (2) 8.5 (8) 2.5 (3) 4.3 (3) 9.9 (3) 3.6 (3) 2.8 (3) 16.8 (4) 9.3 (4) 8.8 (4) 6.3 (4) 5.9 (4) 4.2 (4) 1.2 (4)
57.6
PIP179
Fragment No.
Size’
1’ 2 3 4 5 6 7’ 8 9 10 11’ 12’ 13
22.6 (3) 11.6 (3) 9.7 (3) 8.8 (3) 7.7 (3) 6.2 (3) 3.7 (3) 3.4 (3) 1.6 (3) 1.3 (3) 0.75 (1) 0.6 (1) 0.55 (1)
Added size
78.5
Fragment No.
Size’
1” 2 3” 4 5” 6” 7” 8” 9” lo” 1I” 12 13 14”
30.5 (3) 11.6 (3) 9.0 (3) 8.6 (4) 4.9 (4) 3.7 (4) 2.4 (4) 2.0 (4) 1.6 (4) 1.3 (4) 1.3 (3) 0.75 (1) 0.6 (1) 0.55 (1)
Added size
78.8
Note. Number and letter designations are as explained in the text (cf., under Results: Restriction endonuclease analysis of plasmid DNA). * pIP135:57.6 + A + B + C = 79.3 kb. b plP135-1:57.6 + B + D = 70.4 kb. ‘pIP151:57.6 + B + E + F + G = 82.4 kb. dpTHl:57.6 + H + I + J + K + L + M + N = 110.1 kb. eThe size of the fragments (in kilobases) was estimated after agarose gel electrophoresis using Xc1857 DNA digested by HindIII, EcoRI + Hind111(Thomas and Davis, 1975; Robinson and Landy, 1977),and SmaI (Rosenvold and Honigman, 1977) as molecular size standard. The values are means from the number of determinations indicated in parentheses.
donucleasesand the resulting fragments were separated by agarose gel electrophoresis, denatured in situ, transferred to a nitrocellulose filter (driver), and hybridized to in vitro 32P-labeledplasmid or bacteriophage DNA or complementary RNA (cRNA) probes. The distribution of homologous sequences among the different fragments of plasmid DNA was then determined by autoradiography. The properties of the template DNAs for probes used to localize the R determinants and search for the presence of insertion sequences (IS) are summarized in Table 4. In each experiment, the reaction with PIP 135 cRNA probe which hybridizes with a majority of DNA fragments was used as an internal
standard. The results of the hybridization experiments are shown in Figs. 4 and 5, and summarized in Table 5.
(1) Comparison of the Inc 7-M Plasmids Plasmid PIP 135-1 has the smallest size and encodes the fewest resistances (Tc and Hg only). The probe made from this plasmid was therefore used for the comparison of the Inc 7-M transfer factors. Comparison of the electrophoretic mobilities of the EcoRI-generated plasmid DNA fragments (Fig. 3) with those of fragments which hybridized to 32P-labeled pIP135-1 cRNA (Fig. 4) showed that pIP135-1 shared
222
LABIGNE-ROUSSEL, WITCHITZ, AND COURVALIN TABLE 4 F’LAWIDS AND BACFERIOPHAGES USED AS PROBES FOR HYBRIDIZATION
Plasmid or bacteriophage
Relevant genotype”
Source or reference
ColE I pSF2 124 pCHl3’ ColEl::Tn’l pLHIC psc101 PRT~~ pBR325 Xe A: :Tn6’ X: :Tn601g X::ISlh PlCm PIP111
b&Tern- I ) aad(3@)(9),sul aad(3?9), dfr1 aad(3”)(9), sac(3)-I, sul tet(c1as.sC) tef(class B) bla(Tem- 1), cat, tet(class C) aph(3’)(5”)-I aph(Y)(S”)-I cat IS15
Bamral and Helinski ( 1972) So et al. (1975) J. Davies N. Datta Kagan and Davies ( 1980) Cohen and Chang ( 1973) Jorgensen and ReznikofF (1979) Bolivar (1978) Fiandt et al. (1977) Berg et al. (1975) Davies et al. ( 1977) P. Starlinger Kondo et al. (1962) Rouse1 et al. ( i 979) Labigne-Roussel and Courvalin (1982)
’ Genetic symbols are according to Novick et al. (1976), Davies and Smith (1978), and Mendez et al. (1980). bpCH13, ColElQ {aad(3”)(9), A}. ’ Plasmid pLH1 consists in pIPl35 EcoRI fragment A cloned in ColE I [ColElQ { aad(3”)(9), aad3)-I, sul}]. d pRT2, pVH5 1fl { tef(class B)}. e A, hcz857. /X::Tn6, Xb5156519 ~I857 Sam7::Tn6. gX::Tn601, Xb515b519 ~1857Sam7::Tn601. h X::ISl, Xl, Adgal cI857 St68-OP306::ISl.
DNA sequenceswith the 16 common fragments of pIP135, as expected, but also with those of pIP 151 and pTH 1. Plasmid PIP 135 1 labeled cRNA also hybridized to pIP135 &RI-generated DNA fragments B and C, to pIPl5 1 fragments B, F, and G and to pTH1 fragments H, K, L, and, possibly, I and N. The two latter fragments corn&rate with common EcoRI fragments 2 and 13, respectively. Plasmid pIPl35-1 cRNA hybridized to 9 pIP69 and 11 pIP179 EcoRI fragments. (2) Localization of the R Determinants and the Insertion Sequences Tetracycline R determinant. Plasmid pSClO1 (Tc) probe hybridized to the common EcoRI fragment four. The tetracycline determinant in pIP135, pIP135-1, pIP151, and pTH 1 therefore belongs to classC (Mendez et al., 1980).
Streptomycin, sulfonamide, and gentamitin R determinants. Probes made from pCHl3 (SmSu), pLH 1 (GmSmSu), and ColE 1 allowed us to assign the streptomycin, sulfonamide, and gentamicin R determinants to EcoRI fragment A of PIP 135, E of pIP151, and I of pTH1. Trimethoprim and streptomycin R determinants. The trimethoprim R determinant was localized with ColE 1::Tn7 (SmTp) cRNA probe on pTH1 EcoRI fragments H and L. The transposable element Tn7 (Barth et al., 1976) confers resistance to trimethoprim by synthesis of a dihydrofolate reductase type I (Pattishall et al., 1977) and to streptomycin by synthesis of an aad(3”)(9). There is an EcoRI site in Tn7 (Richards and Nugent, 1979) and HindIII generates two DNA fragmerits of 2.6 and 2.1 kb (Datta et al., 1981, Fig. 5E). Plasmid pTH 1 digested with Hind111 also gave rise to two DNA fragments of 2.6
IN VIVO EVOLUTION OF GENTAMICIN
R PLASMIDS
223
activities in plasmid pTH1 are structurally related and that, under our conditions, the gene encoding streptomycin resistance in Tn7 is not transcribed in vitro. Ampicillin R determinant. Plasmid pTH 1 encodes a TEM- 1 P-lactamase (Table 1). Datta et al. (1979) found that at least part of the ampicillin R determinant in pTH1 is located in EcoRI fragment K (fragment number five in their nomenclature) and in our experiments the pSF2 124 (Ap) probe hybridized to fragments J and K of pTH1. The widespread Tn3 element contains a BamHI site, but no EcoRI site (Heffron and McCarthy, 1979). In order to discriminate between the presence in pTH1 of two ampiPIP PIP DRIVERS PIP PIP PIP pTH1 69 179 135 135-1 151 (EcoRI) cillin R determinants as in the case of streptomycin resistance, or the occurrence of an FIG. 4. Analysis of plasmid DNA by hybridization. Ten micrograms of the driver plasmid DNAs indicated EcoRI site in the region associated with the on the bottom line were digested with EcoRI restriction P-lactamase, pSF2 124 (Ap) cRNA was hyendonuclease. The resulting DNA fragments were frac- bridized with pTH1 DNA digested with tionated by electrophoresis through horizontal slab gels BamHI or BamHI plus EcoRI (Fig. 5E). The (0.7% agarose, 18 X 13 X 0.4 cm) for 12 h at 3 V/cm, transferred to a nitrocellulose sheet, and hybridized to pSF2 124 (Ap) probe hybridized to two fragin vitro 32P-labeledplasmid PIP 135-1 cRNA as indicated ments of pTH1 digested with BamHI and on the top line (probe). The autoradiogram was exposed three fragments after digestion with BamHI for a period of 12 h. Letter and number designations are plus EcoRI, whereas the presence of two amas explained in the text (cf., Results: Restriction endopicillin R determinants should result in the nuclease analysis of plasmid DNA). hybridization with four pTH 1 BamHI-generated DNA fragments. The EcoRI site in, or close to, the gene specifying the P-lactaand 2.1 kb which shared homology with Tn7 mase could be due to the insertion, in the (Fig. 5E). This observation (Datta et al., Tn3 element, of a DNA fragment possessing 1981) confirms that, as suggested (Datta et a recognition site for this restriction endoal., 1979) on the basis of molecular weight nuclease. and restriction enzyme analysis of plasmid Chloramphenicol R determinant and ISI. DNA, the entire Tn7 element is present in Bacteriophages PlCm (Cm ISl) and h::ISl plasmid pTH 1. Plasmid pTH 1 therefore en- cRNAs hybridized to pTH1 EcoRI DNA codestwo a&( 3”)(9) activities. Hybridization fragments J and M (Fig. 5D) indicating the of pCH 13 (SmSu) and, probably, pLH1 presence in this plasmid of two copies of a (GmSmSu) probes to pTH 1 EcoRI fragment sequence homologous to IS1 separated by a L is due to the presence, in this fragment, of minimum of one EcoRI recognition site. The the portion of Tn7 coding for the adenyly- pBR325 (ApCmTc) probe hybridized to lating enzyme. Conversely, because of the pTH1 EcoRI fragments 4 (because of the presence in these fragments of the strepto- presence in this fragment of the tetracycline mycin R determinant, plasmid ColEl ::Tn7 resistance gene), J and K (because of honick-translated DNA also hybridized to mology with the ampicillin determinant) and EcoRI fragments A of pIPl35, E of pIPl5 1, M showing that at least part of the chlorand, most likely, I of pTH1 whereas the amphenicol determinant is located in this ColEl::Tn7 cRNA probe did not. These ob- fragment (data not shown). servations indicate that the two adenylylating DNA from plasmids pTH1 and ColEl:: PROBE 0
plP135-1
224
LABIGNE-ROUSSEL, WITCHITZ, AND COURVALIN
PROBES
ORIVERS FIG. 5. AP&& of p&,Mid DNA by hybridization. Ten micrograms of the driver plasmid DNAs were digested with restriction eedonucleases as indicated on the bottom line. The resulting DNA fragments were fractionated by electrophoresis (same conditions as in Fig. 31,transferred to a nitiocellulose sheet, and hybridized to in vitro 32P-laheIedplasmid cRNA as indicated on the top line (probes). The autora-
IN VW0 EVOLUTION OF GENTAMICIN
Tn9 (Cm ISl) was digested with PstI restriction endonuclease, compared by agarosegel electrophoresis, and hybridized, following transfer on a nitrocellulose sheet, to X::ISl, pBR325 (ApCmTc), and pSF2124 (Ap) probes. Transposable element Tn9 (Rosner and Gottesman, 1977) consists of a chloramphenicol determinant flanked by two copies of IS1 in direct orientation. Insofar as there is a single EcoRI recognition site in the structural gene for the chloramphenicol acetyltransferase and IS 1 contains a unique P.stI restriction site, Tn9 DNA generates upon digestion a 1.75kb PstI fragment and two EcoRI/PstI fragments 1.2 and 0.55 kb in size. Plasmid pTH1 restricted with PstI did not give rise to the characteristic Tn9 fragment but to a slightly larger one ( 1.95 kb) identified on the basis of homology with X::ISl and pBR325 (ApCmTc) but not with pSF2 124 (Ap) (data not shown). Plasmid pTH1 DNA was further digested by PstI plus EcoRI and hybridized, after electrophoresis in an agarose gel, with the same three probes. Since they each contain a copy of IS 1, and therefore a PstI site, both EcoRI fragments J and M disappeared in this analysis. Two new smaller fragments hybridized with X::ISl and pBR325 (ApCmTc) but not with the pSF2 124 (Ap) probe demonstrating that the structural gene for chloramphenicol resistance, as in Tn9 (Rosner and Gottesman, 1977), has one EcoRI recognition site and is flanked by two copies of IS 1. In conclusion, plasmid pTH1 contains a transposon closely related to but distinct from Tn9. The function, if any, coded for by the additional 200 bp in the central portion of this element remains unknown. Kanamycin R determinant and IS1.5. The kanamycin determinant was localized with X::Tn60 1 and XcRNA probes on EcoRI fragment J of pTH 1. Transposable elements Tn6 (Berg et al., 1975) and Tn601 (Davies et al.,
R PLASMIDS
225
1977), later designated Tn903 (Nomura et
al., 1978) (Table 4), confer resistance to kanamycin by synthesis of an amino glycoside phosphotransferase (3’)( 5”) type I (aph(3’)(5”) I) (Haas and Dowding, 1975), similar to that encoded by pTH1 (Table 2). The structural gene for the phosphotransferase in the 3.15-kb Tn60 1 element is flanked by two inverted copies of IS903, 1.05 kb in length (Kopecko et al., 1976; Courvalin et al., 1979). The 4. I-kb Tn6 element contains two terminal directly repeated copies of IS 15 (Labigne-Roussel et al. (1981) and (1982). Analysis of heteroduplexes by electron microscopy and hybridization experiments (Courvalin et al., 1979) indicated that the 1.05-kb structural genes for the phosphotransferases in Tn601 and Tn6 are homologous. As expected, the X::Tn6 probe hybridized to the kanamycin R determinant of pTH1, but, as opposed to X::Tn60 1 cRNA also hybridized to EcoRI fragments K of pTH1, C of pIP135, D of pIP135-1, and F and G of PIP 151. The existence of IS 15 in EcoRI-generated DNA fragments D of pIPl35-1 and J and K of pTH 1 was confirmed with a probe made from PIP 111. This IncIl plasmid does not confer resistance to kanamycin (Chabbert and Gerbaud, 1974) and contains one copy of IS15 (LabigneRoussel and Courvalin, 1982). There is a Hind111recognition site in the kanamycin R determinant of Tn6 (Berg et al., 1975). Plasmid pTH1 DNA was digested with EcoRI + Hind111and hybridized with X::Tn60 1 and pIPll1 probes (data not shown). The two pTH1 fragments which shared homology with Tn60 1 also hybridized with PIP 111 indicating the presence of two copies of IS 15 and therefore of an entire Tn6 element in pTH1 EcoRI fragment J. Bacteriophage Xc1857and Colicin E 1 factor cRNAs, used as controls, and probe from pRT2 did not hybridize to any of the plasmids studied.
diograms were exposed for a period of 12 h. Letter and number designations are as explained in the text (cf., Results: Restriction endonuclease analysis of plasmid DNA). EcoRI-generated DNA fragments 14 to 16, 11’ to 13’, and 12” to 14” are not seen in this particular analysis.
+ ? + + ?
H I J K L M N
pTH 1
+ + + -
+ +
-
-
-
+ + -
+ + -
,,-.,-
-
+ + -
+ +
+
+
A: :Tn6 (KmIS1522)
+ + -
ND ND ND ND
+
ND ND ND
PIP111 (IS15)
---
---
), indicates a weak hybridization relative
+ -
-
-
-
-
X::Tn601 (Km)
X::ISl
’ f, Positive result; -, negative result; ?, indicates comigration with an EcoRI fragment sharing homology with PIP 135-1; ( to the others; ND, not determined. b ColEl, pRT2, and Xc1857cRNA probes gave negative results. ’ Result positive with the nick-translated DNA probe and negative with the cRNA probe.
(+) -
+ -
+ + -
+ ? + + ?
+ + + +
B E F G
pIP151
($ + -
+c -
+ -
+ -
+ + +
+ +
B D
pIP135-1 -
-
-
+
-
+ +
-
-
-
+’ -
+ -
PlCm (CmISl)
pBR325 (ApCmTc)
pSF2124 (AP)
+ -
ColEl::Tn7 (Sm’W
+ +
pLH 1 (GmSmSu)
+ + +
pCH13 (SmSu)
pIP135-1
pIP135
A B C
EcoRI fragment
pIP135
Driver
Probe (Relevant characters)h
SUMMARY OF THE RESULTSOF THE HYBRIDIZATIONS~
TABLE 5
g T F 2
5
E 3 P E 1 2 =i “N
5
5
F
IN VW0 EVOLUTION OF GENTAMICIN
(3) Evolutionary Relationships among the 7-M Plasmids Plasmid pIPl35- 1 is a spontaneous derivative of pIP135 obtained in the laboratory (Witchitz and Gerbaud, 1972). This plasmid arose from an 8.9-kb deletion which fused pIP135 EcoRI fragments A (10.7 kb) and C (2.5 kb) to generate pIP135-1 EcoRl fragment D (4.3 kb) (Fig. 2). As already mentioned, this new DNA fragment retained the pIP 135 EcoRI fragment C copy of IS 15 and similar adjacent deletions induced by this insertion sequence have already been observed (Labigne-Roussel et al., 1981). Plasmid pLH1 (GmSmSu) cRNA hybridized to pIP135-1 EcoRI fragment D, but probes from pCH 13 (SmSu) and ColEl::Tn7 (SmTp) did not. Plasmid pIPl35- 1 does not confer resistance to gentamicin (nor to streptomycin and sulfonamide) and we did not detect any acetylating activity in cells harboring this plasmid. It appears therefore that a vestige of the PIP 135 EcoRI fragment A gene encoding the AAC(3) I enzyme is present in pIP135-I EcoRI fragment D but is not expressed in vivo. The fact that pIPl35- 1 cRNA did not hybridize to pIP135 EcoRI fragment A is probably due to a similar lack of transcription in vitro. As opposed to PIP 135-1, plasmid PIP 151 (82.4 kb) is larger than pIP135 (79.3 kb). Plasmid pIPl5 1 EcoRI fragment E which contains the gentamicin, streptomycin, and sulfonamide R determinants is smaller (by 0.8 kb) than pIP135 EcoRI fragment A (Fig. 3). The filiation between these two DNA fragments is confirmed by the fact that they both contain two BamHI recognition sites flanking DNA fragments which precisely differ in size by 0.8 kb (data not shown). In pIPl51, plasmid pIP135 EcoRI fragment C is replaced by the larger fragments F and G which share homology with PIP 135. This change in the EcoRI-generated fragment pattern of plasmid DNA may be due to IS15 mediated rearrangements within fragment C associated with a duplication of this insertion sequence. Presumably, for the same reason as for
R PLASMIDS
221
PIP 135 EcoRI fragment A, plasmid pIP 1351 cRNA did not hybridize to PIP 151 EcoRI fragment E. Plasmid pTH1 is also larger, by 30.8 kb, than plasmid pIP135 and seven additional EcoRI-generated DNA fragments replace the three noncommon EcoRI fragments of pIP135 (Fig. 3). Plasmid pTH1 EcoRI fragment I is responsible for resistance to gentamicin, streptomycin, and sulfonamide and has a size reduced by 1.4 kb compared to that of pIP135 EcoRI fragment A. The two BamHI recognition sites in fragment I delimit a DNA segment with migration properties indistinguishable from those of the BamHI fragment which originates from pIPl5 1 EcoRI fragment E. This latter fragment could therefore represent an intermediate step between pIP135 EcoRI fragment A and pTH1 EcoRI fragment I. The second deletion leading from E to I must be located outside the internal BamHI fragment. Based on molecular weight determinations, the most likely candidate for integration of the transposable element Tn7 (14 kb, Table 4) which overlaps fragments H (16.8 kb) and L (5.9 kb) is PIP 135 EcoRI fragment B (8.5 kb, Table 3). Compatible with this event is the fact that fragments H and L share homology with PIP 135 cRNA. Integration of Tn7 in PIP 135 EcoRI fragment B rules out, by lack of space,the presencein pTH 1 EcoRI fragment L of genes mediating resistance to gentamicin and sulfonamide. Because plasmids pCH 13 (SmSu) and pLH 1 (GmSmSu) carry aad(3”)(9) encoding genes homologous to that of Tn7 in EcoRI fragment L we could not eliminate the presence, also in this DNA fragment, of gentamicin and sulfonamide R determinants. The mapping of the remaining four noncommon EcoRI-generated fragments of plasmid pTH 1 is shown in Fig. 1 and was based on the following observations. -Acquisition of the R determinants results from the insertion of a DNA fragment into pIP135 EcoRI fragment C, which disappears. -As already discussed, EcoRl fragments
228
LABIGNE-ROUSSEL, WITCHITZ, AND COURVALIN
A and C are contiguous in plasmid pIPl35, and pTH1 EcoRI fragment I derives from PIP 135 EcoRI fragment A. Therefore EcoRI fragment I is adjacent to the insert in plasmid pTH1. -As found in pIP135 EcoRI fragment C, plasmid pTH1 EcoRI fragment K contains a copy of IS15 and thus is next to fragment I. -Fragments K and J are adjacent because Tn3 hybridized to both DNA fragments. -Fragments J and M are also adjacent in so far as the structural gene for the chloramphenicol acetyltransferase overlaps these two fragments. Plasmid pTH1 EcoRI fmgment J contains two copies of IS 15 but, being flanked by fragments K and M, this fragment cannot be adjacent to fragment I. -Plasmid pIP135 cRNA did not hybridize to pTH1 EcoRI fragment M thus indicating that M is not a junction fragment, i.e., one of the two restricted fragments composed of DNA from the insert fused with that of the pIP135 receptor. The cryptic EcoRI fragment N, which likely shares homology with plasmid pIP135, is therefore the secondjunction fragment. In all the plasmids studied, because we have no probe to trace this R determinant, the location of the genesconferring resistance to mercury remains unknown.
known plasmid of this type (Witchitz and Gerbaud, 1972). The second plasmid, PIP 151 (Roussel and Chabbert, 1978) confers to the host an antibiotic resistance phenotype indistinguishable from that due to PIP 135 and was isolated a year later in a different hospital. The third plasmid, pTH 1, was detected more recently (Casewell et al., 1977) in Great-Britain and mediates resistance to an expanded number of antibiotics. Plasmids pIPl35, pIPl5 1, and pTH 1, encode aminocyclitol acetylating enzymes with identical site specificity and indistinguishable substrate profiles (Fig. 2). Comparison by restriction endonuclease-generated patterns followed by nucleic acid hybridizations indicated that these three plasmids were similar (Figs. 3 and 4) and that they were related (Fig. 4), but distinct (Fig. 3) from the other Inc 7-M plasmids (e.g., pIP69 and pIP179). This finding confirms an earlier observation (Roussel and Chabbert, 1978) based on quantitative DNA annealing studies and constitutes further support for the notion of plasmid epidemic (Chabbert et al., 1974, 1979). Plasmid pTH 1 could derive from plasmid pIP135 following at least four distinct events: two sequential deletions in EcoRI fragment A and stepwiseacquisition of Tn7 and a large insert responsible for the remaining resistances. Assuming that plasmid pTH 1 harbors the entire Tn6 and Tn7 transposable eleDISCUSSION ments, and also a Tn9-like element, we can We have compared plasmids conferring account for 25.7 kb out ofthe 29.4 additional resistance to gentamicin by synthesis of an kb compared to plasmid pIP135. The funcacetylating enzyme and belonging to incom- tion(s), if any, encoded by the remaining 3.9 patibility group 7-M with other plasmids of kb is unknown. In any case, whatever the the same group. We focused our attention on explanation may be for the remaining structhe study of the epidemiology of this mech- tural differences, plasmid pIP135 is not the anism of resistance to aminocyclitols because direct progenitor of pTH1 and plasmid it first appeared among enterobacteria in our pIPI 1 appears as the fossil of an intermecountry (Witchitz and Gerbaud, 1972) and diate state in this in vivo modular evolution. we were therefore able to trace its subsequent Such a modular evolution has been proposed spread. The three Inc 7-M plasmids mediat- in other systems(Sharp et al., 1973; Kopecko ing acetylation of gentamicin included in this et al., 1976; Susskind and Botstein, 1978). The transition from plasmid pIPI to study were isolated from different bacterial hosts (Table 1) and were selected on the fol- plasmid pTH 1 provides an example of struclowing criteria. Plasmid pIP135 is the first tural stability combined with genetic flexi-
IN VIVO EVOLUTION OF GENTAMICIN
bility under natural conditions. The striking similarity between pIPl35, pIPl5 1, and pTH 1 reflects a high degree of stability of the transfer factor part of PIP135 in vivo over at least 6 years. By contrast, acquisition, during the same period of time, of various genesled to resistance toward major groups of antibiotics (certain @-lactams,chloramphenicol, the remaining aminocyclitols, and trimethoprim). The new selective advantages conferred by pTH1 represent an addition to the potential of plasmid pIPl35: there was no insertion-inactivation of any preexisting R determinant nor of any transfer gene upon acquisition of the new genetic information. With the exception of transposable element Tn7, the additional R determinants are clus’ tered on the DNA molecule (Fig. 1). As op- posed to pIP135, plasmid pTH1 possesses two copies of a gene encoding a streptomytin-spectimomycin adenylyltransferase (Fig. 5E), an enzyme which confers low-level resistance toward the two antibiotics. The adenylyltransferase levels in cells harboring pTH1 are two to three times higher than those found in cells harboring pIP135. The MICs of streptomycin and spectinomycin for E. coli containing pTH1 are higher than when it contains pIP135. These variations could also be explained by differences between the copy numbers of the two plasmids. However this eventually seemsmost unlikely since the acetyltransferase levels, and subsequent aminocyclitol resistance phenotypes, mediated by pIP135 and pTH1 are identical (data not shown). The resistance phenotype conferred by pTH1, compared to that due to the presence of plasmid pIPl35, is therefore not only qualitatively but also quantitatively altered. Similar gene dosage effects have already been reported for aminocyclitol-modifying enzymes (Courvalin and Fiandt, 1980). Gentamicin resistance by acetylation of the 3-amino group of the antibiotic molecule has long been associated exclusively with Inc 7-M plasmids in our observations. This stable linkage was not in favor of the location of this resistance gene on a transposable ele-
R PLASMIDS
229
ment (Calos and Miller, 1980). In fact, we have no evidence for transposition of gentamicin resistance from the Inc 7-M plasmids studied. However, recently, four transposons conferring resistance to gentamicin by a similar mechanism have been described (Rubens et al., 1979a,b; Datta et al., 1981). It is likely that the Inc 7-M plasmids, which already contain a minimum of one copy of the ubiquitous insertion sequence IS 1522 (LabigneRoussel and Courvalin, 1982) represent the pool of origin of these transposable elements. In order to test this hypothesis it will be of interest to determine the structures of these various transposons and to compare them with that of the corresponding portion of a putative Inc 7-M ancestor. Theoretically, any structural gene can be located on a transposon, provided that selective pressures are exerted. This prerequisite is obviously fulfilled in certain ecosystemsfor gentamicin resistance and may well be at the “origin” of these presumably recently evolved transposable elements. ACKNOWLEDGMENTS We thank N. Datta and S. Schaeffler for gift of strains, C. Carlier for the assaysof the aminocyclitol-modifying enzymes, E. Collatz for isoelectric point determination, 0. Rouelland for secretarial assistance,and Y. A. Chabbert for material support. This work was supported by a grant (ATP 72.79.104) from the Institut National de la Sante et de la Recherche Medicale (INSERM).
REFERENCES BARTH, P. T., DA?TA, N., HEDGES,R. W., AND GRINTER,N. J. ( 1976).Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons. J. Bacterial. 125, 800-8 10. BAZARAL,M., AND HELINSKI, D. R. (1972). Characterization of multiple circular DNA forms of colicinogenie factor El from Proteus mirabilis. Biochemistry 7,3513-3519. BERG, D. E., DAVIES, J., ALLET, B., AND ROCHAIX, J. D. (1975). Transposition of R factor genes to bacteriophage X. Proc. Nat. Acad. Sci. USA 72, 36283632. BOLIVAR, F. (1978). Construction and characterization of new cloning vehicles. Gene 4, 12I- 136.
230
LABIGNE-ROUSSEL, WITCHITZ, AND COURVALIN
CALOS, M. P., AND MILLER, J. H. (1980). Transposable
elements. Cell 20, 519-595. CASEWELL, M. W., DALTON, M. T., WEBSTER,M., AND PHILLIPS, I. (1977). Gentamicin-resistant Klebsiella aerogenes in a urological ward. Lancet 2, 444-446. CHABBERT,Y. A., AND GERBAUD, G. R. (1974). Surveillance tpidemiologique des plasmides responsables de la resistance au chloramphenicol de Salmonella typhi. Ann. Microbial. (Inst. Pasteur) 125A, 153- 166. CHABBERT,Y. A., ROUSSEL,A., WITCHITZ, J. L., SANSON-LE PORS, M. J., AND COURVALIN, P. (1979). Restriction endonuclease generated patterns of plasmids belonging to incompatibility groups 11, C, M, and N: Application to plasmid taxonomy and epidemiology. In “Plasmids of Medical, Environmental, and Commercial Importance” (Timmis and Piihler, eds.), pp. 183-193. Elsevier/North-Holland, Amsterdam/New York/Oxford. CHABBERT,Y. A., SCAVIZZI,M. R., WITCHITZ, J. L., GERBAUD,G. R., AND BOUANCHAUD,D. H. (1972). Incompatibility groups and the classification of Firesistance factors. J. Bacterial. 112, 666-675. CHABBERT,Y. A., WITCHITZ, J. L., AND GERBAUD, G. R. (1974). Epidemics of R factors mediating gentamicin resistance and belonging to different incompatibility groups. In “Progress in Chemotherapy-I” (G. K. Daikos, ed), pp. 22-28. Hellenic Society of Chemotherapy, Athens. COHEN, S. N., AND CHANG, A. C. Y. (1973). Recircularization and autonomous replication of sheared Rfactor DNA segments in Escherichia coli transforman@. Prof. Nat. Acad. Sci. USA 70, 293-297. COURVALIN, P., AND FIANDT, M. (1980). AminogJycoside-modifying enzymes of Staphylococcus aureus: Expression in Escherichia coli. Gene 9, 247-269. COURVALIN, P., FIANDT, M., AND DAVIES, J. (1979). DNA relationships between genes coding for aminoglycoside-modifying enzymes from antibiotic-producing bacteria and R plasmids. Zn “Microbiology1978” (D. Schlessinger,ed.), pp. 262-266. Amer. Sot. for Microbial., Washington, D. C. COURVALIN,P., WEISBLIJM,B., AND DAVIES,J. (1977). Aminoglycoside-modifying enzyme of an antibioticproducing bacterium acts as a determinant of antibiotic resitance in Escherichia coli. Proc. Nat. Acad. Sci. USA 74, 999-1003. DATTA, N. (1975). Epidemiology and classification of plasmids. In “Microbiology-1974” (D. Schlessinger, ed.), pp. 9-15. Amer. Sot. for Microbial., Washington, D. C. DAT~A, N., HUGHES, V. M., NUGENT, M. E., AND RICHARDS,H. (1979). Plasmids and transposons and their stability and mutability in bacteria isolated during an outbreak of hospital infection. Plasmid 2, I82196. DATTA, N., NUGENT, M., AND RICHARDS,H. (1981). Transposons encoding trimethoprim or gentamicin resistancein medically important bacteria. Cold Spring Harbor Symp. Quant. Biol. 45, 45-5 1. DAVIES, J., BERG, D., JORGENSEN,R., FIANDT, M.,
HUANG, T. S. R., COURVALIN,P., AND SEHLOFF,J. ( 1977). Transposable neomycin phosphotransferases. In “R-factors: Their properties and possible control” (Drews and Hiigenauer, eds.), pp. 101-l 10. SpringerVerlag, Wien/New York. DAVIES,J., AND SMITH, D. I. (1978). Plasmid-mediated resistance to antimicrobial agents. Annu. Rev. Microbiol. 32, 469-5 18. DENHARDT,D. T. ( 1966). A membrane-tilter technique for the detection of complementary DNA. B&hem. Biophys. Res. Commun. 23, 641-646. FIANDT, M., HONIGMAN, A., ROSENVOLD,E. C., AND SZYBALSKI,W. (1977). Precise measurement of the b2 deletion in coliphage lambda. Gene 2, 289-293. HAAS, M. J., AND DOWDING,J. E. (1975). Aminoglycoside modifying enzymes. In “Methods in Enzymology” (J. H. Hash, ed.), Vol. 43, pp. 6 1l-628. Academic Press, New York. HEFFRON,F., AND MCCARTHY, J. M. (1979). DNA sequence analysis of the transposon Tn3: Three genes and three sites involved in transposition of Tn3. Cell 8, 1153-I 163. JORGENSEN,R. A., AND REZNIKOFF,W. S. (1979). Organization of structural and regulatory genes that mediate tetracycline resistance in transposon TnlO. J. Bacterial. 138, 705-7 14. KAGAN, S. A., AND DAVIES, J. E. (1980). Enzymatic modification of aminocychtol antibiotics: Mutations affecting the expression of aminocyclitol acetyltransferase-3. Plasmid 3, 3 12-3 18. KUNDO, E., HARADA, K., AND MITSUHASHI,S. (1962). Drug-resistance of enteric bacteria. 12. Transduction of the transmissible drug resistance factor by bacteriophage Pl kc. Japan J. Exp. Med. 32, 139-147. KOPECKO,D. J., BREVET,J., AND COHEN,S. N. (1976). Involvement of multiple translocating DNA segments and recombinational hotspots in the strnctural evolution of bacterial plasmids. J. Mol. Biol. 108, 333360.
LABIGNE-ROUSSEL,A., GERBAUD, G. R., AND COURVALIN, P. (198 1). Translocation of sequencesencoding antibiotic resistance from the chromosome to a receptor plasmid in Salmonella ordonez. Mol. Gen. Genet. 182, 390-408. LABIGNE-ROUSSEL,A., BRIAUX-GERBAUD, S., AND COURVALIN,P. (1982). Tn1525, a kanamycin R determinant flanked by two direct copies of IS 15. Mol. Gen. Genet., in press. LABIGNE-ROUSSEL, A., AND COURVALIN, P. (1982). A new insertion sequence widely spread in R plasmids of gram negative bacteria. Mol. Gen. Genet., in press. MANIATIS, T., JEFFREY,A., AND KLEID, D. G. (1975). Nucleotide sequence of the rightward operator of phage h. Proc. Nat. Acad. Sci. USA 72, 1184-I 188. MENDEZ, B., TACHIBANA,~., AND LEVY, S. B.(1980). Heterogeneity of tetracycline resistance determinants. Plasmid 3, 99- 108. NOMURA, N., YAMAGISHI, H., AND OKA, A. (1978). Isolation and characterization of transducing coli-
IN VZVO EVOLUTION OF GENTAMICIN phage fd carrying a kanamycin resistance gene. Gene 3, 39-5 1. NOVICK, R. P., CL~WES,R. C., COHEN,S. N., CURTIS% R., III, DATTA, N., AND FALKOW,S. (1976). Uniform nomenclature for bacterial plasmids: A proposal. Butteriol. Rev. 40, 168-189. PATTISHALL, K. H., ACAR, J. F., BURCHALL, J. J., GOLDSTEIN,F. W., AND HARVEY, R. J. (1977). Two distinct types of Tmp-resistant dihydrofolate reductase specified by R. plasmids of different compatibility groups. J. Biol. Chem. 252, 2319-2323. RICHARDS,H., AND NUGENT, M. (1979). The incidence and spread of transposon Tn7. In “Plasmids of Medical, Environmental and Commercial Importance” (Timmis and Puhler, eds.), pp. 195-198. Elsevier/ North-Holland, Amsterdam/New York/Oxford. ROBINSON, L. H., AND LANDY, A. (1977). HindII, Hind111 and HpaI restriction fragment maps of bacteriophage X DNA. Gene 2, l-3 1. ROSENVOLD,E. C., AND HONIGMAN, A. (1977). Map ping of AvaI and XmuI cleavage sites in bacteriophage DNA including a new technique of DNA digestion in agarose gels. Gene 2, 273-288. ROSNER,J. L., AND GO~MAN, M. M. (1977). Transposition and deletion of Tn9: A transposable element carrying the gene for chloramphenicol resistance. In “DNA Insertion Elements, Plasmids and Episomes” (A. I. Bukhari, J. A. Shapiro, and S. C. Adhya, eds.), pp. 2 13-2 18. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. ROUSSEL,A., CARLIER, C., GERBAUD,G., CHABBERT, Y. A., CROISSANT,O., AND BLANGY, D. (1979). Reversible translocation of antibiotic resistance determinants in Salmonella ordonez. Mol. Gen. Genet. 169, 13-25.
ROUSSEL,A. F., AND CHABBERT,Y. A. (1978). Taxonomy and epidemiology of Gram-negative bacterial plasmids studied by DNA-DNA filter hybridization in formamide. J. Gen. Microbial. 104, 269-276. RUBENS,G. E., MCNEILL, W. F., AND FARRAR,W. E., JR. (1979a). Transposable plasmid deoxyribonucleic acid sequence in Pseudomonas aeruginosa which mediates resistance to gentamicin and four other antimicrobial agents. J. Bacterial. 139, 877-882. RUBENS,G. E., MCNEILL, W. F., AND FARRAR,W. E., JR. (I 979b). Evolution of multiple-antibiotic-resis-
R PLASMIDS
231
tancc plasmids mediated by transposable plasmid deoxyribonucleic acid sequence. J. Bacterial. 140, 7 13719. SHARP,P. A., COHEN,S. N., AND DAVIDSON,N. (1973). Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. J. Mol. Biol. 75, 235-255. SHINNICK,T. M., LUND, E., SMITHIES,O., AND BLATTNER, F. R. (1975). Hybridization of labeled RNA to DNA in agarosegels. Nucl. Acids Res. 2, 1911-1929. So, M., GILL, R., AND FALKOW,S. (1975). The generation of a ColE I -Ap cloning vehicle which allows detection of insert DNA. Mol. Gen. Genet. 142, 239249.
SOUTHERN,E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-5 17. SUMEGI, J., BREEDVELD,D., HOSSENLOPP,P., AND CHAMBON, P. (1977). A rapid procedure for purification of EcoRl endonuclease. Biochem. Biophys. Res. Commun. 16, 78-85. SUSSKIND,M. M., AND BOTSTEIN,D. (1978). Molecular genetics of bacteriophage P22. Microb. Rev. 42, 385413. TANAKA, T., AND WEISBLUM,B. (1975). Construction of colicin El-R factor composite plasmid in vitro: Means for amplification of deoxyribonucleic acid. J. Bacterial. 121, 354-362. THOMAS,M., AND DAVIS, R. W. (1975). Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease. J. Mol. Biol. 91, 315-328. UMEZAWA,H., YAGISAWA,M., MATSUHASHI,Y., NAGANAWA,H., YAMAMOTO, H., KONDO, S., TAKEUCHI, T., AND CHABBERT,Y. A. (1973). Gentamicin acetyltransferase in Escherichia coli carrying R factor. J. Antibiot. (Tokyo) 26, 6 12-6 13. WITCHITZ, J. L. (1972). Plasmid-mediated gentamicin resistance not associated with kanamycin in Enterobacteriaceae. J. Antibiot. (Tokyo) 25, 622-624. WITCHITZ, J. L., AND CHABBERT,Y. A. (197 1). Rbistance transferable & la gentamicine. I. Expression de la resistance.Ann. Inst. Pasteur 121, 733-742. WITCHITZ, J. L., AND GERBAUD,G. R. (1972). Classification de plasmides con&ant la resistance & la gentamicine. Ann. Inst. Pasteur 123, 333-339.