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A method of plasmid classification by integrative incompatibility
PLASMID
3,
116- 127 (1980)
A Method CHIHIRO
of Plasmid SASAKAWA,
Classification
by Integrative
NORIMASA TAKAMATSU,’ HIROFUMI MASANOSUKE YOSHIKAWA
Incompatibility DANBARA,~
AND
Department of Bacterial Infection, Institute of Medical Science, University of Tokyo, Minato-ku, Shiroganedai-machi, Tokyo 108, Japan Received July 24, 1979 A method of plasmid classification by integrative incompatibility has been developed. The characteristics of this system are as follows: (i) The conventional plasmids usually used as standards for incompatibility grouping were integrated into the host chromosome to increase stability and to minimize recombination with the superinfecting plasmid. Strains were constructed by integrative suppression which was in some cases facilitated by the introduction of Tn5 into the plasmid. (ii) The resulting Hfr strains were made deficient in the recA function to eliminate homologous recombination between the resident and the superinfecting plasmids. A test plasmid is introduced into these recA Hfr test strains in the stationary phase of growth. In an incompatible cross, the number of transconjugant colonies was usually less than 1O-2 of that in a compatible cross. Occasionally, an inhibitory mechanism, other than incompatibility wascoded by the resident plasmid [e.g., restriction in R124 (inc FIV)]. Thiscomplicated the interpretation, but did not invalidate the experiment. The colonies arising in incompatible crosses were shown to carry drug resistance determinants coded by both the resident and superinfecting plasmids. These were presumably the result of ret-independent integration of all or part of the superinfecting plasmid into the host chromosome. Thus the reduced frequency of superinfectant formation in an incompatible cross is usually the consequence of incompatibility between the resident and the superinfecting plasmids. This integrative incompatibility system should be useful for epidemiological studies of R plasmids.
Numerous naturally occurring plasmids must be classified into descriptive groups in epidemiological studies on R plasmids. Among various phenotypes of plasmids characterized for this purpose, plasmid incompatibility, the inability of closely related plasmids to be stably comaintained (Novick et al., 1976), has usually been considered to be an indication of the relatedness of two plasmids (Datta, 1974). The determinant of incompatibility has been shown to be required for miniplasmid formation together with other functions, ori and rep, and so believed to be required for maintenance of the replicon autonomously in host bacteria (Timmis et al., 1978; Manis and Kline, 1978; Synenki et al., 1979). These findings have 1 Present address: Kanebo Institute of Pharmacy, Miyakojima-ku, Osaka, Japan. z Present address: Max-Planck-Institut fur Molekulare Genetik, 1 Berlin 33, Ihnestrasse, 63-73 (Dahlem), F. R. G. 0147-619X/80/020116-12$02.00/0 Copyright 8 1960 by Academic Press, Inc. All rights of reproduction in any form reserved.
116
enabled plasmid incompatibility phenotypes to be utilized as indicators of plasmid relatedness for the purpose of classification in epidemiological investigations on the spread of clinically important plasmids. Among enteric plasmids more than 20 incompatibility groups have been classified. Although membership in a particular incompatibility group is defined on the basis of incompatibility between autonomous plasmids, incompatibility also occurs between an integrated and autonomous plasmid (De Vries et al., 1974). Despite the utility and the distinct feature of the phenotypes as described above, the classification of plasmids by incompatibility has not been easily applicable in epidemiological studies due to requirements for complicated genetic manipulations in determining plasmid incompatibility. These difficulties seem to arise from the following two facts. First, loss of the resident plasmid may be due to inherent instability
INTEGRATIVE
PLASMID
INCOMPATIBILITY
rather than to super-infection by an incompatible plasmid. The second difficulty originates from the ease with which two plasmids form a stable recombinant carrying resistance markers derived from both the resident and the superinfecting plasmid. Such a recombinant gives a phenotype similar to that of two stably coexisting compatible plasmids. To distinguish these two situations, a further characterization, which is rather troublesome in large-scale epidemiological studies, is required. We have developed an integrative incompatibility method to classify plasmids by the use of stationary phase cultures of recAdeficient strains with various integrated plasmids as recipients. These Hfr recipients have guaranteed the stability of the plasmid and minimized the possibility of recombination of two plasmids. Thus, the method requires only the counting of colonies superinfected by the plasmid to be tested. MATERIALS
AND METHODS
Bacterial strains and plasmids. The bacterial strains and plasmids used in these experiments are shown in Tables 1 and 2. Media and drugs. Penassay broth (Difco Laboratories, Detroit Mich.) supplemented with 10 pg of thymine per ml (PAB-Thy) was used as the complete liquid medium. EMB agar with 0.5% glucose (Lederberg, 1950; Hirota, 1960) and EM sugar agar (Lederberg, 1950; Hirota, 1960) supplemented with appropriate nutritional requirements were used as the complete and the minimal agar, respectively. Antibiotics were added to agar media at following concentrations: chloramphenicol (Cm; Sankyo, Tokyo) 25 CLg/ml; tetracycline (Tc; Nihon Lederle, Tokyo) 25 &ml; ampicillin sodium salt (Ap; Takeda, Tokyo) 50 pg./ml; kanamycin sulfate (Km; Meiji-seika, Tokyo) 75 &ml; streptomycin sulfate (Sm; Kaken-kagaku, Tokyo) 12.5 E.Lg/ml; and rifampicin (Rif; Sigma, St. Louis) 50 pg/ml. L-broth and L-agar (Lennox, 1955) were used for transduction by Plvir phage. M-9 minimal medium (Miller, 1972) supplemented with 0.2% glu-
GROUPING
117
cose, 0.5% casein hydrolysate (Difco Laboratories, Detroit, Mich.), 10 pg of vitamin Bl, and 2 pg of thymine per ml was used for labeling DNA. [3H]Thymine was purchased from the Radiochemical Centre, Amersham, U. K. Conjugative plasmid transfer. This method was described previously (Sasakawa and Yoshikawa, 1978). Construction of Tn5 inserted-derivatives. To transpose Tn5 to a plasmid, we obtained chromosomal Tn5 insertions by infection of C600 with Xb221~1857rex::Tn5, which was kindly supplied by D. Berg. The method described by Berg (1977) was used. Among 200 kanamycin resistant derivatives, one isoleucine-valine requiring mutant was isolated and the insertion site of the Tn5 transposon was determined at 84 min on the linkage map ofE. coli K12 (Bachmannet al., 1976) by crosses with various F’ strains. YC1216 (see Table 1) was constructed by transducing the ilv::Tn5 segment by Plvir from YC1210 to YC1214, a mal+ derivative of CRT46. One such transductant clone reconfirmed for the DnaA- phenotype and isolucine-valine requirement was designated as YC1216. Hfr construction. The construction of Hfr derivatives by integrative suppression with various plasmids was as described by Nishimura et al. (1973). Plasmid-carrying strains of CRT46 were made by conjugative transfer to CRT46 of individual piasmids from our stock strains carrying representative plasmids belonging to various incompatibility groups. Many of them were kindly given by N. Datta at Royal Postgraduate Medical School, London and by E. M. Lederberg at Plasmid Reference Center, Stanford University . Plasmid-containing strains were plated on EMB glucose agar plates which were then incubated at 42°C for 2 days. The resulting thermoresistant clones were examined for efficiency of plating at 42°C. The clones which showed the highest plating efficiency at 42” were stocked and further characterized for their Hfr natures.
118
SASAKAWA
ET AL.
TABLE BACTERIAL
Strain LC193 JC1569 KL16-99 C600 CRT46 YC258 YC259 YC1210 YC1211 YC1214 YC1216 YC1217 YC1218 YC1221 YC1222 YC2004 YC2005 YC2010 YC2011 YC2012 YC2014 YC2019 YC2020 YC2024 YC202.5 YC2026 YC2028 YCl125 YCll30 YCl135 YC1140 YCl145 YCl150 YC1155 YC1160 YC1165 YC1170 YC1180
STRAINS,
1
PROPERTIES,
AND
ORIGINS
Properties
Origin
F-, i/v, metA, leu, purE, trp, argG, thi, proA or B, x.vl, mtl, ara, lac, gal, malA, str F-, recAl, leu, his, at-g, met, lac, ma/, gal, man Hfr, recA/ , thi F-, thr, leu, thi, lac F-, thy, thr, leu, thi, malA, lac, dnaA(Ts) Rifampicin-resistant derivative of CR34; F-, thy, thr, leu, thi, lac recA derivative of YC258 C600 ilv::TnS As YC1210 but carrying R64drdll Mal+ transductant of CRT46 (dnaA(Ts)) ilv::TnS transductant of YC1214 (dnaA(Ts)) YC1214 carrying pMYll21 (dnaA(Ts)) YC1216 carrying pMYlI21 (dnaA(Ts)) CR34 carrying Rtsl CR34 Nal’ (R6-5) CRT46 carrying R124 CRT46 carrying R27 CRT46 carrying R471a CRT46 carrying R446b CRT46 carrying N3 CRT46 carrying R478 Temperature-resistant revertant of YC2004 Temperature-resistant revertant of YC2005 Temperature-resistant revertant of YC2010 Temperature-resistant revertant of YC2011 Temperature-resistant revertant of YC2012 Temperature-resistant revertant of YC2014 RIP, recA derivative of KL1699 RIP, recA derivative of Hfr (RlOO-1)#2 RiP, recA derivative of YC2019 Rif, recA derivative of YC2024 RiP, recA derivative of YC2025 RiP, recA derivative of YC2026 RiP, recA derivative of YC2028 RIP, recA derivative of YC2020 RIP, recA derivative of Hfr (R6K) RIP, recA derivative of Hfr (Rtsl) RiP, recA derivative of a Hfr strain of YC1218
Demonstration of Hfr characters. Hfr derivatives of CRT46 carrying RlOO-1 (Yoshikawa, 1974), R6K (Sotomura and Yoshikawa, 1973, and Rtsl (Yoshimoto and Yoshikawa, 1975) had been isolated and well characterized previously. Other Hfr candidates isolated in this study were examined for their ability to transfer chromosomal markers sequentially, essentially as described previously (Nishimuraet al., 1971; Sotomura and
Yoshikawa., 1975; Yoshimoto kawa., 1975).
Y. Hirota K. Suzuki H. Ikeda Our collection Y. Hirota This paper This paper This paper This paper This paper This paper This paper This paper Our collection K. Timmis This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper
and Yoshi-
Test forplasmid DNA in Hfr strains. This was made by the alkaline sucrose gradient method (Freifelder et al., 1971). Construction of recA derivatives from Hfr rifampicin-resistant strains. Spontaneous
derivatives (above 100 &ml) were isolated from each of the Hfr strains. Then each of these Rif’ Hfr strains were made recA-defi-
INTEGRATIVE
PLASMID
INCOMPATIBILITY TABLE
CHARACTERISTICS
Plasmid
Incompatibility group
R386 RlOO-1 Rl R6-5 R124 RWrd 11 pMYll21 R27 R478 R47la R446b R69-2 N3 pJA4733 RPC3 Rtsl R401 R6K pA03
FI FII FII FII FIV Io Ia HI H2 L M M N N N T T X -
2
OF THE PLASMIDS
Resistant pattern Tc Tc, Cm, Sm, Su Cm, Sm, Su, Km, Ap Cm, Sm, Su, Km Tc Tc Tc Tc Tc, Cm, Km AP Tc, Su Ap,Km
Tc, Sm, Su Cm, Sm, Ap Km Km AP, Sm
AP
119
GROUPING
USED~
Origin
Source
Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Derepressed mutant of R64 Tn5:insertion mutant of R64drdll Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate ColE 1 derivative
N. Datta Y. Hirota E. M. Lederberg K. Timmis N. Datta N. Datta This paper N. Datta N. Datta N. Datta N. Datta E. M. Lederberg N. Datta T. Arai T. Arai Y. Terawaki N. Datta N. Datta A. Oka
n Abbreviation for plasmid-determined drug resistance markers are as proposed by Novick er al. (1976). Tc, tetracycline; Sm, streptomycin; Cm, chloramphenicol; Su, sulfonamides; Ap, aminobenzylpenicillin; and Km, kanamycin.
cient by mating with KL16-99 for 30 min at 30°C followed by selection for thymine nonrequirement and rifampicin resistance. The recA-deficiency was confirmed by uv sensitivity. Integrative incompatibility test. Rifampicin resistant, recA-Hfr strains were superinfected by a test plasmid in a uecA-deficient strain. Overnight 30°C cultures of the recipient (Hfr) were diluted 20-fold with PAB-Thy and further grown at 30°C for about 22 h with shaking to reach the late stationary phase. On the basis of previous experience, this is expected to minimize the entry exclusion (Lederberg, 1952; Willetts and Maule, 1974) exerted by resident plasmids. A recAdeficient donor strain should be used because the Rif’ gene on the recipient chromosome could be transferred to the donor strain resulting in formation of Rif’ recombinants derived from the donor. A test plasmid is scored as belonging to the same incompatibility group as the resident when the frequency of superinfectant formation is reduced at
least loo-fold in comparison to recipients containing a compatible plasmid or no plasmid. RESULTS Isolation and Characterization of Hfr Strains with Various Plasmids in an Integrated State
Several Hfr strains were isolated by integrative suppression as described in Materials and Methods; others were already available (Yoshikawa, 1974; Sotomura and Yoshikawa, 1975; Yoshimoto and Yoshikawa, 1975). Five temperature;resistant revertants of plasmid-bearing dnaA(Ts) strains were isolated for each plasmid, mated with a multiauxotrophic recipient, LC193, and examined for their ability to form recombinants for four chromosomal markers, metA, argG, his, and proA (or proB) and also for antibiotic resistance markers derived from the plasmid. Table 3 shows the results of a quantitative mating with representative temperature-resistant
120
SASAKAWA
revertants obtained with plasmids R124, R27, R471a, R446b, N3, and R478, respectively. As controls, parental strains carrying the respective plasmids autonomously were used. In this case, only the antibiotic resistance markers were transferred. With the temperature-resistant revertants the transfer frequency for antibiotic resistance markers was usually lower and the recombination frequency for at least one of four chromosomal markers was higher than with the parental strains with an autonomous plasmid. The relative frequencies of recombination for the four markers revealed that the chromosome was transferred sequentially. These results suggest that each thermoresistant isolate was an Hfr with a fixed origin and a sequential gradient of transfer. To prove further that the plasmid exists in an integrated state in these integratively suppressed strains, Hfr derivatives and their plasmid-carrying parents were grown in M-9 glucose medium with 2 pg of thymine per ml and [3H]thymine (6- 10 &i/ml) for about two to three generations at 30°C. Lysates were prepared and analyzed by the alkaline sucrose gradient
ET AL.
method (Freifelderet al., 1971). As shown in Fig. 1, all the DNA preparations from Hfr derivatives showed no detectable peak of radioactivity at the position where the parent carrying the same plasmid autonomously gave a peak. Hfr Construction by Transposon Mediated Plasmid Integration
Although all the conjugative plasmids so far tested conferred upon the dnaA(Ts) host an ability to give temperature-resistant revertants at a frequency higher than that without the plasmid, neither the integrative suppression method (Nishimura et al., 1971) nor the directed transposition method (Ippen et al., 1971) gave stable Hfr strains with some conjugative plasmids including R64 and mini ColEl. To overcome this, we attempted to obtain transposon-mediated integration of plasmids (Kleckner et al., 1977; Danilevich et al., 1978) into the host chromosome. An example involving R64drd 11, belonging to incompatibility group Ia is described. YC1216 (CRT46ilv::TtG) was first
TABLE SEQUENTIAL
GENE
TRANSFER
(Tn5)-
BY THERMORESISTANT
3 DERIVATWES
FROM
CRT46(R+)
STRAINS”
Phenotype selected Plasmid
Incompatibility group
Strain
Properties
Met+
Arg+
His+
Pro+
Drug
R124
FIV
YC2004 YC2019
Autonomous T revertant
(1 200
4000 80
R2J
Hl
YC2005 YC2020
Autonomous T revertant
400
R4Jla
L
YC2010 YC2024
Autonomous T revertant
70000
R446b
M
YC2011 YC2025
Autonomous T revertant
10000 100
N3
N
YC2012 YC2026
Autonomous T revertant
8000 10
R478
H2
YC2014 YC2028
Autonomous T revertant
7
100
200 30
o Frequency of recombination in the crosses of plasmid-bearing dnuA(Ts) strains (autonomous) or their temperature resistant revertants (T revertant) and a recipient, LC193. Mating conditions were described in Materials and Methods. Figures show the number of colonies yielded by a unit volume of mating mixtures.
INTEGRATIVE
PLASMID
INCOMPATIBILITY
121
GROUPING
-6
-4
1 D)
6-
-1 R27 1
“0 ;; It
4
F
2 J;‘k
R6-5 1
0
F)
(E)
I
I 6
I FRACTION
NUMBER
FIG. 1. Alkaline sucrose gradient analysis of DNAs extracted from thermoresistant strains. Cells were grown at 30°C in M-9 glucose medium supplemented with a nutritional requirements and containing 6-10 &i of rH]thymine per ml. At a cell density of about 5 x 1oRper ml, lysates were prepared and layered onto 5 to 20% linear alkaline sucrose gradients, which were centrifuged in an SW65 rotor (Beckman) at 40,000 rpm for 18 min at 18°C. Lysates were cosedimented with a reference DNA in the following combinations. (A) Lysates from YC2012 (N3 autonomously) and YC1221; (B) lysates from YC2026 (N3 integrated) and YC1221; (C) lysates from YC2005 (R27 autonomously) and YC1222; (D) lysates from YC2020 (R27 integrated) and YC1222; (E) lysates from YC2014 (R478 autonomously) and YC1222; (F) lysates from YC2028 (R478 integrated) and YC1222. Note the peak of sheared DNA from the host chromosome on the right of each panel plotted on a different scale. Panels A, C, and E are the results with strains carrying autonomous plasmids, whereas panels B, D, and F are those with Hfr strains with respective plasmids.
constructed and a plasmid, pMY 1121 (R64drdl 1: :Tn5), was then transferred into YC1216 and YC1214 (transposon-free) by
conjugation. The respective transconjugants, designated as YC1218 and YC1217, respectively, were examined for the frequency of
122
SASAKAWA
formation of thermoresistant revertants at 42°C. Table 4 shows a marked increase in number of thermoresistant revertants of YC1217aswellasYC1218ascomparedwith YC1214. However, there was no difference in the frequency of thermoresistant revertant formation between YC1217 and YC1218 (Table 4). Several thermoresistant revertants of YC1218 were picked and examined for the Hfr character. They were shown to be Hfrs but the site of integration was not at ilv (84 min) but near his (44 min). Thus, Hfr clones with an integrated R64drdll plasmid could be constructed by a combination of transposon-mediated integration and integrative suppression. It should be possible to construct Hfr strains by this method with any plasmid as long as the plasmid carries the genetic potentiality to provoke integrative suppression. In addition, we also succeeded (Sasakawa and Yoshikawa, in preparation) in isolating clones with integrated ColE 1 or its mini-derivative, pA03 (Oka et al., 1978) mediated by Tn5. Integration of ColEl itself had not been observed previously (Sotomura and Yoshikawa, 1975) by ordinary suppressive integration. However, a dnaA(Ts) host strain with Tn5 at the ilv locus and ColEl or pA03 inserted by the same transposon did produce integratively suppressed strains efficiently. Superinfectant Formation with Respect to the State of Existence of the Resident Plasmid and Recombination Proficiency and Deficiency. One of the reasons why the resident plasmid has been integrated is to guarantee TABLE EFFECTOFT~SON
ET AL.
its stability. However, even with these Hfr strains, the plasmid may be detached from the chromosome and cured. On the other hand, two plasmids belonging to the same incompatibility groups often share extensive sequence homology (Falkow et al., 1974) and hence easily recombine. These two problems, the detachment and curing of the resident plasmid and homologous recombinant formation in superinfectants, would be minimized by using Hfr strains and making them recombination-deficient. Thus, a study was made of superinfectant formation as a function of the state of the resident plasmid and of proficiency and deficiency in generalized recombination. As shown in Table 5, in incompatible crosses with the same plasmid pairs, the frequency of superinfectant formation was lower when an Hfr was used as the recipient than when a strain carrying the same plasmid autonomously was used. The introduction of recA deficiency was also effective in decreasing the frequency of establishment of the selective marker of the superinfecting piasmid, as shown in Table 5. Demonstration of the Simplicity Reliability of this Integrative Incompatibility Test
Five Rif’ recA Hfr derivatives having integrated plasmids belonging to various incompatibility groups were used as recipients, and examined for the formation of superinfectants. As shown in Table 6, in the crosses between incompatible plasmids (italics) superinfectant formation was always reduced to a frequency at least 100-fold lower than 4
FORMATIONOFTHERMORESISTANTREVERTANTS Properties
strain
Chromosome
YC1214 YC1217 YC1218
ilv::TnS
and
Colony counts per ml at Plasmid R64drdll::Tn5 Rbldrdll::Tn5
30°C
42°C
8.4 x lo8 3.6 x lo8 5.0 x 108
4.2 x lo* 4.2 x lo5 7.2 x 105
INTEGRATIVE
PLASMID
INCOMPATIBILITY TABLE
SIJPERINFECTANT
123
GROUPING
5
FORMATION IN INCOMPATIBLE CROSSES WITH REFERENCE TO THE STATE OF EXISTENCE OF THE PLASMID AND RECOMBINATION PROFICIENCY AND DEFICIENCY”
Relative frequency of superinfectant formations* State
recA + + +
Hfr Hfr Autonomous’ -
(RlOO-1):Rl’
(Rtsl):R401d
(N3):RPC3’
<7 x 10-S 0.03 0.46 1.00
1 x 10-a 0.02 0.23 1.00
<5 x 10-S 0.01 0.21 1.00
<3 x 10-Z 0.10 NT’ 1.00
a JC1569 with various plasmids to be tested was used as the donor and mating conditions were as described in Materials and Methods. * Figures are expressed as a ratio of the frequency of superinfectant formation in each cross relative to that in the cross with a plasmid-free strain, YC258. The resident plasmid in each cross is given in parentheses followed by the code of the superinfecting plasmid. c Rii and Km were used for selection of superinfectants. d Rif and Ap were used for selection of superinfectants. e YC258 was used as the host strain. ’ NT, not tested.
that in compatible crosses. These results suggest that the decrease in superinfectant formation was mainly due to incompatibility between the resident and the superinfecting plasmid. Consequently, this system should be applicable to the classification of conjugative plasmids by incompatibility, especially in epidemiological surveys of R plasmids. Thus far, we have constructed 11 recombination-deficient Hfr strains belonging to representative incompatibility groups for this purpose (Table 7). Under appropriately TABLE DEMONSTRATION
controlled conditions, the mating mixtures of many crosses may be scored on a single plate. DISCUSSION
The use of incompatibility to classify large numbers of naturally occurring plasmids isolated in an epidemiological survey requires a test that is simple as well as scientifically accurate. However, the standard method in general use is slow, laborious, 6
OF INTEGRATIVE
INCOMPATIBILITY~
Superinfected plasmid
Strain
Resident plasmid
YCl125 YCll30 YCll50 YCll40 YC1155 YC259
F RlOO-1 N3 Rts 1 R446b -
R386
Rl
pJA4733
R401
R69-2
Incompatibility
FI
FII
N
T
M
FI FII N T M -
0.08 NT NT 1.8 NT 1.0
1.2 co.02 3.7 NT 2.0 1.0
>l >II >l 1.0
0.9 0.5 2.6 5 x IO-” 0.8 1.0
1.0 1.1 1.4 1.2 0.01 1.0
n The experimental conditions were the same as in Table 5, except that YC259 was used as the plasmid-free control.
124
SASAKAWA
ET AL.
TABLE RIFAMPICIN-RESISTANT, recA Hfr INTEGRATIVE
Strain codes of rif-r, recA Hfr YC112.5 YC1130 YC1135 YC1160 YC1140 YCI 14.5
YCl150 YC1155 YC1165 YC1170 YCll80
Plasmid
Incompatibility FI FII FIV Hl L M
N3
N
R478 R6K Rts 1
H2 x T Iff
11
STRAINS
INCOMPATIBILITY
F RlOO-1 R124 R27 R471a R446b
R64drd
7 TO BE USED
AS RECIPIENT
FOR
TESTING
Resistance patterns Tc Cm Sm Su Tc Tc AP Tc Su Tc Sm Su Cm Tc Km AP Km Tc
Leading marker in chromosome transfer thyA metA metA metA
argG his metA his metA argG his
and sometimes difficult to interpret. Thus, suggesting that a marked reduction in superinstability may result in superinfectants ap- infectants may occur due only to incompatiparently lacking the resident plasmid be- bility. However, we have to take into account cause of spontaneous curing rather than other plasmid-determined factors that may incompatibility; recombination may complireduce the number of superinfectants. The cate the scoring of colonies having resistance most obvious example is the degradation of markers derived from both the resident and the superinfecting replicon (Yoshikawa and superinfecting plasmids (Falkow et al., Akiba, 1962). In fact, we have sometimes 1974). The use of recA strains with the observed a reduction in superinfectants due reference plasmids integrated, as recipients, to the Hsp 1 phenotype when an Hfr recipient appears to have largely overcome these with R124 was used. Other, as yet unknown, problems. mechanisms may also exist. The use of recA In analogy to the already known examples donors to prevent colony formation by of the F plasmid (Lederberg et al., 1952) chromosomal Rif’ recombinants appears to and those belonging to the FI and FII in- involve a troublesome additional step in strain construction. However, in any epicompatibility groups such as ColV2, R538-1, demiological survey, it is the first job to ColVBtrp, Rldrdl9, RlOO-1, and R136 (Wilprove that field isolates carry a conjugative letts and Maule, 1974) the Hfr recipients were grown up to late stationary phase to R plasmid. For this purpose the best apminimize the effect of entry exclusion. How- proach is to transfer the drug resistance to ever, the entry exclusion mechanism (Novick a recA strain. After this has been proven, the resulting recA strain carrying the plaset al., 1976) may not always be overcome by the use of stationary phase; this may mid isolated from a field specimen may be result in reduction of superinfectants mainly now used as the donor for an integrative incompatibility test. by entry exclusion rather than by incomIn incompatible crosses with recA recipipatibility. In this respect it is noteworthy ents, a few colonies have occasionally been that a marked reduction of superinfectants occurred in the cross of RIOO-1 with RI observed. These were further characterized (Table 5). These plasmids belong to the same and shown to have emerged presumably by transposition of various incompatibility group but to different entry recA-independent parts of the superinfecting plasmid most exclusion systems (Willetts and Maule, 1974),
INTEGRATIVE
PLASMID
INCOMPATIBILITY
likely to the host choromosome (data not shown). The standard incompatibility grouping test in use at present is based on the interaction between two autonomously replicating plasmids and in this point differs from our modified method which is based on the interaction of an autonomous with an integrated plasmid. These two may be different phenomena but are in any case based on the relatedness of two plasmids (Scaife and Gross, 1962; Echols, 1963; de Vries et al., 1975; Yoshikawa, 1975; Novick and Schwesinger, 1976; Pfister et al., 1976; Sasakawa et al., 1979). In fact, the phenomenon of incompatibility was first found in the introduction of F’ plasmids into Hfr cells (Scaife and Gross, 1962; Echols, 1963). Various conjugative plasmids have been shown to give rise to Hfr derivatives by integrative suppression (Nishimura et al., 1971; Lindahletal., 1971; Moody and Runge, 1972; Nishimura et al., 1973; Goebel, 1974; Yoshikawa, 1974; Sotomura and Yoshikawa, 1975; Yoshimoto and Yoshikawa, 1975; Datta and Barth, 1976; Chesney and Scott, 1978). In this paper we have extended the list of these to include R124, R471a, R446b, N3, R27, and R478 (Table 3). Although we observed a marked increase in the frequency of thermoresistant (dnaA (Tr)) revertants with all the plasmids tested, in some cases, namely, plasmids of incompatibility groups Inc Ia, Inc W, Inc P, and Inc A-C, we were unable to isolate stable Hfr derivatives. Similar results have been obtained previously with some of these (Datta and Barth, 1976; Martin et al., Abstr. Annu. Meet. Amer. Sot. Microbial. 1975, H52, p. 104). We tried in vain to isolate stable Hfr strains with these plasmids also by the directed transposition method (Ippen et al., 1971). However, plasmid integration has been shown to be facilitated by the presence of a transposon, Tnl, on both plasmid and chromosome (Danilevich ef al., 1978). In our attempts to demonstrate this phenomenon with R64drd 11 and Tn5, we found that a copy of Tn5 on the plasmid greatly increased
GROUPING
125
the frequency of stable Hfr’s but that a chromosomal copy had no effect (Table 4); indeed, when Tn5 was present on both, insertion took place at a chromosomal site far distant from the location of the transposon. Thus, integrative suppression is evidently not due to TnS-mediated homologous recombination; rather, the plasmid-carried Tn5 accelerated illegitimate recombination elsewhere on the chromosome. In any case, the introduction of a transposon is likely to permit the construction of Hfr strains probably with any plasmid. It also appears that any plasmid that can replicate autonomously and does not require dnuA for its own replication may potentially be suppressively integrated in a dnaA (Ts) strain. Thus, the simplicity and reliability of this integrative incompatibility test method has been suggested by the experimental results presented in this paper. In addition, several Japanese members of the Plasmid Epidemiology Group have initiated an examination of the validity of this method. According to personal communication from some of them (Terakado, National Institute of Animal Health, Tsukuba; Nakaya et al., Tokyo Medical and Dental University), all of their clinically isolated plasmids so far tested were shown to give the same results when judged both by the standard autonomousto-autonomous method and by our autonomous-to-integrated method. ACKNOWLEDGMENTS The authors wish to thank Dr. N. Datta and Dr. E. M. Lederberg for their generous gift of plasmids, Dr. D. E. Berg for giving the phage of Ab22Zc1857rex::Tn5, an editor and a referee for valuable suggestions, and Dr. Terakado and Dr. Nakaya and their collaborators for generously communicating with us their unpublished data. This work was supported in part by a grant provided by the Ministry of Education, Science and Culture of the Japanese Government (Grant numbers 211901, 311202 and 410701).
REFERENCES B.J.,Low,K.B., ANDTAYLOR, (1976). Recalibrated linkage map of Escherichia K-12. Bacterial. Rev. 40, 116- 167.
BACHMANN,
A. L. coli
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SASAKAWA
BERG, D. (1977). Insertion and excision of the transposable kanamycin resistance determinant Tn5. In “DNA Insertion Elements, Plasmids, and Episomes” (A. I. Bukhari, J. A. Shapiro, and S. L. Adhya, eds.), pp. 205-212. Cold Spring Harbor Laboratory. CHESNEY, R. H., AND SCOTT, J. R. (1978). Suppression of a thermosensitive dnaA mutation of Escherichia coli by bacteriophage Pl and W. Plasmid 1, 145163. DANILEVICH, V. N., STEPHANSHIN, Y. G., VOLOZHANTSEV, N. V., AND BOLUB, E. I. (1978). Transposon-mediated insertion of R factor into bacterial chromosome. Molec. Gen. Genet. 161, 337-339. DATTA, N. (1974). Epidemiology and classification of plasmids. In “Microbiology-1974” (D. Schlessinger, ed.), pp. 9- 15. American Society of Microbiology, Washington, D. C. DATTA, N., AND BARTH, P. T. (1976). Hfr formation by I pilus-determining plasmids in Escherichia coli K-12. J. Bacterial. 125, 811-817. DEVRIES, J. K., PFISTER, A., HAENNI, C., PALCHAUDHURI, S., AND MAAS, W. K. (1974). Fincompatibility. In “Microbiology-1974” (D. Schlessinger, eds.). pp. 166- 170. American Society of Microbiology, Washington, D. C. ECHOLS, H. (1963). Properties of F’ strains of Escherichia coli superinfected with F-lactose and F-galactose episomes. .I. Bacterial. 85, 262-268. FALKOW, S., GUERRY, P., HEDGES, R. W., AND DATTA, N. (1974). Polynucleotide sequence relationships among plasmids of the I compatibility complex. J. Gen. Miclobiol. 83, 65-76. FREIFELDER, D., FOLKMANIS, A., AND KIRSCHNER, I. (1971). Studies on Escherichia co/i sex factors: evidence that covalent circles exist within cells and the general problem of isolation of covalent circles. .I. Bacterial. 105, 722-727. Cioaa~L, W. (1974). Integrative suppression of temperature-sensitive mutants with a Iesion in the initiation of DNA replication. Eur. J. Biochem. 43, 125130. HIROTA, Y. (1960). The effect of acridine dyes on mating type factors in E. coli. Proc. Nut. Acad. Sci. U. S. A. 46, 57-64. IPPEN, K., SHAPIRO, J., AND BECKWITH, J. (1971). Transposition of the lac region to the gal region of the Escherichia coli chromosome: isolation of Alac transducing bacteriophages. J. Bacferiol. 108, 5-9. KLECKNER, N., ROTH, J., AND BOTSTEIN, D. (1977). Genetic engineering in viva using translocatable drug-resistance elements: new methods in bacterial genetics. J. Mol. Biol. 116, 125-159. LEDERBERG, J. (1950). Isolation and characterization of biochemical mutants of bacteria. Methods. Med. Res. 3, 5-22. LEDERBERG, J., CAVALLI, L. L., AND LEDERBERG, E. M. (1952). Sex compatibility in Escherichia coli. Genetics 37, 720-730. LENNOX, E. S. (1955). Transduction of linked genetic
ET AL. characters of the host by bacteriophage Pl. Virology 1, 190-206. LINDAHL, G., HIROTA, Y., AND JACOB, F. (1971). On the process of cellular division in Escherichia coli: replication of the bacterial chromosome under control of prophage P2. Proc. Nat. Acad. Sci. U. S. A. 68, 2407-2411. MANIS, J. J., AND KLINE, B. C. (1978). F plasmid incompatibility and copy number genes: their map location and interactions. Plasmid 1, 492-507. MILLER, J. H. (1972). “Experiments in Molecular Genetics.” Cold Spring Harbor Laboratory. MOODY, E. E. M., AND RUNGE, R. (1972). The integration of autonomous transmissible plasmids into the chromosome of Escherichia co/i K12. Genet. Res. 19, 181-186. NISHIMURA, Y., CARO, L., BERG, C. M., AND HIROTA, Y. (1971). Chromosome replication in Escherichia coli. IV. Control of chromosome replication and cell division by an integrated episome. J. Molec. Biol. 55, 441-456. NISHIMURA, A., NISHIMURA, Y., AND CARO, L. (1973). Isolation of Hfr strains from R+ and ColV2+ strains of Escherichia coli and derivation of an R’lac factor by transduction. J. Bacterial. 116, 1 lO7- 1112. NOVICK, R. P., AND SCHWESINGER, M. (1975). Independence of plasmid incompatibility and replication control functions in Staphylococcus aureus. Nature (London) 262, 623-626. NOVICK, R. P., CLOWES, R. C., COHEN, S. N., CURTISS, R., III, DATTA, N., AND FALKOW, S. (1976). Uniform nomenclature for bacterial plasmid: A proposal. Bacreriol. Rev. 40, l68- 189. OKA, A., NOMURA, N., SUGIMOTO, K., SUGISAKI, H., AND TAKANAMI, M. (1978). Nucleotide sequence at the insertion site of a kanamycin transposon. Nature (London) 276, 845-847. PFISTER, A., DEVRIES, J. K., AND MASS, W. K. (1976). Expression of a mutation affecting F incompatibility in the integrated but not the autonomous state of F. J. Bacterial. 127, 348-353. SASAKAWA, C., TSUCHIYA, T., AND YOSHIKAWA, M. (1979). Isolation and characterization of plasmiddeletion derivatives from an Hfr made with Rtsl carrying a chromosomal mutation, p/t. Molec. Gen. Gene?. (in press). SASAKAWA, C., AND YOSHIKAWA, M. (1978). Requirements for suppression of a dnaG mutation by an Itype plasmid. J. Bacreriol. 133, 485-491. SCAIFE, J., AND GROSS, J. (1%2). Inhibition of multiplication of an F’-lac factor in Hfr cells OfEscherichia co/i K-12. Biochem. Biophys. Res. Commun. 7, 403-407. SOTOMURA, M., AND YOSHIKAWA, M. (1975). Reinitiation of chromosome replication in the presence of chloramphenicol under an integratively suppressed state by R6K. J. Bacterial. 122, 623-628. SYNENKI, M. R., NORDHEIM, A., AND TIMMIS, K. N. (1979). Plasmid replication functions. III. Origin and
INTEGRATIVE
PLASMID
INCOMPATIBILITY
direction of replication of a “mini” plasmid derived from R6-5. Molec. Gen. Genet. 168, 27-36. TIMMIS, K. N., ANDRES, I., AND SLOCOMBE, P. M. (1978). Plasmid incompatibility: cloning analysis of an inc FII determinant of R6-5. Nature (London) 273, 27-32. WILLETTS, N., AND MAULE, J. (1974). Interactions between the surface exclusion systems of some Flike plasmids. Genet. Res. 24, 81-89. YOSHIKAWA, M. (1974). Identification and mapping of the replication genes of an R factor, RlOO-1, integrated into the chromosome ofEscherichia coli K-12. J. Bacteriol. 118, 1123- 113 1.
GROUPING
127
YOSHIKAWA, M. (1975). Genetic loci responsible for incompatibility in a co-integrate plasmid, RlOO-1. J. Bacterial. 124, 1097- 1100. YOSHIKAWA, M., AND AKIBA, T. (1962). Studies on transferable drug resistance in bacteria. IV. Suppression of plaque formation of phages by the resistance factor. Japan. J. Microbial. 6, 121-132. YOSHIMOTO, H., AND YOSHIKAWA, M. (1975). Chromosome-plasmid interaction in Eschen’chia co/i K-12 carrying a thermosensitive plasmid, Rtsl, in autonomous and integrated state. .I. Bacterial. 124, 661-667.
3,
116- 127 (1980)
A Method CHIHIRO
of Plasmid SASAKAWA,
Classification
by Integrative
NORIMASA TAKAMATSU,’ HIROFUMI MASANOSUKE YOSHIKAWA
Incompatibility DANBARA,~
AND
Department of Bacterial Infection, Institute of Medical Science, University of Tokyo, Minato-ku, Shiroganedai-machi, Tokyo 108, Japan Received July 24, 1979 A method of plasmid classification by integrative incompatibility has been developed. The characteristics of this system are as follows: (i) The conventional plasmids usually used as standards for incompatibility grouping were integrated into the host chromosome to increase stability and to minimize recombination with the superinfecting plasmid. Strains were constructed by integrative suppression which was in some cases facilitated by the introduction of Tn5 into the plasmid. (ii) The resulting Hfr strains were made deficient in the recA function to eliminate homologous recombination between the resident and the superinfecting plasmids. A test plasmid is introduced into these recA Hfr test strains in the stationary phase of growth. In an incompatible cross, the number of transconjugant colonies was usually less than 1O-2 of that in a compatible cross. Occasionally, an inhibitory mechanism, other than incompatibility wascoded by the resident plasmid [e.g., restriction in R124 (inc FIV)]. Thiscomplicated the interpretation, but did not invalidate the experiment. The colonies arising in incompatible crosses were shown to carry drug resistance determinants coded by both the resident and superinfecting plasmids. These were presumably the result of ret-independent integration of all or part of the superinfecting plasmid into the host chromosome. Thus the reduced frequency of superinfectant formation in an incompatible cross is usually the consequence of incompatibility between the resident and the superinfecting plasmids. This integrative incompatibility system should be useful for epidemiological studies of R plasmids.
Numerous naturally occurring plasmids must be classified into descriptive groups in epidemiological studies on R plasmids. Among various phenotypes of plasmids characterized for this purpose, plasmid incompatibility, the inability of closely related plasmids to be stably comaintained (Novick et al., 1976), has usually been considered to be an indication of the relatedness of two plasmids (Datta, 1974). The determinant of incompatibility has been shown to be required for miniplasmid formation together with other functions, ori and rep, and so believed to be required for maintenance of the replicon autonomously in host bacteria (Timmis et al., 1978; Manis and Kline, 1978; Synenki et al., 1979). These findings have 1 Present address: Kanebo Institute of Pharmacy, Miyakojima-ku, Osaka, Japan. z Present address: Max-Planck-Institut fur Molekulare Genetik, 1 Berlin 33, Ihnestrasse, 63-73 (Dahlem), F. R. G. 0147-619X/80/020116-12$02.00/0 Copyright 8 1960 by Academic Press, Inc. All rights of reproduction in any form reserved.
116
enabled plasmid incompatibility phenotypes to be utilized as indicators of plasmid relatedness for the purpose of classification in epidemiological investigations on the spread of clinically important plasmids. Among enteric plasmids more than 20 incompatibility groups have been classified. Although membership in a particular incompatibility group is defined on the basis of incompatibility between autonomous plasmids, incompatibility also occurs between an integrated and autonomous plasmid (De Vries et al., 1974). Despite the utility and the distinct feature of the phenotypes as described above, the classification of plasmids by incompatibility has not been easily applicable in epidemiological studies due to requirements for complicated genetic manipulations in determining plasmid incompatibility. These difficulties seem to arise from the following two facts. First, loss of the resident plasmid may be due to inherent instability
INTEGRATIVE
PLASMID
INCOMPATIBILITY
rather than to super-infection by an incompatible plasmid. The second difficulty originates from the ease with which two plasmids form a stable recombinant carrying resistance markers derived from both the resident and the superinfecting plasmid. Such a recombinant gives a phenotype similar to that of two stably coexisting compatible plasmids. To distinguish these two situations, a further characterization, which is rather troublesome in large-scale epidemiological studies, is required. We have developed an integrative incompatibility method to classify plasmids by the use of stationary phase cultures of recAdeficient strains with various integrated plasmids as recipients. These Hfr recipients have guaranteed the stability of the plasmid and minimized the possibility of recombination of two plasmids. Thus, the method requires only the counting of colonies superinfected by the plasmid to be tested. MATERIALS
AND METHODS
Bacterial strains and plasmids. The bacterial strains and plasmids used in these experiments are shown in Tables 1 and 2. Media and drugs. Penassay broth (Difco Laboratories, Detroit Mich.) supplemented with 10 pg of thymine per ml (PAB-Thy) was used as the complete liquid medium. EMB agar with 0.5% glucose (Lederberg, 1950; Hirota, 1960) and EM sugar agar (Lederberg, 1950; Hirota, 1960) supplemented with appropriate nutritional requirements were used as the complete and the minimal agar, respectively. Antibiotics were added to agar media at following concentrations: chloramphenicol (Cm; Sankyo, Tokyo) 25 CLg/ml; tetracycline (Tc; Nihon Lederle, Tokyo) 25 &ml; ampicillin sodium salt (Ap; Takeda, Tokyo) 50 pg./ml; kanamycin sulfate (Km; Meiji-seika, Tokyo) 75 &ml; streptomycin sulfate (Sm; Kaken-kagaku, Tokyo) 12.5 E.Lg/ml; and rifampicin (Rif; Sigma, St. Louis) 50 pg/ml. L-broth and L-agar (Lennox, 1955) were used for transduction by Plvir phage. M-9 minimal medium (Miller, 1972) supplemented with 0.2% glu-
GROUPING
117
cose, 0.5% casein hydrolysate (Difco Laboratories, Detroit, Mich.), 10 pg of vitamin Bl, and 2 pg of thymine per ml was used for labeling DNA. [3H]Thymine was purchased from the Radiochemical Centre, Amersham, U. K. Conjugative plasmid transfer. This method was described previously (Sasakawa and Yoshikawa, 1978). Construction of Tn5 inserted-derivatives. To transpose Tn5 to a plasmid, we obtained chromosomal Tn5 insertions by infection of C600 with Xb221~1857rex::Tn5, which was kindly supplied by D. Berg. The method described by Berg (1977) was used. Among 200 kanamycin resistant derivatives, one isoleucine-valine requiring mutant was isolated and the insertion site of the Tn5 transposon was determined at 84 min on the linkage map ofE. coli K12 (Bachmannet al., 1976) by crosses with various F’ strains. YC1216 (see Table 1) was constructed by transducing the ilv::Tn5 segment by Plvir from YC1210 to YC1214, a mal+ derivative of CRT46. One such transductant clone reconfirmed for the DnaA- phenotype and isolucine-valine requirement was designated as YC1216. Hfr construction. The construction of Hfr derivatives by integrative suppression with various plasmids was as described by Nishimura et al. (1973). Plasmid-carrying strains of CRT46 were made by conjugative transfer to CRT46 of individual piasmids from our stock strains carrying representative plasmids belonging to various incompatibility groups. Many of them were kindly given by N. Datta at Royal Postgraduate Medical School, London and by E. M. Lederberg at Plasmid Reference Center, Stanford University . Plasmid-containing strains were plated on EMB glucose agar plates which were then incubated at 42°C for 2 days. The resulting thermoresistant clones were examined for efficiency of plating at 42°C. The clones which showed the highest plating efficiency at 42” were stocked and further characterized for their Hfr natures.
118
SASAKAWA
ET AL.
TABLE BACTERIAL
Strain LC193 JC1569 KL16-99 C600 CRT46 YC258 YC259 YC1210 YC1211 YC1214 YC1216 YC1217 YC1218 YC1221 YC1222 YC2004 YC2005 YC2010 YC2011 YC2012 YC2014 YC2019 YC2020 YC2024 YC202.5 YC2026 YC2028 YCl125 YCll30 YCl135 YC1140 YCl145 YCl150 YC1155 YC1160 YC1165 YC1170 YC1180
STRAINS,
1
PROPERTIES,
AND
ORIGINS
Properties
Origin
F-, i/v, metA, leu, purE, trp, argG, thi, proA or B, x.vl, mtl, ara, lac, gal, malA, str F-, recAl, leu, his, at-g, met, lac, ma/, gal, man Hfr, recA/ , thi F-, thr, leu, thi, lac F-, thy, thr, leu, thi, malA, lac, dnaA(Ts) Rifampicin-resistant derivative of CR34; F-, thy, thr, leu, thi, lac recA derivative of YC258 C600 ilv::TnS As YC1210 but carrying R64drdll Mal+ transductant of CRT46 (dnaA(Ts)) ilv::TnS transductant of YC1214 (dnaA(Ts)) YC1214 carrying pMYll21 (dnaA(Ts)) YC1216 carrying pMYlI21 (dnaA(Ts)) CR34 carrying Rtsl CR34 Nal’ (R6-5) CRT46 carrying R124 CRT46 carrying R27 CRT46 carrying R471a CRT46 carrying R446b CRT46 carrying N3 CRT46 carrying R478 Temperature-resistant revertant of YC2004 Temperature-resistant revertant of YC2005 Temperature-resistant revertant of YC2010 Temperature-resistant revertant of YC2011 Temperature-resistant revertant of YC2012 Temperature-resistant revertant of YC2014 RIP, recA derivative of KL1699 RIP, recA derivative of Hfr (RlOO-1)#2 RiP, recA derivative of YC2019 Rif, recA derivative of YC2024 RiP, recA derivative of YC2025 RiP, recA derivative of YC2026 RiP, recA derivative of YC2028 RIP, recA derivative of YC2020 RIP, recA derivative of Hfr (R6K) RIP, recA derivative of Hfr (Rtsl) RiP, recA derivative of a Hfr strain of YC1218
Demonstration of Hfr characters. Hfr derivatives of CRT46 carrying RlOO-1 (Yoshikawa, 1974), R6K (Sotomura and Yoshikawa, 1973, and Rtsl (Yoshimoto and Yoshikawa, 1975) had been isolated and well characterized previously. Other Hfr candidates isolated in this study were examined for their ability to transfer chromosomal markers sequentially, essentially as described previously (Nishimuraet al., 1971; Sotomura and
Yoshikawa., 1975; Yoshimoto kawa., 1975).
Y. Hirota K. Suzuki H. Ikeda Our collection Y. Hirota This paper This paper This paper This paper This paper This paper This paper This paper Our collection K. Timmis This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper This paper
and Yoshi-
Test forplasmid DNA in Hfr strains. This was made by the alkaline sucrose gradient method (Freifelder et al., 1971). Construction of recA derivatives from Hfr rifampicin-resistant strains. Spontaneous
derivatives (above 100 &ml) were isolated from each of the Hfr strains. Then each of these Rif’ Hfr strains were made recA-defi-
INTEGRATIVE
PLASMID
INCOMPATIBILITY TABLE
CHARACTERISTICS
Plasmid
Incompatibility group
R386 RlOO-1 Rl R6-5 R124 RWrd 11 pMYll21 R27 R478 R47la R446b R69-2 N3 pJA4733 RPC3 Rtsl R401 R6K pA03
FI FII FII FII FIV Io Ia HI H2 L M M N N N T T X -
2
OF THE PLASMIDS
Resistant pattern Tc Tc, Cm, Sm, Su Cm, Sm, Su, Km, Ap Cm, Sm, Su, Km Tc Tc Tc Tc Tc, Cm, Km AP Tc, Su Ap,Km
Tc, Sm, Su Cm, Sm, Ap Km Km AP, Sm
AP
119
GROUPING
USED~
Origin
Source
Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Derepressed mutant of R64 Tn5:insertion mutant of R64drdll Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate Natural isolate ColE 1 derivative
N. Datta Y. Hirota E. M. Lederberg K. Timmis N. Datta N. Datta This paper N. Datta N. Datta N. Datta N. Datta E. M. Lederberg N. Datta T. Arai T. Arai Y. Terawaki N. Datta N. Datta A. Oka
n Abbreviation for plasmid-determined drug resistance markers are as proposed by Novick er al. (1976). Tc, tetracycline; Sm, streptomycin; Cm, chloramphenicol; Su, sulfonamides; Ap, aminobenzylpenicillin; and Km, kanamycin.
cient by mating with KL16-99 for 30 min at 30°C followed by selection for thymine nonrequirement and rifampicin resistance. The recA-deficiency was confirmed by uv sensitivity. Integrative incompatibility test. Rifampicin resistant, recA-Hfr strains were superinfected by a test plasmid in a uecA-deficient strain. Overnight 30°C cultures of the recipient (Hfr) were diluted 20-fold with PAB-Thy and further grown at 30°C for about 22 h with shaking to reach the late stationary phase. On the basis of previous experience, this is expected to minimize the entry exclusion (Lederberg, 1952; Willetts and Maule, 1974) exerted by resident plasmids. A recAdeficient donor strain should be used because the Rif’ gene on the recipient chromosome could be transferred to the donor strain resulting in formation of Rif’ recombinants derived from the donor. A test plasmid is scored as belonging to the same incompatibility group as the resident when the frequency of superinfectant formation is reduced at
least loo-fold in comparison to recipients containing a compatible plasmid or no plasmid. RESULTS Isolation and Characterization of Hfr Strains with Various Plasmids in an Integrated State
Several Hfr strains were isolated by integrative suppression as described in Materials and Methods; others were already available (Yoshikawa, 1974; Sotomura and Yoshikawa, 1975; Yoshimoto and Yoshikawa, 1975). Five temperature;resistant revertants of plasmid-bearing dnaA(Ts) strains were isolated for each plasmid, mated with a multiauxotrophic recipient, LC193, and examined for their ability to form recombinants for four chromosomal markers, metA, argG, his, and proA (or proB) and also for antibiotic resistance markers derived from the plasmid. Table 3 shows the results of a quantitative mating with representative temperature-resistant
120
SASAKAWA
revertants obtained with plasmids R124, R27, R471a, R446b, N3, and R478, respectively. As controls, parental strains carrying the respective plasmids autonomously were used. In this case, only the antibiotic resistance markers were transferred. With the temperature-resistant revertants the transfer frequency for antibiotic resistance markers was usually lower and the recombination frequency for at least one of four chromosomal markers was higher than with the parental strains with an autonomous plasmid. The relative frequencies of recombination for the four markers revealed that the chromosome was transferred sequentially. These results suggest that each thermoresistant isolate was an Hfr with a fixed origin and a sequential gradient of transfer. To prove further that the plasmid exists in an integrated state in these integratively suppressed strains, Hfr derivatives and their plasmid-carrying parents were grown in M-9 glucose medium with 2 pg of thymine per ml and [3H]thymine (6- 10 &i/ml) for about two to three generations at 30°C. Lysates were prepared and analyzed by the alkaline sucrose gradient
ET AL.
method (Freifelderet al., 1971). As shown in Fig. 1, all the DNA preparations from Hfr derivatives showed no detectable peak of radioactivity at the position where the parent carrying the same plasmid autonomously gave a peak. Hfr Construction by Transposon Mediated Plasmid Integration
Although all the conjugative plasmids so far tested conferred upon the dnaA(Ts) host an ability to give temperature-resistant revertants at a frequency higher than that without the plasmid, neither the integrative suppression method (Nishimura et al., 1971) nor the directed transposition method (Ippen et al., 1971) gave stable Hfr strains with some conjugative plasmids including R64 and mini ColEl. To overcome this, we attempted to obtain transposon-mediated integration of plasmids (Kleckner et al., 1977; Danilevich et al., 1978) into the host chromosome. An example involving R64drd 11, belonging to incompatibility group Ia is described. YC1216 (CRT46ilv::TtG) was first
TABLE SEQUENTIAL
GENE
TRANSFER
(Tn5)-
BY THERMORESISTANT
3 DERIVATWES
FROM
CRT46(R+)
STRAINS”
Phenotype selected Plasmid
Incompatibility group
Strain
Properties
Met+
Arg+
His+
Pro+
Drug
R124
FIV
YC2004 YC2019
Autonomous T revertant
(1 200
4000 80
R2J
Hl
YC2005 YC2020
Autonomous T revertant
400
R4Jla
L
YC2010 YC2024
Autonomous T revertant
70000
R446b
M
YC2011 YC2025
Autonomous T revertant
10000 100
N3
N
YC2012 YC2026
Autonomous T revertant
8000 10
R478
H2
YC2014 YC2028
Autonomous T revertant
7
100
200 30
o Frequency of recombination in the crosses of plasmid-bearing dnuA(Ts) strains (autonomous) or their temperature resistant revertants (T revertant) and a recipient, LC193. Mating conditions were described in Materials and Methods. Figures show the number of colonies yielded by a unit volume of mating mixtures.
INTEGRATIVE
PLASMID
INCOMPATIBILITY
121
GROUPING
-6
-4
1 D)
6-
-1 R27 1
“0 ;; It
4
F
2 J;‘k
R6-5 1
0
F)
(E)
I
I 6
I FRACTION
NUMBER
FIG. 1. Alkaline sucrose gradient analysis of DNAs extracted from thermoresistant strains. Cells were grown at 30°C in M-9 glucose medium supplemented with a nutritional requirements and containing 6-10 &i of rH]thymine per ml. At a cell density of about 5 x 1oRper ml, lysates were prepared and layered onto 5 to 20% linear alkaline sucrose gradients, which were centrifuged in an SW65 rotor (Beckman) at 40,000 rpm for 18 min at 18°C. Lysates were cosedimented with a reference DNA in the following combinations. (A) Lysates from YC2012 (N3 autonomously) and YC1221; (B) lysates from YC2026 (N3 integrated) and YC1221; (C) lysates from YC2005 (R27 autonomously) and YC1222; (D) lysates from YC2020 (R27 integrated) and YC1222; (E) lysates from YC2014 (R478 autonomously) and YC1222; (F) lysates from YC2028 (R478 integrated) and YC1222. Note the peak of sheared DNA from the host chromosome on the right of each panel plotted on a different scale. Panels A, C, and E are the results with strains carrying autonomous plasmids, whereas panels B, D, and F are those with Hfr strains with respective plasmids.
constructed and a plasmid, pMY 1121 (R64drdl 1: :Tn5), was then transferred into YC1216 and YC1214 (transposon-free) by
conjugation. The respective transconjugants, designated as YC1218 and YC1217, respectively, were examined for the frequency of
122
SASAKAWA
formation of thermoresistant revertants at 42°C. Table 4 shows a marked increase in number of thermoresistant revertants of YC1217aswellasYC1218ascomparedwith YC1214. However, there was no difference in the frequency of thermoresistant revertant formation between YC1217 and YC1218 (Table 4). Several thermoresistant revertants of YC1218 were picked and examined for the Hfr character. They were shown to be Hfrs but the site of integration was not at ilv (84 min) but near his (44 min). Thus, Hfr clones with an integrated R64drdll plasmid could be constructed by a combination of transposon-mediated integration and integrative suppression. It should be possible to construct Hfr strains by this method with any plasmid as long as the plasmid carries the genetic potentiality to provoke integrative suppression. In addition, we also succeeded (Sasakawa and Yoshikawa, in preparation) in isolating clones with integrated ColE 1 or its mini-derivative, pA03 (Oka et al., 1978) mediated by Tn5. Integration of ColEl itself had not been observed previously (Sotomura and Yoshikawa, 1975) by ordinary suppressive integration. However, a dnaA(Ts) host strain with Tn5 at the ilv locus and ColEl or pA03 inserted by the same transposon did produce integratively suppressed strains efficiently. Superinfectant Formation with Respect to the State of Existence of the Resident Plasmid and Recombination Proficiency and Deficiency. One of the reasons why the resident plasmid has been integrated is to guarantee TABLE EFFECTOFT~SON
ET AL.
its stability. However, even with these Hfr strains, the plasmid may be detached from the chromosome and cured. On the other hand, two plasmids belonging to the same incompatibility groups often share extensive sequence homology (Falkow et al., 1974) and hence easily recombine. These two problems, the detachment and curing of the resident plasmid and homologous recombinant formation in superinfectants, would be minimized by using Hfr strains and making them recombination-deficient. Thus, a study was made of superinfectant formation as a function of the state of the resident plasmid and of proficiency and deficiency in generalized recombination. As shown in Table 5, in incompatible crosses with the same plasmid pairs, the frequency of superinfectant formation was lower when an Hfr was used as the recipient than when a strain carrying the same plasmid autonomously was used. The introduction of recA deficiency was also effective in decreasing the frequency of establishment of the selective marker of the superinfecting piasmid, as shown in Table 5. Demonstration of the Simplicity Reliability of this Integrative Incompatibility Test
Five Rif’ recA Hfr derivatives having integrated plasmids belonging to various incompatibility groups were used as recipients, and examined for the formation of superinfectants. As shown in Table 6, in the crosses between incompatible plasmids (italics) superinfectant formation was always reduced to a frequency at least 100-fold lower than 4
FORMATIONOFTHERMORESISTANTREVERTANTS Properties
strain
Chromosome
YC1214 YC1217 YC1218
ilv::TnS
and
Colony counts per ml at Plasmid R64drdll::Tn5 Rbldrdll::Tn5
30°C
42°C
8.4 x lo8 3.6 x lo8 5.0 x 108
4.2 x lo* 4.2 x lo5 7.2 x 105
INTEGRATIVE
PLASMID
INCOMPATIBILITY TABLE
SIJPERINFECTANT
123
GROUPING
5
FORMATION IN INCOMPATIBLE CROSSES WITH REFERENCE TO THE STATE OF EXISTENCE OF THE PLASMID AND RECOMBINATION PROFICIENCY AND DEFICIENCY”
Relative frequency of superinfectant formations* State
recA + + +
Hfr Hfr Autonomous’ -
(RlOO-1):Rl’
(Rtsl):R401d
(N3):RPC3’
<7 x 10-S 0.03 0.46 1.00
1 x 10-a 0.02 0.23 1.00
<5 x 10-S 0.01 0.21 1.00
<3 x 10-Z 0.10 NT’ 1.00
a JC1569 with various plasmids to be tested was used as the donor and mating conditions were as described in Materials and Methods. * Figures are expressed as a ratio of the frequency of superinfectant formation in each cross relative to that in the cross with a plasmid-free strain, YC258. The resident plasmid in each cross is given in parentheses followed by the code of the superinfecting plasmid. c Rii and Km were used for selection of superinfectants. d Rif and Ap were used for selection of superinfectants. e YC258 was used as the host strain. ’ NT, not tested.
that in compatible crosses. These results suggest that the decrease in superinfectant formation was mainly due to incompatibility between the resident and the superinfecting plasmid. Consequently, this system should be applicable to the classification of conjugative plasmids by incompatibility, especially in epidemiological surveys of R plasmids. Thus far, we have constructed 11 recombination-deficient Hfr strains belonging to representative incompatibility groups for this purpose (Table 7). Under appropriately TABLE DEMONSTRATION
controlled conditions, the mating mixtures of many crosses may be scored on a single plate. DISCUSSION
The use of incompatibility to classify large numbers of naturally occurring plasmids isolated in an epidemiological survey requires a test that is simple as well as scientifically accurate. However, the standard method in general use is slow, laborious, 6
OF INTEGRATIVE
INCOMPATIBILITY~
Superinfected plasmid
Strain
Resident plasmid
YCl125 YCll30 YCll50 YCll40 YC1155 YC259
F RlOO-1 N3 Rts 1 R446b -
R386
Rl
pJA4733
R401
R69-2
Incompatibility
FI
FII
N
T
M
FI FII N T M -
0.08 NT NT 1.8 NT 1.0
1.2 co.02 3.7 NT 2.0 1.0
>l >I
0.9 0.5 2.6 5 x IO-” 0.8 1.0
1.0 1.1 1.4 1.2 0.01 1.0
n The experimental conditions were the same as in Table 5, except that YC259 was used as the plasmid-free control.
124
SASAKAWA
ET AL.
TABLE RIFAMPICIN-RESISTANT, recA Hfr INTEGRATIVE
Strain codes of rif-r, recA Hfr YC112.5 YC1130 YC1135 YC1160 YC1140 YCI 14.5
YCl150 YC1155 YC1165 YC1170 YCll80
Plasmid
Incompatibility FI FII FIV Hl L M
N3
N
R478 R6K Rts 1
H2 x T Iff
11
STRAINS
INCOMPATIBILITY
F RlOO-1 R124 R27 R471a R446b
R64drd
7 TO BE USED
AS RECIPIENT
FOR
TESTING
Resistance patterns Tc Cm Sm Su Tc Tc AP Tc Su Tc Sm Su Cm Tc Km AP Km Tc
Leading marker in chromosome transfer thyA metA metA metA
argG his metA his metA argG his
and sometimes difficult to interpret. Thus, suggesting that a marked reduction in superinstability may result in superinfectants ap- infectants may occur due only to incompatiparently lacking the resident plasmid be- bility. However, we have to take into account cause of spontaneous curing rather than other plasmid-determined factors that may incompatibility; recombination may complireduce the number of superinfectants. The cate the scoring of colonies having resistance most obvious example is the degradation of markers derived from both the resident and the superinfecting replicon (Yoshikawa and superinfecting plasmids (Falkow et al., Akiba, 1962). In fact, we have sometimes 1974). The use of recA strains with the observed a reduction in superinfectants due reference plasmids integrated, as recipients, to the Hsp 1 phenotype when an Hfr recipient appears to have largely overcome these with R124 was used. Other, as yet unknown, problems. mechanisms may also exist. The use of recA In analogy to the already known examples donors to prevent colony formation by of the F plasmid (Lederberg et al., 1952) chromosomal Rif’ recombinants appears to and those belonging to the FI and FII in- involve a troublesome additional step in strain construction. However, in any epicompatibility groups such as ColV2, R538-1, demiological survey, it is the first job to ColVBtrp, Rldrdl9, RlOO-1, and R136 (Wilprove that field isolates carry a conjugative letts and Maule, 1974) the Hfr recipients were grown up to late stationary phase to R plasmid. For this purpose the best apminimize the effect of entry exclusion. How- proach is to transfer the drug resistance to ever, the entry exclusion mechanism (Novick a recA strain. After this has been proven, the resulting recA strain carrying the plaset al., 1976) may not always be overcome by the use of stationary phase; this may mid isolated from a field specimen may be result in reduction of superinfectants mainly now used as the donor for an integrative incompatibility test. by entry exclusion rather than by incomIn incompatible crosses with recA recipipatibility. In this respect it is noteworthy ents, a few colonies have occasionally been that a marked reduction of superinfectants occurred in the cross of RIOO-1 with RI observed. These were further characterized (Table 5). These plasmids belong to the same and shown to have emerged presumably by transposition of various incompatibility group but to different entry recA-independent parts of the superinfecting plasmid most exclusion systems (Willetts and Maule, 1974),
INTEGRATIVE
PLASMID
INCOMPATIBILITY
likely to the host choromosome (data not shown). The standard incompatibility grouping test in use at present is based on the interaction between two autonomously replicating plasmids and in this point differs from our modified method which is based on the interaction of an autonomous with an integrated plasmid. These two may be different phenomena but are in any case based on the relatedness of two plasmids (Scaife and Gross, 1962; Echols, 1963; de Vries et al., 1975; Yoshikawa, 1975; Novick and Schwesinger, 1976; Pfister et al., 1976; Sasakawa et al., 1979). In fact, the phenomenon of incompatibility was first found in the introduction of F’ plasmids into Hfr cells (Scaife and Gross, 1962; Echols, 1963). Various conjugative plasmids have been shown to give rise to Hfr derivatives by integrative suppression (Nishimura et al., 1971; Lindahletal., 1971; Moody and Runge, 1972; Nishimura et al., 1973; Goebel, 1974; Yoshikawa, 1974; Sotomura and Yoshikawa, 1975; Yoshimoto and Yoshikawa, 1975; Datta and Barth, 1976; Chesney and Scott, 1978). In this paper we have extended the list of these to include R124, R471a, R446b, N3, R27, and R478 (Table 3). Although we observed a marked increase in the frequency of thermoresistant (dnaA (Tr)) revertants with all the plasmids tested, in some cases, namely, plasmids of incompatibility groups Inc Ia, Inc W, Inc P, and Inc A-C, we were unable to isolate stable Hfr derivatives. Similar results have been obtained previously with some of these (Datta and Barth, 1976; Martin et al., Abstr. Annu. Meet. Amer. Sot. Microbial. 1975, H52, p. 104). We tried in vain to isolate stable Hfr strains with these plasmids also by the directed transposition method (Ippen et al., 1971). However, plasmid integration has been shown to be facilitated by the presence of a transposon, Tnl, on both plasmid and chromosome (Danilevich ef al., 1978). In our attempts to demonstrate this phenomenon with R64drd 11 and Tn5, we found that a copy of Tn5 on the plasmid greatly increased
GROUPING
125
the frequency of stable Hfr’s but that a chromosomal copy had no effect (Table 4); indeed, when Tn5 was present on both, insertion took place at a chromosomal site far distant from the location of the transposon. Thus, integrative suppression is evidently not due to TnS-mediated homologous recombination; rather, the plasmid-carried Tn5 accelerated illegitimate recombination elsewhere on the chromosome. In any case, the introduction of a transposon is likely to permit the construction of Hfr strains probably with any plasmid. It also appears that any plasmid that can replicate autonomously and does not require dnuA for its own replication may potentially be suppressively integrated in a dnaA (Ts) strain. Thus, the simplicity and reliability of this integrative incompatibility test method has been suggested by the experimental results presented in this paper. In addition, several Japanese members of the Plasmid Epidemiology Group have initiated an examination of the validity of this method. According to personal communication from some of them (Terakado, National Institute of Animal Health, Tsukuba; Nakaya et al., Tokyo Medical and Dental University), all of their clinically isolated plasmids so far tested were shown to give the same results when judged both by the standard autonomousto-autonomous method and by our autonomous-to-integrated method. ACKNOWLEDGMENTS The authors wish to thank Dr. N. Datta and Dr. E. M. Lederberg for their generous gift of plasmids, Dr. D. E. Berg for giving the phage of Ab22Zc1857rex::Tn5, an editor and a referee for valuable suggestions, and Dr. Terakado and Dr. Nakaya and their collaborators for generously communicating with us their unpublished data. This work was supported in part by a grant provided by the Ministry of Education, Science and Culture of the Japanese Government (Grant numbers 211901, 311202 and 410701).
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