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Demonstration of a third incompatibility function on plasmids already incompatible with group P and group I plasmids
PLASMID
9, 191-200 (1983)
Demonstration of a Third Incompatibility Function on Plasmids Already Incompatible with Group P and Group I Plasmids PHILLIP Department of Microbiology,
I. BIRD
AND JAMES
PITTARD
University of Melbourne, Parkville 3052, Victoria, Australia
Received September 20, 1982; revised November
18, 1982
The addition of extra antibiotic resistance determinants to plasmids of a series previously shown to be incompatible in an atypical fashion with group I and group P plasmids, has enabled the existence of a third and major incompatibility function determined by these plasmids to be demonstrated. The in vitro construction of a plasmid consisting of the replication region of one of the plasmids, linked to the genes of the galactose operon, facilitated the identification of this third incompatibility function as similar to IncB plasmids.
We have previously described a series of R plasmids (pMU 700 to 707) which were isolated from the fecal Escherichia coli of domestic livestock. These plasmids are conjugative, of similar molecular weight (120 kb), determine resistance to streptomycin and sulphathiazole, and are incompatible with plasmids of both the I and the P incompatibility groups (Grant et aZ.,1980). The nature of these incompatibility interactions is unusual because plasmid loss from cells containing an incompatible pair is very slow, unilateral, and in some cases host dependent. Since these interactions are markedly different from those usually observed between incompatible plasmids, and because any model for incompatibility must explain the basis for every observation, we have been interested in investigating the incompatibility properties of the pMU plasmids. An integral part of such a study is to determine whether the pMU plasmids possessfurther genetic loci which would mediate a conventional result if two pMU plasmids were tested for incompatibility. To date, the lack of appropriate distinguishing markers on different plasmids of the series has prevented such tests from being performed. This is a common problem encountered in incompatibility testing (Datta, 1979), and can often prevent the unambiguous assignation of an unknown plasmid to a particular incompatibility group. The availability of transposable
antibiotic resistance determinants exemplified by Tn3 (ampicillin) and Tn5 (kanamytin) has allowed us to circumvent this problem by constructing derivatives of the pMU plasmids which carry either transposon, and which can be distinguished in a test situation. Although such derivatives may enable us to detect a third incompatibility function coded for by the pMU plasmids, the presence of at least two antibiotic resistance determinants on each plasmid may prevent the identification of the function by limiting the number of standard plasmids that may be used in such tests. To overcome this, we have taken advantage of an approach recently developed in this laboratory by which the replication and incompatibility genes of a plasmid of interest are linked to the genes coding for the fermentation of galactose (Davey and Pittard, manuscript in preparation). Such a plasmid would not be expected to exhibit entry exclusion, and the availability of different indicator media (Davey and Pittard, manuscript in preparation; Miller, 1972) enables its maintenance or loss from a Gal-deficient strain to be easily monitored. As the gal operon is not usually plasmid associated, such an artificially constructed replicon may be tested with every available standard plasmid without encountering problems with a lack of distinguishing markers. In this report, we describe experiments 191
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which indicate the existence of a third major incompatibility determinant on the pMU plasmids that is similar to those on plasmids of the IncB group. MATERIALS
AND
METHODS
Bacterial strains and plasmids. The strains of E. coli K 12 used in this study are described in Table 1. The plasmids, with the exception of the current incompatibility exemplars, are
also listed in Table 1. Referenceplasmids used included pIP55 (A=C=E); R16(B=0); R7 1lb(D); R386(FI); Rl(FI1); pSUl04(FIII); R124(FIV); pGL61 lA(FV1); R27(Hl); R478(H2=S); pIP233(H3); R64 drd 1l(1,); TPl14(1& R391 (J); R387(K); R446b(L=M); R46(N); pIE509(0F=FV); RP4(P); Rtsl(T); R1460(U); S-a(W); R6K(X); pIP231a(Y); and pIP7 la(9). These have been described by Jacob et al. ( 1977) except R7 1lb (Datta, 1979),
TABLE 1 BACTERIALSTRAINSANDPLASMIDS
Designation Strain
Plasmid
Characteristics”
JP777
thr, leu, rpoB, gal, lacy
JP990
argE3, his4, ilvC7, proA2, thi-1, supE44, gaik2, A-, nalA368 thi- 1, X-, rpoB35 1
JP995 PB1907 KA56 pMU700 pMU705 pMU706 pMU707 pMU715 pMU716 pMU717 pMU718 pMU719 pMU720 R144-3 R648 R621a R621a-1 R805a TP113 pBR322 pRBD13
polA 1, nalA HfrH, galE, rel-1, spoTI, A-, thi-1 Incly, IncP, IncB Sm Su Incla, IncP, IncB Sm Su Incly, IncP, IncB Sm Sp Su Incla, IncP, IncB Sm Su Inclr, IncP, IncB Sm Su Ap Incla, IncP, IncB Sm Su Ap IncB su Km Incla, IncP, IncB Su Km Inch, IncB, ColE’ SmTc Incla, IncB Gal Km Collb Inclol Inclo Ap Sm Km Incly” Tc Ap Km Cm Tc IncIyd IncIlb Km IncB Km ColE’ Ap Tc Tc Gal ColE’
Source or Reference Derived from JFM43 (Smith et al., 1982) Derived from AB 1157 (Grant and Pittard, 1974) Derived from W3 110 (Grant and Pittard, 1974) Obtained from P. L. Bergquist (Russell and Pittard, 1971) (Grant ef al., 1980) (Grant et al., 1980) (Grant et al., 1980) (Grant et al., 1980) pMU7OO::Tn3-this study pMU705: :Tn3-this study pMU707::Tn5-this study pMU707: :TnS-this study This study-derived from pMU707 Derived from pMU707 (Meynell and Datta, 1967) (Hedges and Datta, 1973) (Hedges and Datta, 1973) (Hedges, 1974) (Datta and Olarte, 1974) (GrindIey et al., 1973) (Rodriguez et al., 1976) pBR322 with gal t’iagment inserted in the PstI site (Davey and Pittard, manuscript in preparation)
‘See footnote b, Table 2. bE. Lederberg of the Plasmid Reference Center (personal communication) has recently recommended the withdrawal of these incompatibility designations to rationalize the I complex. For convenience we shall use the old designations. ’ Refers to incompatibility function. dThe incompatibility functions of this plasmid are similar to those of R621a (Bird and Pittard, 1982).
MULTIPLE
INCOMPATIBILITY
OF THE pMU PLASMIDS
193
pSU104 and pIP233 (De La Cruz et al., 1979), the appropriate media. These conditions were pIP23la (Roussel and Chabbert, 1978), and found to allow sufficient transfer of those plasmids with low transfer efficiencies or temperpGL61 IA (E. Lebek, unpublished). Antibiotics, chemicals, enzymesand media. ature-sensitive transfer functions. Incompatibility tests. The conventional Most of the chemicals, culture media, and antibiotics used have already been described methods used in this laboratory have been in detail (Grant et al., 1980; Grant and Pit- described previously (Grant et al., 1980). The tard, 1974). The indicator medium used to test involves checking appropriate transconscreen clones for their Gal status has been jugants for the maintenance of plasmid markdescribed (Davey and Pittard, manuscript in ers after four successivesubculturings-twice preparation), but basically consisted of a ga- on selective media and twice on nonselective lactose, tryptone, agar (Oxoid No. 1) basal media. Clones giving equivocal results were medium with either 25 &ml tetrazolium salts subcultured a further four times on unselecor a mixture of bromothymol blue (0.5% w/ tive media and retested. The stability of the v) and bromocresol purple (1.5% w/v) dyes. resident plasmids was checked in each case. Incompatibility tests involving the GalThe PstI and BamHI restriction endonucleasesand the T4 DNA ligase were obtained plasmid derived from pMU707 were performed by mating the standard plasmids with from Boehringer-Mannheim. Plasmid transfer. Conjugation was gener- a Gal-deficient strain (JP777) harboring the ally carried out asdescribed previously (Grant Gal-plasmid, as described above. On the meet al., 1980) and in all cases selection was dia utilized (basal plus tetrazolium salts), cells made for the incoming plasmid alone. Some- unable to ferment galactose gave rise to red times transfer was found to be more efficient colonies. Hence, an incompatible interaction if mating was allowed to occur on a solid sur- between a standard plasmid and the Gal plasface, as described by Bradley et al. (1980). mid gave rise to red colonies, while a comWhere entry exclusion prevented transfer of patible interaction gave rise to white colonies. Transposition of Tn3 and Tn5 to the pMlJ the donor plasmid, the recipient cells were grown under conditions that were shown to plasmids. The transposons were carried on reduce entry exclusion (Jacob and Wollman, separate derivatives of the multicopy, non1961). Such recipients were grown to station- conjugative, nonmobilizable DNA polymerary phase in nutrient broth (NB)’ overnight ase I-dependent plasmid, pMB8 (Rodriguez at 37°C. These cells were concentrated ten- et al., 1976), which were maintained in recA fold and added to a concentrated culture of backgrounds. (These strains were obtained donor cells (in log phase) so that a ratio of from P. L. Bergquist.) To translocate a trans1:10, donor to recipient, was achieved in a poson to a conjugative plasmid, the “target” volume of 4 ml, to give a total population of plasmid was crossedinto a cell containing the approximately lo8 CPU/ml. Mating was al- pMB8: :Tn plasmid. Subsequently, a pool of lowed to occur for 2 h at 37°C; then the mix- transconjugants derived from this cross was used as a donor in conjugation to a polA retures were plated to the appropriate media. In experiments where a large number of cipient (PB 1907) selecting for both the transstandard plasmids were crossed to recipients poson and the target plasmid. Transconjuharboring a Gal-plasmid derived from gants resulting from this cross were purified pMU707 (pMU720), mating was allowed to and transferred to JP990 selecting for the taroccur for 4 h at 37OC, followed by an over- get plasmid alone. Transconjugants showing night incubation at 20°C. Ten-microliter al- 100% coinheritance of the transposon were iquots from each mixture were spotted onto screened for plasmid DNA content to ensure that no ColE-type plasmid had been cotransferred by mobilization (Warren et al., 1978) ’ Abbreviations used: NB, nutrient broth, CFU, color cointegration (Crisona et al., 1980). ony-forming units.
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DNA manipulations. Small-scale preparations of DNA were obtained by the method of Bimboim and Doly ( 1979),while large-scale preparations of either multicopy (Clewell and Helinski, 1969) or low copy (Hansen and Olsen, 1978) plasmid DNA were made by centrifuging cleared lysates on cesium chlorideethidium bromide gradients. Transformations were performed by the method of Kushner (1978). Agarose gel electrophoresis and visualization of DNA restriction fragments followed standard procedures (Davis et al., 1980),while restriction endonucleasesand T4 DNA ligase were used as recommended by the manufacturers. RESULTS
Construction and Properties of TransposonCarrying Derivatives of the pMiJ Plasmids Derivatives of pMU700 and pMU705 which carried Tn3 (ampicillin), and of pMU707 which carried Tn5 (kanamycin) were isolated as outlined above. To ensure that the known incompatibility functions had not been altered as a result of the transpositional event, the derivatives were tested with RP4 (IncP), R 144-3or R648 (IncIa), and R62 1a or R62 1al (IncIy). The results shown in Table 2 indicated that with the exception of pMU7 17, the new plasmids were unaltered in their known compatibility functions. However pMU715 and 716 apparently had Tn3 inserted in gene(s)whose loss of function led to an increase of lo- to 1OO-foldin the plasmids’ transfer efficiencies, while pMU7 18 apparently had its streptomycin resistance function inactivated by the insertion of Tn5. The plasmid pMU7 17 was no longer incompatible with plasmids of the IncP or IncIcv groups, whereas a derivative of pMU707 should be incompatible with these plasmids (Table 2). This loss of incompatibility functions was accompanied by loss of the strep tomycin resistance function. The simultaneous loss of distinct functions is most readily explained by postulating a transposon-mediated deletion event, similar to those previ-
PITTARD
ously observed in other systems, associated with Tn5 (Berg, 1977). The isolation of a plasmid deficient in both known incompatibility functions was useful in establishing the existence of a third independent incompatibility function encoded by the pMU plasmids. Incompatibility between the pMU Plasmids The plasmids pMU7 15, 7 16, 7 17, and 7 18 were tested where possible for incompatibility with each other and with pMU706 (which has a distinguishable spectinomycin resistance determinant). The results are shown in Table 3. Initially it proved impossible to transfer one of these plasmids into a host containing another, presumably due to strong entry exclusion. However, by growing the recipient cells under conditions designed to reduce entry exclusion (as described above), and by obtaining an increased efficiency of transfer by mating on a solid surface, a significant number of transconjugants were obtained and tested for the maintenance of plasmid markers. Even under these optimum conditions, a second pMU plasmid could not be introduced into a cell already harboring pMU717. The incompatibility interactions observed between the pMU plasmids resemble those seenbetween strongly incompatible plasmids such as R144-3 and R62 la (Bird and Pittard, 1982), and are markedly different from those previously observed between the pMU plasmids and exemplars of the I and the P incompatibility groups (Grant et al., 1980). The interactions described in Table 3 were not dependent on the host strain used in the test (data not shown), were reciprocal (except when pMU 7 17 was the resident plasmid), and appeared to be unconnected with the I and the P incompatibility functions of the plasmids, as illustrated by the interaction of pMU717 with the other pMU plasmids. The use of a single plasmid selection prior to incompatibility testing means that there is no means of ensuring that every recipient cell initially contained a resident plasmid. When entry exclusion occurs, there may be a strong bias of transfer of the incoming plasmid to
MULTIPLE
INCOMPATIBILITY
OF THE
pMU
195
PLASMIDS
TABLE 2 INTERACTIONS OF THE pMU DERIVATIWS WITH GROUP I AND GROUP P PLASMIDS” Incompatibilityd Transfer frequencyC
Recipient
Selection6
JF’990‘(pMU7 15) JF990 (pMU7 16) JF990 (pMU7 17) JF990 (pMU718) JF990
Km Nx Km Nx Ap Nx Ap Nx Km Nx
6.3 X 1.3 x 1.0 x 2.2 x 2.5 X
Jp995 (R144-3)
JP990 (pMU7 15) JP990 (pMU7 16) JF990
Jp995 (R648)
First testing
Second testing
IO-’ 10-S 10-G 10-h 10-s
KmTc(40/4O)SmSu(16/40) KmTc(40/4O)SmSu(8/40) ApWWWn(40/40) Ap(40/4O)Su(16/40) NT’
KmTc(40/4O)SmSu(O/40) KmTc(40/4O)SmSn(0/40) AdWWSdWJ’-J) Ap(40/4O)Sn(0/40) NT
Km Nx Km Nx Km Nx
1.3 x 10-I 8.8 x 10-Z 2.0 x 10-l
Km(40/40)SmSnAp(35/40) Km(40/4O)SmSnAp(8/40) NT
Km(40/4O)SmSuAp(30/40) Km(40/40)Kmsn(0/40) NT
JF990 (pMU7 17) JP990 (pMU7 18) JP990
Ap Nx Ap Nx Ap Nx
1.0 x 10-5 1.4 x 10-4 1.1 x 104
JP995 (R621a)
JP990 (pMU7 15) JP990 (pMU7 16) Ji’990
Tc Nx Tc Nx Tc Nx
4.7 x 10-a 1.3 x 10-s 1.6 X lo-’
Tc@O/4O)SmSuAp(6/40) Tc@O~4O)SmSnAp(37/) NT
Tc(40/4O)SmSuAp(O/40) Tc(40/4O)SmSuAp(37/40) NT
JF995 (R621a-I)
JP990 (pMU7 17) JF990 (pMU7 18) JP990
Ap Nx Ap Nx Ap Nx
9.5 x 10-5 2.9 X IO-’ 8.0 X IO-’
(Rp4)
0 The reciprocal crossesare not shown, but the results obtained were consistent with these, and with those described previously (Grant and F’ittard, 1974). b Abbreviations used: Km, kanamycin; Ap, ampicillin; Tc, tetracycline; Nx, nalidixic acid, Sm, streptomycin; Su, sulphathiazole; Sp, spectinomycin. c Defined as the number of transconjugants per donor per hour. d Incompatibility results are shown as the markets displayed by the 40 clones tested in each case.Those clones showing cc-existence of both setsof plasmid markers following the second testing were used as donors in further cmssesto plasmid-free recipients and were shown to be able to transfer each plasmid’s markers separately and independently. =Not tested.
plasmid-free recipients, which could lead to the false conclusion that the plasmids were incompatible. Our results indicate there is a lo- to 500-fold decreasein transfer efficiency of the incoming plasmid if the recipient pop ulation harbors a resident plasmid (Table 3). Assuming that transfer of the incoming plasmid only occurs to plasmid-free cells within this population (and not to others because of entry exclusion), such cells would have to appear at a frequency of between 10-i and 5 x lo-*. Accordingly, cells harboring pMU718 were grown under the same conditions used for the recipients in the experiments outlined in Table 3 and 1200 independent clones were checked for the maintenance of plasmid markers. No loss was observed, implying that resident plasmid instability did
not occur at high enough frequencies to influence the results. Construction and Properties of a Derivative of pMU707 that Carries the gal Operon Although the pMU plasmids have been tested previously with plasmids of the A, C, FI, FII, FIV, H, Icu, Iy, J, K, L, M, N, P, S, T, and W incompatibility groups (Grant et al., 1980), the existence of a third incompatibility function determined by the pMU plasmids, as well as the expansion and reorganization of the recognized incompatibility groups, necessitatedthe retesting of the pMU plasmids. To facilitate this aim and to prevent such problems as entry exclusion and overlapping markers, we decided to link the rep-
196
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AND
PITTARD
TABLE 3 INTERACTIONS
OF THE pMU PLASMIDS
Selection”
AND THEIR
DERIVATIVES
Transfer frequency b
Incompatibility’: First testing
Donor strain
Recipient strain
JP995 (pMU7 17)
JP990 (pMU706) JP990 (pMU7 16) JF990 (pMU7 15) JP990
Km Km Km Km
Nx Nx Nx Nx
1x 2x 4 x 2 x
lo-5 lo-5 lo-6 lo-4
Km(40/40)Sp(0/40) Km(40/40)Ap(0/40) Km(40/40)Ap(0/40) NTd
JP995 (pMU7 18)
JP990 (pMU706) JP990 (pMU7 16) JP990(pMU7 15) JF990
Km Km Km Km
Nx Nx Nx Nx
8 8 2 6
x x x x
1O-5 IO-’ 1o-4 IO-’
Km(40/40)Sp(0/40) Km(40/40)Ap(0/40) Km(40/40)Ap(0/40) NT
JP995 (pMU706)
JP990 JP990 JP990 JP990 JP990
(pMU7 17) (pMU7 18) (pMU716) (pMU7 15)
Sp Sp Sp Sp Sp
Nx Nx Nx Nx Nx
5 1 7 3
NT Sp(40/4O)Km(0/40) Sp(40/40)Ap(0/40) Sp(40/40)Ap(0/40) NT
JF995 (pMU7 16)
JP990 (pMU7 17) JP990 (pMU7 18) JP990 (pMU706) JP990
Ap Ap Ap Ap
Nx Nx Nx Nx
NT Ap(40/4O)Km(0/40) Ap(40/40)Sp(0/40) NT
JP995 (pMU7 15)
JP990 (pMU7 17) JF990 (pMU7 18) JP990 (pMU706) JP990
Ap Ap Ap Ap
Nx Nx Nx Nx
NT Ap(40/40)Km(0/40) Ap(40/40)$(0/40) NT
‘.‘,‘,‘See footnotes b, c, d, and e, respectively, Table 2.
lication genesof pMU707 to those of the gal operon, on the assumption that the major (unknown) incompatibility genesof pMU707 were linked to those directing replication. The major advantage of this approach is that the status of the Gal plasmid in an incompatibility test may be monitored simply by utilization of appropriate indicator media (see Materials and Methods). The construction of such a plasmid involved a two step procedure utilizing an artificially derived plasmid, pMU7 19, as a source of the replication genes of pMU707. pMU7 19 was derived by ligating DNA fragments of pMU707, generated by a partial digest with PSI, to PstI-generated fragments of pBR322 DNA and transforming JP777 to streptomycin resistance. Although the aim of this experiment was to clone either the IncP or Inc Ia locus of pMU707, on the assumption (based on experience with
pMU7 17) that these loci were linked to its streptomycin resistance determinant, pMU7 19, as well as having the Inc ICXlocus, was also incompatible with pMU7 17 and was able to replicate in a a& host (data not shown). These results indicated the presence of a replication system on pMU7 19 distinct from that of pBR322, presumably that of pMU707. Given the smaller size, higher copy number, and hence greater ease of manipulation of pMU7 19 compared to pMU707, we used the former plasmid as a source of the replication genesof the latter. To construct a miniplasmid consisting of the replication system of pMU707 linked to Gal, we partially digested pMU7 19 DNA with PstI and ligated these fragments to those resulting from a complete BarnHI and PstI double digest of pRBDl3 (pRBD13 is described in Table 1). Since BumHI cuts pRBD13 once, not in the
MULTIPLE
INCOMPATIBILITY
Gal fragment, this enzyme was also used to digest pRBDl3 DNA in order to lower the probability of obtaining reformed pRBD 13 molecules on ligation and transformation of the recipient strain to Gal+. Selection for plasmids carrying gal was made by transforming KA56 (a guE strain) with the ligation mixture and plating onto a galctose-tryptone-bromthymol blue/bromocresol purple plate. Since the g& mutation is lethal in the presence of galactose,clones harboring Gal plasmids were selected and could be easily separated from the slight background growth obtained on theseplates becauseof their strong yellow coloration. Forty Gal+, tetracycline-sensitive clones were picked and screened for their plasmid DNA content. Two clones containing the smallest plasmid species were chosen and small-scale DNA isolated from them was used to transform JP990 and JP777 to Gal+. Analysis of the restriction patterns of the two plasmids, generated by PA, indicated they were similar and consisted of one large fragment (Gal), and two smaller fragments both of which are necessaryfor replication (Bird and Pittard, in preparation) to give a total size of approximately 7.9 kb. Both plasmids were incompatible with pMU707 and pMU7 17 in JP990 and JP777, and with R144-3 in JP990 (pMU plasmids with an Ia function only display it in JP990 (Grant et al., 1980)), but were compatible with RP4 in both hosts. They were nonconjugative and no entry exclusion was observed with pMU707, pMU717, R144-3, or RP4 (data not shown). These results indicate that the genesdetermining both the unknown incompatibility and Ia incompatibility functions of pMU707 are associated with or near its replication genes, while the P incompatibility function is quite distinct. One of the two isolates, pMU720, was used in further studies. IdentiJication of the Unknown Incompatibility Determinant of pMU720 Rifampicin-sensitive strains containing reference plasmids of known incompatibility
OF THE pMU PLASMIDS
197
groups were usedas donors in crossesto JP777 and to JP777 harboring pMU720. On the basis of the color reaction on the selective media (galactose-tryptone-tetrazolium salts; see Materials and Methods) displayed by the transconjugants resulting from each mating, pMU720 was found to be incompatible with R16(IncB). To confirm this, we predicted that pMU720 should also be incompatible with the plasmids R805a (Datta and Olarte, 1974), and TP113 (Grindley et al., 1973) both of which have been shown to be incompatible with R16. We found pMU720 to be incompatible with TP113 and R805a. It was compatible with the exemplars of every other incompatibility group tested, namely, groups A, D, FI, FII, FIR, FVI, Hl, H2, H3, I,, IZ, J, K, L, N, OF, P, T, U, W, X, Y, and 9. Under the conditions used for these tests, the weak interaction of pMU720 with the I, (Ia) plasmids was not apparent. DISCUSSION
Two recurring problems encountered in classical incompatibility testing are the lack of suitable markers on each plasmid to allow their distinction in a test, and the difficulty in telling whether in some circumstances the apparent nonexistence of clones containing both plasmids is due to entry exclusion, incompatibility or a combination of both effects. To circumvent a problem such as no distinguishing markers, one approach is simply to add extra markers, in the form of antibiotic resistance determinants, to a particular plasmid and this is conveniently done by utilizing transposons (De La Cruz et al., 1979; Royle and Holloway, 1980). However some care is necessaryto ensurethat the transposon (which can act as a mutagen) does not alter the incompatibility function of the target plasmid. The probability of this occurring may be reduced by choosing plasmids that are inactivated in a particular function such as antibiotic resistance or transfer (pMU7 15, 7 16, and 7 18), although as illustrated by pMU7 17, a transpositional event may lead to the loss of several functions.
198
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The transposons themselves do not appear to influence the outcome of incompatibility tests, provided that a single plasmid selection is used to construct cells containing the plasmids to be tested. However in those cases where entry exclusion or incompatibility between the plasmids under test leads to a significant reduction in the number of clones available for testing, and especially when a doubIe selection is used in such circumstances to attempt to increase the numbers of clones available, transposition of markers from replicon to replicon may complicate the interpretation of the results (D. Jans, B.Sc. (Hons.) thesis, University of Melbourne, 1980). While the use of transposons as extra markers in incompatibility testing does not necessarily overcome the problem of distinguishing the effectsof entry exclusion from incompatibility, the second approach we have used overcomesboth problems. In this method, we have linked the replication genesof a plasmid of interest to genes that endow a selectable phenotype on cells containing the new plasmid, expecting, on the basisof experience with other well-studied plasmid systems, that the incompatibility genes would be closely associated with the genesdirecting replication. The major advantages of this method are that the genes coding for entry exclusion should no longer be present, the marker used (Gal) is not commonly associated with plasmids and is easily distinguishable, and the utilization of indicator media allows the presence or absence of the Gal plasmid in a strain to be continually monitored. Using this system we have easily and unambiguously assigned the unknown incompatibility determinant of pMU707 to the IncB group. However there are some considerations to be made when utilizing this approach to incompatibility testing. First, it is assumed that the incompatibility genesand replication genesare closely linked. In the light of recent molecular analyses of plasmids of the FI and FII incompatibility groups (Lane, 1981; Timmis, 1981) it appears that there may be more than one incompatibility locus, and hence the isolation of replication genesmay not necessarily ensure the
PITTARD
isolation of all the incompatibility functions. For example, in our case the derivative of pMU707, pMU720, no longer displays the IncP function of its parent. Another consideration is that the genes essential for replication and stable maintenance may not be clustered in one segment of the genome, as is the case for the IncP plasmid RR2 (Thomas et al., 1980), and the construction of miniplasmid derivatives by in vitro cloning techniques may prove to be extremely difficult. These problems may be overcome by constructing a bank of Gal plasmids, each carrying a defined incompatibility locus. Although the initial construction of such a bank would be hindered by the same problems as outlined above, once obtained, it could be used to quickly and unequivocally assign an unknown plasmid to a particular incompatibility group without the need for extensive in vivo and in vitro manipulations. The finding that pMU720 interacts with plasmids of the I and the B incompatibility groups implies that this plasmid has complex replication and/or incompatibility systems. Several plasmids which interact with members of more than one incompatibility group have been described (Grant et al., 1980; Guerry et al., 1974; Monti-Bragadin and Samer, 1975; Smith et al., 1973), and although this multiple incompatibility in some cases has been attributed to similarities in genetic or biophysical characteristics between plasmids of the shared groups (Falkow et al., 1974), in most cases, the basis of multiple incompatibility hasyet to be explained. In our case,it is likely that the IncB function of the pMU plasmids is the major incompatibility function, and since the IncP and IncIa functions may be lost from pMU7 17 without apparently affecting its stability in the cell, and because they are qualitatively different from “typical” incompatibility functions, it may be that they represent the involvement of separate mechanisms in the incompatibility of the pMU plasmids. We would prefer not to classify the pMU plasmids as typical IncB plasmids becausethe basis for their interactions with IncI plasmids
MULTIPLE
INCOMPATIBILITY
and IncP plasmids is not yet understood, and as these interactions are unusual we would hesitate to call them IncI or IncP plasmids. It is our intention to study the pMU plasmids further to determine the basesfor their multinle incomDatibiIitv and to establish whether they resemble othe; well-characterized plasmids in replication and incompatibility functions. ACKNOWLEDGMENTS We thank E. Lederberg of the Plasmid Reference Center for providing the incompatibility exemplars and information on the current status of the incompatibility classification scheme,as well as P. Bergquist for providing PB 1907 and the pMB8: :Tn plasmids. This investigation was supported by a grant from the National Health and Medical ResearchCouncil. P.B. acknowledgesthe receipt of a Commonwealth Postgraduate Research Award.
REFERENCES BERG,D. E. ( 1977).Insertion and excision of transposable kanamycin resistancedeterminant Tn5. In “DNA Insertion Elements, Plasmids and Episomes” (A. J. Bukhari, J. A. Shapiro, and S. L. Adhya, eds.), pp. 2052 12.Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. BIRD, P. I., AND PACKARD,J. ( 1982). An unexpected incompatibility interaction between two plasmids of the I compatibility complex. Plusmid 8, 21 l-214. BIRNBOIM,H. C., AND DOLY, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Rex 1, 1513-1523. BRADLEY, D. E., TAYLOR, D. E., AND COHEN, D. R. (1980). Specification of surface mating systemsamong conjugative drug resistance plasmids in E. coli K-12. J. Bacterial. 143, 1466-1470. CLEWELL,D. B., AND HELINSKI, D. R. (1969). Supercoiled circular DNA-protein complex in E. coli: Purification and induced conversion to an open circular DNA form. Proc. Nat. Acad. Sci. USA 62, 1159-l 166. CRISONA,N. J., NOWAK,J. A., NAGA~SHI,H., ANDCLARK, A. J. (1980). Transposon-mediated conjugational transmission of non-conjugative plasmids. J. Bacterial. 142.701-713.
DATTA, N. (1979). Plasmid classification: Incompatibility grouping. In “Plasmids of Medical, Environmental, and Commercial Importance” (K. N. Timmis and A. Ptlhler, eds.), pp. 3- 12. Elsevier/North-Holland Biomedical Press,Amsterdam. DATTA, N., AND OLARTE,J. (1974). R factors in strains of Salmonella typhi and Shigelra dysenteriae isolated during epidemics in Mexico: Classification and compatibility. Antimicrob. Agents Chemother. 5, 3 10-3 17.
OF THE pMU PLASMIDS
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DAVEY, R. B., AND P~T~ARD,J., manuscript in preparation. DAVIS, R. W., BOTSTEIN,D., AND ROTH, J. R. ( 1980). “Advanced Bacterial Genetics.” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. DE LA CRUZ,F., ZABALA,J. C.. AND ORTIZ.J. M. ( 1979). Incompatibility among ol-Hemolytic plasmids studied a&r inactivation of the cY-Hemolysin gene by transposition of Tn802. Plasmid 2,507-5 19. FALKOW,S., GUERRY,P., HEDGES,R. W., AND DA-I-~A, N. ( 1974).Polynucleotide sequencerelationships among plasmids of the I compatibility complex. J. Gen. Microbiol. 85, 65-76. GRANT, A. J., BIRD, P. I., AND P~TTARD,J. (1980). Naturally occurring plnsmidsexhibiting incompatibility with members of incompatibility groups I and P. J. Bacteriol. 144, 758-765. GRANT, A. J., AND PITTARD, J. (1974). Incompatibility reactions of R plasmids isolated from Escherichia coii of animal origin. J. Bacterial. 120, 185-190. GRINDLEY,N. D. F., HUMPHREY&G. O., AND ANDERSON,E. S. (1973). Molecular studies of R factor compatibility groups. J. Bucteriol. 115, 387-398. GUERRY,P., FALKOW,S., AND DATTA, N. (1974). R62, a naturally occurring hybrid R-plasmid. J. Bacterial. 119, 144-151. HANSEN,J. B., AND OLSEN,R. H. (1978). Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5. J. Bacterial. 135, 227-238. HEDGES,R. W. (1974). R-factors from Providence.J. Gen. Microbial. 81, 171-181. HEDGES,R. W., AND DAT~A, N. (1973). Plasmids determining I pili constitute a compatibility complex. J. Gen. Microbial. 11, 19-25. JACOB,F., AND WOLLMAN,E. L. (1961). ‘Sexuality and the Genetics of Bacteria,” p. 44. Academic Press,New York/London. JACOB,A. E., YAMAMOTO, L., SMITH, D. I., COHEN, S. N., AND BERG,D. (1977). Plasmids studied in Escherichia coli and other enteric bacteria. In “DNA Insertion Elements, Plasmids and Episomes” (A. I. Bukhari, J. A. Shapiro, and S. L. Adhya, eds.), pp. 607638. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. KUSHNER,S. R. (1978). An improved method for transformation of Escherichia coli with Co1E 1 derived plasmids. In “Genetic Engineering” (H. W. Boyer and S. Nicosia, eds.), pp. 17-23. Elsevier/North-Holland Biomedical Press,Amsterdam. LANE, H. E. D. (1981). Replication and incompatibility of F and plasmids in the Inc FI group. Plasmid 5, lOO126. MEYNELL, E., AND DATTA, N. (1967). Mutant drug R factors of high transmissibility. Nature (London) 214, 885-887. MILLER, J. H. (1972). “Experiments in Molecular Genetics.” Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
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MONTI-BRAGADIN,C., AND SAMER,L. (1975). Compatibility of pTM89, a new F-like R factor, and of derivative plasmids. J. Bucteriol. 124, 1132-l 136. RODRIGUEZ,R. L., BOLIVAR, F., GOODMAN, H. M., BOYER,H. W., AND BETLACH,M. (1976). Construction and characterization of cloning vehicles. In “Molecular Mechanisms in the Control of Gene Expression” (D. P. Nierlich, W. J. Rutter, and C. F. Fox, eds.), pp. 471-477. Academic Press,New York. 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. ROYLE,P. L., AND HOLLOWAY,B. W. (1980). Relationships between R and FP plasmids in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 17, 293297.
RUSSELL,R. R. B., AND PITTARD,A. J. (1971). Mutants of Escherichia coli unable to make protein at 42°C. J. Bacterial. 108, 790-798. SMITH, D. R., ROOD,J. I., BIRD, P. I., SNEDDON,M. K.,
CALVO, J. M., AND MORRISON,J. F. (1982). Amplification and modification of dihydrofolate reductasein Escherichiu coli: Nucleotide sequenceoffol genesfrom mutationally altered plasmids.J. Biol. Chem. 257,90439048.
SMITH, H. R., GRINDLEY,N. D. F., HUMPHREYS,G. O., AND ANDERSON,E. S. (1973). Interactions of group H resistance factors with the F-factor. J. Bacterial. 115, 623-628.
THOMAS,C. M., MEYER,R., AND HELINSKI,D. R. ( 1980). Regions of broad host range plasmid RK2 which are essential for replication and maintenance. J Bucteriol. 141,213-222.
TIMMIS, K. N., DANBARA, H., BRADY, G., AND Lutz, R. ( 1981). Inheritance functions of group Inc FII transmissible antibiotic resistanceplasmids. Plasmid 5, 5375.
WARREN,G. J., TWIGG, A. J., AND SHERRATT,D. J. (1978). ColEI plasmid mobility and relaxation complex. Nature (London) 274, 259-26 1.
9, 191-200 (1983)
Demonstration of a Third Incompatibility Function on Plasmids Already Incompatible with Group P and Group I Plasmids PHILLIP Department of Microbiology,
I. BIRD
AND JAMES
PITTARD
University of Melbourne, Parkville 3052, Victoria, Australia
Received September 20, 1982; revised November
18, 1982
The addition of extra antibiotic resistance determinants to plasmids of a series previously shown to be incompatible in an atypical fashion with group I and group P plasmids, has enabled the existence of a third and major incompatibility function determined by these plasmids to be demonstrated. The in vitro construction of a plasmid consisting of the replication region of one of the plasmids, linked to the genes of the galactose operon, facilitated the identification of this third incompatibility function as similar to IncB plasmids.
We have previously described a series of R plasmids (pMU 700 to 707) which were isolated from the fecal Escherichia coli of domestic livestock. These plasmids are conjugative, of similar molecular weight (120 kb), determine resistance to streptomycin and sulphathiazole, and are incompatible with plasmids of both the I and the P incompatibility groups (Grant et aZ.,1980). The nature of these incompatibility interactions is unusual because plasmid loss from cells containing an incompatible pair is very slow, unilateral, and in some cases host dependent. Since these interactions are markedly different from those usually observed between incompatible plasmids, and because any model for incompatibility must explain the basis for every observation, we have been interested in investigating the incompatibility properties of the pMU plasmids. An integral part of such a study is to determine whether the pMU plasmids possessfurther genetic loci which would mediate a conventional result if two pMU plasmids were tested for incompatibility. To date, the lack of appropriate distinguishing markers on different plasmids of the series has prevented such tests from being performed. This is a common problem encountered in incompatibility testing (Datta, 1979), and can often prevent the unambiguous assignation of an unknown plasmid to a particular incompatibility group. The availability of transposable
antibiotic resistance determinants exemplified by Tn3 (ampicillin) and Tn5 (kanamytin) has allowed us to circumvent this problem by constructing derivatives of the pMU plasmids which carry either transposon, and which can be distinguished in a test situation. Although such derivatives may enable us to detect a third incompatibility function coded for by the pMU plasmids, the presence of at least two antibiotic resistance determinants on each plasmid may prevent the identification of the function by limiting the number of standard plasmids that may be used in such tests. To overcome this, we have taken advantage of an approach recently developed in this laboratory by which the replication and incompatibility genes of a plasmid of interest are linked to the genes coding for the fermentation of galactose (Davey and Pittard, manuscript in preparation). Such a plasmid would not be expected to exhibit entry exclusion, and the availability of different indicator media (Davey and Pittard, manuscript in preparation; Miller, 1972) enables its maintenance or loss from a Gal-deficient strain to be easily monitored. As the gal operon is not usually plasmid associated, such an artificially constructed replicon may be tested with every available standard plasmid without encountering problems with a lack of distinguishing markers. In this report, we describe experiments 191
0147-619X/83
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which indicate the existence of a third major incompatibility determinant on the pMU plasmids that is similar to those on plasmids of the IncB group. MATERIALS
AND
METHODS
Bacterial strains and plasmids. The strains of E. coli K 12 used in this study are described in Table 1. The plasmids, with the exception of the current incompatibility exemplars, are
also listed in Table 1. Referenceplasmids used included pIP55 (A=C=E); R16(B=0); R7 1lb(D); R386(FI); Rl(FI1); pSUl04(FIII); R124(FIV); pGL61 lA(FV1); R27(Hl); R478(H2=S); pIP233(H3); R64 drd 1l(1,); TPl14(1& R391 (J); R387(K); R446b(L=M); R46(N); pIE509(0F=FV); RP4(P); Rtsl(T); R1460(U); S-a(W); R6K(X); pIP231a(Y); and pIP7 la(9). These have been described by Jacob et al. ( 1977) except R7 1lb (Datta, 1979),
TABLE 1 BACTERIALSTRAINSANDPLASMIDS
Designation Strain
Plasmid
Characteristics”
JP777
thr, leu, rpoB, gal, lacy
JP990
argE3, his4, ilvC7, proA2, thi-1, supE44, gaik2, A-, nalA368 thi- 1, X-, rpoB35 1
JP995 PB1907 KA56 pMU700 pMU705 pMU706 pMU707 pMU715 pMU716 pMU717 pMU718 pMU719 pMU720 R144-3 R648 R621a R621a-1 R805a TP113 pBR322 pRBD13
polA 1, nalA HfrH, galE, rel-1, spoTI, A-, thi-1 Incly, IncP, IncB Sm Su Incla, IncP, IncB Sm Su Incly, IncP, IncB Sm Sp Su Incla, IncP, IncB Sm Su Inclr, IncP, IncB Sm Su Ap Incla, IncP, IncB Sm Su Ap IncB su Km Incla, IncP, IncB Su Km Inch, IncB, ColE’ SmTc Incla, IncB Gal Km Collb Inclol Inclo Ap Sm Km Incly” Tc Ap Km Cm Tc IncIyd IncIlb Km IncB Km ColE’ Ap Tc Tc Gal ColE’
Source or Reference Derived from JFM43 (Smith et al., 1982) Derived from AB 1157 (Grant and Pittard, 1974) Derived from W3 110 (Grant and Pittard, 1974) Obtained from P. L. Bergquist (Russell and Pittard, 1971) (Grant ef al., 1980) (Grant et al., 1980) (Grant et al., 1980) (Grant et al., 1980) pMU7OO::Tn3-this study pMU705: :Tn3-this study pMU707::Tn5-this study pMU707: :TnS-this study This study-derived from pMU707 Derived from pMU707 (Meynell and Datta, 1967) (Hedges and Datta, 1973) (Hedges and Datta, 1973) (Hedges, 1974) (Datta and Olarte, 1974) (GrindIey et al., 1973) (Rodriguez et al., 1976) pBR322 with gal t’iagment inserted in the PstI site (Davey and Pittard, manuscript in preparation)
‘See footnote b, Table 2. bE. Lederberg of the Plasmid Reference Center (personal communication) has recently recommended the withdrawal of these incompatibility designations to rationalize the I complex. For convenience we shall use the old designations. ’ Refers to incompatibility function. dThe incompatibility functions of this plasmid are similar to those of R621a (Bird and Pittard, 1982).
MULTIPLE
INCOMPATIBILITY
OF THE pMU PLASMIDS
193
pSU104 and pIP233 (De La Cruz et al., 1979), the appropriate media. These conditions were pIP23la (Roussel and Chabbert, 1978), and found to allow sufficient transfer of those plasmids with low transfer efficiencies or temperpGL61 IA (E. Lebek, unpublished). Antibiotics, chemicals, enzymesand media. ature-sensitive transfer functions. Incompatibility tests. The conventional Most of the chemicals, culture media, and antibiotics used have already been described methods used in this laboratory have been in detail (Grant et al., 1980; Grant and Pit- described previously (Grant et al., 1980). The tard, 1974). The indicator medium used to test involves checking appropriate transconscreen clones for their Gal status has been jugants for the maintenance of plasmid markdescribed (Davey and Pittard, manuscript in ers after four successivesubculturings-twice preparation), but basically consisted of a ga- on selective media and twice on nonselective lactose, tryptone, agar (Oxoid No. 1) basal media. Clones giving equivocal results were medium with either 25 &ml tetrazolium salts subcultured a further four times on unselecor a mixture of bromothymol blue (0.5% w/ tive media and retested. The stability of the v) and bromocresol purple (1.5% w/v) dyes. resident plasmids was checked in each case. Incompatibility tests involving the GalThe PstI and BamHI restriction endonucleasesand the T4 DNA ligase were obtained plasmid derived from pMU707 were performed by mating the standard plasmids with from Boehringer-Mannheim. Plasmid transfer. Conjugation was gener- a Gal-deficient strain (JP777) harboring the ally carried out asdescribed previously (Grant Gal-plasmid, as described above. On the meet al., 1980) and in all cases selection was dia utilized (basal plus tetrazolium salts), cells made for the incoming plasmid alone. Some- unable to ferment galactose gave rise to red times transfer was found to be more efficient colonies. Hence, an incompatible interaction if mating was allowed to occur on a solid sur- between a standard plasmid and the Gal plasface, as described by Bradley et al. (1980). mid gave rise to red colonies, while a comWhere entry exclusion prevented transfer of patible interaction gave rise to white colonies. Transposition of Tn3 and Tn5 to the pMlJ the donor plasmid, the recipient cells were grown under conditions that were shown to plasmids. The transposons were carried on reduce entry exclusion (Jacob and Wollman, separate derivatives of the multicopy, non1961). Such recipients were grown to station- conjugative, nonmobilizable DNA polymerary phase in nutrient broth (NB)’ overnight ase I-dependent plasmid, pMB8 (Rodriguez at 37°C. These cells were concentrated ten- et al., 1976), which were maintained in recA fold and added to a concentrated culture of backgrounds. (These strains were obtained donor cells (in log phase) so that a ratio of from P. L. Bergquist.) To translocate a trans1:10, donor to recipient, was achieved in a poson to a conjugative plasmid, the “target” volume of 4 ml, to give a total population of plasmid was crossedinto a cell containing the approximately lo8 CPU/ml. Mating was al- pMB8: :Tn plasmid. Subsequently, a pool of lowed to occur for 2 h at 37°C; then the mix- transconjugants derived from this cross was used as a donor in conjugation to a polA retures were plated to the appropriate media. In experiments where a large number of cipient (PB 1907) selecting for both the transstandard plasmids were crossed to recipients poson and the target plasmid. Transconjuharboring a Gal-plasmid derived from gants resulting from this cross were purified pMU707 (pMU720), mating was allowed to and transferred to JP990 selecting for the taroccur for 4 h at 37OC, followed by an over- get plasmid alone. Transconjugants showing night incubation at 20°C. Ten-microliter al- 100% coinheritance of the transposon were iquots from each mixture were spotted onto screened for plasmid DNA content to ensure that no ColE-type plasmid had been cotransferred by mobilization (Warren et al., 1978) ’ Abbreviations used: NB, nutrient broth, CFU, color cointegration (Crisona et al., 1980). ony-forming units.
194
BIRD
AND
DNA manipulations. Small-scale preparations of DNA were obtained by the method of Bimboim and Doly ( 1979),while large-scale preparations of either multicopy (Clewell and Helinski, 1969) or low copy (Hansen and Olsen, 1978) plasmid DNA were made by centrifuging cleared lysates on cesium chlorideethidium bromide gradients. Transformations were performed by the method of Kushner (1978). Agarose gel electrophoresis and visualization of DNA restriction fragments followed standard procedures (Davis et al., 1980),while restriction endonucleasesand T4 DNA ligase were used as recommended by the manufacturers. RESULTS
Construction and Properties of TransposonCarrying Derivatives of the pMiJ Plasmids Derivatives of pMU700 and pMU705 which carried Tn3 (ampicillin), and of pMU707 which carried Tn5 (kanamycin) were isolated as outlined above. To ensure that the known incompatibility functions had not been altered as a result of the transpositional event, the derivatives were tested with RP4 (IncP), R 144-3or R648 (IncIa), and R62 1a or R62 1al (IncIy). The results shown in Table 2 indicated that with the exception of pMU7 17, the new plasmids were unaltered in their known compatibility functions. However pMU715 and 716 apparently had Tn3 inserted in gene(s)whose loss of function led to an increase of lo- to 1OO-foldin the plasmids’ transfer efficiencies, while pMU7 18 apparently had its streptomycin resistance function inactivated by the insertion of Tn5. The plasmid pMU7 17 was no longer incompatible with plasmids of the IncP or IncIcv groups, whereas a derivative of pMU707 should be incompatible with these plasmids (Table 2). This loss of incompatibility functions was accompanied by loss of the strep tomycin resistance function. The simultaneous loss of distinct functions is most readily explained by postulating a transposon-mediated deletion event, similar to those previ-
PITTARD
ously observed in other systems, associated with Tn5 (Berg, 1977). The isolation of a plasmid deficient in both known incompatibility functions was useful in establishing the existence of a third independent incompatibility function encoded by the pMU plasmids. Incompatibility between the pMU Plasmids The plasmids pMU7 15, 7 16, 7 17, and 7 18 were tested where possible for incompatibility with each other and with pMU706 (which has a distinguishable spectinomycin resistance determinant). The results are shown in Table 3. Initially it proved impossible to transfer one of these plasmids into a host containing another, presumably due to strong entry exclusion. However, by growing the recipient cells under conditions designed to reduce entry exclusion (as described above), and by obtaining an increased efficiency of transfer by mating on a solid surface, a significant number of transconjugants were obtained and tested for the maintenance of plasmid markers. Even under these optimum conditions, a second pMU plasmid could not be introduced into a cell already harboring pMU717. The incompatibility interactions observed between the pMU plasmids resemble those seenbetween strongly incompatible plasmids such as R144-3 and R62 la (Bird and Pittard, 1982), and are markedly different from those previously observed between the pMU plasmids and exemplars of the I and the P incompatibility groups (Grant et al., 1980). The interactions described in Table 3 were not dependent on the host strain used in the test (data not shown), were reciprocal (except when pMU 7 17 was the resident plasmid), and appeared to be unconnected with the I and the P incompatibility functions of the plasmids, as illustrated by the interaction of pMU717 with the other pMU plasmids. The use of a single plasmid selection prior to incompatibility testing means that there is no means of ensuring that every recipient cell initially contained a resident plasmid. When entry exclusion occurs, there may be a strong bias of transfer of the incoming plasmid to
MULTIPLE
INCOMPATIBILITY
OF THE
pMU
195
PLASMIDS
TABLE 2 INTERACTIONS OF THE pMU DERIVATIWS WITH GROUP I AND GROUP P PLASMIDS” Incompatibilityd Transfer frequencyC
Recipient
Selection6
JF’990‘(pMU7 15) JF990 (pMU7 16) JF990 (pMU7 17) JF990 (pMU718) JF990
Km Nx Km Nx Ap Nx Ap Nx Km Nx
6.3 X 1.3 x 1.0 x 2.2 x 2.5 X
Jp995 (R144-3)
JP990 (pMU7 15) JP990 (pMU7 16) JF990
Jp995 (R648)
First testing
Second testing
IO-’ 10-S 10-G 10-h 10-s
KmTc(40/4O)SmSu(16/40) KmTc(40/4O)SmSu(8/40) ApWWWn(40/40) Ap(40/4O)Su(16/40) NT’
KmTc(40/4O)SmSu(O/40) KmTc(40/4O)SmSn(0/40) AdWWSdWJ’-J) Ap(40/4O)Sn(0/40) NT
Km Nx Km Nx Km Nx
1.3 x 10-I 8.8 x 10-Z 2.0 x 10-l
Km(40/40)SmSnAp(35/40) Km(40/4O)SmSnAp(8/40) NT
Km(40/4O)SmSuAp(30/40) Km(40/40)Kmsn(0/40) NT
JF990 (pMU7 17) JP990 (pMU7 18) JP990
Ap Nx Ap Nx Ap Nx
1.0 x 10-5 1.4 x 10-4 1.1 x 104
JP995 (R621a)
JP990 (pMU7 15) JP990 (pMU7 16) Ji’990
Tc Nx Tc Nx Tc Nx
4.7 x 10-a 1.3 x 10-s 1.6 X lo-’
Tc@O/4O)SmSuAp(6/40) Tc@O~4O)SmSnAp(37/) NT
Tc(40/4O)SmSuAp(O/40) Tc(40/4O)SmSuAp(37/40) NT
JF995 (R621a-I)
JP990 (pMU7 17) JF990 (pMU7 18) JP990
Ap Nx Ap Nx Ap Nx
9.5 x 10-5 2.9 X IO-’ 8.0 X IO-’
(Rp4)
0 The reciprocal crossesare not shown, but the results obtained were consistent with these, and with those described previously (Grant and F’ittard, 1974). b Abbreviations used: Km, kanamycin; Ap, ampicillin; Tc, tetracycline; Nx, nalidixic acid, Sm, streptomycin; Su, sulphathiazole; Sp, spectinomycin. c Defined as the number of transconjugants per donor per hour. d Incompatibility results are shown as the markets displayed by the 40 clones tested in each case.Those clones showing cc-existence of both setsof plasmid markers following the second testing were used as donors in further cmssesto plasmid-free recipients and were shown to be able to transfer each plasmid’s markers separately and independently. =Not tested.
plasmid-free recipients, which could lead to the false conclusion that the plasmids were incompatible. Our results indicate there is a lo- to 500-fold decreasein transfer efficiency of the incoming plasmid if the recipient pop ulation harbors a resident plasmid (Table 3). Assuming that transfer of the incoming plasmid only occurs to plasmid-free cells within this population (and not to others because of entry exclusion), such cells would have to appear at a frequency of between 10-i and 5 x lo-*. Accordingly, cells harboring pMU718 were grown under the same conditions used for the recipients in the experiments outlined in Table 3 and 1200 independent clones were checked for the maintenance of plasmid markers. No loss was observed, implying that resident plasmid instability did
not occur at high enough frequencies to influence the results. Construction and Properties of a Derivative of pMU707 that Carries the gal Operon Although the pMU plasmids have been tested previously with plasmids of the A, C, FI, FII, FIV, H, Icu, Iy, J, K, L, M, N, P, S, T, and W incompatibility groups (Grant et al., 1980), the existence of a third incompatibility function determined by the pMU plasmids, as well as the expansion and reorganization of the recognized incompatibility groups, necessitatedthe retesting of the pMU plasmids. To facilitate this aim and to prevent such problems as entry exclusion and overlapping markers, we decided to link the rep-
196
BIRD
AND
PITTARD
TABLE 3 INTERACTIONS
OF THE pMU PLASMIDS
Selection”
AND THEIR
DERIVATIVES
Transfer frequency b
Incompatibility’: First testing
Donor strain
Recipient strain
JP995 (pMU7 17)
JP990 (pMU706) JP990 (pMU7 16) JF990 (pMU7 15) JP990
Km Km Km Km
Nx Nx Nx Nx
1x 2x 4 x 2 x
lo-5 lo-5 lo-6 lo-4
Km(40/40)Sp(0/40) Km(40/40)Ap(0/40) Km(40/40)Ap(0/40) NTd
JP995 (pMU7 18)
JP990 (pMU706) JP990 (pMU7 16) JP990(pMU7 15) JF990
Km Km Km Km
Nx Nx Nx Nx
8 8 2 6
x x x x
1O-5 IO-’ 1o-4 IO-’
Km(40/40)Sp(0/40) Km(40/40)Ap(0/40) Km(40/40)Ap(0/40) NT
JP995 (pMU706)
JP990 JP990 JP990 JP990 JP990
(pMU7 17) (pMU7 18) (pMU716) (pMU7 15)
Sp Sp Sp Sp Sp
Nx Nx Nx Nx Nx
5 1 7 3
NT Sp(40/4O)Km(0/40) Sp(40/40)Ap(0/40) Sp(40/40)Ap(0/40) NT
JF995 (pMU7 16)
JP990 (pMU7 17) JP990 (pMU7 18) JP990 (pMU706) JP990
Ap Ap Ap Ap
Nx Nx Nx Nx
NT Ap(40/4O)Km(0/40) Ap(40/40)Sp(0/40) NT
JP995 (pMU7 15)
JP990 (pMU7 17) JF990 (pMU7 18) JP990 (pMU706) JP990
Ap Ap Ap Ap
Nx Nx Nx Nx
NT Ap(40/40)Km(0/40) Ap(40/40)$(0/40) NT
‘.‘,‘,‘See footnotes b, c, d, and e, respectively, Table 2.
lication genesof pMU707 to those of the gal operon, on the assumption that the major (unknown) incompatibility genesof pMU707 were linked to those directing replication. The major advantage of this approach is that the status of the Gal plasmid in an incompatibility test may be monitored simply by utilization of appropriate indicator media (see Materials and Methods). The construction of such a plasmid involved a two step procedure utilizing an artificially derived plasmid, pMU7 19, as a source of the replication genes of pMU707. pMU7 19 was derived by ligating DNA fragments of pMU707, generated by a partial digest with PSI, to PstI-generated fragments of pBR322 DNA and transforming JP777 to streptomycin resistance. Although the aim of this experiment was to clone either the IncP or Inc Ia locus of pMU707, on the assumption (based on experience with
pMU7 17) that these loci were linked to its streptomycin resistance determinant, pMU7 19, as well as having the Inc ICXlocus, was also incompatible with pMU7 17 and was able to replicate in a a& host (data not shown). These results indicated the presence of a replication system on pMU7 19 distinct from that of pBR322, presumably that of pMU707. Given the smaller size, higher copy number, and hence greater ease of manipulation of pMU7 19 compared to pMU707, we used the former plasmid as a source of the replication genesof the latter. To construct a miniplasmid consisting of the replication system of pMU707 linked to Gal, we partially digested pMU7 19 DNA with PstI and ligated these fragments to those resulting from a complete BarnHI and PstI double digest of pRBDl3 (pRBD13 is described in Table 1). Since BumHI cuts pRBD13 once, not in the
MULTIPLE
INCOMPATIBILITY
Gal fragment, this enzyme was also used to digest pRBDl3 DNA in order to lower the probability of obtaining reformed pRBD 13 molecules on ligation and transformation of the recipient strain to Gal+. Selection for plasmids carrying gal was made by transforming KA56 (a guE strain) with the ligation mixture and plating onto a galctose-tryptone-bromthymol blue/bromocresol purple plate. Since the g& mutation is lethal in the presence of galactose,clones harboring Gal plasmids were selected and could be easily separated from the slight background growth obtained on theseplates becauseof their strong yellow coloration. Forty Gal+, tetracycline-sensitive clones were picked and screened for their plasmid DNA content. Two clones containing the smallest plasmid species were chosen and small-scale DNA isolated from them was used to transform JP990 and JP777 to Gal+. Analysis of the restriction patterns of the two plasmids, generated by PA, indicated they were similar and consisted of one large fragment (Gal), and two smaller fragments both of which are necessaryfor replication (Bird and Pittard, in preparation) to give a total size of approximately 7.9 kb. Both plasmids were incompatible with pMU707 and pMU7 17 in JP990 and JP777, and with R144-3 in JP990 (pMU plasmids with an Ia function only display it in JP990 (Grant et al., 1980)), but were compatible with RP4 in both hosts. They were nonconjugative and no entry exclusion was observed with pMU707, pMU717, R144-3, or RP4 (data not shown). These results indicate that the genesdetermining both the unknown incompatibility and Ia incompatibility functions of pMU707 are associated with or near its replication genes, while the P incompatibility function is quite distinct. One of the two isolates, pMU720, was used in further studies. IdentiJication of the Unknown Incompatibility Determinant of pMU720 Rifampicin-sensitive strains containing reference plasmids of known incompatibility
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groups were usedas donors in crossesto JP777 and to JP777 harboring pMU720. On the basis of the color reaction on the selective media (galactose-tryptone-tetrazolium salts; see Materials and Methods) displayed by the transconjugants resulting from each mating, pMU720 was found to be incompatible with R16(IncB). To confirm this, we predicted that pMU720 should also be incompatible with the plasmids R805a (Datta and Olarte, 1974), and TP113 (Grindley et al., 1973) both of which have been shown to be incompatible with R16. We found pMU720 to be incompatible with TP113 and R805a. It was compatible with the exemplars of every other incompatibility group tested, namely, groups A, D, FI, FII, FIR, FVI, Hl, H2, H3, I,, IZ, J, K, L, N, OF, P, T, U, W, X, Y, and 9. Under the conditions used for these tests, the weak interaction of pMU720 with the I, (Ia) plasmids was not apparent. DISCUSSION
Two recurring problems encountered in classical incompatibility testing are the lack of suitable markers on each plasmid to allow their distinction in a test, and the difficulty in telling whether in some circumstances the apparent nonexistence of clones containing both plasmids is due to entry exclusion, incompatibility or a combination of both effects. To circumvent a problem such as no distinguishing markers, one approach is simply to add extra markers, in the form of antibiotic resistance determinants, to a particular plasmid and this is conveniently done by utilizing transposons (De La Cruz et al., 1979; Royle and Holloway, 1980). However some care is necessaryto ensurethat the transposon (which can act as a mutagen) does not alter the incompatibility function of the target plasmid. The probability of this occurring may be reduced by choosing plasmids that are inactivated in a particular function such as antibiotic resistance or transfer (pMU7 15, 7 16, and 7 18), although as illustrated by pMU7 17, a transpositional event may lead to the loss of several functions.
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The transposons themselves do not appear to influence the outcome of incompatibility tests, provided that a single plasmid selection is used to construct cells containing the plasmids to be tested. However in those cases where entry exclusion or incompatibility between the plasmids under test leads to a significant reduction in the number of clones available for testing, and especially when a doubIe selection is used in such circumstances to attempt to increase the numbers of clones available, transposition of markers from replicon to replicon may complicate the interpretation of the results (D. Jans, B.Sc. (Hons.) thesis, University of Melbourne, 1980). While the use of transposons as extra markers in incompatibility testing does not necessarily overcome the problem of distinguishing the effectsof entry exclusion from incompatibility, the second approach we have used overcomesboth problems. In this method, we have linked the replication genesof a plasmid of interest to genes that endow a selectable phenotype on cells containing the new plasmid, expecting, on the basisof experience with other well-studied plasmid systems, that the incompatibility genes would be closely associated with the genesdirecting replication. The major advantages of this method are that the genes coding for entry exclusion should no longer be present, the marker used (Gal) is not commonly associated with plasmids and is easily distinguishable, and the utilization of indicator media allows the presence or absence of the Gal plasmid in a strain to be continually monitored. Using this system we have easily and unambiguously assigned the unknown incompatibility determinant of pMU707 to the IncB group. However there are some considerations to be made when utilizing this approach to incompatibility testing. First, it is assumed that the incompatibility genesand replication genesare closely linked. In the light of recent molecular analyses of plasmids of the FI and FII incompatibility groups (Lane, 1981; Timmis, 1981) it appears that there may be more than one incompatibility locus, and hence the isolation of replication genesmay not necessarily ensure the
PITTARD
isolation of all the incompatibility functions. For example, in our case the derivative of pMU707, pMU720, no longer displays the IncP function of its parent. Another consideration is that the genes essential for replication and stable maintenance may not be clustered in one segment of the genome, as is the case for the IncP plasmid RR2 (Thomas et al., 1980), and the construction of miniplasmid derivatives by in vitro cloning techniques may prove to be extremely difficult. These problems may be overcome by constructing a bank of Gal plasmids, each carrying a defined incompatibility locus. Although the initial construction of such a bank would be hindered by the same problems as outlined above, once obtained, it could be used to quickly and unequivocally assign an unknown plasmid to a particular incompatibility group without the need for extensive in vivo and in vitro manipulations. The finding that pMU720 interacts with plasmids of the I and the B incompatibility groups implies that this plasmid has complex replication and/or incompatibility systems. Several plasmids which interact with members of more than one incompatibility group have been described (Grant et al., 1980; Guerry et al., 1974; Monti-Bragadin and Samer, 1975; Smith et al., 1973), and although this multiple incompatibility in some cases has been attributed to similarities in genetic or biophysical characteristics between plasmids of the shared groups (Falkow et al., 1974), in most cases, the basis of multiple incompatibility hasyet to be explained. In our case,it is likely that the IncB function of the pMU plasmids is the major incompatibility function, and since the IncP and IncIa functions may be lost from pMU7 17 without apparently affecting its stability in the cell, and because they are qualitatively different from “typical” incompatibility functions, it may be that they represent the involvement of separate mechanisms in the incompatibility of the pMU plasmids. We would prefer not to classify the pMU plasmids as typical IncB plasmids becausethe basis for their interactions with IncI plasmids
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and IncP plasmids is not yet understood, and as these interactions are unusual we would hesitate to call them IncI or IncP plasmids. It is our intention to study the pMU plasmids further to determine the basesfor their multinle incomDatibiIitv and to establish whether they resemble othe; well-characterized plasmids in replication and incompatibility functions. ACKNOWLEDGMENTS We thank E. Lederberg of the Plasmid Reference Center for providing the incompatibility exemplars and information on the current status of the incompatibility classification scheme,as well as P. Bergquist for providing PB 1907 and the pMB8: :Tn plasmids. This investigation was supported by a grant from the National Health and Medical ResearchCouncil. P.B. acknowledgesthe receipt of a Commonwealth Postgraduate Research Award.
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