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The use of mini-Gal plasmids for rapid incompatibility grouping of conjugative R plasmids
PLASMID
11, 234-242 (1984)
The Use of Mini-Gal Plasmids for Rapid Incompatibility Grouping of Conjugative R Plasmids R. BRENT DAVEY,' PHILLIP I. BIRD, SUZANNE M. NIKOLETTI, JUDYTA PRASZKIER,ANDJAMES PITTARD~ Department of Microbiology, University of Melbourne, Paddle,
Victoria 3052, Australia
ReceivedNovember9, 1983; revised January 10, 1984 The galactose operon of Escherichia coli K-12 has been used as a phenotypic marker for miniplasmids derived in vitro from R plasmids representing six incompatibility groups. This has enabled the development of a rapid incompatibility typing scheme in which the miniplasmids are used as incompatibility exemplars, their presence in strains being monitored on galactose fermentation indicator media.
Incompatibility between two plasmids is shown by their inability to coexist stably in the same cell, in the absence of selective pressure (Novick et al., 1976). All plasmids studied to date exhibit incompatibility and this has been used as the basis of a classification system, on the assumption that naturally occurring incompatible plasmids derive from a common ancestor (Datta, 1979). The usefulness of incompatibility in typing particular plasmids may be limited by several factors. These include the presence of overlapping or identical markers on the test and reference plasmids, difficulty in establishing the test and reference plasmids in the same cell due to entry exclusion, the existence of more than one incompatibility function on either plasmid, and the time and amount of media required to perform a test. For these reasons incompatibility testing is not widely used in clinical microbiology laboratories as a routine epidemiological tool. In this paper we describe the development of a system designed to overcome most of the practical difficulties encountered in incompatibility testing. We have constructed a series ’ Current address: Fairheld Hospital, Yarra Rend Road, Fairfield, Victoria 3078, Australia. ’ To whomreprint requests and general inquiries should
be addressed. 0147-619X/84
$3.00
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form resewed.
of miniplasmids (representing six major incompatibility groups) which consist of the replication and incompatibility genes of particular plasmids linked to the genes of the galactose operon. These plasmids no longer determine resistance to antibiotics or code for entry exclusion functions. The presence or absence of these mini-Gal plasmids in a strain is easily monitored by the color of colonies on suitable indicator media. Incompatibility is indicated by the appearance of Gall transconjugants following transfer of an appropriate conjugative R plasmid into a strain containing a Gal plasmid. MATERIALS
AND METHODS
Bacterial strains and plasmids. The strains of Escherichia coli K12 used in this study, as well as plasmids other than incompatibility exemplars, are listed in Table 1. The plasmids used as incompatibility standards were RA 1, R686, R40a (A = C = E); R16, TPI 13, R724 (B = 0); R386 (FI); Rldrd19, Rldrd16, RlOOdrdl (FII); pSU104 (FVI); R124 (FIV); pIE509 (FV); TP116, TP124, R27 (Hl); R478, pHH1058a (H2 = S); R64drdl1, R648, R483 (I,); R621a (1~); TP114 (I& R721 (16); R805a (In; R391 (J); R387 (K); R47la, R446b (L = M); R46 (N); RP4 (P); Rtsl, R394 (T); R1460, pIE420 (U); S-a, RA3 (W); R6K (X); pIP231a (Y); pIP71 (9); and pIE545 (Z).
234
235
MINI-Gal PLASMIDS IN RAPID Inc TESTING TABLE 1 BACTERIALSTRAINSANDPLASMIDS
Designation strain/plasmid
Source or reference
Characteristics”
KA56
HfrH, galE45, reMI, spoTI, thil, X-
A. Rorsch
CA13
HfrH, galTl18, relA1, spoT1, h-
B. Bachmann
JP777
thrl, leul, lacY1, gal35 1, supE44, tonA2, hsdR4, rpoB364
Spontaneous 2deoxygalactose- and rifampicin-resistant derivative of JFM43 (Smith et al., 1982)
JP798
F-, ga1354,polA 1, gyrA, thyA
Spontaneous 2deoxygalactose and nalidixic acid-resistant derivative of PB1906 (P. Bergquist)
argE3, his4, ilvC7, proA2, thil, supE44, gala, X-, gyrA368
(Grant et al., 1980)
pBR322
Ap Tc IncColE
(Rodriguez et al., 1976)
pMU407. I
Ap,6 Cm, Km, Gm, Sm, Sp, Su, Tc IncL/M
(Davey and Pittard, 1977)
pMU700
Sm, Su, IncB/O, IncIy, IncP
(Grant et al., 1980)
R64drdll
Sm, Tc IncI,
(Jacob et al., 1977)
pIP7 1
Ap, Cm, Sm, Su, Tc Inc9
(Jacob et al., 1977)
R46
Ap (OXA-2), Sm, Sp, Su, Tc IncN
(Jacob et al., 1977)
R6-5
Cm, Km, Nm, Sm, Su IncFII
(Jacob et al., 1977)
pMU106a
Ap IncN
Derived from R46 (P. Bird)
pRBD13
Tc, Gal IncColE
This study
pMU600
Gal IncFII
This study-derived from R6-5
pMU60 1
Gal Inc9
This study-derived from pIP7 1
pMU602
Gal Inc B/O
This study-derived from pMU700
pMU603
Gal IncN
This study-derived from pMU 106a
pMU604
Gal IncL/M
This study-derived from pMU407. I
pMU605
Gal IncI,
This study-derived from R64drdll
’ Abbreviations used: Ap, ampicillin resistance;Cm, chloramphenicol resistance;Gm, gentamicin resistance; Km, kanamycin resistance;Nm, neomycin resistance; Sp, spectinomycin resistance; Sm, streptomycin resistance; Su, sulfathiazole resistance;Tc, tetracycline resistance; Gal, galactose fermentation. b Unless specified ampicillin resistance refers to the high-level TEM type.
These have been described by Jacob et al. ( 1977) except pSU104 (De La Cruz et al., 1979), pIP231a (Roussel and Chabbert, 1978), pHH1058a, R1460, pIE545, pIE509, and pIE420 (E. Lederberg, personal communication). The status of the incompatibility groups is according to the Plasmid Reference
Center (E. Lederberg, personal communication). Antibiotics, chemicals, and media. Most of the chemicals and media used in this study have been described previously (Grant and Pittard, 1974; Grant et al., 1980). Antibiotics were used at the following concentrations:
236
DAVEY -El
ampicillin (25 clglml), tetracycline ( 15 &ml), chloramphenicol (10 pg/ml), kanamycin (20 pg/ml), streptomycin (5 pg/ml), spectinomycin ( 15 pg/ml), sulfathiazole ( 125 pg/ml), trimethoprim (10 pg/ml), gentamicin (10 pg/ ml), neomycin (20 pg/ml), rifampicin (100 dml). Galactose-tetrazolium indicator (T)3 agar was prepared as follows: a base medium was prepared containing 0.9% (w/v) tryptone (Oxoid L42); 0.25% (w/v) yeast extract (Oxoid L2 1); 0.125% (w/v) Lab Lemco (Oxoid L29); 2.5 IIIM MgC12in 15 mM Mops3 pH 7.0; 2.2% (w/v) agar (Oxoid No 1, Ll 1) was added to half the base medium. After sterilization of the basemedium 1%(v/v) galactose [substantially glucose-free, Sigma Chemical Co.], appropriate antibiotics and triphenyltetrazolium chloride (Sigma, 25 &ml) were added aseptically from stock solutions to the half without agar. Both halves of the medium were then combined, mixed thoroughly, and used to pour plates. On this medium Gal+ colonies were white, and Gal- colonies red (Miller, 1972). Galactose-bromocresol purple/bromothymol blue indicator (B) plates were prepared similarly with the exception that the basemedium contained 5 mrvr Mops. The indicator usedwas a combination of bromocresol purple (55 cLg/ml) and bromothymol blue (17.5 pg/ ml). On this medium Gal+ colonies were yellow and Gal- colonies blue. Conjugation and incompatibility testing. Unless specifically mentioned, conjugations and incompatibility testing were performed as describedpreviously (Grant et al., 1980). Plate matings were performed using the technique of Low (1973). Recombinant DNA techniques. Purified plasmid DNA and bacteriophageDNA for use in cloning procedures were isolated as described previously (Bird and Pittard, 1983; Davis et al., 1980). Otherwise DNA was prepared according to the method of Bimboim
‘* AL.
and Doly (1979). Methods for the use of restriction endonucleases,T4 DNA ligase, and agarosegel electrophoresishave been described by Davis et al. (1980). Enzymes were purchased from Boehringer-Mannheim, Amersham, or BethesdaResearchLaboratories, and used in the buffers prescribed by the manufacturer. Transformation was performed by the method of Kushner (1978). RESULTS
Cloning of DNA containing the gal operon. The initial source of gal operon DNA used in the construction of the Gal plasmids was Xpgal8 (Feisset al., 1972). To generate a more convenient source of gal operon DNA it was decided to clone the gal genes into pBR322. Since no physical map of Xpgal8 was available, a “shotgun” approach to cloning the gal operon from the phage to pBR322 was taken. PhageDNA was digestedwith PstI and ligated to P&cleaved pBR322 DNA, and the mixture was used to transform JP777 to tetracycline resistanceon B medium. Gal+ colonies arising from this transformation were screened for ampicillin sensitivity. The plasmid content of ampicillin-sensitive, Gal+ clones was determined by restriction analysis of appropriate DNA preparations. A tetracycline-resistant, ampicillin-sensitive, Gal+ clone containing a small plasmid (pRBD 13), which had an extra 5-kb PstI-generatedrestriction fragment when compared to pBR322, was chosen for further characterization. DNA was prepared from a strain containing pRBD13 and used to transform strains carrying various mutations in their gal genes. The plasmid could complement mutations in the galK (JP990), galE (KA56), and galT (CAM3) genes. It also complemented a Gal deficiency in a derivative of KA56 with a deletion spanning hatt, gal, aroG, and nadA. These results indicate that the entire gal operon is carried by pRBD 13. The absenceof a functional DNA pokindependent replication system on the 5-kb Gal 3Abbreviations used:T, galactose-tetrazolium indicator; fragment was indicated by the failure of Mops, 4-morpholinepropanesulfonic acid; B, galactosepRBD13 to transform a polA strain (JP798). bromocresol purple/bromothymol blue.
237
MINI-Gal PLASMIDS IN RAPID Inc TESTING
The location and orientation of the gal operon on pRBDl3 as shown in Fig. 1 was deduced from previously published data (Busby et al., 1982). This plasmid was used as the source of Gal marker DNA for subsequent plasmid constructions. Construction of mini referenceplasmids currying the gal operon. The construction of the IncB, In&II, IncI, IncM, and Inc9 Gal plasmids (derived from pMU700, R6-5, R64drdl1, pMU407.1, and pIP71, respectively) was performed by ligating parental plasmid DNA partially cleaved with PstI to &I- and BumHI-cleaved pRBDl3 DNA.
(pRBDl3 was also cleaved with BamHI to lower the frequency at which recircularized pRBD 13 molecules were obtained in the experiment, as BarnHI cleavesthe pBR322 portion of pRBDl3 but not the Gal fragment.) The ligation mixtures were then used to transform KA56 (galE) to Gal+. Strains carrying a galE mutation are sensitive to galactose so Gal+ transformants could be selected on B medium without encountering problems with background growth. The Gal+ clones resulting from each ligation were checked for tetracycline sensitivity, and in each case plasmid DNA from about
Hc Hc H
P
pRBD13
9.3kb
pMU600
7.7kb
pMU601
7.1 kb
pMU602
8.4 kb
pMU603
9.2kb
pMU604
7.1 kb
pMU605
9.8kb
*----K--T--EGAL H
P Bg
P I
SP
*--REP----INCS
*--REP--INC H
P
s
Ba PP
PS
-----REP----lNCIi
P
Ba
P I
Bg *--REP--* ‘--,NC--‘
H
PPP
P
‘---REPTh?CC
H
P
E
P I
-
PS
e-----REP-I
1 kb
PP
Bg ____
-+
-lNC-
FIG. 1. Maps of the Gal plasmids used in this study. The plasmids are represented as linear molecules opened at one PstI site, which is depicted at the right-hand end of each map. Broken arrows indicate loci that have not been fully delineated. The source replicon for each of the Gal plasmids is as follows: pMU600 from R6-5 (In&B); pMU601 from pIP7 1 (In&Q pMU602 from pMU700 (IncB/O); pMU603 from pMU 106a (IncN); pMU604 from pMU407.1 (IncL/M); and pMU605 from R64drdll (Incl,). Abbreviations used: REP, replication region; INC, incompatibility region; GAL, galactose operon; Ba, BarnHI; Bg, BglII; E, EcoRI; Hc, HincII; H, HindIII; Hp, HpaI; P, &I; Pv, PvuII; S, SalI; Ss, SstII. Not all these restriction endonucleaseswere used to map each plasmid, and with the exception of HindIII, the recognition sites in the pBR322derived portion of pRBD13 are not shown.
238
DAVEY
20 Gal+, tetracycline-sensitive clones was screened after digestion with PstI. A variety of plasmid sizes (from approximately 50 to 10 kb) was observed. On the assumption that the smaller the plasmid the lesslikely it would be to retain parental entry exclusion or antibiotic resistance functions, DNA from several clones harboring small plasmid species (lessthan 20 kb) was used to transform JP777 and JP798 to Gal’. The most stable, DNA p&-independent plasmids derived from each of the parent plasmids were chosen for further characterization. The IncN Gal plasmid (pMU603) was derived from R46, which has been mapped and characterized (Brown and Willetts, 1981). In the course of the construction of cloning vectors derived from R46, an intermediate plasmid, pMU 106a, was made. This consists of a 2.2-kb portion of the BgZE (replication) fragment and 0.5 kb ofthe BglD fragment of R46, linked to a 1.5-kb portion of Tn.? coding for ampicillin resistance(P. Bird, unpublished results). The plasmid pMU106a has a unique PstI site within the ampicillin resistancegene, so a Gal+, ampicillin-sensitive derivative of pMU106a was constructed by ligating PstIcleaved pMU 106a DNA to PstI- and BamHIcleaved pBRDl3 DNA and transforming IL456 to Gal+. Plasmid DNA from ampicillinsensitive Gal+ clones was analyzed, and DNA from clones giving the predicted restriction pattern after cleavage with PstI was used to transform JP798 and JP777. Properties of the Gal plasmids. Maps of the Gal plasmids are shown in Fig. 1. With the exception of pRBD 13, each plasmid determines DNA polLindependent replication functions as judged by their ability to successfully transform JP798 to Gal+. The approximate locations of the replication and incompatibility regions of pMU60 1 (Inc9), pMU602 (IncB), pMU604 (IncM), and pMU605 (IncI) were deduced in the following fashion. In each case,Gal plasmid DNA partially cleaved with PstI was ligated to PstIcleaved pBR322 DNA and used to transform JP777 harboring the corresponding Gal plasmid to tetracycline resistance on T medium.
ET AL.
Those clones that received a pBR322 derivative not carrying the Gal fragment but which expressedincompatibility against the resident Gal plasmid were Gall on T medium. Both Gall and Gal+ clones arising from the transformation were screened for ampicillin sensitivity and plasmid DNA from such ampicillin-sensitive clones was screened after digestion with PstI. In this way a number of clones containing pBR322 derivatives with one or more extra Pst fragments was obtained from each subcloning experiment. The locations of the incompatibility and replication loci of each Gal plasmid were assigned by testing its pBR322 derivatives for the ability to express incompatibility against the corresponding Gal plasmid (as described above), and for the ability to transform and hence to replicate in a polA strain. In all cases the locus involved in the expression of incompatibility could be assigned to a single PstI fragment, while the locus involved in DNA PO/I-independent replication spanned several PstI fragments, including the fragment containing the incompatibility locus (Fig. 1). The locations of the replication and incompatibility regions of the IncFII and IncN Gal plasmids are according to maps of the parent plasmids. All the DNApolI-independent Gal plasmids are unstable,judging by the percentageof Gall clones arising from an overnight culture (in nonselective broth) of JP777 harboring the appropriate Gal plasmid. The degree of instability observed-IncB (2%), IncFII (16%), IncI (lo%), IncM (7%), IncN ( l%), Inc9 (0.1%)-did not complicate the interpretation of the results when the plasmids were used in the incompatibility testing scheme described below. None of the Gal plasmids carried any functional antibiotic resistance determinant in comparison to their parents. None were found to be conjugative, and, with the exception of the IncN plasmid, none appeared to exhibit entry exclusion against the parental plasmids. Entry exclusion was tested by comparing the conjugational transfer frequency of a reference plasmid into JP777 harboring its derivative
MINI-Gal PLASMIDS IN RAPID Inc TESTING
Gal plasmid, to its transfer frequency into JP777 alone. Entry exclusion is indicated when a significant decreaseis observed in the former frequency compared to the latter. The transfer frequency of R46 or N3 into JP777 harboring the IncN Gal plasmid was lo- to loo-fold lessthan the corresponding frequency of transfer to JP777 alone. Since the IncN Gal plasmid does not contain any portion of the known transfer loci of R46 [entry exclusion appears to be linked to transfer functions (Achtman et al., 1971)] it is likely that this decreasein transfer efficiency reflects another type of interaction between the two plasmids. Further work suggeststhat pMU603 has two to three times the copy number of pMU106a (P. Bird, unpublished results). The reason for this is not clear but we postulate that due to the increased copy number of pMU603 the frequency of establishment of R46 or N3 in JP777 harboring pMU603 is lower than expected, leading to an apparent decrease in transfer efficiency. This does not appear to affect the results when the IncN Gal plasmid is tested against R46 in the incompatibility grouping scheme described below. All the Gal plasmids were tested by conventional means for incompatibility with their parents. These tests were performed using the Gal plasmids as residents in JP777 and the parent plasmids were introduced by conjugation, selecting for the incoming plasmid alone. Each Gal plasmid was incompatible with its parent, but pMU602 no longer displayed the secondary, weak IncP and IncIy functions of pMU700 (data not shown). Use of the Gal plasmids in a rapid incompatibility grouping scheme. JP777 strains har-
bomg the Gal plasmids were used as recipients in plate matings with a panel of rifampicinsensitive donor strains each containing a reference R plasmid (listed under Materials and Methods). These recipient straints were grown in galactose-minimal medium prior to the matings to minimize the effectsof Gal plasmid instability. The optimum mating time in most caseswas determined to be between 4 and 8 h at 37°C. Transfer of temperature-sensitive plasmids such as R27 or Rtsl was allowed to
239
occur at 30°C for 8 h. After mating on nonselective plates transconjugants were selected on T medium containing rifampicin and antibiotics to which the incoming plasmids specified resistance. Where the two plasmids were incompatible the transconjugant patch was colored red, usually after overnight incubation at the appropriate temperature (Fig. 2). The efficiency of tetrazolium asan indicator has been observed to decreasein situations of high cell density (Miller, 1972). Thus the intensity of color of the Gal- transconjugant patchesvaried with the number of cells making up the patch, so that a Gal- patch appeared Gal+ when it consisted of a confluent, heavy growth of cells. This was overcome by replica plating the mating plate to two successiveselective plates, discarding the velvet, and finally replica plating the second selective plate to a third. The result was then read from a transconjugant patch showing light, nonconfluent growth. It should be noted that under no circumstances were false Gal- results observed. With the exception of pMU60 1 (Inc9), each Gal plasmid was incompatible only with plasmids of the group to which its parent belonged. As well as being incompatible with pIP7 1, pMU60 1 was incompatible with R 1drd 16 or Rldrd19 (IncFII), but was compatible with RlOOdrdl (In&II). In conventional tests pMU601 was found to be incompatible with R ldrdl6 and Rldrdl9, but was compatible with RlOOdrdl and R6-5. On the other hand, pMU600 (derived from R6-5) was compatible with pIP71. On the basis of these results it would appear that the R 1 derivatives are atypical FII plasmids, although more work (such as the construction of a Gal plasmid derived from Rl) is necessaryto confirm this. Alternatively, this interaction between the Rl derivatives and pMU60 1 may be a consequence of the secondary replication functions associated with the kanamycin resistance determinant of Rldrdl9 (Clerget et al., 1982). DISCUSSION
The problems with overlapping markers and entry exclusion in incompatibility testing have led to the use of several plasmids within
240
DAVEY
ET AL.
MINI-Gal PLASMIDS IN RAPID Inc TESTING
each incompatibility group as exemplars. However, work done on I complex and IncFI plasmids indicates that plasmids belonging to a particular incompatibility group may show heterogeneous interactions and that one particular plasmid may not be representative of the whole group (Hedges and Datta, 1973; Bird and Pittard, 1982; Bergquist et al., 1982). We have attempted to overcome such difficulties by constructing reference plasmids in vitro which do not code for antibiotic resistance or entry exclusion functions, and which have defined replication and incompatibility loci. Where detailed physical and genetic maps do not exist for a particular plasmid, we have adopted a “shotgun” approach to the-isolation of its replication and incompatibility regions, on the assumption that these functions are linked on the plasmid genome. For a plasmid such as RR2 (Thomas et al., 1980) where the regions essential for replication are scattered around the genome, this approach would probably fail, and if so, sutiable maps would have to be derived. Secondary replication or incompatibility functions are also unlikely to be isolated using the “shotgun” method. Indeed pMU602 lacks the weaker IncP and IncIy functions of its parent pMU700, and it appearsthat all the Gal plasmids lack stability (perhaps partitioning) functions. The use of Gal as a marker on these miniplasmids has led to the development of a rapid incompatibility test in which loss of the Gal plasmid from a strain is easily monitored without the need for extensive subculturing and replica plating. In its final form the test will allow each unknown plasmid to be tested againsta large panel of referenceGal plasmids. The stability of those Gal plasmids in the re-
241
cipient strains would be checked by replica plating the recipient patches to T medium just before the mating; and the inclusion of JP777 as a recipient would act as a control on both the transfer of the unknown plasmid and the indicator in the selective medium. It should be noted that our incompatibility testing method differs from the conventional in that although selection is made for the incoming plasmid alone, there is no subsequent repetition of the test following the reciprocal cross.This is usually done in casethe plasmids display unilateral incompatibility, which can be explained by instability of the unselected plasmid in one cross or by the presence of secondary replication functions on either plasmid. Given our intention to construct referenceplasmids having unique replication and incompatibility functions, and the control on the Gal plasmids’ stability in the test we have outlined, we do not consider the reciprocal test to be necessary.Nevertheless an incompatible interaction could be confirmed by transforming JP777 harboring the unknown plasmid with DNA of the appropriate Gal plasmid, but it would not be practical to do this where compatible results are evident. We are presently constructing Gal plasmids derived from IncP, IncA, IncT, IncX, IncQ, IncIz, and IncW reference plasmids, which will be described in detail later. We envisage the eventual construction of a panel of reference Gal plasmids comprising exemplars of the major incompatibility groups, which will facilitate plasmid classification, particularly in diagnostic laboratories. Such reference plasmids may also facilitate the clarification of anomalies in the existing incompatibility grouping scheme.
FIG. 2. Representative plates from a typical rapid incompatibility test showing transconjugant patches on galactose-tetrazolium (T) indicator medium. An incompatible result is shown by dark (Gal-) patches while a compatible result is shown by light (Gal+) patches. In this experiment the recipient was JP777/ pMU602 (IncB). Top: dark patches represent incompatible interactions with R724 (upper right) and RI6 (lower left). Light patchesrepresentcompatible interactions with R46, R386, pIP7 1, R62 1a, RP4, R64drdl1, N-3, pMU407.1, R446b, R478, RA 1, R1460. Bottom: dark patches represent incompatible interactions with TPll3 (left) and R805a (right). Light patchesrepresent compatible interactions with R144drd3, TP114, RP4, pMU407.1, R478, TPl16, R1460.
DAVEY ET AL.
242 ACKNOWLEDGMENTS
We wish to thank E. Lederbergof the Plasmid Reference Center for providing most of the reference plasmids used in this study, and P. Bergquist for PB1906 and X pgal8. We acknowledge the technical assistanceof Y. Jackson and L. Vizard. This project is supported by the National Health and Medical ResearchCouncil. P.I.B. and S.M.N. are recipients of Commonwealth Postgraduate Research Awards.
DE LA CRUZ,F., ZABALA,J. C., AND ORTIZ,J. M. (1979). Incompatibility among cr-hemolytic plasmids studied after inactivation of the cY-hemolysingene by transposition of Tn802. Plasmid 2, 507-S 19. FEISS,M., ADHYA, S., AND COURT,D. L. (1972). Isolation of plaque-forming galactose-transducingstrains of phage lambda. Genetics 71, 189-20 1. GRANT, A. J., BIRD, P. I., AND PITTARD,J. (1980). Naturally occurring plasmidsexhibiting incompatibility with members of incompatibility groups I and P. J. Bacterial. 144, 758-765.
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BERCQUIST,P. L., LANE, H. E. D., MALCOLM, L., AND MWNARD, R. A. (1982). Molecular homology and incompatibility in the IncFI plasmid group. J. Gen. Microbiol. 128, 223-238.
BIRD, P. I., AND PITTARD,J. (1982). An unexpected incompatibility interaction between two plasmids of the I compatibility complex. Plasmid 8, 2 11-2 14. BIRD, P. I., AND PITTARD, J. (1983). Demonstration of a third incompatibility function on plasmids already incompatible with group P and group I plasmids. Plasmid 9, 191-200.
BIRNBOIM,H. C., AND DOLY, J. (1979). A rapid alkaline extraction procedure for screeningrecombinant plasmid DNA. Nucl. Acids Res. 1, 1513-1523. BROWN,A. M. C., ANDWILLE-IX, N. S.(1981).A physical and genetic map of the IncN plasmid R46. Plasmid 5, 188-201. BUSBY,S., IRANI, M., AND DECROMBRUCGHE, B. (1982). Isolation of mutant promoters in the Escherichia co/i galactose operon using local mutagenesis on cloned DNA fragments. J. Mol. Biol. 154, 197-209. CLERGET,M., CHANDLER,M., AND CARO, L. (1982). Isolation of the kanamycin resistanceregion (Tn2350) of plasmid Rldrd19 as an autonomous replicon. J. Bacterial. 151, 924-93 1. DATTA, N. (1979). Plasmid classification: Incompatibility grouping. In “Plasmids of Medical, Environmental, and Commercial Importance” (K. N. Timmis and A. Piihler, eds.), pp. 3-12. Elsevier/North-Holland Biomedical Press,Amsterdam. DAVEY, R. B., AND PITTARD, J. (1977). Plasmids mediating resistance to gentamicin and other antibiotics in Enterobacteriaceae from four hospitals in Melbourne. Aust. J. Exp. Biol. Med. Sci. 55, 299-307.
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GRANT, A. J., AND PITTARD,J. (1974). Incompatibility reactions of R-plasmids isolated from Escherichia coli of animal origin. J. Bacterial. 120, 185-190. HEDGES,R. W., AND DATTA, N. (1973). Plasmids determining I pili constitute a compatibility complex. J. Gen. Microbial.
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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, N. Y. KUSHNER,S. R. (1978). An improved method for transformation of Escherichia coli with ColEl derived plasmids. In “Genetic Engineering” (H. W. Boyer and S. Nicosia, eds.), pp. 17-23. Elsevier/North-Holland Biomedical Press,Amsterdam. Low, K. B. (1973). Rapid mapping of conditional and auxotrophic mutations in E. coli K-12. J. Bacterial. 113,798-812.
MILLER, J. H. (1972). “Experiments in Molecular Genetics.” Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. NOVICK, R. P. , CLOWES,R. C., COHEN,S. N., CURTIS& R., III, DATTA, N., AND FALKOW,S. (1976). Uniform nomenclature for bacterial plasmids: A proposal. Bacteriol. Rev. 40, 168-189. 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., ANDCHABBERT,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. 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 reductase in Escherichia coli. J. Biol. Chem. 257, 9043-9048.
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11, 234-242 (1984)
The Use of Mini-Gal Plasmids for Rapid Incompatibility Grouping of Conjugative R Plasmids R. BRENT DAVEY,' PHILLIP I. BIRD, SUZANNE M. NIKOLETTI, JUDYTA PRASZKIER,ANDJAMES PITTARD~ Department of Microbiology, University of Melbourne, Paddle,
Victoria 3052, Australia
ReceivedNovember9, 1983; revised January 10, 1984 The galactose operon of Escherichia coli K-12 has been used as a phenotypic marker for miniplasmids derived in vitro from R plasmids representing six incompatibility groups. This has enabled the development of a rapid incompatibility typing scheme in which the miniplasmids are used as incompatibility exemplars, their presence in strains being monitored on galactose fermentation indicator media.
Incompatibility between two plasmids is shown by their inability to coexist stably in the same cell, in the absence of selective pressure (Novick et al., 1976). All plasmids studied to date exhibit incompatibility and this has been used as the basis of a classification system, on the assumption that naturally occurring incompatible plasmids derive from a common ancestor (Datta, 1979). The usefulness of incompatibility in typing particular plasmids may be limited by several factors. These include the presence of overlapping or identical markers on the test and reference plasmids, difficulty in establishing the test and reference plasmids in the same cell due to entry exclusion, the existence of more than one incompatibility function on either plasmid, and the time and amount of media required to perform a test. For these reasons incompatibility testing is not widely used in clinical microbiology laboratories as a routine epidemiological tool. In this paper we describe the development of a system designed to overcome most of the practical difficulties encountered in incompatibility testing. We have constructed a series ’ Current address: Fairheld Hospital, Yarra Rend Road, Fairfield, Victoria 3078, Australia. ’ To whomreprint requests and general inquiries should
be addressed. 0147-619X/84
$3.00
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form resewed.
of miniplasmids (representing six major incompatibility groups) which consist of the replication and incompatibility genes of particular plasmids linked to the genes of the galactose operon. These plasmids no longer determine resistance to antibiotics or code for entry exclusion functions. The presence or absence of these mini-Gal plasmids in a strain is easily monitored by the color of colonies on suitable indicator media. Incompatibility is indicated by the appearance of Gall transconjugants following transfer of an appropriate conjugative R plasmid into a strain containing a Gal plasmid. MATERIALS
AND METHODS
Bacterial strains and plasmids. The strains of Escherichia coli K12 used in this study, as well as plasmids other than incompatibility exemplars, are listed in Table 1. The plasmids used as incompatibility standards were RA 1, R686, R40a (A = C = E); R16, TPI 13, R724 (B = 0); R386 (FI); Rldrd19, Rldrd16, RlOOdrdl (FII); pSU104 (FVI); R124 (FIV); pIE509 (FV); TP116, TP124, R27 (Hl); R478, pHH1058a (H2 = S); R64drdl1, R648, R483 (I,); R621a (1~); TP114 (I& R721 (16); R805a (In; R391 (J); R387 (K); R47la, R446b (L = M); R46 (N); RP4 (P); Rtsl, R394 (T); R1460, pIE420 (U); S-a, RA3 (W); R6K (X); pIP231a (Y); pIP71 (9); and pIE545 (Z).
234
235
MINI-Gal PLASMIDS IN RAPID Inc TESTING TABLE 1 BACTERIALSTRAINSANDPLASMIDS
Designation strain/plasmid
Source or reference
Characteristics”
KA56
HfrH, galE45, reMI, spoTI, thil, X-
A. Rorsch
CA13
HfrH, galTl18, relA1, spoT1, h-
B. Bachmann
JP777
thrl, leul, lacY1, gal35 1, supE44, tonA2, hsdR4, rpoB364
Spontaneous 2deoxygalactose- and rifampicin-resistant derivative of JFM43 (Smith et al., 1982)
JP798
F-, ga1354,polA 1, gyrA, thyA
Spontaneous 2deoxygalactose and nalidixic acid-resistant derivative of PB1906 (P. Bergquist)
argE3, his4, ilvC7, proA2, thil, supE44, gala, X-, gyrA368
(Grant et al., 1980)
pBR322
Ap Tc IncColE
(Rodriguez et al., 1976)
pMU407. I
Ap,6 Cm, Km, Gm, Sm, Sp, Su, Tc IncL/M
(Davey and Pittard, 1977)
pMU700
Sm, Su, IncB/O, IncIy, IncP
(Grant et al., 1980)
R64drdll
Sm, Tc IncI,
(Jacob et al., 1977)
pIP7 1
Ap, Cm, Sm, Su, Tc Inc9
(Jacob et al., 1977)
R46
Ap (OXA-2), Sm, Sp, Su, Tc IncN
(Jacob et al., 1977)
R6-5
Cm, Km, Nm, Sm, Su IncFII
(Jacob et al., 1977)
pMU106a
Ap IncN
Derived from R46 (P. Bird)
pRBD13
Tc, Gal IncColE
This study
pMU600
Gal IncFII
This study-derived from R6-5
pMU60 1
Gal Inc9
This study-derived from pIP7 1
pMU602
Gal Inc B/O
This study-derived from pMU700
pMU603
Gal IncN
This study-derived from pMU 106a
pMU604
Gal IncL/M
This study-derived from pMU407. I
pMU605
Gal IncI,
This study-derived from R64drdll
’ Abbreviations used: Ap, ampicillin resistance;Cm, chloramphenicol resistance;Gm, gentamicin resistance; Km, kanamycin resistance;Nm, neomycin resistance; Sp, spectinomycin resistance; Sm, streptomycin resistance; Su, sulfathiazole resistance;Tc, tetracycline resistance; Gal, galactose fermentation. b Unless specified ampicillin resistance refers to the high-level TEM type.
These have been described by Jacob et al. ( 1977) except pSU104 (De La Cruz et al., 1979), pIP231a (Roussel and Chabbert, 1978), pHH1058a, R1460, pIE545, pIE509, and pIE420 (E. Lederberg, personal communication). The status of the incompatibility groups is according to the Plasmid Reference
Center (E. Lederberg, personal communication). Antibiotics, chemicals, and media. Most of the chemicals and media used in this study have been described previously (Grant and Pittard, 1974; Grant et al., 1980). Antibiotics were used at the following concentrations:
236
DAVEY -El
ampicillin (25 clglml), tetracycline ( 15 &ml), chloramphenicol (10 pg/ml), kanamycin (20 pg/ml), streptomycin (5 pg/ml), spectinomycin ( 15 pg/ml), sulfathiazole ( 125 pg/ml), trimethoprim (10 pg/ml), gentamicin (10 pg/ ml), neomycin (20 pg/ml), rifampicin (100 dml). Galactose-tetrazolium indicator (T)3 agar was prepared as follows: a base medium was prepared containing 0.9% (w/v) tryptone (Oxoid L42); 0.25% (w/v) yeast extract (Oxoid L2 1); 0.125% (w/v) Lab Lemco (Oxoid L29); 2.5 IIIM MgC12in 15 mM Mops3 pH 7.0; 2.2% (w/v) agar (Oxoid No 1, Ll 1) was added to half the base medium. After sterilization of the basemedium 1%(v/v) galactose [substantially glucose-free, Sigma Chemical Co.], appropriate antibiotics and triphenyltetrazolium chloride (Sigma, 25 &ml) were added aseptically from stock solutions to the half without agar. Both halves of the medium were then combined, mixed thoroughly, and used to pour plates. On this medium Gal+ colonies were white, and Gal- colonies red (Miller, 1972). Galactose-bromocresol purple/bromothymol blue indicator (B) plates were prepared similarly with the exception that the basemedium contained 5 mrvr Mops. The indicator usedwas a combination of bromocresol purple (55 cLg/ml) and bromothymol blue (17.5 pg/ ml). On this medium Gal+ colonies were yellow and Gal- colonies blue. Conjugation and incompatibility testing. Unless specifically mentioned, conjugations and incompatibility testing were performed as describedpreviously (Grant et al., 1980). Plate matings were performed using the technique of Low (1973). Recombinant DNA techniques. Purified plasmid DNA and bacteriophageDNA for use in cloning procedures were isolated as described previously (Bird and Pittard, 1983; Davis et al., 1980). Otherwise DNA was prepared according to the method of Bimboim
‘* AL.
and Doly (1979). Methods for the use of restriction endonucleases,T4 DNA ligase, and agarosegel electrophoresishave been described by Davis et al. (1980). Enzymes were purchased from Boehringer-Mannheim, Amersham, or BethesdaResearchLaboratories, and used in the buffers prescribed by the manufacturer. Transformation was performed by the method of Kushner (1978). RESULTS
Cloning of DNA containing the gal operon. The initial source of gal operon DNA used in the construction of the Gal plasmids was Xpgal8 (Feisset al., 1972). To generate a more convenient source of gal operon DNA it was decided to clone the gal genes into pBR322. Since no physical map of Xpgal8 was available, a “shotgun” approach to cloning the gal operon from the phage to pBR322 was taken. PhageDNA was digestedwith PstI and ligated to P&cleaved pBR322 DNA, and the mixture was used to transform JP777 to tetracycline resistanceon B medium. Gal+ colonies arising from this transformation were screened for ampicillin sensitivity. The plasmid content of ampicillin-sensitive, Gal+ clones was determined by restriction analysis of appropriate DNA preparations. A tetracycline-resistant, ampicillin-sensitive, Gal+ clone containing a small plasmid (pRBD 13), which had an extra 5-kb PstI-generatedrestriction fragment when compared to pBR322, was chosen for further characterization. DNA was prepared from a strain containing pRBD13 and used to transform strains carrying various mutations in their gal genes. The plasmid could complement mutations in the galK (JP990), galE (KA56), and galT (CAM3) genes. It also complemented a Gal deficiency in a derivative of KA56 with a deletion spanning hatt, gal, aroG, and nadA. These results indicate that the entire gal operon is carried by pRBD 13. The absenceof a functional DNA pokindependent replication system on the 5-kb Gal 3Abbreviations used:T, galactose-tetrazolium indicator; fragment was indicated by the failure of Mops, 4-morpholinepropanesulfonic acid; B, galactosepRBD13 to transform a polA strain (JP798). bromocresol purple/bromothymol blue.
237
MINI-Gal PLASMIDS IN RAPID Inc TESTING
The location and orientation of the gal operon on pRBDl3 as shown in Fig. 1 was deduced from previously published data (Busby et al., 1982). This plasmid was used as the source of Gal marker DNA for subsequent plasmid constructions. Construction of mini referenceplasmids currying the gal operon. The construction of the IncB, In&II, IncI, IncM, and Inc9 Gal plasmids (derived from pMU700, R6-5, R64drdl1, pMU407.1, and pIP71, respectively) was performed by ligating parental plasmid DNA partially cleaved with PstI to &I- and BumHI-cleaved pRBDl3 DNA.
(pRBDl3 was also cleaved with BamHI to lower the frequency at which recircularized pRBD 13 molecules were obtained in the experiment, as BarnHI cleavesthe pBR322 portion of pRBDl3 but not the Gal fragment.) The ligation mixtures were then used to transform KA56 (galE) to Gal+. Strains carrying a galE mutation are sensitive to galactose so Gal+ transformants could be selected on B medium without encountering problems with background growth. The Gal+ clones resulting from each ligation were checked for tetracycline sensitivity, and in each case plasmid DNA from about
Hc Hc H
P
pRBD13
9.3kb
pMU600
7.7kb
pMU601
7.1 kb
pMU602
8.4 kb
pMU603
9.2kb
pMU604
7.1 kb
pMU605
9.8kb
*----K--T--EGAL H
P Bg
P I
SP
*--REP----INCS
*--REP--INC H
P
s
Ba PP
PS
-----REP----lNCIi
P
Ba
P I
Bg *--REP--* ‘--,NC--‘
H
PPP
P
‘---REPTh?CC
H
P
E
P I
-
PS
e-----REP-I
1 kb
PP
Bg ____
-+
-lNC-
FIG. 1. Maps of the Gal plasmids used in this study. The plasmids are represented as linear molecules opened at one PstI site, which is depicted at the right-hand end of each map. Broken arrows indicate loci that have not been fully delineated. The source replicon for each of the Gal plasmids is as follows: pMU600 from R6-5 (In&B); pMU601 from pIP7 1 (In&Q pMU602 from pMU700 (IncB/O); pMU603 from pMU 106a (IncN); pMU604 from pMU407.1 (IncL/M); and pMU605 from R64drdll (Incl,). Abbreviations used: REP, replication region; INC, incompatibility region; GAL, galactose operon; Ba, BarnHI; Bg, BglII; E, EcoRI; Hc, HincII; H, HindIII; Hp, HpaI; P, &I; Pv, PvuII; S, SalI; Ss, SstII. Not all these restriction endonucleaseswere used to map each plasmid, and with the exception of HindIII, the recognition sites in the pBR322derived portion of pRBD13 are not shown.
238
DAVEY
20 Gal+, tetracycline-sensitive clones was screened after digestion with PstI. A variety of plasmid sizes (from approximately 50 to 10 kb) was observed. On the assumption that the smaller the plasmid the lesslikely it would be to retain parental entry exclusion or antibiotic resistance functions, DNA from several clones harboring small plasmid species (lessthan 20 kb) was used to transform JP777 and JP798 to Gal’. The most stable, DNA p&-independent plasmids derived from each of the parent plasmids were chosen for further characterization. The IncN Gal plasmid (pMU603) was derived from R46, which has been mapped and characterized (Brown and Willetts, 1981). In the course of the construction of cloning vectors derived from R46, an intermediate plasmid, pMU 106a, was made. This consists of a 2.2-kb portion of the BgZE (replication) fragment and 0.5 kb ofthe BglD fragment of R46, linked to a 1.5-kb portion of Tn.? coding for ampicillin resistance(P. Bird, unpublished results). The plasmid pMU106a has a unique PstI site within the ampicillin resistancegene, so a Gal+, ampicillin-sensitive derivative of pMU106a was constructed by ligating PstIcleaved pMU 106a DNA to PstI- and BamHIcleaved pBRDl3 DNA and transforming IL456 to Gal+. Plasmid DNA from ampicillinsensitive Gal+ clones was analyzed, and DNA from clones giving the predicted restriction pattern after cleavage with PstI was used to transform JP798 and JP777. Properties of the Gal plasmids. Maps of the Gal plasmids are shown in Fig. 1. With the exception of pRBD 13, each plasmid determines DNA polLindependent replication functions as judged by their ability to successfully transform JP798 to Gal+. The approximate locations of the replication and incompatibility regions of pMU60 1 (Inc9), pMU602 (IncB), pMU604 (IncM), and pMU605 (IncI) were deduced in the following fashion. In each case,Gal plasmid DNA partially cleaved with PstI was ligated to PstIcleaved pBR322 DNA and used to transform JP777 harboring the corresponding Gal plasmid to tetracycline resistance on T medium.
ET AL.
Those clones that received a pBR322 derivative not carrying the Gal fragment but which expressedincompatibility against the resident Gal plasmid were Gall on T medium. Both Gall and Gal+ clones arising from the transformation were screened for ampicillin sensitivity and plasmid DNA from such ampicillin-sensitive clones was screened after digestion with PstI. In this way a number of clones containing pBR322 derivatives with one or more extra Pst fragments was obtained from each subcloning experiment. The locations of the incompatibility and replication loci of each Gal plasmid were assigned by testing its pBR322 derivatives for the ability to express incompatibility against the corresponding Gal plasmid (as described above), and for the ability to transform and hence to replicate in a polA strain. In all cases the locus involved in the expression of incompatibility could be assigned to a single PstI fragment, while the locus involved in DNA PO/I-independent replication spanned several PstI fragments, including the fragment containing the incompatibility locus (Fig. 1). The locations of the replication and incompatibility regions of the IncFII and IncN Gal plasmids are according to maps of the parent plasmids. All the DNApolI-independent Gal plasmids are unstable,judging by the percentageof Gall clones arising from an overnight culture (in nonselective broth) of JP777 harboring the appropriate Gal plasmid. The degree of instability observed-IncB (2%), IncFII (16%), IncI (lo%), IncM (7%), IncN ( l%), Inc9 (0.1%)-did not complicate the interpretation of the results when the plasmids were used in the incompatibility testing scheme described below. None of the Gal plasmids carried any functional antibiotic resistance determinant in comparison to their parents. None were found to be conjugative, and, with the exception of the IncN plasmid, none appeared to exhibit entry exclusion against the parental plasmids. Entry exclusion was tested by comparing the conjugational transfer frequency of a reference plasmid into JP777 harboring its derivative
MINI-Gal PLASMIDS IN RAPID Inc TESTING
Gal plasmid, to its transfer frequency into JP777 alone. Entry exclusion is indicated when a significant decreaseis observed in the former frequency compared to the latter. The transfer frequency of R46 or N3 into JP777 harboring the IncN Gal plasmid was lo- to loo-fold lessthan the corresponding frequency of transfer to JP777 alone. Since the IncN Gal plasmid does not contain any portion of the known transfer loci of R46 [entry exclusion appears to be linked to transfer functions (Achtman et al., 1971)] it is likely that this decreasein transfer efficiency reflects another type of interaction between the two plasmids. Further work suggeststhat pMU603 has two to three times the copy number of pMU106a (P. Bird, unpublished results). The reason for this is not clear but we postulate that due to the increased copy number of pMU603 the frequency of establishment of R46 or N3 in JP777 harboring pMU603 is lower than expected, leading to an apparent decrease in transfer efficiency. This does not appear to affect the results when the IncN Gal plasmid is tested against R46 in the incompatibility grouping scheme described below. All the Gal plasmids were tested by conventional means for incompatibility with their parents. These tests were performed using the Gal plasmids as residents in JP777 and the parent plasmids were introduced by conjugation, selecting for the incoming plasmid alone. Each Gal plasmid was incompatible with its parent, but pMU602 no longer displayed the secondary, weak IncP and IncIy functions of pMU700 (data not shown). Use of the Gal plasmids in a rapid incompatibility grouping scheme. JP777 strains har-
bomg the Gal plasmids were used as recipients in plate matings with a panel of rifampicinsensitive donor strains each containing a reference R plasmid (listed under Materials and Methods). These recipient straints were grown in galactose-minimal medium prior to the matings to minimize the effectsof Gal plasmid instability. The optimum mating time in most caseswas determined to be between 4 and 8 h at 37°C. Transfer of temperature-sensitive plasmids such as R27 or Rtsl was allowed to
239
occur at 30°C for 8 h. After mating on nonselective plates transconjugants were selected on T medium containing rifampicin and antibiotics to which the incoming plasmids specified resistance. Where the two plasmids were incompatible the transconjugant patch was colored red, usually after overnight incubation at the appropriate temperature (Fig. 2). The efficiency of tetrazolium asan indicator has been observed to decreasein situations of high cell density (Miller, 1972). Thus the intensity of color of the Gal- transconjugant patchesvaried with the number of cells making up the patch, so that a Gal- patch appeared Gal+ when it consisted of a confluent, heavy growth of cells. This was overcome by replica plating the mating plate to two successiveselective plates, discarding the velvet, and finally replica plating the second selective plate to a third. The result was then read from a transconjugant patch showing light, nonconfluent growth. It should be noted that under no circumstances were false Gal- results observed. With the exception of pMU60 1 (Inc9), each Gal plasmid was incompatible only with plasmids of the group to which its parent belonged. As well as being incompatible with pIP7 1, pMU60 1 was incompatible with R 1drd 16 or Rldrd19 (IncFII), but was compatible with RlOOdrdl (In&II). In conventional tests pMU601 was found to be incompatible with R ldrdl6 and Rldrdl9, but was compatible with RlOOdrdl and R6-5. On the other hand, pMU600 (derived from R6-5) was compatible with pIP71. On the basis of these results it would appear that the R 1 derivatives are atypical FII plasmids, although more work (such as the construction of a Gal plasmid derived from Rl) is necessaryto confirm this. Alternatively, this interaction between the Rl derivatives and pMU60 1 may be a consequence of the secondary replication functions associated with the kanamycin resistance determinant of Rldrdl9 (Clerget et al., 1982). DISCUSSION
The problems with overlapping markers and entry exclusion in incompatibility testing have led to the use of several plasmids within
240
DAVEY
ET AL.
MINI-Gal PLASMIDS IN RAPID Inc TESTING
each incompatibility group as exemplars. However, work done on I complex and IncFI plasmids indicates that plasmids belonging to a particular incompatibility group may show heterogeneous interactions and that one particular plasmid may not be representative of the whole group (Hedges and Datta, 1973; Bird and Pittard, 1982; Bergquist et al., 1982). We have attempted to overcome such difficulties by constructing reference plasmids in vitro which do not code for antibiotic resistance or entry exclusion functions, and which have defined replication and incompatibility loci. Where detailed physical and genetic maps do not exist for a particular plasmid, we have adopted a “shotgun” approach to the-isolation of its replication and incompatibility regions, on the assumption that these functions are linked on the plasmid genome. For a plasmid such as RR2 (Thomas et al., 1980) where the regions essential for replication are scattered around the genome, this approach would probably fail, and if so, sutiable maps would have to be derived. Secondary replication or incompatibility functions are also unlikely to be isolated using the “shotgun” method. Indeed pMU602 lacks the weaker IncP and IncIy functions of its parent pMU700, and it appearsthat all the Gal plasmids lack stability (perhaps partitioning) functions. The use of Gal as a marker on these miniplasmids has led to the development of a rapid incompatibility test in which loss of the Gal plasmid from a strain is easily monitored without the need for extensive subculturing and replica plating. In its final form the test will allow each unknown plasmid to be tested againsta large panel of referenceGal plasmids. The stability of those Gal plasmids in the re-
241
cipient strains would be checked by replica plating the recipient patches to T medium just before the mating; and the inclusion of JP777 as a recipient would act as a control on both the transfer of the unknown plasmid and the indicator in the selective medium. It should be noted that our incompatibility testing method differs from the conventional in that although selection is made for the incoming plasmid alone, there is no subsequent repetition of the test following the reciprocal cross.This is usually done in casethe plasmids display unilateral incompatibility, which can be explained by instability of the unselected plasmid in one cross or by the presence of secondary replication functions on either plasmid. Given our intention to construct referenceplasmids having unique replication and incompatibility functions, and the control on the Gal plasmids’ stability in the test we have outlined, we do not consider the reciprocal test to be necessary.Nevertheless an incompatible interaction could be confirmed by transforming JP777 harboring the unknown plasmid with DNA of the appropriate Gal plasmid, but it would not be practical to do this where compatible results are evident. We are presently constructing Gal plasmids derived from IncP, IncA, IncT, IncX, IncQ, IncIz, and IncW reference plasmids, which will be described in detail later. We envisage the eventual construction of a panel of reference Gal plasmids comprising exemplars of the major incompatibility groups, which will facilitate plasmid classification, particularly in diagnostic laboratories. Such reference plasmids may also facilitate the clarification of anomalies in the existing incompatibility grouping scheme.
FIG. 2. Representative plates from a typical rapid incompatibility test showing transconjugant patches on galactose-tetrazolium (T) indicator medium. An incompatible result is shown by dark (Gal-) patches while a compatible result is shown by light (Gal+) patches. In this experiment the recipient was JP777/ pMU602 (IncB). Top: dark patches represent incompatible interactions with R724 (upper right) and RI6 (lower left). Light patchesrepresentcompatible interactions with R46, R386, pIP7 1, R62 1a, RP4, R64drdl1, N-3, pMU407.1, R446b, R478, RA 1, R1460. Bottom: dark patches represent incompatible interactions with TPll3 (left) and R805a (right). Light patchesrepresent compatible interactions with R144drd3, TP114, RP4, pMU407.1, R478, TPl16, R1460.
DAVEY ET AL.
242 ACKNOWLEDGMENTS
We wish to thank E. Lederbergof the Plasmid Reference Center for providing most of the reference plasmids used in this study, and P. Bergquist for PB1906 and X pgal8. We acknowledge the technical assistanceof Y. Jackson and L. Vizard. This project is supported by the National Health and Medical ResearchCouncil. P.I.B. and S.M.N. are recipients of Commonwealth Postgraduate Research Awards.
DE LA CRUZ,F., ZABALA,J. C., AND ORTIZ,J. M. (1979). Incompatibility among cr-hemolytic plasmids studied after inactivation of the cY-hemolysingene by transposition of Tn802. Plasmid 2, 507-S 19. FEISS,M., ADHYA, S., AND COURT,D. L. (1972). Isolation of plaque-forming galactose-transducingstrains of phage lambda. Genetics 71, 189-20 1. GRANT, A. J., BIRD, P. I., AND PITTARD,J. (1980). Naturally occurring plasmidsexhibiting incompatibility with members of incompatibility groups I and P. J. Bacterial. 144, 758-765.
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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. Bacterial. 141, 2 13-222