Incompatibility group Y member relationships: pIP231 and plasmid prophages P1 and P7

PLASMID

8,

307-3 11 (1982)

SHORT COMMUNICATION Incompatibility Group Y Member Relationships: plP231 and Plasmid Prophages PI and P7 MIKE A. CAPAGE, JAMES K. GOODSPEED, AND JUNE R. SCOTT Department

of Microbiology

and Immunology,

Emory University

School of Medicine, Atlanta, Georgia 30322

Received November IO, 1981; revised July 6, 1982 The plasmid pIP231 exhibits stronger incompatibility with both plasmid prophages Pl and P7 than Pl and P7 exhibit with each other. DNA-DNA hybridization experiments showed that pIP23 1 is strongly homologous with both PI and P7 in the central region of the prophage genomeswhich contains genesdetermining replication, incompatibility, and maintenance functions. In addition, a region on the left of P7 which contains Tn902 showed some homology with pIP23 1, as did the region of Pl carrying El.

The 60-kb Escherichia coli plasmid, pIP23 1, carries determinants for tetracycline resistance and hydrogen sulfide production and is classified as a member of the incompatibility group Y (2). This group also includes pl5B and the related plasmid prophages Pl and P7 (3). Twenty-two percent of the pIP231 genome is homologous with about 15% of the Pl chromosome (1). Two loci (IncA and IncB) located near the middle of the Pl map (Fig. 2) have been implicated in determination of incompatibility of plasmids Pl and P7 (4, 18). Other plasmids in the Y group should share homology with one or both of these regions. Marker rescue tests performed by phage infection indicate that pIP231 contains the wild-type allele of the two Pl amber mutations 135 and 180 (I) which are located near IncA and B. These observations suggested that the central region of the PI genome and the closely related phage P7, which shares 90% DNA homology with Pl (5), might be physically homologous with pIP23 1. We wished to test this hypothesis and to determine whether additional regions of homology exist among these plasmids. P 1 and P7 plasmid DNA was isolated from N99(Pl) and N99(P7) and purified by equilibrium centrifugation through ethidium brmide-CsCl gradients (6). For each plasmid, the fragments generated by digestion with

BamHI, BglII, and EcoRI were separated by electrophoresis on an 0.8% agarose gel (Fig. la). The fragments were denatured, neutralized, transferred to nitrocellulose paper, and hybrdized at 65°C for 18-24 h with denatured pIP231 DNA (8) labeled in vitro with 32P by nick translation (9). The limit of detectable homology in this system is less than 500 base pairs since the probe clearly hybridized to an EcoRI fragment of pIP23 1 of that size. Figure 1B is an autoradiogram of the Pl and P7 restriction fragments that hybridize to pIP23 1. Conclusions from this autoradiogram are presented in the map in Fig. 2. In all the digests, the restriction fragments from the center of the Pl chromosome show the most intense hybridization with the pIP23 1 probe. Because of the large number of EcoRI sites in this region, the EcoRI digest provides the most information about the extent of homology. PlEcoRI fragments 8, 15, and 18 hybridize with pIP23 1. Since PI EcoRI-5, which maps between these, is the same size as fragments 4 and 6, it is not clear which piece of Pl is responsible for hybridization in this region of the gel. Because there is no apparent hybridization with P 1BamHI2, it is unlikely that EcoRI-6, contained within this BamHI fragment, is responsible for the hybridization seen in this gel region. Absence of homology between pIP231 and

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0 147-619X/82/060307-05$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

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b

FIGS. la and b. Restriction digest patterns of PI and P7 prophage plasmids and hybridization of each restriction digest with pIP23 1. (a) Lanes 1, 2, and 3 are Pl DNA digested, respectively, with BumHI, BgBI, and EcoRI. Lanes 4, 5, and 6 are P7 DNA digested, respectively, with BumHI, BgllI, and EcoRI. Lane 7 is pIFl1 (20) cleaved with BgBI, and lane 8 is pIP23 1 cleaved with EcoRI. (b) Autoradiogram of the agarose gel shown in (a) after transfer of the restriction fragments to nitrocellulose paper and hybridization with pIP231 DNA labeled with 32P.The small plasmid pIFl1 (lane 7) was included as a negative control to be sure that the labeled pIP23 1 DNA was not hybridizing indiscriminately. Surprisingly, the smaller BgflI fragment of this plasmid hybridized with pIP23 1. This fragment contains a small portion of the F genome and a kanamycin resistance determinant from pSC 105 (20). We tested pIP23 1 for possible presence of kanamycin resistance but none was found. The hybridization may result from the presence of whole or partial insertion sequencescommon to both the smaller BgfiI fragment of pIFl1 and pIP23 1. The absence of hybridization with the larger BgflI fragment of pIF I 1 indicates that pIP23 1 is not hybridizing indiscriminately.

PlEcoRI-6 was confirmed by lack of homology of the probe with this fragment in an EcoRI digest of pMAC 102, a P 1 derivative carrying only the Bg/II-1 fragment (7). On the other hand, homology between the probe and P 1EcoRI-4 is likely since the overlapping PI fragments BgZII-2 and BamHI-3 hybridize with pIP23 1. This region of PI carries IS1 (II). The tetracycline resistance element of pIP23 1 (Tn 1523) forms cointegrate intermediates during transposition (2). Since cointegrate formation is often mediated by IS elements, it seemed possible that IS1 was responsible for the homology observed between pIP23 1 and this region of P 1. The pres-

ence of IS1 in pIP231 was confirmed by hybridizing it with an EcoRI digest of pMOB45 (21) which carries IS1 at its unique EcoRI site. Since PlEcoRI-4 shows homology with pIP23 1, we are unable to demonstrate directly whether P 1EcoRI-5 also hybridizes with this probe. The same general picture of strong DNA homology in the central region is observed with P7 (Figs. 1 and 2). In addition, homology is displayed by P7 BarnHI fragment 7 and/or 8 and by BglII-3 and/or 4 (both pairs run as doublets). The map location of BgiII4 overlaps BarnHI- and thus suggeststhat this left end region is responsible for the hy-

A -i

C

D

E

F,G

EcoRI

HindJII

BamHI

BglII

BamHI

BglII

FIG. 2. Restriction maps of PI and P7. This figure is a composite of one in Baumstark and Scott (14), Chesney et al. (16), and Cowan and Scott (4). The Pl restriction map is’ adapted from Bachi and Arber (IO) with additions based on information of Mural et al. (15) and Iida et al. (II). Digest fragments are identified by numbers assigned according to size with number 1 being the largest. The P7 restriction map is from Chesney et al. (16) and Cowan and Scott (4). The Tn902 transposon expressing ampicillin resistance is not present in PI. Shown above the Pl-EcoRl map are the locations of the incA and incB loci (19) and IS1 (II). The Pl amber mutations (15, 17) which were tested for marker rescue with pIP231 by Briaux et al. (I) are shown connected by vertical lines to the EcoRI fragment on which they are located. The large letters above the Pl map and below the P7 map identify the regions of nonhomology between the two phages and the invertible “C” segment; the lines underneath the letters indicate the approximate sizes of the regions of nonhomology (II, 4). Restriction fragments which hybridize with pIP23 1 are indicated as follows: ( m, ) for strong hybridization, (///////////// ) for weaker hybridization, (*) for uncertainty caused by the presence of other fragments of similar size, and no shading for no detectable hybridization. The Hind111 map of P7 (13) is included for discussion purposes although no hybridization was performed to fragments produced with this enzyme.

Pl

5 8 2

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bridization seen between P7 and pIP231. This was confirmed with EcoRI digests. Our EcoRI digest of P7 shows strong pIP231 hybridization with fragments 1 and 8 and weaker hybridization with fragments 7, 17, and 19. Since an EcoRI map of P7 is not yet available we cannot locate the position of all of these fragments on the P7 genome. However, we were able to deduce the location of EcoRI fragments 1 and 7 by performing double digestions with EcoRI and Hind111 or PstI since the Hind111 and PstI maps of P7 have been published (13). The electrophoretic mobility of EcoRI fragment 1 was altered in a double digestion of P7 with EcoRI and HindIII, so it must contain a Hind111 site. Of the three Hind111 sites in P7 (Fig. 2), only the one separating Hind111fragments 1 and 3 is overlapped by hybridizing fragments from both the BanzHI digest (fragment 3) and the BglII digest (fragment 1). Therefore, EcoRI fragment 1 is probably located within the region occupied by the junction of the Hind111 fragments 1 and 3. There are four PstI sites in P7 (13). Three of these sites are within Tn902, and the fourth is located within P7 BglII fragment 3. In a double digest of P7 with EcoRI and PstI, EcoRI fragment 7 disappeared while a new fragment with the same electrophoretic mobility as P7 PstI fragment 3, which is located within Tn902, was generated. This indicates that P7 EcoRI fragment 7 contains at least two of the three PstI sites located within Tn902. Thus EcoRI fragment 7, which hybridizes with pIP23 1, overlaps the Tn902 element. This result is consistent with our assignment of homology to BamHI fragment 7 and BgnI fragment 4 and suggeststhat pIP231 shares DNA homology with Tn902. This homology might reside in Tnl523, in the H2S determinant of pIP23 1 (1,2), or it might be elsewhere in the plasmid. Incompatibility has been extensively studied in group Y only between Pl and P7. Two incompatibility determinants have been defined (Fig. 2). IncA seems to be responsible for group-specific incompatibility while IncB is mostly plasmid specific (18). Furthermore, incompatibility between homologous plas-

mids (PI-PI or P7-P7) is much stronger than incompatibility between heterologous plasmids (Pl-P7) (4). When a homologous plasmid is introduced into a cell carrying P 1 or P7, colonies composed of cells carrying both plasmids cannot be isolated. Those colonies which grow on double selective medium at a very low frequency (I 1 X 1Om5)are recombinants in which one plasmid’s marker has transposed. When a heterologous PI or P7 plasmid is introduced, most cells retaining the incoming plasmid long enough to form a colony on selective medium also retain the resident. However, the two heterologous plasmids cannot be maintained together in the same cell and are eventually segregated to different daughter cells. Therefore, two PI or two P7 plasmids cannot be established in the same cell while Pl and P7 can be established together but cannot be comaintained (4). Thus, we wished to determine whether pIP23 1 behaves like either P 1 or P7 or shows a new “type specificity.” Using the procedure devised by Cowan and Scott (4) the recA strain N 100 carrying pIP23 1 was infected with Pl Ap, P lCm, P7Ap, and P7Cm in turn (Table 1). Following infection, the frequency of cells carrying markers of both plasmids is at least 4.5 x lo2 times lower in each case than the frequency of cells carrying only the incoming plasmid marker. The rare colonies with both plasmid markers occur at a frequency expected for transposition of one of the markers (4) and probably do not contain heteroplasmid cells. Thus, although both Pl and P7 can replace pIP231, neither plasmid can be established in a cell still retaining the resident. This behavior is in striking contrast to the Pl -P7 heteroplasmid experiments in which the incoming plasmid resides in the same cell with the resident at least long enough to produce a colony [Table 1 and (4)]. It thus appears that pIP231 exhibits stronger incompatibility with both PI and P7 than PI and P7 do with each other. From studies on P 1 and P7, only two loci have been implicated in determination of incompatibility. The strong incompatibility effect determined by IncB is plasmid specific, i.e., PI plasmids deleted for the other determinant,

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SHORT COMMUNICATION TABLE 1 INCOMPATIBILITYOF pIP23 1 WITH Pl AND P7 AND INCOMPATIBILITYOF PI WITH P7 Frequency” of resistant cells Line

Infecting phage

Resident plasmid

1 2 3 4 5 6 7

PlAP PlCm P7Ap P7Cm PlAp PlCm P7Ap P7Cm

pIP23 1 pIP23 1 pIP23 1 pIP23 1 P7Cm P7Ap PlCm PlAp

8

Incoming plasmid 2.5 x 3.6 X 1.6 X 9.4 x 4.5 x 1.8 x 1.8 X 2.4 x

lo-* 1O-4 10-l 1o-2 1o-2 1om3 10-l 1O-2

Both 5.5 x 13.7 x 6.7 X
1o-5 lo-’ 1O-6 10-6 lo-* 10-3 10-l 1O-2

Note. Log phase cells of the recA- strain N 100 carrying the indicated resident plasmid were infected with phage at a moi of 5. Free phage was removed by centrifugation, the cells were diluted at least IO-fold into prewarmed (37°C) LB medium containing 50 mM citrate to prevent further phage adsorption and incubated at 37°C for 15 min to allow expression of antibiotic resistance. The infected cells were plated on LB medium with or without 20 &ml of the indicated antibiotic(s). Citrate was added to all plates to prevent infection by free phage. The data showing incompatibility between PI and P7 (lines 5, 6, 7, and 8) are from Cowan and Scott (4). ’ Frequency = (viable cell titer on antibiotic medium)/(viable cell titer on nonselective medium).

IncA, show little incompatibility with P7. (The reciprocal experiment has not been done.) pIP23 1, however, shows strong incompatibility with both Pl and P7 although they do not show strong incompatibility with each other. This suggeststhat pIP23 1 has one or more Inc loci that is/are intermediate between that of P 1 and P7 and is/are recognized by both plasmids as being “self.” This could be either IncA, IncB, both, or an additional locus that has not yet been detected. Further experiments are required to test this.

4. COWAN,J., AND SCOTT,J. R., Plasmid 6,202-22 1 (1981). 5. YUN, T., AND VAPNEK, D., Virology 77, 376-385 (1977). 6. CAPAGE,M., AND HILL, C. W., J. Mol. Biol. 127, 73-87 (1979).

7. CAPAGE,M., AND SCOTT,J. R., unpublished. 8. SOUTHERN,E. M., J. Mol. Biol. 98,503-5 17(1975). 9. RIGBY, P. W., DIECKMANN, M., RHODES,C., AND BERG, P., J. Mol. Biol. 113, 237-251 (1977 10. BACHI, B., AND ARBER,W., Mol. Gen. Genet. 153, 31 l-324 (1977). II.

IIDA, S., MEYER, J., AND ARBER, W., Plasmid 1,

357-365 (1978). 12. WATKINS,C., AND SCOTT,J. R., Virology 110,302-

317 (1981).

ACKNOWLEDGMENTS We thank Kathleen Tatti for assistancewith Southern hybridizations, Susan Hollingshead for the gift of pMOB45 and June Harris for excellent secretarial assistance. This work was supported by Grant AI 17538 from the National Institute of Allergy and Infectious Disease.

13. IIDA, S., AND ARBER, W., Mol. Gen. Genet. 177,

261-270 (1979). 14. BAUMSTARK,B. R., AND SCOTT,J. R., J. Mol. Biol. 140,47 l-480 ( 1980).

15. MURAL, R. J., CHESNEY, R. H., VAPNEK, D., KROPF,M. M., AND SCOTT,J. R., Virology 93, 387-397 (1979). 16. CHESNEY,R. H., SCOTT,J. R., AND VAPNEK, D. J. Mol. Biol. 130, 161-173 (1979).

17. STERNBERG,N. C., Virology 96, 129-142 (1979). 18. STERNBERG, N., AND AUSTIN, S., Plasmid 5,20-3 1 1. BRIAUX, S., GERBAUD, G., AND J~FFE-BRACHET, (1981). A., Mol. Gen. Genet. 170, 3 19-325 (1979). 19. YARMOLINSKI,Y., submitted for publication. 2. BRIAUX-GERBAUD,S., GERBAUD,G., AND J&FE20. KAHN, M., FIRGURSKI,D., ITO, L., AND HELINSKI, BRACHET,A., Gene 15, 139-149 (1981). D. R., Cold Spring Harbor Symp. Quant. Biol. 3. HEDGES,R. W., JACOB,A. E., BARTH, P. T., AND 43,99-103 (1979). GRINTER, N. J. Mol. Gem Genet. 141, 263-267 21. BI~NER, M., AND VAPNEK, D., Gene 15,319-329 (1975). (1981).

REFERENCES