Identification of the P1 compatibility and plasmid maintenance locus by a mini P1 lac+-plasmid

96, 32-37 (1979)

VIROLOGY

Identification of the Pi Compatibility and Plasmid Maintenance Locus by a Mini Pl kc+-Plasmid AVIGDOR SHAFFERMAN, Israel

Institute

TANIA GELLER,’

for Biological Research, Ness-Ziona, Bar-Ilan

AND

ISRAEL HERTMAN”

and Department Ramat-Gan, Israel

University,

Accepted January

of Life Sciences

26, 1979

pIH1972 is a 23.GMdal lac+ plasmid derived from Pldlw. This plasmid is stably maintained and it exhibits absolute incompatibility with Pl in RecA bacteria. A cluster of Pl-EcoRI DNA fragments 15,17,18,21, and 23 and part of fragments 14 and 5 are identified in the Pl DNA region carried by pIH1972. It is suggested that Pl compatibility and plasmid maintenance genes are carried on the identified 10% segment of the Pl genome. INTRODUCTION

Pldlw , a defective derivative of Pl carrying the lac region, was isolated by Luria et al. (1960). We characterized Pldlw in stable single lysogens by marker rescue experiments and isolation of prophage DNA (Shafferman et al., 1978) and studied the excision of the lac region from Pldlw prophage. The excision of lac from Pldlw produces two Pl-immune Zac--segregant types. Type I probably carries the entire Pl genome and type II is deleted of genes 34, 35, and 36. In a different type of excision of lac from Pldlw a lac+ plasmid may be derived (Hertmanet al., 1972). These Zac+plasmids, pIH1972, derived in both Ret+ or RecA hosts, are identical in size, 23.6 * 1.0 Mdal, and show no rescue for all the Pl alleles tested (Shafferman et al., 1978). In the present communication we demonstrate the incompatibility of pIH1972 with Pl, and identify the “silent” Pl DNA region carried by this plasmid. Strains Escherichia coli K12 strains (Hertman and Scott, 1973; Shafferman et al., 1978). Pldlw and derivatives (Shafferman et al., ’ In partial fulfillment of the requirements for the Ph.D. thesis at Bar-Ilan University. * Author to whom reprint requests should be addressed. 0042-6822/79/090032-06$02.00/O Copyright All rights

8 19’79 by Academic Press, Inc. of reproduction in any form reserved.

32

1978). Superinfection by PlCm and transduction by HFT lysates were described previously (Hertman et al., 1972). Marker rescue experiments were carried out according to Chesney and Scott (1975). Gel electrophoresis separation of DNA fragments was carried out in agarose 1.2% slabs (Green et al., 1975). Restriction enzymes EcoRI (prepared according to Tanaka and Weisblum, 1975). BamI, BgZII, and Hind111 are from Biolabs. Maintenance and Compatibility of pIHl972 pIH1972, carrying the Zac+ marker, is stably maintained for at least 15 generations in a ret+ host (Fig. 1). Similar results were obtained with a recA (pIH1972) strain (data not shown). To test for incompatibility two types of reciprocal experiments were carried out: (a) super-infection of recA (pIH1972) with PlCm lysate, (b) superinfection ofrecA (PlCm) with pIH1972 lysate. In both types of experiments we were not able to isolate transductants which harbor both the two replicons-PlCm and pIH1972 (Table 1, last column). Establishment of PlCm excludes the resident pIH1972 plasmid and vice versa. We conclude therefore that pIH1972 and PlCm are absolutely incompatible. Note however that the transduction frequency of PlCm in recA (pIH1972) or recA hosts is similar while the transduction frequency of pIH1972 in a

Pl COMPATIBILITY

33

AND PLASMID MAINTENANCE

IO6IO’* 106d 8 + lO55

I I03

IO4 TOTAL

Id

IO6

VIABLE

I07

IO8

CELLS

FIG. 1. pIH19’72 maintenance. A single K204 (pIH1972) colony isolate was resuspended and diluted in LB broth to lo3 cells/ml. Cells were allowed to grow for 6 hr at 37”. Samples were withdrawn at 20 min intervals and plated on LZ-lactose tetrazolium agar (Hertman et al., 1972), which allow determination of total bacterial counts and number of Lac- segregants.

TABLE 1 COMPATIBILITY OF pIH1972 WITH PlCm” Transduction frequency Recipient

Expt No.

RecA

m.0.i.

PlCm-lysate

pIH1972-lysate

No. of transductants retaining resident plaamid per total No. of transductants screened

3.7 0.5 1.30 0.8”

1.2 x 10-s 1.0 x 10-z -

-

-

6.8 x 10-S 1.0 x 10-a

1.3 x 10-a 1.4 x IO-3 6.6 x 1O-4

RecA(pIH1972)

1 2 3

2.4 0.4 5.0

RecA(PlCm)

1 2

3.6” 1.9”

-

3.0 x lo-’ 1.0 x 10-7

O/1.6 x lo5 o/4.4 x 104 o/2.2 x 105 o/1.1 x 102’ o/1.3 x 10*c

a Bacteria growing exponentially in LB were harvested resuspended to l-2 x log cells/ml and infected with the lysates at desired multiplicity. After 30 min the culture was centrifuged, resuspended to original volume, and plated. The lac character was screened on EMB-lactose and LZ plates (Hertman et al., 1972). The CMR character was screened by addition of chloramphenicol (12.5 pg/ml) to these plates. RecA (pIH1972) cultures in LB contained before infection l-2% Lac- segregants, this value did not change significantly after transduction. Sixty percent of bacteria survived in transduction experiments at multiplicities greater than 1. RecA (PlCm) cultures were grown in the presence of chloramphenicol (12.5 pg/ml) prior to infection. b Multiplicity of infection of viable phage in lysate. c The Lac+ CMS character was reconfirmed by streaking the colonies on MacConkey plates with and without chloramphenicol added.

SHAFFERMAN,

34

I 2

GELLER,

1234

5

AND HERTMAN

67

B

A I

8910

2

I

PI

plli1972

PI

ECORI

EccRl

ECdll

2 PI Bgl II

3

4

5

6

9

IO

PI

plHl972

plHl972

pl+972

p11972

pmS72

#HI972

pW972

Bani

EeaM+ HindlU

EwR*+ BtjlI

EmR1+ Earn1

HindlI

Born1

EcoRI

BglP

7

B

OS-

;= 5--l ,s.*JC

GI4-

L-

II2-

,-

1.2-

2-

w3=-

.-

4-

6-

a---

I=

SC=

:=

7,

,

6.

?.o---

a.

FIG. 2. Gel electrophoretic 16 x 20 cm, 2.5 m&cm].

patterns

of Pl and pIH1972 DNA fragments

,.

,,

T-

[Agarose

slab gels (1.2%)

Pl COMPATIBILITY

AND PLASMID MAINTENANCE

recA (PlCm) host is significantly lower than that found in the nonlysogenic isogenic strain. This assymetrical behavior of incompatibility is now further studied. Comparative Endonuclease and pIHl972

Digest

of Pl

Pl DNA sequences in pIH1972 were identified by comparative gel electrophoresis of fragments produced after treatment of Pl and pIH1972 DNA with EcoRI, BgZII, HindIII, and BamI. The analysis is based on the known (Bachi and Arber, 1977) cleavage maps of Pl with these four restriction enzymes. EcoRI digest of pIH1972 produces seven visible bands (Fig. 2A, lane 2). The EcoRI-3, -4, -5, -6, and -7 fragments of pIH1972 comigrate with the Pl-EcoRI15, -17, -18, -21, and -23 fragments respectively (cf. Fig. 2A, lanes 1 and 2). The latter fragments are clustered together on the physical map of Pl (Fig. 4). If the pIH1972 EcoRI-3, -4, -5, -6, and -7 fragments indeed

originate from the Pl region carried by pIH1972 then a double digest of the Zac plasmid with EcoRI + Bum1 or EcoRI + Hind111 should not affect these fragments since neitherBarn nor Hind111 have sites in the Pl-EcoRI-15, -17, -18, -21, and -23 fragments (Fig. 4). Indeed such double digests of pIH1972 (Fig. 2B, lanes 7 and 9) do not affect the EcoRI fragment in question. Moreover pIH1972 has no site for Barn1 Fig. 2B, lane 6) and only a single Hind111 site (Fig. 2B, lane lo), located in either the EcoRI-1 or EcoRI-2 fragments of pIH1972 (Fig. 2B, lane 7). On the other hand BgZII has a site within the Pl EcoRI17 fragment (Fig. 4) and thus in a double digest of EcoRI + BgZII the EcoRI-4 fragment of pIH1972, which comigrates with Pl-EcoRI-17 should disappear and instead of it a new fragment should appear with a mobility slightly faster than that of PlEcoRI-18 (or pIH1972-EcoRI-5) as is indeed observed (cf. Fig. 2B, lane 8 to lane 4). BgZII digest of pIH1972 (Fig. 2B, lane 5)

32P-P,

32P-pIH1972

I-

-

32E it--

-

PI -RglII

PI - Born1

PI - EcoRI

4,5,6x 67-

35

32P-Pi

32P-pIH1972

21jr

-

5,6-

6’--

log=

6-

II12,13-

7-

6.9,10,11 -

7.6 1415-

-

S,lO-

Ib17,16-

-

ISII,12 -

3

3’

2 2’

I

I’

FIG. 3. Hybridization of [32P]pIH1972 DNA (4.10’ cpmipg) and [32P]P1 DNA (3.107 cpmipg) to “cold” Pl DNA treated with either EcoRI, Bum1 or BglII. [32P]DNA was obtained by the “nicktranslation” procedure (Kelly et al., 1970).

36

SHAFFERMAN,

GELLER, AND HERTMAN

produces a fragment, BgZII-2, which comigrates with the Pl-BgZII-4. The latter overlaps the region of the Pl-EcoRI-15, -18, -23 and partly EcoRI-5 and EcoRI-17 (Fig. 4). It follows that if pIH1972-BgZII-2 is identical with Pl-BgZII-4 then a digest of pIH1972 with BgZII followed by a digest with EcoRI should result in disappearance of pIH1972-BgZII-2 fragment and the appearance of new bands: (a) those comigrating with Pl-EcoRI-15, -18, and -23 (cf. Fig. 2B, lane 8 to lane l), (b) a new band (the remainder of the Pl-BgZII-4 EcoRI cleaved fragment) which is 5.1% the length of the Pl-genome, like the new BgZII:EcoRI-3 fragment (Fig. 2B, lane 8). To conclude, the above analyses indicate that pIH1972 carries at least 10% of the Pl genome, overlapping the Pl-EcoRI-15, 17, 18,21,23 fragments and part of Pl-EcoRI-5 fragment (Fig. 4). Hybridization Pl-DNA

of 32P-pIH1972-DNA

I

with

In order to find out what are the exact boundaries of the Pl DNA segment carried on pIH1972 and to check for the possible existence of some other Pl DNA sequences on the pIH1972, DNA hybridization experiments by the Southern (1975) technique were carried out. For this purpose [3zP]pIH1972 DNA was hybridized to Pl DNA fragments produced with either EcoRI, BgZII, or BamI. It is clear from Fig. 3, lane 3 and 3’ that pIH1972 DNA hybridizes to the Pl-EcoRI fragments 14, 15, 17, 18, as well as to one (or more) of the EcoRI fragments 4,5, and 6. Based on the fact that [32P]pIH1972 DNA hybridizes only to PlBgZII-3 and -4 and to Pl-BamI-1 and from the known cleavage maps of Pl (Fig. 4), we conclude that it is the Pl-EcoRI-5 fragment to which the pIH1972 hybridizes and not to Pl-EcoRI-4 or -6. Thus the “silent” Pl region carried on pIH1972 includes the entire Pl-EcoRI fragments 15, 17, 18, 21, 23, and part of fragments 5 and 14. The results from both the comparative digests (Fig. 2) and hybridization experiments (Fig. 3) suggest that the part of the Pl EcoRI-5 fragment carried by pIH1972 overlaps the entire Pl-BgZII-4 fragment. The fact that the Pl-BgZII-1

FIG. 4. Cleavage maps of Pl are taken from Bachi and Arber (1977). Genetic map drawn according to Scott and Kropf (19’77),Yarmolinski (1977), and Shafferman et al. (1978). Shaded bars designate the Pl DNA sequence carried by pIH1972.

(Fig. 3) does not hybridize to [32P]pIH1972 DNA indicates that the right-side boundary (Fig. 4) of the Pl segment carried on pIH1972 is very close to the Z3gZII site lo-

Pl COMPATIBILITY

AND PLASMID MAINTENANCE

cated within the Pl-EcoRI-5 fragment. The left side boundary of this Pl region lies within the Pl-EcoRI-14 fragment. The present identification of the Pl DNA content in pIH1972 together with the approximate aligned genetic and physical Pl maps (Yarmolinsky, 1977) may provide an explanation for our inability to show rescue of Pl allels by pIH1972 (Shafferman et al., 1978). In the latter study no markers between 7.14 and vir (or 3.21) were tested. Quantitative rescue experiments carried out recently (Scott and Kropf, unpublished) show that pIH1972 is able to rescue 180, 115, and ~7, and that wild-type allels carried by pIH1972 are derepressed. The c7 gene was already suggested by Scott and Kropf (1977) to be involved in Pl plasmid maintenance. Surprisingly, the Pl region on pIH1972, is not identical with the Pl region identified by marker rescue to be adjacent to the Zac insertion (94 -36~Zac-35) in the progenitor Pldlw (Shafferman et al., 1978). To conclude, pIH1972, a derivative of Pldlw, is stably maintained as an autonomous replicon, with an estimated copy number of one per chromosome (data not shown), similar to the Pl plasmid copy number prophage, and it is absolutely incompatible with Pl. We therefore suggest that the Pl region identified on pIH1972 is the locus for plasmid maintenance and incompatibility. This region is about 10% of the Pl genome and some 30% of pIH1972 DNA content. ACKNOWLEDGMENTS We wish to thank S. Lavi and E. Israeli for their assistance in the hybridization experiments. REFERENCES BACHI, B., and ARBER, W. (1977). Physical mapping of Bg111,BamHI, EcoRI, Hind111 and PstI restric-

37

tion fragments of bacteriophage Pl DNA. Mol. Gen. Genet. 153, 311-324. CHESNEY, R. H., and SCOTT,J. R. (1975). Superinfection immunity and prophage repression in phage PI. II. Mapping of the immunity difference and ampicillin resistance loci of PI and +np. Virology 67, 375-384.

GREEN, P. J., BETLACH, M. C., and BAYER, H. N. (1974). In “Methods in Molecular Biology” (R. B. Wichner ed.), Vol. 7, pp. 87-111. HERTMAN, I., ZEHAVI, A., and RUBINSTEIN, S. (1972). A bacterial recombination function involved in lyzogenization by transducingphage Pldlw. Virology 48, 182-192. HERTMAN, I., and SCOTT,J. R. (1973). Recombination of phage Pl in recombination deficient hosts. Virology 53, 468-470.

KELLY, R. B., COZZARELLI, N. R., DEUTSCHER, M. P., LEHMAN, I. R. and KORNBERG,A. (1970). Enzymatic synthesis of deoxyribonucleic acid. J. Biol. Chem. 245, 39-45.

LURIA, S. E., ADAMS, J. N., and TING, R. C. (1960). Transduction of lactose-utilizing ability among strains of E. coli and S. dysenteriae and properties of transducing phage particles. Virology 12, 348390.

SCOTT,J. R., and KROPF, M. M. (1977). Location of new clear plaque genes on the Pl map. Virology 82, 362-368.

SHAFFERMAN, A., GELLER, T., and HERTMAN, I. (1978). Genetic and physical characterization of Pldlw and its derivatives. Virology 86, 115-126. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. TANAKA, T., and WEISBLUM, B. (1975). Construction of colicine El-R factor composite plasmid in vitro: Means for amplification of deoxyribonucleic acid. J. Bacterial. 121, 354-362. YARMOLINSKY, M. B. (1977). Genetic and physical structure of bacteriophage Pl DNA. In “DNA Insertion Elements, Plasmids and Episomes,” pp. 721732. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. WALKER, D. H., and WALKER, J. ‘T. (1975). Genetic studies of coliphage Pl. I. Mapping by use of prophage deletions. J. Viral. 16, 525-534.