Lambda transducing phages carrying plasmid R100 replication genes

Gene, 14(1981) 321-324 Elsevier/North-HollandBiomedicalPress

321

Short Communications Lambda transducing phages carrying plasmid RIO0 replication genes (Dual replicon, R100-~. cointegrates) Sarah A. Mclntire a and Walter B. Dempsey a,b a VeteransAdministration Medical Center, Dallas, TX 75216, and b Department of Biochemistry, Universityof Texas Health Science Center, Dallas, TX 75235 (U.&A.)

(Recei~,edMarch 20th, 1981) (Accepted April 2nd, 1981)

SUMMARY VAX73, a ~, bacteriophage carrying R100 replication genes, maintains itself as an independent plasmid under conditions in which the ~, replication genes are inactive.

The replication genes of R100 have been studied in considerable detail and mapped just counterclockwise of ISlb in EcoRI fragment B (Fig. IA). Yoshikawa (1974) located a replication gene, repA, in this region and Silver et al. (1977) mapped the origin of replication, orlV, at kb coordinate 85.9. Taylor and Cohen (1979), using a copy number mutant of R100, cloned the minimum region required for expression of R100 incompatibility and replication (83.7 to 85.9) and identified three sites (84.4, 84.7 and 84.9) as essential for RI00 replication. Another gene, stb, is not required for replication but is necessary for stable maintenance of R100. This gene has been mapped just clockwise of ISla (Mild et al., 1980) and was first identified and called stb by Hashimoto et al. (1969); it also has been called repB O'oshikawa, 1974). We have previously described VAZ73 (Dempsey and Mclntire, 1979), a phage which has RI00 DNA from coordinate 81.3 through the antibiotic resistance determinant including ISla (Fig. 1A). VA~73 was derived from an RlOO::~b5196515cI8575am7 coinAbbreviations: Cml, chloramphenicol; EtBr, ethidium bromide; kb, kilobase pairs; LFT, low frequency Uansducing; Met, mercuric ion;or/, originof replication;Spc, specfinomytin.

tegrated molecule in which the complete X had integrated into the finO gene at R100 coordinate 81.3. The LFT lysate produced by temperature induction of this strain was screened for phage carrying R100 antibiotic resistance genes. Representative types (Mere; MerRSpcR; MereSpcRCml R) were purified from transductants carrying a chromosomal wild.type helper phage (see Dempsey and Mclntire, 1979). These phage all proved to be the classical ~ a l type (left.arm substitution) as shown for VA~73 in Fig. 1B. Since this substitution included the region required for RI00 replication, it seemed likely that the phage might replicate via these R100 genes and be mainmined in the cell as plasmids. The experiments described here show that strains transduced with phage VA~73 do contain plasmid DNA and that it is maintained via RIO0 replication genes. VA~73 can transduce the antibiotic resistance markers it carries into both attX* and attT~- strains with equal frequency (approx. 10.4 for Cmlg). The transductants, however, are uastable if the selecting agent is removed. This instabtity is expected if replication is via wild.type RI00 genes because VA~73 lacks the stability gene (stb) mentioned above. Therefcre, in the experiments described below, cells are always grown in media containing spectinomycin.

0378-1119/81/0000-0000/502.50 O 1981 ElsevieffNorth-HollandBiomedice/Press

322

A • B

~

l

S

I

b

89.3/0.0

~r~

mer L

tra

K

i

t.

sul

C

G

s clstr fu$

cml

J

stb ISla

tet D A B •

ISla

ISlb o~r'~/inc

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'-°T- t °l Fig. 1. (A) Map of plasmid R100. EcoRl fragments (Tanaka et ~1., 1976} are indicated by letters A - M outside the circle; 89,3/0.0 marks the terminus/orion of the R100 coordinate system; the arrow at coordinate 81.3 indicates the k insertion site in the parental R100::?, cointegrate (see tex0; the segment of R100 DNA carried by VA?,73 is shown on the right side of the map; hatched squares are ISla and ISlb; the bracketed 83.7-85.9 area represents the R100 replication region. Inside the circle are shown the genes for transfe~ (tra), fertility inhibition ffinO), autonomous replication (rep), stability (stb) and resistances to mercuric ion (met), sulfonamides (su/), spectinomycin/sUeptomycin (spc/str), fusidic acid flus), chloramphanicol (cml) and tetracycline (tet). (B) Schematic diagram showing the orientation of R100 DNA (heavy line) in VAk73 (Dempsey and Mc!ntire, 1979). EcoRl cleavage sites in R100 (Tanaka et al., 1976) and ?, (Murray and Murray, 1974) ate indicated by arrows. ~ t t indicates the phage attP' region: locations of ?, genes O and P ate shown.

323 If the transductants contain a VA~73 plasmid and, in addition, are X4mmune, replication cannot be via the ~ system since genes O and P are repressed. Accordingly, the simple isolation of VA?~73 as a plasmid would prove that it can replicate via the RIO0 genes it carries. To show conclusively that a VA~73 transductant carries plasmid DNA identical to phage VA~73 DNA we have isolated plasmid DNA and compared its restriction patterns to those of the parental phage. Strain ED2149 [A(niagalattX bio)lacAU124] was transduced with VA~73 as previously described (Dempsey and Willetts, 1976) and plated at 33°C on ML agar containing spectinomycin. A single colony was grown at 33°C in L broth containing spectinomycin (0.1 mg/ml) and harvested at late exponential phase. Plasmid DNA was isolated by EtBr4~sCl centrifugation after cell lysis and DNA precipitation by the method of Humphreys et al. (1975). EtBr was removed by extraction with isopentyl alcohol, CsCI was removed by dialysis, and DNA was precipitated with ethanol. VA~,73 phage DNA was prepared by phenol extraction of purified phage as described (Dempsey and Mclntire, 1979). 2.5/zg portions of plasmid DNA and VAX73 phage DNA were hydrolyzed with the restriction endonucleases EcoRI, HindIII and Sail. The digested DNA fragments were separated by electrophoresis on 0.7% agarose, stained with EtBr and photographed. Methods for endonuclease digestions and agarose electrophoresis have been described (Dempsey and Mclntire, 1979). Fig. 2 shows that the plasmid DNA isolated from the cell is the circular form of VA?~73 DNA. With each endonuclease, the linear phage DNA and the circular plasmid DNA show identical patterns except for the two phage end fragments that are ligated in the plasmid DNA. The fusion of the phage cohesive ends to make a new band in the plasmid is most clearly seen in the Sall digest shown in Fig. 2. The second and third bands from the top in the phage track are mbsing in the plasmid track, which has, instead, a new top band. In the EcoRl digest of plasmid DNA, the new fragment is about the same size as the third band from the top and thus increases the intensity of that ~ d in the plasmid track. Similarly, in the Hindlll digest, the new plasmid fragment is about the same si~e as the second band. As shown conclusively (Dempsey and Mclntire, 1979) and as seen by intensity in Fig. 2, the third Hfndlll band in the phage is

!

Fig, 2. Agaxose gel electrophotesis of endonuclease treated samples of VAk73isolated as plasmid (P) or from phage (~). The symbol× to the fight of each plasmid track indicates the band that is composed of the two ligated end fragments. The two end fragments are indicated by white dots next to the phage tracks. Agaxoseconcentration wa~ 0.7%.

composed of two identically sized fiagrnents, one of which is the left end. Thus the increased intensity of the second plasmid band corresponds to the decreased intensity of the third phage band and the loss of the fourth phage band. This analysis proves that the VA~73 transductant has extrachromosomal plasmid DNA that is identical to the DNA isolated from the phage VAX73.

324 TABLE I

AC~O~E~E~NT

Spot tests for determining expression of k genes in VA~73 L-an~uctant Purified VA~73 tmmductant (ED2149 selected for SpcR) was overlaid on spectinomycin nutrient agar and 104 phage were applied in spots. Plates were incubated at the indicated temperatures and scored after 16-18h. ( - ) = n o lysis; (+) = lysis. In control experiments, a non-transduced ED2149 was(+) with Xv/rand Ab2¢- and ( - ) with XOam and ~,Pam at both tem,-,eratures.

This work was supported b y funds from the Research Service of the United States Veterans Administration. We thank Dr. D. Berg for phage strains and K.R. Sims for technical assistance.

REFERENCES X mutants tested

Lysis at 33°C

37°C

vir b2c-

+ -

+ +

Oam Pam

-

+ +

Evidence that the X repfication genes are repressed at 33°C comes from testing the capacity o f some ), mutants to form plaques on the VAA73 transductant used for the plasmid analysis. Since VAA73 carries the temperature~'nsitive c1857 mutation, its repressor functions normally at 33°C but is partially inactivated at 37°C. When functioning normally, the c1857 repressor will prevent transcription of all incoming homoimmune ), phage. We tested homoimmune M,2c- and two heteroin~,:ane ), replication mutants ~ i m m 2 1 O a m a n d ~ n m 2 1 P a m for plaqueforming ability at 33°C and 37°C. The results (Table I, line 2) show that at 33°C the VA~73 transductant is el* as expected and (lines 3 and 4) the ), replication genes O and P are not expressed. At 37°C the repressor is partially inactivated and the O a n d P genes are expressed, leading to marker rescue of the Oam and Pare mutants. The data presented here show conclusively that strains transduced with phage VAA73 contain circular plasmid DNA that is identical to VA},73 DNA and is replicated in the cell via the R100 genes it carries. This easily manipulated phage and others like it (Dempsey and Mclntire, 1979) should be useful tools in studies of R100 replication.

Dempsey, W.B. and Mclntire, S.A.: Lambda transducing phages derived from a f i n O - RI00::X cointegrate plasmid: Proteins encoded by the R100 replication/incompatibility region and the antibiotic msis~tancedetermL,tant. MoL Gen. Genet. 176 (1979) 319-334. Dempsey, W.B. and Willetts, N.S.: Plasmid co-integrates of prophage lambda and R factor RIO0. J. Bacteriol. 126 (1976) 166-176. Hashimoto, H., lyobe, S. and Mitsuhashi, S.: Unstable mutants of R factor. Japan J. Microbiol. 13 (1969) 343349. Humphreys, G.O., Wilhhaw, G.A. and Anderson, E.S.: A simple method for the preparation of large quantifies of pure plasmid DNA. Biochim. Biophys. Acta 383 (1975) 457-463. Miki, T., Easton, A.M. and Rownd, R.H.: Cloning of replication, incompatibiliW, and stability functions of R plasmid NRI. J. Bacteriol. 141 (1980) 87-99. Murray, N.E. and Murray, K.: Manipulation of restriction targets in phage ~, to form receptor chromosomes for DNA fragments. Nature (London) 251 (1974) 476-481. Silver, L., Chandler, M., Boy de Ia Tour, E. and Caw, L.: Origin and direction of replication of the drug resistance plasmid RI00-1 and of a resistance transfer factor deftvative in synchronized culture. J. BacterioL 131 (1977) 929 -942. Tanaka, N., Cramer, J.H. and Rownd, RI-I.: E c o R l restriction endonuclease map of composite R plasmid NR1. J. Bacteriol. 127 (1976) 619-636. Taylor, D.P. and Cohen, S.N.: Structural and functional analysis of cloned DNA segments containing the repficafion and incompatibility regions of a miniplasmid derived from a copy number mutant of NRI. J. BacterioL 137 (1979) 92-104. Yoshikawa, M.: Identification and mapping of the replication genes of an R factor, RI00-1, integrated into the chromosome of Escherichia coli K-12. J. Bacteriol. 118 (1974) 1123-1131. Communicated by M. Lieb.