Three mutants of bacteriophage P1 affected in their ability to maintain the
lysogenic ... cause prophage instability suggests that P1 codes for at least some
of the.
Vol. 23, No. 3 Printed in U.S.A.
JOURNAL OF VIROLOGY, Sept. 1977, P. 476-482 Copyright © 1977 American Society for Microbiology
Maintenance of Bacteriophage P1 Plasmid ALINE JAFF9-BRACHET' AND RICHARD D'ARI* Unite de Ggnitique Microbienne, Centre National de la Recherche Scientifique, Institut de Biologie Moleculaire, UniversitW de Paris VII, 75221 Paris Cedex 05, France Received for publication 30 November 1976
P1 plasmid consists of covalently closed circular duplex DNA molecules of molecular weight about 60 x 106 (10, 12, 36). It is normally present in lysogenic cells in a number of copies per chromosome that is close to unity (12). Prophage loss is a rare event, occurring at frequencies of less than 10-4 per division (26). The fact that this element is not frequently lost in growing populations implies the existence of a strict control mechanism governing its replication and segregation to daughter cells at cell division.
Two classes of models have been proposed for the control of plasmid replication. In 1963, Jacob et al. postulated that the regulation of replication is under positive control, dependent on a replicon-specific initiator active only at some specific time in the cell cycle (13). To explain the accurate segregation into daughter cells at cell division, they suggested that replicons may be attached to a site on the cell membrane that duplicates in such a way that at least one copy, with replicon attached, is passed to each daughter cell. In 1969, Pritchard proposed a negative control of replication under which a repliconspecific inhibitor of replication, synthesized at the moment of initiation, prevents initiation of a further cycle of replication until the inhibitor concentration falls below a critical value due to cell volume increase during growth (24). We have chosen to study the coordination of replication and segregation of the plasmid phage P1 through the isolation and characteri1 Present address: Bact6riologie M6dicale, Institut Pasteur, 75015 Paris, France.
zation of mutants that cannot be maintained stably as prophage. A preliminary characterization of three such mutants is described here. These plasmid stability mutations fall into two classes on the bases of their genetic location, their effect on the capacity to lysogenize recA bacteria, and their suppressibility by ant mutations; the P1 ant gene, as shown by Chesney and Scott (3) and in the accompanying article (5), appears to code for an antirepressor. Lysogens carrying these mutant prophage are unstable and give rise to nonlysogens at an abnormally high frequency. Cultures of these lysogens contain elongated cells, and in some cases considerable filamentation is observed. This partial interference with normal cell division has been reported for cells harboring maintenance mutants of other plasmids-ColEl (14), ColVB (17), R1 (9)-and suggests that certain plasmid-linked mutations may also affect some bacterial process related to cell division. The fact that mutations in P1 can cause lysogen instability suggests that part of the control mechanism(s) governing the maintenance and segregation of this extrachromosomal replicon is coded for by the plasmid itself. MATERIALS AND METHODS Bacterial strains. Table 1 lists the nonlysogenic Escherichia coli K-12 derivatives used. Sh16 (19) is a nonsuppressing streptomycin-resistant strain of Shigella dysenteriae furnished by J. L. Rosner. Bacteriophage strains. All P1 phages are derivatives of Plkc (18). The different mutations used are listed in Table 2, except for seg-1 and seg-5, which are described in the text. Recombinants were ob-
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Three mutants of bacteriophage P1 affected in their ability to maintain the lysogenic state stably are described here. These mutants were normal in lytic growth, but lysogenic derivatives segregated nonlysogens at abnormally high rates (1 to 30% per division). Cells harboring these mutant prophages were elongated or filamentous. The mutations responsible for this prophage instability fell into two classes on the bases of their genetic location, their effect on the ability to lysogenize recA bacteria, and their suppressibility by ant mutations eliminating antirepressor activity. The two mutants that were able to form recA lysogens showed the same prophage instability and partial inhibition of cell division in recA as in rec+ lysogens. The fact that plasmid-linked mutations can cause prophage instability suggests that P1 codes for at least some of the functions determining its own autonomy and segregation.
VOL. 23, 1977
MAINTENANCE OF PHAGE P1 PLASMID
TABLz 1. E. coli K-12 strains used Strain
Relevant characteristics
Source or reference
C600 Q1008
Str' supE44 Str' sup+ dnaB27 dnaB70 Strr sup+ gal Str' sup+ gal recA mutD5 galU Pir
1 6 6 6 W3102, parent of N100 (35) Strain 152 (35)
Q1504
Q1i08 N99 N100 Q205 Q206
GY2168
mutD5gal+ P1'
lacYl mal/F'2-
tained from the progeny of appropriate crosses. P7 (34) is the 4)amp+ of H. W. Smith (31). Media and diluents; growth and assay of phages. For a description of these materials and procedures, see D'Ari et al. (6). Phage mutagenesis. Mutagenized stocks of PlCm were prepared by confluent lysis of the P1-sensitive mutD mutator strain Q206. Cultures of Q206 were grown overnight in glucose-minimal salts medium at 370C and then diluted 100-fold into LB broth supplemented with 50 ,ug of thymidine per ml and aerated for 3 h at 370C before use. LMC plates were seeded with 5 x 108 bacteria and 108 PlCm, incubated at 370C for 7 h and then scraped; the lysates, after chloroform treatment, were centrifuged. The number of clear plaques on Shl6 was taken as a measure of the extent of mutagenesis; mutagenized stocks typically contained about 3% clear mutants, compared with less than 0.1% in unmutagenized stocks. Phage crosses. For a description of phage crosses, see D'Ari (5). Scoring of markers. (i) The seg markers. The seg markers in a cl+ genetic background were scored by measuring the stability of the resulting lysogens, as described below. In a c1.100 background, the following rapid test for seg was devised. C600 cells were lysogenized at 300C with the progeny from a cross; after 2 nights of incubation, isolated colonies were picked with sterile toothpicks and inoculated into 0.5 ml of LB broth containing citrate in nylon microculture containers, which were incubated overnight at 300C to obtain saturated cultures. The following day the cultures were diluted about 100-fold with stainless-steel prongs into 0.7 ml of LB broth containing citrate, incubated for 90 min at 420C to induce the lysogens, and then replicated onto LBcitrate plates, which were incubated overnight at 420C. A confluent spot of bacterial growth indicated Plseg lysogens, which segregate many temperatureresistant nonlysogens; no growth indicated Plseg+ lysogens.
(ii) Amber markers. Lysates of the phage to be tested, prepared as for the seg test, were replicated onto LMC plates containing streptomycin and seeded with the sup+ Sti' strain Q1008 and 3 x 107 amber tester phage; absence of lysis indicated the presence of the tester amber allele.
Lysogen stability. Lysogens of PlCm and derivatives, identified as chloramphenicol-resistant colonies, were restreaked twice on selective media before use. Measurements of lysogen stability were made on exponential cultures at 300C in LB broth containing chloramphenicol (25 pug/ml) and citrate (5 x 10-3 M); the former prevents growth of nonlysogenic segregants without affecting their viability, and the latter, by blocking Ca2+-dependent P1 adsorption (2), prevents relysogenization of segregants by free phage. Cultures were plated on citrate plates and incubated for 2 nights at 300C; about 200 isolated colonies were picked and replicated onto plates with and without chloramphenicol and incubated overnight at 3000. For thermoinducible lysogens, the ratio of colonies on citrate plates at 420C to colonies at 300C is also used as a measure of lysogen stability. Immunity tests. Immunity was tested by spotting Plvire onto an LMC plate seeded with the strain to be tested. For cultures containing nonlysogenic segregants, chloramphenicol was added to the plates. Phage concentrations ranging from 104 to 108 per ml were used; at the higher concentrations, nonimmune strains show clear circles of lysis, even recA strains, on which P1 does not make visible plaques.
RESULTS Stability of the Cm insertion. To isolate mutants of P1 affected in their maintenance, PlCm was used; its cat gene (chloramphenicol acetyl transferase), derived from an R factor (15), confers chloramphenicol resistance to lysogens and thus serves as a convenient prophage marker. To check the stability of the Cm insertion in lysogens, a culture of Q1008 (PlCm) was grown and plated at 300C without drug selection, and colonies were picked and replicated to plates with or without chloramphenicol. Of 1,500 clones tested, only one was chloramphenicol sensitive; analysis showed it to be a defective lysogen, immune to superinfecting P1 but deleted for several prophage genes. Thus, the loss of Cm from a lysogen is seen to be a rare event. This is in striking contrast to the instaTABLz 2. P1 genetic markers Name cat(Cml)
ci. 100 c5.482 virs dan-1 sud-2
ant-i bac-i ban-i am3.6 am8.13 am34.62 )a
am56.32
amM
Reference Description 15 Chloramphenicol acetylease insertion 26 Temperature-sensitive repressor Double mutant (see text) Virulent Suppressor of c.i100 Suppressor of dan-i Antirepressor negative ban negative L
Lethal
embers,
premed bysupE
sup-
28 27 5 5 5 6 27 27 33
29, 33
33
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lac +
7 Spontaneousgal+ mutant of Q205 mal derivative of 200PS/ F'2-lac (8) from J. George
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J. VIROL.
latter phage against PlCm am56.32 cl.100; an am+ phage that segregates nonlysogens at high frequency was recovered. This recombinant is shown below to have the genotype Cm c5 seg-5 cl.100 (direct detection of the c5 marker is obscured by the cl.100 mutation). Lysogens for PlCm c5 seg-5 cl.100 are thermoinducible; when plated at 42°C on citrate plates, survival is about 10-2 and the survivors are mainly nonlysogens; PlCm cl.100 lysogens give rise to about 10-5 survival. This permits simple detection ofthe seg marker (see Materials and Methods). Both lysogens give survival frequencies of about 10-5 when plated on chloramphenicolcitrate plates at 420C. The clear mutation of Pic5 seg-5 lies in the same region as the ban-i mutation, which is located between am3.6 and am56.32 (6). The P1 ban protein, analogous to the bacterial dnaB, is able to replicate bacterial and phage DNA (23, 6); ban mutants, unlike wild-type P1, depend on functional dnaB product to replicate their DNA and cannot grow lyrically in dnaB (ts) hosts at nonpermissive temperatures. To see whether ban function is normal in P1c5 seg-5, lytic growth was measured in dnaB70 (Q1508) and dnaB27 (Q1504) hosts. Scott's original P1c5 seg-5 and the recombinants PlCm c5 seg-5 and PlCm c5 seg-5 cl.100 were all ban (burst sizes
