JOURNAL OF VIROLOGY, Apr. 1982, p. 283-291 Vol. 42, No. 1 0022-538X/82/040283-09$02.00/0

4,, a Temperate Phage of Agrobacterium tumefaciens, Is Mutagenic DOMINIQUE EXPERT* AND JACQUES TOURNEUR Laboratoire de Biologie Cellulaire, Centre National de Recherches Agronomiques, 78000 Versailles, France Received 28 September 1981/Accepted 21 December 1981

A new temperate (phage '1) ofAgrobacterium tumefaciens strain B91 is described. Many octopine-utilizing strains of A. tumefaciens harbor prophage 4 or a phage that is similar if not identical to it. This phage has a very narrow host range, and we found that its growth is strongly reduced in strains which carry an octopine pTi . When sensitive bacteria are infected with 4, 1 to 3% of the survivors carry mutations on the chromosome, as well as on the pTi plasmid. This phenomenon appears to be a direct consequence of lysogenization. The possible mechanisms whereby such 14-induced mutations occur are discussed.

The first lysogenic system described in Agro- medium (MM) contained 2 g of (NH4)2SO4 per liter, 6 g bacterium tumefaciens involved temperate of KH2PO4 per liter, 14 g of K2HPO4 per liter, 1 g of phage omega, which was isolated from strain B6 trisodium citrate per liter, 0.2 g of MgSO4-7H2O per (1). Later, several other reports liter, and 5 g of glucose per liter. When necessary, L- (3, 16, 25) amino acids (100 ,ug/ml), bases (100 ,ug/ml), or vita- described the occurrence of temperate phages in mins (50 ,ug/ml) were added. The synthetic media used a wide variety of A. tumefaciens strains. All of for assays of catabolic pTi functions (19) contained these phages had similar morphological, biologi- 13.6 g of KH2PO4 per liter, 0.2 g of MgSO4-7H2O per cal, and biophysical properties and the same liter, 0.005 g of FeSO4 per liter, 0.01 g of CaCl2 per immunity. liter, 0.002 g of MnCl2 per liter, and 2 g of glucose per In 1975, using electron microscopy, Vervliet liter; the nitrogen source was (NH4)2SO4 (2 g/liter) for et al. (22) demonstrated the occurrence of SM medium, arginine (0.1 g/liter) for ASM medium, or "phage-like particles" or "defective phages" in octopine (0.3 g/liter; a gift from J. Tempd) for OSM mitomycin C-treated cultures of strains B6-806, medium. The pH of each of these media was adjusted to 7.2. For pTi plasmid transfer, glucose was omitted, B6S3, B2As, and CV1. All of these particles and octopine (2 g/liter) was used as the carbon and were morphologically identical. The particles nitrogen source (14). When required, the media were released by strain B6-806 were shown to contain solidified by using Merieux agar (16 g/liter for plates a DNA molecule which had a molecular weight and slants and 6 g/liter for soft overlays). When of 25.2 x 106. Hybridization by the heteroduplex necessary, the following antibacterial drugs were add- method revealed no homology between this ed to the media: 500 pLg of streptomycin per ml, 500 ,ug DNA and that of phage Ql. of spectinomycin per ml, and 50 jig of rifampin per ml. We report here that the phage released by the Except when specified otherwise, all incubations strains mentioned above is not defective and is were carried out at 27°C. Preparation of phage stocks. A 30-ml culture of the harbored by several other octopine-utilizing permissive strain growing logarithmically (absorbance strains of A. tumefaciens. This phage is not at 600 nm, 0.25) in NB was inoculated with a single related to Ql, and we have called it phage 4. We plaque picked with a Pasteur pipette. After 12 h of describe the general characteristics of this tem- incubation at 27°C, chloroform was added, the culture perate phage and demonstrate that it has two was centrifuged, and the resulting supernatant was unusual properties. First, it frequently induces filtered with a membrane filter (0.45 ,um; Millipore mutations in its host during lysogenization; and Corp.). The phage stock was kept over a chloroform second, its growth is inhibited in strains which phase. carry an octopine pTi plasmid. Induction of a lysogenic strain. Lysogenic cells were grown in NB, harvested by centrifugation in early log phase (absorbance at 600 nm, 0.25), washed, and MATERIALS AND METHODS suspended in Tris buffer (10 mM Tris-hydrochloride, Strains and media. The bacterial strains used in this 10 mM MgSO4, pH 7.2) to a density of 5 x 107 cells per work are described in Tables 1, 2, and 5. Phages Ql and ml. A 5-ml sample of this suspension was irradiated * were obtained from lysates produced by UV induc- with a General Electric model G8T5 germicidal lamp. tion of strains B6 and B91 (Table 2), respectively. The The energy was measured with a Latarjet dosimeter. following media were used. Nutrient broth (NB) con- The irradiated suspension was supplemented with 0.5 tained 8 g of Merieux nutrient broth per liter, 5 g of ml of 10-fold-concentrated NB and incubated for 12 h NaCl per liter, and 5 g of glucose per liter. Minimal with shaking in a dark room. Chloroform was then 283 284 EXPERT AND TOURNEUR J. VIROL. TABLE 1. Characteristics of the derivatives of strain B91 used in this worka

Strain Characteristics harboredPhage Plasmid(s) harboredb VBC001 B91 cured of phage 4' and of pTi and pAt after subculturing at 37°C VBC002 VBC001 lysogenic for fl VBC003 VBC001 Rif' Strr Spcrc VBC004 B91 cured of phage 4 after UV irradiation and subsequent pTi B91, pAt B91 growth at 37°C VBC006 Transconjugant from B6-806 x VBC003 pTi B6-806 VBC009 VBC003 lysogenic for 4 4 VBCO1O VBC003 resistant to 4 VBC013 Transconjugant from B91 x VBC009 4 pTi B91 VBC015 Transconjugant from B6-806 x VBC010, acquired prophage 4 4 pTi B6-806 during conjugation VBC050 Transconjugant from B6-806 x VBC010 acquired prophage 4' k4 pTi B6-806, pAt during conjugation B6-806 VBC201 VBC004 cured of plasmid pTi upon 4' lysogenization il pAT B91 VBC203 4i-induced Arc- mutant from VBC004d 4' pTi B91, pAt B91 VBC231 VBC004 cured of plasmids pTi and pAt upon 4' lysogenization 4' VBC238 4'-induced Occ- mutant from VBC004' 4' pTi B91, pAt B91 a Strains B91 and B6-806 are listed in Table 2. b The strains were analyzed for the presence of plasmids by the method of Casse et al. (7). pTi and pAt are specific plasmids of A. tumefaciens strains. In strains B91 and B6-806 both of these plasmids have a molecular weight of approximately 125 x 106. pTi is a tumor-inducing catabolic plasmid (octopine or nopaline catabolism), and pAt is a cryptic plasmid (2, 17, 21, 23, 24). In addition, all of these strains harbor the large cryptic 270- megadalton plasmid which was found to be present in several A. tumefaciens strains by Casse et al. (7). c Rifr, Strr, and Spcr, Rifampin, streptomycin, and spectinomycin resistance, respectively. d Arc-, Inability to utilize arginine as a nitrogen source. e Occ-, Inability to utilize octopine as a nitrogen source. added, and the lysate was centrifuged and filtered as determined by replica-plating immunity, and sensitiv- described above. ity to phage was determined by spotting loopfuls of Prophage curing. Two methods were used. In the varying dilutions of a phage lysate onto a soft agar first method, the lysogenic strain was UV irradiated as overlay seeded with the culture to be tested. described above. The irradiated cells were harvested Isolation and characterization of auxotrophic mu- by centrifugation, suspended in NB containing anti- tants. Sensitive cells grown in NB were infected with 4 phage serum (obtained from J. F. Vieu, Institut Pas- at a multiplicity of infection of 1 to 5, incubated for 12 teur), and incubated at 37°C with shaking. After h, harvested by centrifugation, and plated onto NB growth to stationary phase, the cells were harvested agar. Under these conditions, approximately 50% of by centrifugation, washed, and suspended in NB. the surviving bacteria were lysogens. The colonies Suitable dilutions were plated onto NB agar for isola- were replicated onto MM agar and NB agar succes- tion of cured clones. sively. The auxotrophic mutants were distinguished In the second method, the lysogenic strain was from the prototrophs by their inability to grow on MM grown in NB and infected with 4 during the logarith- agar. After a second isolation, the mutant clones were mic phase by using varying multiplicities of infection characterized by replica-plating or streaking onto MM (10 to 100 PFU/bacterium). After 30 min of incubation, agar containing various growth factors. the infected cells were harvested by centrifugation, Transfer of the pTi plasmid by conjugation. The pTi suspended in NB containing anti-phage serum, and octopine donor strain was grown in octopine conjuga- incubated at 27°C with shaking. When the culture tion medium. When the culture reached an absorbance reached the stationary phase, the bacteria were col- of 600 nm of 0.4 to 0.5, it was mixed with a culture of a lected by centrifugation, washed, and suspended in recipient strain growing exponentially in SM medium. Tris buffer. Suitable dilutions were plated onto NB The proportion was five donor cells per recipient cell. agar for isolation of cured clones. After 1 h of incubation without shaking, varying Determination of adsorption rates. Bacteria growing dilutions of the bacterial mixture were plated onto exponentially in NB were washed and resuspended in octopine conjugation medium containing streptomy- NB at a density of 108 cells per ml. Then they were cin, spectinomycin, and rifampin. After 1 week of infected with phage 4' at a multiplicity of infection of incubation, the plates were scored for octopine-ca- 0.1. The suspension was incubated for 30 min at 27°C tabolizing transconjugants (Occ+) by streaking onto and centrifuged. Suitable dilutions of the supernatant OSM agar supplemented with antibiotics. As a con- were plated by using the overlay method for determin- trol, a culture of the donor strain was spotted onto ing the unadsorbed phage. OSM agar containing antibiotics. Test for lysogeny. Spontaneous phage release was Tumorigenicity test. Virulence assays were carried VOL. 42, 1982 PHAGE %P 285 TABLE 2. Spontaneous phage production in Agrobacterium strains Phage growth in strain:a StrainStrainOrigin B91 VBC001 VBC001 ~~~~~~~~~~~~~~~~~~Phage(s)harbored (270C) (370C) VBC002 A. tumefaciens octopine strains B6 Kent + + + + Q, B2 Kent + + + + Q, Vi Hamilton + + + + Q, CG Ark + + + + Q, B48 Ark + + + + Q, SN Lippincott + + + + Q7, B6-806 Beardsley - + - + ' B6S3 Schell - + - + ' CVi Pootjes - + - + ' R61 Zinca - + - + ' R62 Zinca - + - + ' R63 Zinca - + - + ' B6S Leff - + - + ' B2AS Schell - + - + ' S379 Camus - + - + ' B91 Faivre - + - + ' A. tumefaciens nopaline strains T37 Ricker - - - - C58 Dickey - - - - II-BV7 Braun - - - - A. rubi ATCCb - - - - A. rhizogenes strain Hri Faivre - - - - A. radiobacter strains TR1 De Ley - - - - TR6 De Ley - - - - S1005 De Ley - - - - a The supernatants from the strains were plated onto strains B91, VBC001, and VBC002, which is an Ql lysogen of VBC001. In the case of VBC001, the test was performed at both 27 and 37°C. +, Presence of lysis with some dilutions of the supernatant; -, no lysis even with undiluted supernatant. b ATCC, American Type Culture Collection. out on tobacco plants (Nicotiana tabacum cv. Wiscon- all previously described A. tumefaciens temper- sin 38 or Nicotiana rustica var. pavonii). The tobacco ate phages, because it did not have the same plants were wounded and inoculated with a sterile immunity as Ql. Indeed, it yielded plaques identi- needle previously seeded with a 24-h-old bacterial cal in number and aspect on strain VBC002, an culture grown on a slant. Virulence was scored 3 Ql lysogen of strain VBC001. This new phage weeks after inoculation. was called psi. Unlike phage Ql, which can grow at 37°C, RESULTS phage ' was not able to produce plaques at Origin of phage *. Phage 'p does not grow on temperatures higher than 33°C. Phage *, or at most strains of A. tumefaciens (see below). The least a phage with identical properties (same discovery of this phage resulted from the fortu- plaque morphology, same immunity, same tem- itous isolation of a sensitive host, strain perature range for growth), was found in cul- VBC001. This strain was obtained after several tures of all octopine-degrading strains tested subculturings of strain B91 at 37°C and did not (Table 2). This included strains which also har- contain the two plasmids originally present in bored phage Ql. On the other hand, phage 'p was B91 (i.e., pTi B91 and pAt B91). not found in cultures of three nopaline-degrading When strain VBC001 was plated at 27°C with strains or in strains Agrobacterium rubi, Agro- dilutions of supernatants from B91 cultures, bacterium rhizogenes, and Agrobacterium ra- small turbid plaques were obtained. These diobacter. plaques contained a temperate phage which Phage 'p is probably identical to the phage-like could be used to lysogenize strain VBC001. No particles found by Vervliet et al. in B6-806 plaques were obtained when supernatants from cultures. Indeed, it has the same morphology B91 were plated with the resulting lysogens. The (Fig. 1) and is produced by at least four strains phage released by strain B91 was different from reported to release these particles. 286 EXPERT AND TOURNEUR J. VIROL.

li OI jl:.. .. 4..;.i ;F .Ypr 0,2- p.

FIG. 1. Electron micrograph of phage 'p particles isolated from A. tumefaciens strain B91 after negative straining with 1% phosphotungstic acid. The morphology of the phage 'I particles is similar to that of the defective particles of A. tumefaciens strain B6-806 described by Vervliet et al. (22).

Phage ij seemed to have a very restricted host since they all harbored a 4 prophage. As for the range. It failed to yield plaques on any of the other strains, they were not able to adsorb the strains tested (Table 2). This was not surprising phage. The only sensitive host for phage q; was for the octopine-utilizing A. tumefaciens strains the strain which allowed its discovery, strain VOL. 42, 1982 PHAGE 4i 287 VBC001. Quite obviously, this strain had lost Therefore, we concluded that the of the prophage originally present in B91. Howev- phage 4, could be transferred by conjugation er, as shown below, the fact that this strain also together with the pTi plasmid. had lost the plasmids of strain B91 was also a Effect of pTi on phage * growth. The presence factor in its sensitivity to phage 4,. of plasmid pTi did not affect phage 4, adsorption. Presence of the pTi plasmid affects the efficien- With the partially sensitive strains described cy of plating of phage *. The first suspicion that above, as well as with the fully sensitive strain the pTi plasmid might affect the growth of phage VBC001, more than 80% of the particles were ii stemmed from attempts to cure strain B91 of adsorbed in 30 min at a bacterial concentration its prophage by superinfection in order to obtain of 3 x 108 cells per ml. new sensitive hosts for phage 4,. Two sensitive One-step growth experiments were performed strains were obtained, both of which had lost the with VBC001 and some partially sensitive pTi and pAt plasmids, like strain VBC001. Then strains which carried the pTi plasmid. As Fig. 2 other techniques were used to cure B91 of its shows, the partially sensitive strains did produce prophage. One of these techniques involved UV significant amounts of phage, but the final yield irradiation and subsequent growth at 37°C. Ap- was only 2% of the yield in VBC001. proximately 0.1% of the clones that survived Induction of phage 4, as measured by the this treatment became partially sensitive to appearance of infective centers in lysogenic bac- phage 4P. These clones were lysed at high multi- teria was also affected by the presence of the pTi plicities of infection, but they did not allow the plasmid (Table 3). In plasmid-free strains or in a formation of individual plaques. One of these strain carrying the pAt plasmid, about 10% of partially sensitive clones (VBC004) was ana- the cells spontaneously yielded infective cen- lyzed. It had lost the prophage but retained the ters, and this value could be increased to 100% two plasmids of strain B91. These results sug- by irradiating the cells with UV light (300 ergs/ gested that one or both of the plasmids inhibited 4, development. This possibility was examined further by conjugation experiments. Plasmid pTi was known to be conjugative (11, 15). We found that plasmid pAt was often but not always cotransferred with pTi. Strains B91, B B6-806, and VBC004 were used as pTi donors, and the recipient used was strain VBC003, a Rifr Strr Spcr derivative of VBC001. Octopine- catabolizing transconjugants were selected and tested for sensitivity to phage 4,. When VBC004 8- was used as the donor, the 100 transconjugants -J7. tested displayed the same partial sensitivity to phage 4,. Since 21 of these transconjugants had inherited only pTi and not pAt, this partial 0 sensitivity could be attributed to the presence of pTi. E 0 When strains B91 and B6-806 were used as 0 donors, the result was not so simple. Still, 80 to - 90% of the octopine-positive transconjugants had the same partial sensitivity to phage 4 as o VBC004; some of these transconjugants har- 0- 0cr bored pTi and pAt, whereas the others harbored , 5.

only pTi. However, two other classes of trans- EL- conjugants, approximately equal in number, were also obtained. The first class included 0 strains which displayed 4, sensitivities that were Jc- variable and intermediate between those of VBC001 and VBC004. A possible explanation -03Q 60 90 I20 150 180 for this first class is discussed below. The sec- Minutes of incubation in growth medium ond class included strains which appeared to be resistant In these trans- FIG. 2. One-step growth of phage ti. The numbers totally to phage 4,. fact, of PFU released during jp infection of strains VBC001 conjugants all contained a 4, prophage. The same (-) and VBC004 (0) were determined by plating proportion of lysogenic transconjugants was ob- dilutions of chloroform-treated infected bacteria onto tained when we used a recipient strain that was soft agar overlays seeded with the indicator strain not able to adsorb the phage (strain VBC010). VBC001. 288 EXPERT AND TOURNEUR J. VIROL.

TABLE 3. Effect of plasmid pTi on induction of phage ila Lysogenic strain Plasmid(s) harbored Spontaneous induction (%) UV induction (%) VBC009 5-10 80-100 VBC231 10 80-100 VBC201 pAt B91 6 75-100 B91 pTi B91, pAt B91 0.5 10 VBC013 pTi B91 0.5 10 VBC203 pTi B91, pAt B91 0.5 5 VBC238 pTi B91, pAt B91 1 5 B6-806 pTi B6-806, pAt B6-806 0.05-0.1 1 VBC015 pTi B6-806 2 3 VBC050 pTi B6-806, pAt B6-806 0.1 3 a The results are the percentages of cells that produced infective centers.

mm2). In strains carrying a pTi plasmid, the clones from uninfected cultures were replicated percentage of infective centers ranged from less onto MM agar. than 1% in the absence of induction to 1 to 10% To investigate the possibility that the apparent after UV irradiation. mutagenic effect of phage in fact might have The data described above clearly indicate that resulted from the selective lysogenization of the presence of a pTi plasmid inhibits the intra- preexisting mutants, we used the conditions for cellular development of phage ti after infection, lysogenization described by Taylor (20). The as well as after induction. number of preexisting spontaneous mutants was Phage * induces mutations in the host chromo- reduced by subculturing strain VBC001 several some. When phage +-infected cultures of strain times in MM before infection. Lysogenization VBC001 were plated onto MM agar, slowly was then carried out on NB agar by direct growing clones were observed frequently. After spreading of a mixture of about 103 bacteria in isolation on complete medium, these clones logarithmic phase (multinucleated) with about were tested both for lysogeny and for the ability 108 PFU. Each colony was scored for its content to grow on MM. Several of the clones were of wild-type and auxotrophic bacteria. About found to be auxotrophic mutants requiring spe- 35% of the colonies tested were mixed clones cific nutritional factors. These auxotrophic mu- containing a majority of wild-type cells and 1% tants were always lysogenic for tj. or more auxotrophic mutants. We found no This infection experiment was repeated sever- colony that was composed of mutants exclusive- al times, and on the average, 1 to 3% of the ly, as would have happened if the mutants had survivors carried at least one auxotrophic muta- existed before infection. tion. As Table 4 shows, the different mutants The mutagenic effect of phage ij occurred isolated from a single culture usually had differ- during the establishment of lysogeny but did not ent requirements and, therefore, came from in- occur once the prophage was repressed. Indeed, dependent mutational events. Some mutants re- the frequency of auxotrophic mutants was less quired several nutritional factors and thus were than 10-4 in the cultures of all phage t+ lysogens deficient in more than one biosynthetic path- tested. way. Many of the mutations were leaky. No Phage * induces mutations in the octopine pTi auxotrophic mutant was found when 2,000 plasmid. Phage could also induce specific

TABLE 4. Auxotrophic mutations induced by phage ii No. of survivors No. of auxotrophic Infected strain Culture screened clones Phenotypes characterizeda VBC001 1 300 6 Tyr- Phe-, Met- Chol-, Hyp- 2 400 10 Asp- Pdx- Chol-, His- Pro-, Thi-, Gly- 3 200 3 Thi- His- Thio-, Ilv Control 1,000 0 VBC006 4 300 3 Thi- Met-, Asp- Nic- Pro- 5 300 5 Gly- Thi-, Asp- Ile-, llv- Hyp-, Ile- Trp- Control 1,000 0 a The phenotypes indicated are the phenotypes which could be characterized. For the other auxotrophic clones, the multiplicity of the requirements, the leakiness, and the instability of the mutations complicated characterization. The designations used are those recommended by B. Bachmann, except for Chol, Hyp, Nic, and Thio (choline, hypoxanthine, nicotinic acid, and thiosulfate, respectively). VOL. 42, 1982 PHAGE ji 289 mutations on the pTi plasmid. We looked for any carry a mutation on the pTi plasmid. However, alteration of the following two genetic traits conjugation experiments devised to test this carried by this plasmid: the ability to catabolize possibility gave ambiguous results. About 10% octopine (Occ+ phenotype) (2, 16) and the abili- of the Occ+ transconjugants were indeed Vir- ty to induce tumors in plants (Vir+ phenotype) as expected, and the others were virulent. The (21, 24). Before infection, strain VBC004 was Vir- character is probably carried by the pTi subcultured three times in a synthetic medium plasmid since it can be transferred with this containing octopine as the sole nitrogen source. plasmid, but the high reversion of this character Lysogenization was carried out by direct during conjugation remains to be explained. spreading onto NB agar, as described above. Reversion of *-induced mutations. The stabil- Of the 500 colonies tested, 6 contained some ity of the mutants was highly variable. With very percentage of Occ- mutants. No colony com- few exceptions, all auxotrophic markers, taken posed of mutants exclusively was observed. The separately when coexisting in a same strain, search for Vir- mutants was performed as fol- spontaneously reverted to prototrophy, with fre- lows. A total of 100 colonies from the original quencies ranging from 10-3 to 10-8 (Table 5). lysogenization plate were reisolated, and 100 Reversion frequencies could not be estimated clones, each stemming from a different isolate, for the Vir- character, but a few Vir+ revertants were tested for the ability to induce tumors on spontaneously accumulated in some cultures of tobacco plants. Two Vir- mutants were ob- strain VBC206. All of the prototrophic or Vir+ tained in this way. The six Occ- and two Vir- revertants tested still contained prophage *. mutants were all lysogenic for phage 'p. Two of Conversely, we investigated whether pro- the six Occ- strains were Vir- and in fact had phage curing might be accompanied by rever- lost the pTi plasmid. Two others were unable to sion of the mutations (Table 5). Of the 10 mu- use either arginine or octopine as a nitrogen tants tested, 2 could not be cured. Three clones source. This phenotype (Arc-) resulted from were cured at a very high frequency and con- chromosomal mutations (10). The last two Occ- comitantly had reverted to a wild-type pheno- strains still contained the pTi plasmid, were able type. The five remaining mutants were cured at to grow on arginine as a nitrogen source, and a low frequency, and generally the cured clones therefore almost certainly carried mutations in retained the mutant phenotype. In these cured the pTi plasmid. The two mutants isolated as clones, the mutations reverted at the same fre- Vir- were still Occ+. They were expected to quency as they did before curing.

TABLE 5. Reversion and prophage curing in different mutant strains Frequency of of No. of cured No. of revertants Strain Phenotype Reversiontested spontaneous curedFrequencyclonesb clones analyzed among cured reversiona clonesc VBC120 Tyr- Phe- Tyr Phe 10-5 <0-4 1 0 <10-4 1 0 VBC121 Thi- His- Thio- Thi His Thio 10-4 0.8 50 50 VBC122 His- Pro- His 10-8 <10-4 0 Pro <10-8 VBC123 Thi- Thi <10-8 <10-4 1 0

VBC130 Ile Try Ile 10-7 x 1 0-4 2 0 Try 1- VBC131 Asp- llv- Asp 10-7 <10-4 liv 10-4 1 0 VBC132 Gly- Thi Giy <10-8 d Thi 10-3 ND liv 10-8 ND VBC133 Ilv- Hyp- Hyp103N VBC203 Arc- ND 0.3 50 50 VBC239 Occ- Occ 10-7 10-4 5 1 VBC206 Vir- ND 0.5 50 50 VBC216 Vir- ND 2 x 10-4 4 0 VBC009 Prototroph 5 x 10-4 a All of the revertants tested were still lysogens. b Curing was performed by UV irradiation, followed by subculturing at 37°C. c No revertants means that the cured clones had the same phenotype as the original mutant. When revertants were found, they had the same phenotype as the nonlysogen from which the mutant had been derived. d ND, Not determined. 290 EXPERT AND TOURNEUR J. VIROL. DISCUSSION ly, it is possible that prophage 4 exists as a A. tumefaciens strain B91 harbors a prophage plasmid and can be mobilized by pTi during which we called psi. The active phage particles conjugation. This question of the state of the which are released by B91 spontaneously or prophage inside the cell, which is still unsolved, after UV induction have a very narrow host is intimately related to the question of the mech- range. The only sensitive hosts which we could anism whereby 4 induces mutations in the DNA identify among various strains ofA. tumefaciens of its host. were derivatives of B91 which had lost both the When cultures of the sensitive host were 4, prophage and the pTi plasmid. Several factors infected with 4, 1 to 3% of the survivors carried appear to limit the host range of phage 4,. First, auxotrophic mutations. The mutants were all 4 most of the octopine-utilizing strains ofA. tume- lysogens, and each one carried a different muta- faciens are immune to 4, because they harbor a tion, which could be located on the chromosome prophage which is very similar, if not identical to or on the pTi plasmid. This phenomenon is very it. Second, as discussed below, the octopine pTi reminiscent of the phenomenon observed upon plasmid inhibits the development of phage 4,. infection of with bacteriophage Finally, many strains of A. tumefaciens, A. Mu (20). In fact, since Mu was recently shown to radiobacter, A. rubi, and A. rhizogenes are not infect A. tumefaciens (14), we might have won- able to adsorb the phage. Among the strains dered whether 4, was not actually Mu. This is harboring 4 or a 4,-related prophage was B6-806, clearly not the case. Indeed, *4 does not grow in which had been shown previously to release E. coli, its morphology is different than that of "phage-like particles" (22). These particles Mu (Fig. 1), and the restriction patterns of 4, and were almost certainly phage 4,, for which no Mu DNAs are very different (unpublished data). sensitive host was known at the time. Even though 4, is different from Mu, it might still The production of 4, particles is strongly re- have mutagenized by the same process (i.e., by duced in cells which harbor the pTi plasmid. integrating its chromosome more or less ran- This effect is observed after induction, as well as domly into host DNA). However, the charac- after infection, and therefore presumably con- teristics of 4-induced mutations in A. tumefa- cerns the intracellular development of the phage ciens differ in several respects from those of Mu- rather than its adsorption or the injection of its induced mutations in E. coli. The former are DNA. Examples of plasmids that inhibit phage often leaky and revert at detectable, if not very growth are not unusual (8, 9, 12, 18, 26). The high, frequencies, whereas the latter are usually gene(s) responsible for inhibiting 4 growth could very tight and do not revert readily (5, 6, 13). be useful in the genetic analysis of plasmid pTi. Revertants of Mu-induced mutations have been Mutations that probably affect the gene(s) in obtained in two steps; the first step is the question were obtained during the course of this appearance of the so-called "X mutations" on work. Indeed, when pTi was transferred by the prophage (4). The revertants obtained in this conjugation from a 4 lysogen into a plasmid- way always have lost the Mu prophage. This free, nonlysogenic strain, about 10% of the was not the case with the revertants of 4,- Occ+ transconjugants were more sensitive to 4, induced mutants, which were still lysogens. Fi- than is usual in a strain harboring pTi. There- nally, 4-induced mutants often retained the mu- fore, the plasmid present in these transconju- tant phenotype after they were cured of their gants had been altered in its ability to prevent 4 prophage. Since these cured derivatives still growth. The frequency at which such alterations reverted at the same frequency, they had not occurred was obviously quite high for a muta- acquired a deletion which might have resulted tional event. However, the appearance of 4,- from prophage curing. These results indicate sensitive transconjugants was observed only that the mutagenic effects of 4, and Mu may not when the donor was a *4 lysogen. Therefore, it is have the same cause. The mutations induced by possible that the alterations were induced by 4, 4 may result not from the insertion of 4, DNA which is mutagenic (see below) and may prefer- into various genes but from the action of a entially alter plasmid genes during conjugation. diffusible product encoded by the 4 genome. In the experiments described above, about 5% This would account for all of the observations of the transconjugants which received pTi also reported here but one, namely, that prophage acquired a 4, prophage. This was not due to curing was sometimes accompanied by rever- infection of the transconjugants by phage re- sion to a wild-type phenotype. The hypotheses leased by the donor, since the same phenome- required to explain this fact, as well as other non occurred when the recipient was 4 resistant. facts, would be very speculative. For instance, it One interpretation of this result is that the is possible that 4 carries a prophage was integrated in the pTi plasmid of which can be transposed with or without a gene the donor strain and was induced at a high that controls its excision. Further understanding frequency during conjugal transfer. Alternative- of the mutagenic effect of 4 will require a study VOL. 42, 1982 PHAGE t, 291 of the state of the prophage inside cells. Such 12. Hakura, A., N. Osutji, and Y. Hirota. 1964. A temperate work is in progress. phage specific for female strains of Escherichia coli K12. J. Gen. Microbiol. 35:69-73. 13. Howe, M. M., and E. G. Bade. 1975. 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