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Pseudolysogeny of Azotobacter phages
VIROLOGY
102,
26’7-277 (1986)
Pseudolysogeny
of Azotobacter
Phages
BETTY J. THOMPSON,’ ESTEBAN DOMINGO,* AND ROBERT C. WARNER3 Department
of Moleculur
Biology and Biochemistry, University Irvine, California 92717
of Califbmia,
Accepted December 21, 1979
The establishment of a pseudolysogenic state accompanied by a phenotypic conversion in Azotobacter vinelundii strain 0 by phages A14, A21, A31, and A41 has been identified. Host cells can be recovered from the pseudolysogens by cultivation in phage-specific antiserum. Pseudolysogens continually give rise at a low rate to phage as a result of the occasional initiation of a lytic burst. As a result of the establishment of the pseudolysogenic state the host cells lose their polysaccharide coat, become flagellated and motile and acquire a yellow pigmented appearance. The four phages, although they differ serologically and molecularly, give rise to converted states that are indistinguishable except by the identification of the phage that is produced by it. On repeated subculturingeach of the pseudolysogens will give rise to a stable or permanently converted cell that has the phenotype of the pseudo-lysogen, but from which it is no longer possible to obtain either host cells or phage. INTRODUCTION
mented character. These changes are such that the converted cells can be distinguished The molecular characteristics of a series from those of the host either by microscopic of Azotobacter phages, first isolated by examination of the cells or by inspection Duff and Wyss (1961), have been studied in this laboratory (Domingo et al., 1972; of the colonies. The acapsulated cells were Thompson et al., 1973). One phage from first thought to-be contaminants, but have each of the four serological groups defined been shown to be associated with a pseudoby Duff and Wyss (1961) was examined. lysogenic state. The existence of pseudolyThese phages, A14, A21, A31, and A41, sogeny in the acapsulated, phage-resistant were diverse with respect to their size, cells and the interrelations of the different morphology, serology, and the properties phages producing it are described here. The distinction made in early studies of of their DNAs. There are indications that lysogeny between characteristic examples these phages are temperate as judged by of the phenomenon and variations on it plaque morphology (Duff and Wyss, 1961) classified as pseudolysogeny or the “carrier” and reports of transduction (Wyss and state were reviewed by Lwoff (1953). Like Nimeck, 1962), but lysogeny of them has true lysogeny, pseudolysogeny provides not been studied in detail. an alternative response to a lytic infection During work with these phages we became aware of a striking pseudolyso- and results in a latent state characterized genie conversion phenomenon as a result by resistance to superinfection and by the of which the cells of the host, Azotobacter potential of yielding phage. A common vinelandii strain 0, lose their polysaccha- distinguishing feature was the possibility ride coat, become flagellated and motile of recovering uninfected host cells from phage-infected pseudolysogens. Recent disand the colonies acquire a yellow-pigcussions of more thoroughly studied examples, particularly by Barksdale and ’ Present address: Bruce Lyon Memorial Research Arden (1974), Baess (1971), Romig (1968), Center, Children’s Hospital, Oakland, Calif. 94709. and Bott and Strauss (1965) make it clear *.Present address: Instituto de biologia de1 Desarthat phage-host interactions produced by rollo, Velazques, Madrid, Spain. 3 To whom requests for reprints should be addressed. several disparate mechanisms have been 267
0042-6822/80/060267-ll$OZ.~/O Copyright 0 1980 by Academic Press, Inc. AU rights of reproduction in any form reserved.
THOMPSON,
268
DOMINGO,
classified together as pseudolysogeny. Hayes (1968) distinguished two general types. One, in which host cells and vegetative phage coexist in culture because of the development of a partial resistance of most or all of the cells to infection, is typified by the T7-Shigella interaction studied by Li et al. (1961). In the second type a latent form of the phage exists inside the cell as a carrier and does not necessarily give rise to a lytic infection. In this case, because there is a limited number of particles, host cells without carrier particles may be segregated at cell division as found by Bott and Strauss (1961). However, there are situations in which the mechanisms of the first type include a carrier state in the sense used by Hayes to apply to the second (Barksdale and Arden, 1974). In view of this and of the apparently unique characteristics of each case of pseudolysogeny it does not seem useful to adhere to the distinction made by Hayes. Regardless of the mechanism of establishment of pseudolysogeny, cultivation in the presence of phage antiserum will result in an elimination or decrease in the pseudolysogen and an accumulation of or a replacement by host cells. This response to antiserum serves as an operational definition of pseudolysogeny. It is emphasized by all of the authors quoted above.
AND WARNER
Polysaccharide Depolymerase Assay
the outflow time in a semimicro (l-ml) Cannon viscometer in a water bath regulated at 33 + 0.01”. The polysaccharide was prepared by the method of Cohen and Johnstone (1964) from a culture of A. vinelandii 0. It was dissolved in the medium (DW) described by Duff and Wyss (1961) and was heated to 85” for 15 min to inactivate traces of depolymerase that it contains. It was centrifuged and diluted to a concentration such that the further dilution with 0.09 vol of cell suspension or extract would result in a viscosity relative to that of the DW medium of 2.5. Suspensions of washed cells were prepared having an A,,, = 4.6. Samples were sonicated and centrifuged. An aliquot of 100 ~1 of suspension or of supernatant solution was added to 1100 ~1 of polysaccharide solution, 1 ml of the mixture was placed in the viscometer and the outflow time was determined periodically for 5 to 16 hr. The initial relative viscosities were close to 2.5, but in order to facilitate comparison between experiments all of the relative viscosities for each solution were normalized to an initial nrel of 2.5 by multiplying by 1.5 divided by the specific viscosity at zero time. This procedure is equivalent to assuming that the specific viscosity is proportional to the concentration of polysaccharide over the small difference in concentration among the various experiments. The normalization factor for sonicates was never out of the range of 0.97 to 1.0 and there was no detectable effect on the initial slope of the decrease in viscosity on which the enzyme units depends. Some determinations were also made by measuring the release of reducing groups during the depolymerase reaction by a modification of the method of Somogyi (Dische, 1962). For these assays the enzyme was precipitated from a lysate or sonicate by ammonium sulfate at 75% saturation. This crude fraction from a lysate was also used in the experiments in which the host cells were treated with enzyme.
The enzyme was determined by following the decrease in viscosity of a solution of the partially purified capsular polysaccharide. Viscosities were determined by measuring
The negative staining procedures for the examination of the bacteria and its flagella
MATERIALS
AND METHODS
Phages A14, A21, A31, and A41 have previously been described along with the media and methods used in their cultivation (Domingo et al. , 19’72; Thompson et al., 1973). They were grown on A. vinelandii 0 (ATCC 12518). Phage PAV-1 and an acapsulated host, A. vinelandii OP (ATCC 13705) (Bush and Wilson, 1959) were obtained from Dr. George J. Sorger. This phage was isolated by Sorger and Trofimenkoff (1970) and its properties have been described by Chum1 et al. (1930).
PSEUDOLYSOGENY
269
OF Azotobocter PHAGES
by electron microscopy were those previously employed (Cordon, 19’7.2).Sodium phosphotungsate (pH 5.5) and uranyl acetate stains were used. The presence of a polysaccharide capsule was determined by examination of the bacteria by light microscopy in a dilute suspension of carbon particles. Induction Attempts to induce the converted cells, referred to below as PC cells, were carried out according to the procedure of Jacob and Wollman (1953). A log phase culture was divided into 10 separate aliquots and irradiated in duplicate. The survival rate of the irradiated cultures varied from 1 to 90%. The bacteria were grown overnight. One of the cultures in a pair was plated for colony formers and titered for free phage. The duplicate was treated with chloroform and then titered for free phage. Mitomycin C (Calbiochem B grade) induction was tested according to the procedure of Thomson and Woods (1974). RESULTS
Isolation of Phge-Resistant lysogeny
Cells: Pseudo-
Colonies of yellow, motile cells were first noted as an overgrowth on confluent plaques from lysates of several of the phages. The origin of these cells was examined using phage A21. It was found that when samples of an infected culture were plated as a function of time after infection that a substantial fraction of the cells would yield yellow colonies. The results of such an experiment are shown in Fig. 1. It can be seen that the number of yellow cells at 30 min was about 0.1 the number of host cells at the time of infection and then decreased to about 0.001 at the onset of lysis. Since essentially all of the cells that yielded yellow colonies at 30 min would have lysed had they remained in liquid culture, it is evident that conversion to the yellow form was promoted by plating on a solid medium. Yellow colonies of the same type were present in a great majority when innocula from the turbid zone of a plaque were
0
2 Time,
4 hours
6
FIG. 1. Course of infection of A. vinelandii 0 by phage 21. The total cell density was followed by determination of A,,, (A) and the phage production by determination of PFU (A). At intervals samples were taken and plated for colonies. Those of the host type (0) can no longer be detected when cells are plated ‘/a hr after infection. At this time the only colonies are those having the characteristics of the pseudolysogen and their time course is shown by open circles (0). Colonies of this type cannot be detected before infection. The m.o.i. in this experiment was 10. Similar results were obtained at m.o.i.‘s of 1, 5, 50, and 500.
plated. Numerous isolates from the yellow colonies were selected and propagated. The colonies that survived infection after plating on solid media were colonies with converted characteristics. The cells were acapsulated and motile. If a single colony was subcultured and replated after washing to remove phage, three different types of colonies were seen: 1. Host. The colonies of the host were white, mucoid, opaque, entire, and about 3 mm in diameter. These cells were sensitive to phage. Treatment with phage specific antiserum produced no change in the characteristics. All cells possessed a polysaccharide coat and were nonmotile (Fig. 2A). They could not be distinguished from the parent strain 0 in these respects or in their behavior on reinfection with phage. 2. Pseudolysogen. These colonies were the most prevalent and had the same characteristics as the original single cell isolate
270
THOMPSON, DOMINGO, AND WARNER
FIG. 2. Electron micrographs of Azotobacter winelandii Pseudolysogen of phage A21. Uranyl acetate stain.
0. (A) Host cell. Uranyl acetate stain. (3)
PSEUDOLYSOGENY
OF Azotobactw
FIG. 2. -Continued.
PHAGES
271
272
THOMPSON,DOMINGO,ANDWARNER
from the infection. They were resistant to phage. The colony was a mottled combination of clear yellow and opaque mucoid white with an irregular edge. The mottled appearance was a result of the presence of two types of cells evidently formed by segregation of carrier particles during the growth of the colony. Part of the colony had the same appearance as the colonies of the host and part had a clear, yellow character and consisted of motile, acapsulated cells. Growth in phage-specific antiserum when the colony was subcultured resulted in an increase in the ratio of phagesensitive capsulated nonmotile cells to the acapsulated, motile ones. 3. Permanently converted (PC) cells. The colonies of these cells were clear yellow, entire, and about 0.1 the size of the other types of colonies. The cells were acapsulated, motile and resistant to phage infection (Fig. ZB). Treatment with phage specific antiserum produced no change in the cells. The behavior of the second type of cells identify them as pseudolysogens containing a phage genome in carrier form. The carrier may be segregated on cell division so that daughter cells both with and without carrier are formed. The latter are host cells and are sensitive to phage infection. The segregation can be observed on a plate to take place during the growth of a colony initiated by a single cell. The carrier may also occasionally initiate a lytic infection and produce a burst of phage. As a result of these processes the propagation of a pseudolysogen for several transfers in phage specific antiserum will result in a culture that is chiefly or entirely sensitive cells of the host type. The rate at which this occurs is, in general, variable and has been found to be much greater for some isolates than for others. Some are relatively stable, segregate host cells only occasionally and maintain their converted characteristics over many generations. The kinetics of the segregation process is examined in the accompanying paper (Thompson et al., 1980). Growth without antiserum resulted in a mixture of cells as described above. A culture that was washed and resuspended in fresh broth had a low phage titer that
increased to lo6 PFU ml-’ after several hours of incubation at which time relatively few host cells were present. The high titer of phage can be accounted for by initiation of a lytic response in a fraction of the carrier cells and infection of continually segregating host cells. The colony morphology of pseudolysogens reflects an interaction among resistant and sensitive cells and phage. As the colony grows after a one-day incubation, pseudolysogens arise in the center and after 2 days of incubation host cells begin to grow at the periphery. Only the pseudolysogen is able to survive in the center of the colony where the phage concentration is the highest. The pseudolysogenic behavior of A21 is reflected in its plaque morphology. Plaques consist of a small, central clear area surrounded by a turbid halo or zone. If growth of the plaque is allowed to continue, the lawn beyond the zone becomes partially clear. The zone itself consists almost entirely of pseudolysogens. This. type of morphology could result from an effect of an increasingly high concentration of phage on the initiation of pseudolysogeny and the diffusion of some phage through the turbid zone or its production in the zone by a low level of lysis resulting in a second zone that is clear because of predominance of lytic infections. Permanent Conversion At some frequency dependent on unidentified conditions of culture a pseudolysogen of A21 will be transformed to a different phage-resistant cell which produces the colonies described as type 3 above. It differs from the pseudolysogen in its inability to segregate colonies having the host morphology. When a pseudolysogen was cultured repeatedly either in the presence or absence of antiserum and plated at intervals, the earliest PC colonies to arise usually produced some phage. If cells from them were cultured after washing with antiserum, phage was formed at a very low level. Such phage was identified as A21 by its sensitivity to A21 antiserum and the buoyant density of its DNA in CsCl
PSEUDOLYSOGENYOF Azotobuctm PHAGES of 1.717 g ml-‘. After several passages these cells lost their ability to produce any detectable phage. It is not clear whether the initial low production of phage was a result of a low level of spontaneous lysis or whether it was due to a contamination with pseudolysogen. In an examination of colonies from antiserum-treated cultures of such cells, no host colonies were seen among 20,000 examined. Examination of these cultures in the presence of carbon particles by light microscopy revealed only acapsulated, motile cells. Attempts to induce phage production by uv exposure or treatment with mitomycin C as described in the methods section were unsuccessful. We have referred to cells of this type as permanently converted or PC cells because they do not fit into any established category. Some additional information on their formation from pseudolysogens is given by Thompson et al. (1980). Converted Characteristics All of the pseudolysogen and PC cells had all of the converted characteristics: yellow pigment, absence of polysaccharide coat, and presence of flagella. The PC cells lacked the properties of the pseudolysogen of segregation of host cells and of phage production. Additional study was made of the presence of flagella and of aspects of the absence of the polysaccharide coat. Flagella
Electron micrographs of negatively stained cells are shown in Fig. 2. A host cell obtained from a log phase culture and shown in Fig. 2A has no detectable flagella. The PC cell shown in Fig 2B is flagellated in a peritrichous arrangement, The movement of these cells is consistent with this arrangement of flagella. The pseudolysogen was similarly flagellated. No flagella have been detected in the host cells under any conditions or stages of growth.
Azotobacter
Polysaccharide
Coat
In a normal infection with phage A21 the cells retain their polysaccharide coat until lysis begins at about 7 hr. Partially
273
purified polysaccharide depolymerase (see Materials and Methods) was used to determine the role the polysaccharide coat plays in phage infection. A culture of host was divided and one portion was treated with the enzyme for 30 min at 33”, the cells were washed and resuspended in fresh medium. This treatment completely removed the capsule as determined by microscopic examination in the presence of carbon particles. The other portion was treated similarly, omitting addition of the enzyme. Phage A21 at a multiplicity of infection of 2 was added to the cells and allowed to adsorb for 30 min. Adsorption was determined by centrifuging and assaying the supernatant solution for free phage. In the enzyme-treated culture all of the added phage were recovered while in the control the phage titer dropped to 1% of that added. As expected from the adsorption results no significant phage yield was obtained from the enzyme-treated culture. The capsule is evidently needed for adsorption and its absence from the pseudolysogen and the PC cells provides a sufficient basis for the resistance of these cells to infection. This result is in direct contradiction to the results of Eklund and Wyss (1962), who reported that removal of the capsule increased the rate of adsorption. It has been reported by Eklund and Wyss (1962) and by Barker et al. (1968) that the depolymerase is absent from A. vinelandii 0 and that its production during infection is a result of a phage-specified enzyme. We investigated the presence of the depolymerase in the several cell types described above and its production during infection because of the possibility that the mechanism of this feature of conversion might be clarified. In other phage-host systems with characteristics similar to those of A21A. vinelundii 0 there has also been a failure to detect the depolymerase in the host cells (Adams and Park, 1956; Bartell and Orr, 1969; Sutherland and Wilkinson, 1965; Mare and Smit, 1969; and others). On the other hand Chakrabarty et al. (1967) have shown clearly that the depolymerase of Pseudomonas putida is a host specified enzyme that is evidently induced many-fold by phage infection.
274
THOMPSON, DOMINGO, AND WARNER
1.0 0
I
2 Time , hours
3
4
1.0 0
I Tim.
2 hovrs
3
4
FIG. 3. Change of the viscosity with time of solutions of the capsular polysaccharide ofA. winelundii 0 as it is hydrolyzed by a depolymerase from various sources. (A) Curves for the addition of the indicated volume of a filtered lysate from the infection of A. winekrndii 0 with phage A21. The initial slopes ofthese curves were used, as explained in the text, to define a unit of enzyme activity. (B) Curves for addition of centrifuged sonicates of cultures of several cell types. Curves a and bare controls for no addition and addition of a sonicate of E. coli. Curve c is for the PC cells; curve d, for the host, A. winehndii 0; and curve e, for the pseudolysogen. In all cases 100 ~1 of a sonicate of a suspension of cells having an A,,, = 4.6 was added to 1100 ~1 of polysaccharide solution.
The concentration of depolymerase was determined by following the decrease in viscosity of a solution of the capsular polysaccharide when it is hydrolyzed by the enzyme. The greatest concentration of the enzyme is present in a lysate of infected cells and such a lysate was used to establish a relative enzyme unit. The viscosity curves
for a series of dilutions of a lysate are shown in Fig. 3A. The initial slopes are proportional to the concentration of enzyme as shown by the average value for the slopes of the five curves of 3.2 + 0.4 per unit of enzyme. The slopes are given in qrel units per hour and one unit of enzyme is taken as the amount of enzyme in 1 ~1 of lysate of cells having an Asw = 1. The data on TABLE 1 sonicates of the several cell types are shown CONCENTRATION OFDEPOLYMERASE IN SONICATES in Fig. 3B. There was no change in viscosity ANDA LYSATEFROMDIFFERENTCELLTYPES of the control containing only the heated polysaccharide and that resulting from adding an E. coli sonicate was barely Method detectable. The concentration of enzyme Viscosity Reducing calculated from the initial slope of ‘each (units per group of the other curves is given in Table 1. A AEw unit of (relative clearly detectable amount of enzyme is Source cell density) units) present in each type of cell although at a Lysate loo” 100 much lower level than in the lysate from a A. vinelandii 0 0.021* 0.24 phage infection. The results were confirmed Pseudolysogen 0.16 using sonicates from several different prepPC cells 0.0063 0.09 arations of cells. Similar results were E. coli control 0.0009 Not obtained using suspensions of cells instead detectable of sonicates. In these cases it is not clear a Baaed on one unit/p1 of lysate derived from cells whether the viscosity decrease resulted having A,,, = 1. b (Initial slope)/(3.2 x 4.6). The factor 3.2 was from secretion of the enzyme or from the gradual death and lysis of cells in the culobtained from Fig. 3A as explained in the text; 4.6 = Asw of the cell suspensions from which the ture. The results with cells of A. winelandii 0 sonicates were obtained. were complicated by the contribution to the
PSEUDOLYSOGENY
OF Azotobactw
viscosity of the polysaccharide capsule. As the cells grew, the viscosity increased slightly, but subsequently decreased. Some determinations of the concentration of the depolymerase that hydrolyzes the capsular polysaccharide were made by calorimetric detection of reducing groups liberated in the reaction. These results are included in Table 1. We conclude that depolymerase is present in all three types of cells. Its presence in the uninfected host indicates that it is not specified by the phage. Production of the enzyme at a high level appears to occur only during a lytic infection. The greater activity (Table 1) of depolymerase in the sonicate from the pseudolysogen may not indicate an intrinsically higher activity in these carrier cells, but may result from the occasional induction of a lytic infection in the pseudolysogen (Thompson et al., 1980). Experiments with crude lysates of the type we have done cannot eliminate the possibility that the induced enzyme is different from a depolymerase present in the host cells, although this possibility was eliminated in the case of Pseudomonas putida (Chakrabarty et al., 1967). In spite of the evident requirement of the polysaccharide coat for adsorption, phage is not inactivated by mixing it with polysaccharide. Pseudolysogeny by other Axotobacter Phuges The work described above was carried out with phage A21. Less extensive observations on phages A14, A31, and A41 showed that they produce the same pseudolysogenic conversion of A. vinelandii 0 and pseudolysogens and PC cells similar to those of A21. This was an unexpected result since the four phages are molecularly and serologically unrelated (Domingo et al., 1972; Thompson et al., 1973). The pseudolysogen formed by infection by any one of the four phages showed the same properties with respect to pigmentation, absence of polysaccharide coat, presence of flagella, colony morphology, and resistance to superinfection by any one of the four phages. The four pseudolysogens could be distinguished only by the ability of each to yield the same
PHAGES
275
phage that produced it. We have been unable to find a means for distinguishing the four PC cells from each other. A PC culture isolated after infection by each phage was tested for infectability by and adsorption of each of the four phages. In no case was either detected. We have been unable to obtain clear plaque mutants of A21 in spite of considerable effort. Phage PAV-1 Another Axotobacter phage, PAV-1, described by Sorger and Trofimenkoff (1970), was recently studied in this laboratory (Chum1 et al., 1980). It was found to be molecularly and serologically unrelated to A14, A21, A31, or A41 and in addition to have a different host range from that shared by the four A phages. PAV-1 can infect A. vinelandii 0 and A. vinelandii OP and gives plaques having turbid halos. A. vinelandii OP lacks the polysaccharide coat of A. vinelandii 0 and in pigmentation and flagellation resembles the PC cells derived from A. vinelandii 0. PAV-1 also infects A. vinelandii 0 and produces the same pseudolysogenic conversion as the phages of the A series. In contrast A. vinelandii OP is not infected by any of the A phages. However, PAV-1 can infect all of the pseudolysogens and PC cells of the A phages. DISCUSSION
The Axotobacter phages of the A series interact with their host, A. vinelandii 0, to produce a pseudolysogeny with several unique characteristics. (1) The pseudolysogens acquire a converted phenotypepigmentation, flagellation and loss of polysaccharide capsule -which makes it possible to distinguish them from the host by examination either of the cells or their colonies. (2) The pseudolysogen can subsequently give rise to another type of cell that retains the converted phenotype, but which can no longer either segregate host cells or produce phage. We have termed this cell a permanently converted or PC cell. (3) Phages from four different groups of the A series, unrelated serologically or
276
THOMPSON, DOMINGO, AND WARNER
molecularly, produce pseudolysogens having the same converted phenotype. A fifth phage, PAV-1, which in addition has a different host range, produces a similar pseudolysogen. The phage-resistant cells isolated from infected cultures were originally thought to be mutants or contaminants. Both of these possibilities were eliminated by the repeated isolation of a large fraction of the cells of an infected culture as the resistant pseudolysogens (Fig. 1). In addition they constitute the major number of colonies when cells from the turbid area of a plaque are plated. We have classified them as pseudolysogens by the criteria that they yield host cells by segregation when they are cultivated in antiserum and that they continually produce a low level of phage. The existence of a carrier state is indicated by the ability of such a pseudolysogen to maintain its phenotype through several generations and then segregate host cells. The cell type to which pseudolysogens give rise after repeated transfers we have termed a PC cell. Some information on the nature of this change is given in the accompanying paper (Thompson et al., 1980), but its molecular basis is still obscure. It was first thought to be a form of lysogeny, but since such phage production as initially occurred was always lost and no means of induction was found, this is untenable. More importantly, we have found (Thompson et al., 1980) that no more than a small fraction of a phage genome can be detected in the cell by hybridization methods. The polysaccharide depolymerase does not appear to be present in significantly different amounts in the three cell types. Our results are similar to those of Chakrabarty et al. (1967) in that they indicate that the depolymerase is an enzyme of the host and not of the phage. Its induction to a high concentration is characteristic of a lytic infection. It thus appears that the activity of this enzyme is unrelated to the absence of the polysaccharide capsule in the pseudolysogen and the PC cells. The polysaccharide capsule appears to be essential for phage adsorption although we cannot eliminate the possibility that the crude enzyme preparation used to remove it
produced other changes in the cell wall or membrane than removal of the coat. ACKNOWLEDGMENT This investigation was supported by NIH Research Grant CA12627 from the National Cancer Institute. REFERENCES ADAMS, M. H., and PARK, B. H. (1956). An enzyme produced by a phage-host cell system. II. The properties of the polysaccharide depolymerase. Virology 2, 719-736. BAESS, I. (1971). Report on a pseudolysogenic mycobacterium and a review of the literature concerning pseudolysogeny. Acta Pathol. Microbial. Stand. B 79,428-434.
BARKER, T., EKLUND, C., and WYSS, 0. (1968). Physical differences of virus-associated depolymerases. Biochem. Biophys. Res. Commun. 30, 794-710. BARKSDALE, L., and ARDEN, S. B. (1974). Persisting bacteriophage infections, lysogeny, and phage conversions. Annu. Rev. Microbial. 28, 265-299. BARTELL, P. F., and ORR, T. E. (1969). Origin of polysaccharide depolymeraae associated with bacteriophage infection. J. Viral. 3, 290-296. BUSH, J. A., and WILSON, P. W. (1959). A non-gummy chromogenic strain of Azotobacter &eland% Nature (London) 184, 381. CHAKRABARTY, A. M., NIBLACK, J. F., and GUNSALUS, I. C. (1967). A phage-initiated polysaccharide depolymerase in Pseudomonas putida. Virology 32, 532-534. CHUML, V. A., THOMPSON,B. J., SMIILEY,B. L., and WARNER, R. C. (1980). Properties of Azotobacter phage PAV-1 and its DNA. Virology 102,262-266. COHEN, D. H., and JOHNSTONE,D. B. (1964). Extracellular polysaccharide of Azotobacter vine&&i. J. Bacterial.
88, 329-338.
DISCHE, Z. (1962). Color reactions based on reducing properties of sugars. In “Methods in Carbohydrate Chemistry” (Whistler, R. L., and Wolfrom, M. L., eds.), Vol. 1,512-514. Academic Press, New York. DOMINGO,E., GORDON,C. N., and WARNER, R. C. (1972). Azotobacter phages: Properties of phages A12, A21, A31, A41 and their constituent DNAs. Virology
49, 439-452.
DUFF, J. T., and WYSS, 0. (1961). Isolation and classification of a new series ofAzotobacter bacteriophages. J. Gen. Microbial. 24, 273-289. EKLUND, C., and WYSS, 0. (1962). Enzyme associated with bacteriophage infection. J. Bacterial. 84, 1209- 1215. GORDON,C. N. (1972). The use of octadeconal monolayers as wetting agents in the negative staining technique. J. Ultrastmct. Res. 39, 173-185. HAYES, W. (1968). “The Genetics of Bacteria and
PSEUDOLYSOGENY
OF Azotobacter
Their Viruses” 2nd ed., pp. 448-456. Wiley, New York. JACOB, F., and WOLLMAN, E. (1953). Induction of phage development in lysogenic bacteria. Cold Spring Harbor Symp. Quant. Biol. 18, 101-121. JONES, L. M., MCDUFF, C. R., and WILSON, J. B. (1962). Phenotypic alterations in the colonial morphology of Brucella abortus due to a bacteriophage carrier state. J. Bacterial. 83, 860-866. LI, K., BARKSDALE, L., and GARMISE, L. (1961). Phenotypic alterations associated with the bacteriophage carrier state of Shigella dysenteriae. J. Gen. Microbial.
24, 355-367.
MARE, I. J., and SMIT, J. A. (1969). A capsuledepolymerizing enzyme from Ale&genes fuecalis infected with bacteriophage A6. J. Gen. Viral. 5, 551-552. ROMIG, W. R. (1968). Infectivity of Bacillus subtilis bacteriophage deoxyribonucleic acids extracted from mature particles and from lysogenic hosts. Bacterial. Rev. 32, 349-357.
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SORGER, G. J., and TROFIMENKOFF, D. (1970). Nitrogenaseless mutants of Azotobacter vinelandii. Proc. Nat. Acad. Sci. USA 65, 74-80.
SUTHERLAND, I. W., and WILKINSON, J. F. (1965). Depolymerases for bacterial exopolysaccharides obtained from phage-infected bacteria. J. Gen. Microbial.
39, 373-383.
THOMPSON,B. J., DOMINGO,E., and WARNER, R. C. (1973). Properties of Azotobacter phage Al4 and its DNA. Virology 56, 523-531. THOMPSON,B. J., WAGNER, M. S., DOMINGO,E., and WARNER, R. C. (1980). Pseudolysogenic conversion by Azotobacter phage A21 and the formation of a stably converted form. Virology 102, 278-285. THOMSON,J. A., and WOODS,D. R. (1974). Bacteriophages and cryptic lysogeny in Achromobacter. J. Gen. Viral. 22, 153-157. WYSS, O., and NIMECK, M. W. (1962). Interspecific transduction in Azotobacter. Fed. Proc. 21, 456.
102,
26’7-277 (1986)
Pseudolysogeny
of Azotobacter
Phages
BETTY J. THOMPSON,’ ESTEBAN DOMINGO,* AND ROBERT C. WARNER3 Department
of Moleculur
Biology and Biochemistry, University Irvine, California 92717
of Califbmia,
Accepted December 21, 1979
The establishment of a pseudolysogenic state accompanied by a phenotypic conversion in Azotobacter vinelundii strain 0 by phages A14, A21, A31, and A41 has been identified. Host cells can be recovered from the pseudolysogens by cultivation in phage-specific antiserum. Pseudolysogens continually give rise at a low rate to phage as a result of the occasional initiation of a lytic burst. As a result of the establishment of the pseudolysogenic state the host cells lose their polysaccharide coat, become flagellated and motile and acquire a yellow pigmented appearance. The four phages, although they differ serologically and molecularly, give rise to converted states that are indistinguishable except by the identification of the phage that is produced by it. On repeated subculturingeach of the pseudolysogens will give rise to a stable or permanently converted cell that has the phenotype of the pseudo-lysogen, but from which it is no longer possible to obtain either host cells or phage. INTRODUCTION
mented character. These changes are such that the converted cells can be distinguished The molecular characteristics of a series from those of the host either by microscopic of Azotobacter phages, first isolated by examination of the cells or by inspection Duff and Wyss (1961), have been studied in this laboratory (Domingo et al., 1972; of the colonies. The acapsulated cells were Thompson et al., 1973). One phage from first thought to-be contaminants, but have each of the four serological groups defined been shown to be associated with a pseudoby Duff and Wyss (1961) was examined. lysogenic state. The existence of pseudolyThese phages, A14, A21, A31, and A41, sogeny in the acapsulated, phage-resistant were diverse with respect to their size, cells and the interrelations of the different morphology, serology, and the properties phages producing it are described here. The distinction made in early studies of of their DNAs. There are indications that lysogeny between characteristic examples these phages are temperate as judged by of the phenomenon and variations on it plaque morphology (Duff and Wyss, 1961) classified as pseudolysogeny or the “carrier” and reports of transduction (Wyss and state were reviewed by Lwoff (1953). Like Nimeck, 1962), but lysogeny of them has true lysogeny, pseudolysogeny provides not been studied in detail. an alternative response to a lytic infection During work with these phages we became aware of a striking pseudolyso- and results in a latent state characterized genie conversion phenomenon as a result by resistance to superinfection and by the of which the cells of the host, Azotobacter potential of yielding phage. A common vinelandii strain 0, lose their polysaccha- distinguishing feature was the possibility ride coat, become flagellated and motile of recovering uninfected host cells from phage-infected pseudolysogens. Recent disand the colonies acquire a yellow-pigcussions of more thoroughly studied examples, particularly by Barksdale and ’ Present address: Bruce Lyon Memorial Research Arden (1974), Baess (1971), Romig (1968), Center, Children’s Hospital, Oakland, Calif. 94709. and Bott and Strauss (1965) make it clear *.Present address: Instituto de biologia de1 Desarthat phage-host interactions produced by rollo, Velazques, Madrid, Spain. 3 To whom requests for reprints should be addressed. several disparate mechanisms have been 267
0042-6822/80/060267-ll$OZ.~/O Copyright 0 1980 by Academic Press, Inc. AU rights of reproduction in any form reserved.
THOMPSON,
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DOMINGO,
classified together as pseudolysogeny. Hayes (1968) distinguished two general types. One, in which host cells and vegetative phage coexist in culture because of the development of a partial resistance of most or all of the cells to infection, is typified by the T7-Shigella interaction studied by Li et al. (1961). In the second type a latent form of the phage exists inside the cell as a carrier and does not necessarily give rise to a lytic infection. In this case, because there is a limited number of particles, host cells without carrier particles may be segregated at cell division as found by Bott and Strauss (1961). However, there are situations in which the mechanisms of the first type include a carrier state in the sense used by Hayes to apply to the second (Barksdale and Arden, 1974). In view of this and of the apparently unique characteristics of each case of pseudolysogeny it does not seem useful to adhere to the distinction made by Hayes. Regardless of the mechanism of establishment of pseudolysogeny, cultivation in the presence of phage antiserum will result in an elimination or decrease in the pseudolysogen and an accumulation of or a replacement by host cells. This response to antiserum serves as an operational definition of pseudolysogeny. It is emphasized by all of the authors quoted above.
AND WARNER
Polysaccharide Depolymerase Assay
the outflow time in a semimicro (l-ml) Cannon viscometer in a water bath regulated at 33 + 0.01”. The polysaccharide was prepared by the method of Cohen and Johnstone (1964) from a culture of A. vinelandii 0. It was dissolved in the medium (DW) described by Duff and Wyss (1961) and was heated to 85” for 15 min to inactivate traces of depolymerase that it contains. It was centrifuged and diluted to a concentration such that the further dilution with 0.09 vol of cell suspension or extract would result in a viscosity relative to that of the DW medium of 2.5. Suspensions of washed cells were prepared having an A,,, = 4.6. Samples were sonicated and centrifuged. An aliquot of 100 ~1 of suspension or of supernatant solution was added to 1100 ~1 of polysaccharide solution, 1 ml of the mixture was placed in the viscometer and the outflow time was determined periodically for 5 to 16 hr. The initial relative viscosities were close to 2.5, but in order to facilitate comparison between experiments all of the relative viscosities for each solution were normalized to an initial nrel of 2.5 by multiplying by 1.5 divided by the specific viscosity at zero time. This procedure is equivalent to assuming that the specific viscosity is proportional to the concentration of polysaccharide over the small difference in concentration among the various experiments. The normalization factor for sonicates was never out of the range of 0.97 to 1.0 and there was no detectable effect on the initial slope of the decrease in viscosity on which the enzyme units depends. Some determinations were also made by measuring the release of reducing groups during the depolymerase reaction by a modification of the method of Somogyi (Dische, 1962). For these assays the enzyme was precipitated from a lysate or sonicate by ammonium sulfate at 75% saturation. This crude fraction from a lysate was also used in the experiments in which the host cells were treated with enzyme.
The enzyme was determined by following the decrease in viscosity of a solution of the partially purified capsular polysaccharide. Viscosities were determined by measuring
The negative staining procedures for the examination of the bacteria and its flagella
MATERIALS
AND METHODS
Phages A14, A21, A31, and A41 have previously been described along with the media and methods used in their cultivation (Domingo et al. , 19’72; Thompson et al., 1973). They were grown on A. vinelandii 0 (ATCC 12518). Phage PAV-1 and an acapsulated host, A. vinelandii OP (ATCC 13705) (Bush and Wilson, 1959) were obtained from Dr. George J. Sorger. This phage was isolated by Sorger and Trofimenkoff (1970) and its properties have been described by Chum1 et al. (1930).
PSEUDOLYSOGENY
269
OF Azotobocter PHAGES
by electron microscopy were those previously employed (Cordon, 19’7.2).Sodium phosphotungsate (pH 5.5) and uranyl acetate stains were used. The presence of a polysaccharide capsule was determined by examination of the bacteria by light microscopy in a dilute suspension of carbon particles. Induction Attempts to induce the converted cells, referred to below as PC cells, were carried out according to the procedure of Jacob and Wollman (1953). A log phase culture was divided into 10 separate aliquots and irradiated in duplicate. The survival rate of the irradiated cultures varied from 1 to 90%. The bacteria were grown overnight. One of the cultures in a pair was plated for colony formers and titered for free phage. The duplicate was treated with chloroform and then titered for free phage. Mitomycin C (Calbiochem B grade) induction was tested according to the procedure of Thomson and Woods (1974). RESULTS
Isolation of Phge-Resistant lysogeny
Cells: Pseudo-
Colonies of yellow, motile cells were first noted as an overgrowth on confluent plaques from lysates of several of the phages. The origin of these cells was examined using phage A21. It was found that when samples of an infected culture were plated as a function of time after infection that a substantial fraction of the cells would yield yellow colonies. The results of such an experiment are shown in Fig. 1. It can be seen that the number of yellow cells at 30 min was about 0.1 the number of host cells at the time of infection and then decreased to about 0.001 at the onset of lysis. Since essentially all of the cells that yielded yellow colonies at 30 min would have lysed had they remained in liquid culture, it is evident that conversion to the yellow form was promoted by plating on a solid medium. Yellow colonies of the same type were present in a great majority when innocula from the turbid zone of a plaque were
0
2 Time,
4 hours
6
FIG. 1. Course of infection of A. vinelandii 0 by phage 21. The total cell density was followed by determination of A,,, (A) and the phage production by determination of PFU (A). At intervals samples were taken and plated for colonies. Those of the host type (0) can no longer be detected when cells are plated ‘/a hr after infection. At this time the only colonies are those having the characteristics of the pseudolysogen and their time course is shown by open circles (0). Colonies of this type cannot be detected before infection. The m.o.i. in this experiment was 10. Similar results were obtained at m.o.i.‘s of 1, 5, 50, and 500.
plated. Numerous isolates from the yellow colonies were selected and propagated. The colonies that survived infection after plating on solid media were colonies with converted characteristics. The cells were acapsulated and motile. If a single colony was subcultured and replated after washing to remove phage, three different types of colonies were seen: 1. Host. The colonies of the host were white, mucoid, opaque, entire, and about 3 mm in diameter. These cells were sensitive to phage. Treatment with phage specific antiserum produced no change in the characteristics. All cells possessed a polysaccharide coat and were nonmotile (Fig. 2A). They could not be distinguished from the parent strain 0 in these respects or in their behavior on reinfection with phage. 2. Pseudolysogen. These colonies were the most prevalent and had the same characteristics as the original single cell isolate
270
THOMPSON, DOMINGO, AND WARNER
FIG. 2. Electron micrographs of Azotobacter winelandii Pseudolysogen of phage A21. Uranyl acetate stain.
0. (A) Host cell. Uranyl acetate stain. (3)
PSEUDOLYSOGENY
OF Azotobactw
FIG. 2. -Continued.
PHAGES
271
272
THOMPSON,DOMINGO,ANDWARNER
from the infection. They were resistant to phage. The colony was a mottled combination of clear yellow and opaque mucoid white with an irregular edge. The mottled appearance was a result of the presence of two types of cells evidently formed by segregation of carrier particles during the growth of the colony. Part of the colony had the same appearance as the colonies of the host and part had a clear, yellow character and consisted of motile, acapsulated cells. Growth in phage-specific antiserum when the colony was subcultured resulted in an increase in the ratio of phagesensitive capsulated nonmotile cells to the acapsulated, motile ones. 3. Permanently converted (PC) cells. The colonies of these cells were clear yellow, entire, and about 0.1 the size of the other types of colonies. The cells were acapsulated, motile and resistant to phage infection (Fig. ZB). Treatment with phage specific antiserum produced no change in the cells. The behavior of the second type of cells identify them as pseudolysogens containing a phage genome in carrier form. The carrier may be segregated on cell division so that daughter cells both with and without carrier are formed. The latter are host cells and are sensitive to phage infection. The segregation can be observed on a plate to take place during the growth of a colony initiated by a single cell. The carrier may also occasionally initiate a lytic infection and produce a burst of phage. As a result of these processes the propagation of a pseudolysogen for several transfers in phage specific antiserum will result in a culture that is chiefly or entirely sensitive cells of the host type. The rate at which this occurs is, in general, variable and has been found to be much greater for some isolates than for others. Some are relatively stable, segregate host cells only occasionally and maintain their converted characteristics over many generations. The kinetics of the segregation process is examined in the accompanying paper (Thompson et al., 1980). Growth without antiserum resulted in a mixture of cells as described above. A culture that was washed and resuspended in fresh broth had a low phage titer that
increased to lo6 PFU ml-’ after several hours of incubation at which time relatively few host cells were present. The high titer of phage can be accounted for by initiation of a lytic response in a fraction of the carrier cells and infection of continually segregating host cells. The colony morphology of pseudolysogens reflects an interaction among resistant and sensitive cells and phage. As the colony grows after a one-day incubation, pseudolysogens arise in the center and after 2 days of incubation host cells begin to grow at the periphery. Only the pseudolysogen is able to survive in the center of the colony where the phage concentration is the highest. The pseudolysogenic behavior of A21 is reflected in its plaque morphology. Plaques consist of a small, central clear area surrounded by a turbid halo or zone. If growth of the plaque is allowed to continue, the lawn beyond the zone becomes partially clear. The zone itself consists almost entirely of pseudolysogens. This. type of morphology could result from an effect of an increasingly high concentration of phage on the initiation of pseudolysogeny and the diffusion of some phage through the turbid zone or its production in the zone by a low level of lysis resulting in a second zone that is clear because of predominance of lytic infections. Permanent Conversion At some frequency dependent on unidentified conditions of culture a pseudolysogen of A21 will be transformed to a different phage-resistant cell which produces the colonies described as type 3 above. It differs from the pseudolysogen in its inability to segregate colonies having the host morphology. When a pseudolysogen was cultured repeatedly either in the presence or absence of antiserum and plated at intervals, the earliest PC colonies to arise usually produced some phage. If cells from them were cultured after washing with antiserum, phage was formed at a very low level. Such phage was identified as A21 by its sensitivity to A21 antiserum and the buoyant density of its DNA in CsCl
PSEUDOLYSOGENYOF Azotobuctm PHAGES of 1.717 g ml-‘. After several passages these cells lost their ability to produce any detectable phage. It is not clear whether the initial low production of phage was a result of a low level of spontaneous lysis or whether it was due to a contamination with pseudolysogen. In an examination of colonies from antiserum-treated cultures of such cells, no host colonies were seen among 20,000 examined. Examination of these cultures in the presence of carbon particles by light microscopy revealed only acapsulated, motile cells. Attempts to induce phage production by uv exposure or treatment with mitomycin C as described in the methods section were unsuccessful. We have referred to cells of this type as permanently converted or PC cells because they do not fit into any established category. Some additional information on their formation from pseudolysogens is given by Thompson et al. (1980). Converted Characteristics All of the pseudolysogen and PC cells had all of the converted characteristics: yellow pigment, absence of polysaccharide coat, and presence of flagella. The PC cells lacked the properties of the pseudolysogen of segregation of host cells and of phage production. Additional study was made of the presence of flagella and of aspects of the absence of the polysaccharide coat. Flagella
Electron micrographs of negatively stained cells are shown in Fig. 2. A host cell obtained from a log phase culture and shown in Fig. 2A has no detectable flagella. The PC cell shown in Fig 2B is flagellated in a peritrichous arrangement, The movement of these cells is consistent with this arrangement of flagella. The pseudolysogen was similarly flagellated. No flagella have been detected in the host cells under any conditions or stages of growth.
Azotobacter
Polysaccharide
Coat
In a normal infection with phage A21 the cells retain their polysaccharide coat until lysis begins at about 7 hr. Partially
273
purified polysaccharide depolymerase (see Materials and Methods) was used to determine the role the polysaccharide coat plays in phage infection. A culture of host was divided and one portion was treated with the enzyme for 30 min at 33”, the cells were washed and resuspended in fresh medium. This treatment completely removed the capsule as determined by microscopic examination in the presence of carbon particles. The other portion was treated similarly, omitting addition of the enzyme. Phage A21 at a multiplicity of infection of 2 was added to the cells and allowed to adsorb for 30 min. Adsorption was determined by centrifuging and assaying the supernatant solution for free phage. In the enzyme-treated culture all of the added phage were recovered while in the control the phage titer dropped to 1% of that added. As expected from the adsorption results no significant phage yield was obtained from the enzyme-treated culture. The capsule is evidently needed for adsorption and its absence from the pseudolysogen and the PC cells provides a sufficient basis for the resistance of these cells to infection. This result is in direct contradiction to the results of Eklund and Wyss (1962), who reported that removal of the capsule increased the rate of adsorption. It has been reported by Eklund and Wyss (1962) and by Barker et al. (1968) that the depolymerase is absent from A. vinelandii 0 and that its production during infection is a result of a phage-specified enzyme. We investigated the presence of the depolymerase in the several cell types described above and its production during infection because of the possibility that the mechanism of this feature of conversion might be clarified. In other phage-host systems with characteristics similar to those of A21A. vinelundii 0 there has also been a failure to detect the depolymerase in the host cells (Adams and Park, 1956; Bartell and Orr, 1969; Sutherland and Wilkinson, 1965; Mare and Smit, 1969; and others). On the other hand Chakrabarty et al. (1967) have shown clearly that the depolymerase of Pseudomonas putida is a host specified enzyme that is evidently induced many-fold by phage infection.
274
THOMPSON, DOMINGO, AND WARNER
1.0 0
I
2 Time , hours
3
4
1.0 0
I Tim.
2 hovrs
3
4
FIG. 3. Change of the viscosity with time of solutions of the capsular polysaccharide ofA. winelundii 0 as it is hydrolyzed by a depolymerase from various sources. (A) Curves for the addition of the indicated volume of a filtered lysate from the infection of A. winekrndii 0 with phage A21. The initial slopes ofthese curves were used, as explained in the text, to define a unit of enzyme activity. (B) Curves for addition of centrifuged sonicates of cultures of several cell types. Curves a and bare controls for no addition and addition of a sonicate of E. coli. Curve c is for the PC cells; curve d, for the host, A. winehndii 0; and curve e, for the pseudolysogen. In all cases 100 ~1 of a sonicate of a suspension of cells having an A,,, = 4.6 was added to 1100 ~1 of polysaccharide solution.
The concentration of depolymerase was determined by following the decrease in viscosity of a solution of the capsular polysaccharide when it is hydrolyzed by the enzyme. The greatest concentration of the enzyme is present in a lysate of infected cells and such a lysate was used to establish a relative enzyme unit. The viscosity curves
for a series of dilutions of a lysate are shown in Fig. 3A. The initial slopes are proportional to the concentration of enzyme as shown by the average value for the slopes of the five curves of 3.2 + 0.4 per unit of enzyme. The slopes are given in qrel units per hour and one unit of enzyme is taken as the amount of enzyme in 1 ~1 of lysate of cells having an Asw = 1. The data on TABLE 1 sonicates of the several cell types are shown CONCENTRATION OFDEPOLYMERASE IN SONICATES in Fig. 3B. There was no change in viscosity ANDA LYSATEFROMDIFFERENTCELLTYPES of the control containing only the heated polysaccharide and that resulting from adding an E. coli sonicate was barely Method detectable. The concentration of enzyme Viscosity Reducing calculated from the initial slope of ‘each (units per group of the other curves is given in Table 1. A AEw unit of (relative clearly detectable amount of enzyme is Source cell density) units) present in each type of cell although at a Lysate loo” 100 much lower level than in the lysate from a A. vinelandii 0 0.021* 0.24 phage infection. The results were confirmed Pseudolysogen 0.16 using sonicates from several different prepPC cells 0.0063 0.09 arations of cells. Similar results were E. coli control 0.0009 Not obtained using suspensions of cells instead detectable of sonicates. In these cases it is not clear a Baaed on one unit/p1 of lysate derived from cells whether the viscosity decrease resulted having A,,, = 1. b (Initial slope)/(3.2 x 4.6). The factor 3.2 was from secretion of the enzyme or from the gradual death and lysis of cells in the culobtained from Fig. 3A as explained in the text; 4.6 = Asw of the cell suspensions from which the ture. The results with cells of A. winelandii 0 sonicates were obtained. were complicated by the contribution to the
PSEUDOLYSOGENY
OF Azotobactw
viscosity of the polysaccharide capsule. As the cells grew, the viscosity increased slightly, but subsequently decreased. Some determinations of the concentration of the depolymerase that hydrolyzes the capsular polysaccharide were made by calorimetric detection of reducing groups liberated in the reaction. These results are included in Table 1. We conclude that depolymerase is present in all three types of cells. Its presence in the uninfected host indicates that it is not specified by the phage. Production of the enzyme at a high level appears to occur only during a lytic infection. The greater activity (Table 1) of depolymerase in the sonicate from the pseudolysogen may not indicate an intrinsically higher activity in these carrier cells, but may result from the occasional induction of a lytic infection in the pseudolysogen (Thompson et al., 1980). Experiments with crude lysates of the type we have done cannot eliminate the possibility that the induced enzyme is different from a depolymerase present in the host cells, although this possibility was eliminated in the case of Pseudomonas putida (Chakrabarty et al., 1967). In spite of the evident requirement of the polysaccharide coat for adsorption, phage is not inactivated by mixing it with polysaccharide. Pseudolysogeny by other Axotobacter Phuges The work described above was carried out with phage A21. Less extensive observations on phages A14, A31, and A41 showed that they produce the same pseudolysogenic conversion of A. vinelandii 0 and pseudolysogens and PC cells similar to those of A21. This was an unexpected result since the four phages are molecularly and serologically unrelated (Domingo et al., 1972; Thompson et al., 1973). The pseudolysogen formed by infection by any one of the four phages showed the same properties with respect to pigmentation, absence of polysaccharide coat, presence of flagella, colony morphology, and resistance to superinfection by any one of the four phages. The four pseudolysogens could be distinguished only by the ability of each to yield the same
PHAGES
275
phage that produced it. We have been unable to find a means for distinguishing the four PC cells from each other. A PC culture isolated after infection by each phage was tested for infectability by and adsorption of each of the four phages. In no case was either detected. We have been unable to obtain clear plaque mutants of A21 in spite of considerable effort. Phage PAV-1 Another Axotobacter phage, PAV-1, described by Sorger and Trofimenkoff (1970), was recently studied in this laboratory (Chum1 et al., 1980). It was found to be molecularly and serologically unrelated to A14, A21, A31, or A41 and in addition to have a different host range from that shared by the four A phages. PAV-1 can infect A. vinelandii 0 and A. vinelandii OP and gives plaques having turbid halos. A. vinelandii OP lacks the polysaccharide coat of A. vinelandii 0 and in pigmentation and flagellation resembles the PC cells derived from A. vinelandii 0. PAV-1 also infects A. vinelandii 0 and produces the same pseudolysogenic conversion as the phages of the A series. In contrast A. vinelandii OP is not infected by any of the A phages. However, PAV-1 can infect all of the pseudolysogens and PC cells of the A phages. DISCUSSION
The Axotobacter phages of the A series interact with their host, A. vinelandii 0, to produce a pseudolysogeny with several unique characteristics. (1) The pseudolysogens acquire a converted phenotypepigmentation, flagellation and loss of polysaccharide capsule -which makes it possible to distinguish them from the host by examination either of the cells or their colonies. (2) The pseudolysogen can subsequently give rise to another type of cell that retains the converted phenotype, but which can no longer either segregate host cells or produce phage. We have termed this cell a permanently converted or PC cell. (3) Phages from four different groups of the A series, unrelated serologically or
276
THOMPSON, DOMINGO, AND WARNER
molecularly, produce pseudolysogens having the same converted phenotype. A fifth phage, PAV-1, which in addition has a different host range, produces a similar pseudolysogen. The phage-resistant cells isolated from infected cultures were originally thought to be mutants or contaminants. Both of these possibilities were eliminated by the repeated isolation of a large fraction of the cells of an infected culture as the resistant pseudolysogens (Fig. 1). In addition they constitute the major number of colonies when cells from the turbid area of a plaque are plated. We have classified them as pseudolysogens by the criteria that they yield host cells by segregation when they are cultivated in antiserum and that they continually produce a low level of phage. The existence of a carrier state is indicated by the ability of such a pseudolysogen to maintain its phenotype through several generations and then segregate host cells. The cell type to which pseudolysogens give rise after repeated transfers we have termed a PC cell. Some information on the nature of this change is given in the accompanying paper (Thompson et al., 1980), but its molecular basis is still obscure. It was first thought to be a form of lysogeny, but since such phage production as initially occurred was always lost and no means of induction was found, this is untenable. More importantly, we have found (Thompson et al., 1980) that no more than a small fraction of a phage genome can be detected in the cell by hybridization methods. The polysaccharide depolymerase does not appear to be present in significantly different amounts in the three cell types. Our results are similar to those of Chakrabarty et al. (1967) in that they indicate that the depolymerase is an enzyme of the host and not of the phage. Its induction to a high concentration is characteristic of a lytic infection. It thus appears that the activity of this enzyme is unrelated to the absence of the polysaccharide capsule in the pseudolysogen and the PC cells. The polysaccharide capsule appears to be essential for phage adsorption although we cannot eliminate the possibility that the crude enzyme preparation used to remove it
produced other changes in the cell wall or membrane than removal of the coat. ACKNOWLEDGMENT This investigation was supported by NIH Research Grant CA12627 from the National Cancer Institute. REFERENCES ADAMS, M. H., and PARK, B. H. (1956). An enzyme produced by a phage-host cell system. II. The properties of the polysaccharide depolymerase. Virology 2, 719-736. BAESS, I. (1971). Report on a pseudolysogenic mycobacterium and a review of the literature concerning pseudolysogeny. Acta Pathol. Microbial. Stand. B 79,428-434.
BARKER, T., EKLUND, C., and WYSS, 0. (1968). Physical differences of virus-associated depolymerases. Biochem. Biophys. Res. Commun. 30, 794-710. BARKSDALE, L., and ARDEN, S. B. (1974). Persisting bacteriophage infections, lysogeny, and phage conversions. Annu. Rev. Microbial. 28, 265-299. BARTELL, P. F., and ORR, T. E. (1969). Origin of polysaccharide depolymeraae associated with bacteriophage infection. J. Viral. 3, 290-296. BUSH, J. A., and WILSON, P. W. (1959). A non-gummy chromogenic strain of Azotobacter &eland% Nature (London) 184, 381. CHAKRABARTY, A. M., NIBLACK, J. F., and GUNSALUS, I. C. (1967). A phage-initiated polysaccharide depolymerase in Pseudomonas putida. Virology 32, 532-534. CHUML, V. A., THOMPSON,B. J., SMIILEY,B. L., and WARNER, R. C. (1980). Properties of Azotobacter phage PAV-1 and its DNA. Virology 102,262-266. COHEN, D. H., and JOHNSTONE,D. B. (1964). Extracellular polysaccharide of Azotobacter vine&&i. J. Bacterial.
88, 329-338.
DISCHE, Z. (1962). Color reactions based on reducing properties of sugars. In “Methods in Carbohydrate Chemistry” (Whistler, R. L., and Wolfrom, M. L., eds.), Vol. 1,512-514. Academic Press, New York. DOMINGO,E., GORDON,C. N., and WARNER, R. C. (1972). Azotobacter phages: Properties of phages A12, A21, A31, A41 and their constituent DNAs. Virology
49, 439-452.
DUFF, J. T., and WYSS, 0. (1961). Isolation and classification of a new series ofAzotobacter bacteriophages. J. Gen. Microbial. 24, 273-289. EKLUND, C., and WYSS, 0. (1962). Enzyme associated with bacteriophage infection. J. Bacterial. 84, 1209- 1215. GORDON,C. N. (1972). The use of octadeconal monolayers as wetting agents in the negative staining technique. J. Ultrastmct. Res. 39, 173-185. HAYES, W. (1968). “The Genetics of Bacteria and
PSEUDOLYSOGENY
OF Azotobacter
Their Viruses” 2nd ed., pp. 448-456. Wiley, New York. JACOB, F., and WOLLMAN, E. (1953). Induction of phage development in lysogenic bacteria. Cold Spring Harbor Symp. Quant. Biol. 18, 101-121. JONES, L. M., MCDUFF, C. R., and WILSON, J. B. (1962). Phenotypic alterations in the colonial morphology of Brucella abortus due to a bacteriophage carrier state. J. Bacterial. 83, 860-866. LI, K., BARKSDALE, L., and GARMISE, L. (1961). Phenotypic alterations associated with the bacteriophage carrier state of Shigella dysenteriae. J. Gen. Microbial.
24, 355-367.
MARE, I. J., and SMIT, J. A. (1969). A capsuledepolymerizing enzyme from Ale&genes fuecalis infected with bacteriophage A6. J. Gen. Viral. 5, 551-552. ROMIG, W. R. (1968). Infectivity of Bacillus subtilis bacteriophage deoxyribonucleic acids extracted from mature particles and from lysogenic hosts. Bacterial. Rev. 32, 349-357.
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SORGER, G. J., and TROFIMENKOFF, D. (1970). Nitrogenaseless mutants of Azotobacter vinelandii. Proc. Nat. Acad. Sci. USA 65, 74-80.
SUTHERLAND, I. W., and WILKINSON, J. F. (1965). Depolymerases for bacterial exopolysaccharides obtained from phage-infected bacteria. J. Gen. Microbial.
39, 373-383.
THOMPSON,B. J., DOMINGO,E., and WARNER, R. C. (1973). Properties of Azotobacter phage Al4 and its DNA. Virology 56, 523-531. THOMPSON,B. J., WAGNER, M. S., DOMINGO,E., and WARNER, R. C. (1980). Pseudolysogenic conversion by Azotobacter phage A21 and the formation of a stably converted form. Virology 102, 278-285. THOMSON,J. A., and WOODS,D. R. (1974). Bacteriophages and cryptic lysogeny in Achromobacter. J. Gen. Viral. 22, 153-157. WYSS, O., and NIMECK, M. W. (1962). Interspecific transduction in Azotobacter. Fed. Proc. 21, 456.