JOURNAL OF BACTERIOLOGY, June 1979, p. 962-968 0021-9193/79/06-0962/07$02.00/0
Vol. 138, No. 3
Alteration of Colonial Morphology of Acholeplasma laidlawii and Acholeplasma modicum by Infection with Mycoplasmatales Viruses ALICE L. CONGDON AND GEORGE E. KENNY* Department of Pathobiology, SC-38, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 98195 Received for publication 16 April 1979
Morphologically aberrant colonies resulted from the infection of Acholeplasma laidlawii with two of its three known viruses and from Acholeplasma modicum cells naturally carrying virus. The patterns of colonial alteration differed between cells infected with the two A. laidlawii viruses. Colonies derived from single cells infected with the bullet-shaped virus MV-Li (Mycoplasmatales virus-laidlawii1) had a radial sectoring pattern of intracolonial swellings ("blebs"), whereas cells infected with the tailed icosahedral virus MV-U3 contained bubble-like blebs. Colonies from cells infected with the enveloped virus MV-L2 appeared identical to those obtained from uninfected cells. Aberrant colonies contained 106 colonyforming units of organisms and 106 plaque-forming units of virus serologically identical to the infecting type, indicating that both the virus and host organism were capable of simultaneous replication. Enumeration of virus by means of counting aberrant colonies was 30-fold more sensitive than infectious center assay for MV-Li and 1.2- to 2-fold higher for MV-L3. Furthermore, blebbed colonies were observed with A. modicum cells only at times of concurrent spontaneous plaquing with a new virus specific to A. modicum. Thus, blebbing in colonies provides a valuable marker for detection of the Mycoplasmatales viruses. The order Mycoplasmatales comprises membrane-bounded cells which are the smallest freeliving cells known. Three morphologically distinct viruses have been isolated recently from Acholeplasma laidlawii, one of the organisms in this order: the bullet-shaped MV-Li (MV-L, Mycoplasmatales virus-laidlawii-1; 6,8, 10, 17); the enveloped, spherical MV-L2 (7); and the icosahedral, tailed virus MV-L3 (11). These simple organisms, therefore, are capable of supporting the replication of radically diverse viruses, suggesting that other members of the Mycoplasmatales, including the pathogens, may also contain viruses. The three viruses were isolated from either spontaneous plaques or from washes of lawns of A. laidlawii (18), indicating that the viruses are carried in some cryptic form. As with animal viruses, the development of methods for detecting nonlytic viruses (i.e., those which do not form plaques) may be required for detection of these cryptic viruses. We have observed that strikingly altered and distinct colonies arise from A. laidlawii cells infected with MV-Li or MV-L3 and from cultures of Acholeplasma modicum naturally carrying a new virus MV-Mi (Congdon and Kenny, submitted for publication). Our study 962
focused on determining the virus content of infected colonies, the mechanisms by which they arise, and the numerical relationships of altered colonies to standard measures such as plaques and infectious centers. MATERIALS AND METHODS Viruses and acholeplasmata. MV-Li (7, 8) was obtained from J. Maniloff of the University of Rochester, Rochester, N.Y. MV-L2 (9) and MV-L3 (5) were kindly provided by R. N. Gourlay, Compton, England. Viruses were plaque purified three times. Plaque purification of MV-L2 was carried out in the presence of 1:20 dilution of rabbit antiserum to MV-Li having a kinetic neutralization value (1) of 67,000. A. laidlawii strain BN-1 (19) was obtained from J. Maniloff, and A. laidlawii strain M1353 (3) was from W. Clyde, University of North Carolina, Chapel Hill, N.C. Both strains were cloned three times in our laboratory; serially cultivated in the presence of 1:100 dilution of antisera to MV-LI, MV-L2 and MV-L3; and selected for their efficiency in plaquing the three viruses. A. modicum PG-49 was obtained from J. Tully (23), transferred twice in our laboratory, and frozen at -700C. Media and buffers. Soy peptone broth (14) for growth of cells and virus lysates contained 2% soy peptone (Humko Sheffield, Lyndhurst, N.Y.), 0.5% NaCl, and 20 mM TES [N-tris(hydroxymethyl)-
VOL. 138, 1979
VIRAL DISTORTION OF ACHOLEPLASMIC COLONIES
methyl-2-aminoethane-sulfonic acid], pH 8.0, and was supplemented with 5% fresh yeast dialysate (14), 5% agamma horse serum (North American Biologicals), 80 mM glucose, and 400 U of penicillin per ml. The composition of solid medium was similar, with the omission of both glucose and penicillin and the addition of 0.65% agarose. Agarose medium (12 ml) was placed into each petri dish (60 by 15 mm) and used for the determination of colony-forming units (CFU) and as bottom support for the overlay technique of the plaque assay. The medium for the overlay for plaque assays consisted of 2% tryptose (Difco), 0.5% NaCl, 20 mM TES (pH 8.0), 80 mM glucose, and 0.5% agarose per titer with 5% agamma horse serum. Virus dilutions and storage were carried out in buffer consisting of 0.8% NaCl, 20 mM TES (pH 7.4), and 0.1% gelatin; this buffer was used for all experiments unless otherwise specified. Plaque assay. Plaque-forming units (PFU) and infectious centers were assayed by a modification of the overlay technique (1). Frozen portions of A. laidlawii were thawed, diluted 1:10 in medium without glucose, incubated overnight at 370C in 2.5% C02, diluted 1:3 to 1:8, and incubated in a 37°C water bath with shaking for 5 to 7 h. A 0.1- to 0.2-ml portion of virus dilution was placed on the soy peptone support medium and mixed with 2 ml of overlay containing 0.4 ml of indicator organisms. Lawns of A. modicum were prepared with 2 ml of tryptose overlay medium and 1 ml of broth culture. Plates were incubated at 37°C in 2.5% CO2 for 36 to 48 h. A. laidlawii strain BN-1 was used as indicator for MV-Li, and strain M1353 was used for MV-L2 and MV-L3. Polylysine (molecular weight, 140,000) was added at a concentration of 100 Agg/ml for plaque enhancement with MV-L2. Infection of cells for colony observation. Log-
phase cultures of A. laidlawii strain BN-1 containing 108 to 2 x 108 cells per ml were obtained by 1:10 dilution of overnight cultures and incubation at 370C in a New Brunswick gyratory shaker water bath for several hours. Cultures were infected at a multiplicity of 5 to 10 (PFU/CFU) with either MV-Li, MV-L2, or MV-L3. After a l-min adsorption, cells were diluted in buffer (400), and portions (0.05 ml) were plated in duplicate on plates which were then incubated at 37°C in a 2.5% C02-air atmosphere. Infectious center and blebbing colony assay. Log-phase cells ofA. laidlawii BN-1 strain and M1353 strain at a concentration of 108 to 2 x 108 were exposed to indicated concentrations of MV-Li and MV-L3, respectively. After an adsorption period of 9 min at 370C in the gyratory water bath antiserum was added to the cell suspension at a concentration sufficient to neutralize 98.3 to 99.7% of any remaining free virus during the ensuing 10-min incubation period. For assays of CFU, cells were diluted at 00C, and 0.05-ml portions were plated in triplicate on prewarmed 370C plates, allowing for absorption of the fluid before the end of the latent period for virus release from infected cells, thus preventing the spread of virus. (We found the latent period to be 45 min for MV-Li and 60 min for MV-L3 with the strains of A. laidlawii used as the host.) Infectious centers and input virus were assayed by the overlay technlique described above. To assay for both viable cells and virus, individual colonies were
963
picked with Pasteur pipettes, resuspended in 1 ml of buffer with vigorous pipetting with a 1-ml pipette, diluted, and plated for CFU and PFU. Antisera. Immunogens were prepared by infecting log-phase A. laidlawii grown in broth containing 5% agamma rabbit serum with MV-Li, MV-L2, or MV-L3 at a multiplicity of infection of 0.001 to 0.01. At the end of 16 to 36 h of incubation at 370C in a gyratory water bath, cells were pelleted by centrifugation at 10,400 x g for 30 min. Virus was precipitated from the supernatant by addition of polyethylene glycol (molecular weight, 6,000) and NaCl to final concentrations of 8% and 0.5 M, respectively. Virus was precipitated overnight at 40C, collected by centrifugation at 8,000 x g for 15 min, and dialyzed overnight against buffer without gelatin. Precipitates of MV-Li were treated with 0.2% Nonidet P-40 (final concentration), layered over a 10-ml cushion of 30% sucrose, and centrifuged at 83,000 x g for 4 h in a Beckman SW 25.1 rotor. The resuspended pellet was brought to a density of 1.37 g/ ml with CsCl in 20 mM TES, pH 7.4, and centrifuged at 177,000 x g for 17 h in a Beckman type 65 rotor. The band with viral activity was centrifuged a second time in CsCl at a density of 1.37 g/ml and dialyzed overnight. Precipitated MV-L3 was chloroform extracted, pelleted through a 3-ml 30% sucrose cushion by centrifugation at 83,000 x g for 4.5 h, and banded twice in CsCl at a density of 1.48 g/ml by centrifugation in a Beckman 65 rotor for 18 h at 177,000 x g. The visible band with viral activity was dialyzed overnight. The immunogen for MV-L2 was prepared by dialyzing polyethylene glycol-precipitated virus because a suitable purification scheme had not yet been devised. The immunization scheme was as described previously for mycoplasmata (13). Immunogens contained 10'° to 10" PFU/ml. Sera were obtained within 7 days of the final injection. Neutralization of virus in colonies. Single blebbed colonies were resuspended on day 6 in 1 ml of buffer with vigorous pipetting, and 0.1 ml of the suspension was added to tubes containing 0.2 ml of a 1: 100 dilution of antiserum. Homologous kinetic neutralization values (1) of the antisera were 67,100 (MVLi), 257 (MV-L2), and 40,158 (MV-L3); cross-neutralization was not observed. After 10 min of incubation at 370C, dilutions were made in buffer at 00C and plated in duplicate for PFU. Inhibition of bleb formation. Agarose plates were pretreated with 0.2 ml of a 1:10 dilution of antiserum to MV-Li, MV-L2, or MV-L3. Cells were infected at 370C for 10 min with each of the three viruses, diluted, and plated in duplicate. Colonies (500 minimum) were observed for blebbing on day 8.
RESULTS Alteration of morphology of infected colonies. While enumerating CFU after infection with MV-Li, strikingly altered colonies were observed on plates incubated at 37°C for 5 days or longer. This phenomenon was investigated by infecting broth cultures of A. laidlawii with each of the three viruses (MV-Li, MV-L2, and MV-L3) and observing colonial growth at 24-h
964
CONGDON AND KENNY
intervals (Fig. 1). Colonies seen on days 1 and 2 (Fig. la and lb) resembled those from uninfected controls, but by day 3 (Fig. lc) some colonies were distorted with a blebbed appearance (presence of multiple intracolonial centers and swellings). By day 4 (Fig. ld), radial sectoring characteristic of MV-Ll-infected colonies was easily discernible, and on day 6 (Fig. le) the colonies appeared highly blebbed in a typical pattern of radial sectoring. Blebbing was most marked when colonial density was sparse (500 to 1,000
.
J. BACTERIOL.
colonies per 60-mm plate; Fig. 1). A similar result was obtained after plating cells infected with the icosahedral virus MV-L3. In that case, blebbed colonies again appeared after 72 h, but blebs were larger and developed in a random pattern rather than displaying sectoring (Fig. lf). In contrast, cells infected with MV-L2 gave rise to nonnal fried-egg-appearing colonies (Fig. lg) which were morphologically identical to colonies from uninfected cells (Fig. lh). Rare sectoring was occasionally observed in uninfected colonies
f
...
.:
::
::
..
..
.:
Si
::
::
..:
t
..
..
..
. w
*
.. ...
lS, *. t;. ....
..
*:
..: ... * ....:
FIG. 1. Log-phase A. laidlawii strain BN1 cells were infected at a multiplicity of I to 10 PFU/CFU with each of the three mycoplasmatales viruses. Colonies from cells infected with MV-Li were photographed on days 1 (a), 2 (b), 3 (c), 4 (d), and 6 (e). Colonies from cells infected with MV-L3 (f), MV-L2 (g), and uninfected (h) were photographed on day 6 postinfection. Bar = 0.5 mm.
VIRAL DISTORTION OF ACHOLEPLASMIC COLONIES
VOL. 138, 1979
(Fig. lh), which was easily distinguishable from the severe alteration induced by MV-Li infection (Fig. le). PFU and viable cells within aberrant colonies. Blebbed colonies were examined for virus by resuspending colonies on day 6 with vigorous pipetting in 1 ml of broth, diluting, and plating in agar overlays containing indicator A. laidlawii. Each blebbed colony contained from 105 to 106 PFU serologically identical to the infecting virus (Table 1). (Note that the neutralization constants for all three antisera allowed for greater than 99.9% neutralization of serologically related viruses.) All of 200 blebbed colonies examined contained high-titer virus. Rarely, smooth, normal-appearing colonies were observed in plates prepared from the infected broth cultures; however, all such colonies tested contained high-titer virus. However, colonies obtained from uninfected cultures did not contain virus demonstrable by this method. Colony blebbing was inhibited by pretreatment of plates with antiserum only to the infecting virus. Antisera to the two heterologous viruses had no effect on the blebbed colony formation. Furthermore, the smooth colonies appearing with the homologous antiserum did not contain PFU, whereas high titers of virus were present with heterologous antisera. The aberrant colonies which contained such large numbers of PFU appeared to be products of a single infected cell rather than aggregates of cells. Blebbing was observed in all colonies even when log-phase cells, infected with MV-Li at a multiplicity of 5, were filtered through a polycarbonate filter which decreased the recovery of CFU from 105 to 107.
Temporal sequence of virus appearance. To monitor the simultaneous growth of virus and cells, colonies infected with MV-Li were resuspended with vigorous pipetting and plated for both PFU and CFU (Table 2). Virus was clearly evident in colonies on day 1 with 4.4 x 105 PFU/colony, a value at least 10% of the total TABLE 1. Specific neutralization of virus from blebbed colonies PFU/colony of cells infected with: Treatment' None Nonimmune serum MV-LI antiserum MV-L2 antiserum MV-L3 antiserum
MV-Li 1.45 x 105 3.15 x 105
10'
MV-L2 2.19 x 106 2.98 x 106 6.57 x 106
1.85 x i05 3.05 x 105
4.69 x 106
1
x
1
x
10'
MV-L3 1.68 x 106 2.27 1.11 2.00 9
x x x
106 106
10W
x 10'
Antiserum was diluted 1:100, and neutralization was carried out for 10 min at 37'C. Neutralization constants (K values) were 67,100 (MV-Li), 257 (MV-L2), and 40,158 (MVL3). '
965
virus found in older colonies. Interestingly, blebbed colonies had more PFU than CFU until day 4, the day blebbing became most pronounced. Uninfected colonies contained more CFU initially and reached a higher maximum level of viable cells than infected colonies. Many of the resuspended cells from the blebbed colonies gave rise to colonies which again blebbed (Table 3). The number of aberrant progeny colonies from the original blebbed colony decreased as the age of the parental colony increased. On day 1, 87.5% of the resuspended cells gave rise to blebbed colonies, whereas the percentage dropped drastically to 3.0% by day 9, but all colonies contained virus regardless of their blebbed or smooth appearance. The presence of virus in these smooth progeny colonies was in direct contrast to the lack of virus in smooth colonies from among the original blebbed colonies of the primary infection. Also, at no time was virus isolated from uninfected control colonies. Blebbing in A. modicunL Spontaneous TABLE 2. Numbers of viruses and cells in blebbed colonies of A. laidlawii infected with MV-LIa CFUc Days of icubation
PFUb in MV-
Ll-infected cul- MV-Ll-intures
fected cultures trs
Unnfected cultures
4.4 x 105 1.73 x 105 2.3 x 105 1.53 x 106 8.8 x 105 2.5 x 106 3.64 x 106 2.5 x 106 3.8 x 106 6.2 x 106 2.36 x 106 3.92 x 106 9.2 x 105 5 2.4 X 106 6.9 x 106 6 8.9x105 5.7x106 1.1x106 7 1.16 x 106 3.4 X 106 4.9 x 106 9 5.1 x 105 2.2 x 106 1.1 X 106 aColonies were resuspended by vigorous pipetting, and PFU and CFU were enumerated. The results are the average of four colonies per datum point.
1 2 3 4
b
PFU/colony. Colonies from uninfected cultures
showed no PFU.
'CFU/colony. TABLE 3. Proportion of blebbing in progeny colonies of blebbed colonies Day colony dispersed 1 4 5 7 9
Blebbing" ( 87.5 53.2
Presence of virus' + + + + +
31.5 4.9 3.0 'Colonies were assayed for blebbing on day 7 after resuspension and plating. Three blebbed colonies were
for each time point. resuspended ' Both smooth and blebbed colonies had titers between 8 x i0 and 2 x 106 PFU.
966
CONGDON AND KENNY
J. BACTERIOL.
plaques were observed in overlay lawns of A. modicum PG49 during host range studies with MV-Li, MV-L2, and MV-L3. The plaquing agent was filterable at 0.1 ,um through polycarbonate filter, chloroform labile, and capable of plaquing only on A. modicum but not on A. laidlawii and was not neutralized by potent antisera to each of the three A. laidlawii viruses. This agent appeared to be a new virus which we have named Mycoplasmatales virus-modicum 1, MV-Mi (Congdon and Kenny, submitted for publication). Of particular interest, cultures which gave spontaneous plaques also produced blebbed colonies. All colonies from the frozen stock culture were blebbed, whereas colonies from cells which had been subcultured for several passages were smooth. Closer examination and enumeration revealed a marked transition in colony morphology concurrent with the loss of spontaneous plaques (Table 4). Initially blebbing was observed in 100% of the colonies, decreased to 80% by the day 2 of subculturing, and was followed by a drastic drop to 7 and 2% on days 3 and 4 of subculturing. Spontaneous plaques disappeared in lawns of these subcultures on day 4 and did not reappear upon further subculturing. This transition from blebbed colonies to smooth in cultures of A. modicum which appeared to be naturally carrying a virus and which were somehow induced to productive infection was strikingly similar to the transition seen in laboratory-infected A. laidlawii cells (Table 3). Comparison of virus assay by blebbing, infectious centers, and plaque formation. Since blebbed colonies arose from single infected cells, we compared the number of blebbed colonies with both infectious centers and input PFU
(Fig. 2) to determine the sensitivity of the blebbing assay for virus quantitation. Both the input plaque assay and the infectious center assay (simultaneously performed) for MV-Li were linear and gave nearly identical values (Fig. 2a). Most strikingly, the number of blebbed colonies greatly exceeded the number of PFU or infectious centers at all virus doses. As judged from the linear portion of the blebbed CFU curve (relative viruses doses: 1:8 through 1:64), the blebbing assay was 30-fold more sensitive than the infectious center assay. Therefore, the apparent multiplicity of infection was 3 when judged by blebbing rather than the 0.1 value measured by the PFU/CFU ratio. This greater multiplicity of infection might have influenced the 33% reduction in CFU observed at the two highest virus doses, although even at a multiplicity of 10 PFU/CFU, half of the CFU survived (data not shown). The results of MV-L3 differed from those with MV-Li (Fig. 2b). The PFU assay was linear over a broad range, but as expected at high multiplicities, infectious centers diverged from PFU because the number of cells had become a limiting component (a similar effect was observed with MV-Li at multiplicities of greater than 1 PFU/CFU). The substantial CFU reduction (Fig. 2b) probably contributed to reduced infectious centers. The number of blebbed CFU was always lower than total CFU in contrast to MV-Li, where nearly all colonies blebbed at the highest multiplicities tested. Except for the highest multiplicity, the number of blebbed colonies exceeded the number of infectious centers by a ratio of 1.2 to 2.2.
TABLE 4. Concurrent decrease of blebbing and spontaneous plaquing in A. modicum CFU"
Day
Total Total
% with ~~blebs(
Spontaneoush plaques
3.8 X 106 100 2.9 x 107 93.8 Sparse lawn + 1.02 x 108 79.7 + 9.68 x 10' 6.9 6.1 X 107 1.7 4 Frozen stock culture of A. modicum PG-49 was diluted 1:10 in soy peptone broth on day 0 and subcultured by serial 1:5 dilutions on days 2 through 5. bAppearance of plaques in overlay lawns made with 1-ml portions of the cultures used for the blebbing assay. Lawns were incubated for 36 to 48 h at 37°C. +, Many plaques; -, no plaques, but heavy lawn growth of A. modicum. ' Colonies were examined for blebbed morphology on days 7 to 9 after plating on soy peptone agarose. 0 1 2 3
RELATIVE VIRUS DOSE
RELATIVE VIRUS DOSE
FIG. 2. Comparison of virus enumeration by blebbed colony, PFU, and infectious center assays. Log-phase A. laidlawii cells strain BNJ (a) and strain M1353 (b) were exposed to varying dilutions of MV-LI (a) and MV-L3 (b) for 8 to 9 min at 37°C, diluted into antisera at 0°C for 5 to 8 min, and plated for infectious centers and CFU/ml. The maximum multiplicity of undiluted virus was 0.2 (PFU/CFU) for MV-LI and 1.0 for MV-L3. Blebbed colonies were scored on day 8.
VIRAL DISTORTION OF ACHOLEPLASMIC COLONIES
VOL. 138, 1979
DISCUSSION Results concerning striking colony alterations resulting from persistent infection of procaryotes with viruses are rare in the literature. Size decreases and smooth and rough appearance modifications have been noted with infected bacterial colonies (2), and more strikingly, colonies of Bacillus megaterium had finger-like projections with toothed appearance at the ends when infected with a lysogenic bacteriophage (12). Colonies of A. laidlawii infected with MV-Li have been described as cratered (20), but micrographs did not demonstrate the radical alteration reported here. Furthermore, in the present study, blebs appeared raised when colonies were examined after longitudinal sectioning (unpublished observations). Blebbing appears to result from the generation of new centers of growth in infected colonies, analogous to formation of the yolk portion in normal colonies (15, 22). The new centers could result from selective pressures stimulated by the large numbers of virus within the maturing colony (Tables 1 and 2). The predominant cell type in the colony changes with age, as illustrated by the decreased secondary blebbing of progeny cells resuspended from older infected colonies (Table 3). It is likely that these newly selected cells develop new centers of growth similar to the papillae or sectors in bacterial colonies which form as a result of mutation in aging colonies (4). Our results suggest that the infection of suspended A. laidlawii cells with its virus has several consequences: (i) in an appropriate lawn, plaques and infectious centers are fonned; (i) when plated on the surface of agar plates, colonies carrying virus are produced which may bleb (MV-Li, MV-L3, and MV-Mi) or not (MV-L2); and (iii) reduction in the number of CFU may be observed at high multiplicities. For MV-Li, the number of blebbed colonies exceeded the number of plaques and infectious centers by a factor of 30, suggesting that under the conditions studied, only 3% of the infected cells gave rise to infectious centers. This result might be ascribed to poor survival of infected cells in the agarose overlay when plated for infectious centers; to a delay of synthesis or release of virus resulting in fewer plaques due to overgrowth by indicator cells; or to an enhanced survival of infected cells when plated for CFU on an agarose surface. The close correspondence of blebbing with PFU and infectious centers for MV-L3-infected cells suggests that cells which bleb are also responsible for infectious centers. The use of antiserum in comparison of the infectious center and blebbing assay did not affect the results (Fig. 2) because
967
the PFU assay (necessarily done without antiserum) exactly corresponded to the infectious center assay, indicating that the incubation time at 37°C was sufficient to permit penetration. The increased sensitivity of the blebbing assay over the infectious center assay has important consequences for determination of numbers of functional virus particles. The degree of increased efficiency in enumeration may vary with the strain of A. laidlawii and sensitivity of the plaque assay in various laboratories. Nevertheless, the present data suggest that apparent virus yields per cell may be underestimated. Although particles which induce blebbing might be defective in plaque induction, it is noteworthy that both particle types were neutralized by antiserum and a large number of PFU were present in blebbed colonies. The survival of MV-L3-infected cells is of considerable interest because Liss (16) has shown killing of A. laidlawii by MV-L3, a conclusion in apparent contrast to our results. However, Liss shows paradoxically that cell death only occurred after 30 min of incubation with high (-5 PFU/CFU) multiplicities of MV-L3, whereas plating before 30 min results in colony formation. Our cells were infected, treated with antibody, and plated within 20 to 30 min (see Materials and Methods). Thus, the results are in agreement and suggest that A. laidlawii cells can survive infection with MV-L3 and grow while simultaneously propagating high titers of virus (Table 1), a result previously demonstrated for both MV-Li (17) and MV-L2 (21). We observed reduction in CFU by the highest multiplicities of MV-Li and MV-L3 tested (Fig. 2). The reduction was not proportional to virus dose, i.e., only 50% CFU reduction was observed at a multiplicity of 10 PFU/CFU for MV-Li, therefore cell killing, particularly by lysis from without, may not totally account for this reduction. Other factors such as cell aggregation by excess virus may also influence this result. Detection of blebbed colonies may be a powerful tool in recovering viruses in other Mycoplasma species. A significant example is our observation that A. modicum PG-49, which naturally carried virus, showed blebbing at the time when spontaneous plaquing was observed, but not when the culture had lost its ability to spontaneously plaque (Table 4). The changes in colonial morphology of infected A. laidlawii cells were similar (Table 3), suggesting that blebbing is directly correlated with active virus production. Lastly, the mechanism of viral transfer is unclear. Since antiserum inhibits bleb formation, horizontal transfer appears reasonable. Furthermore, antiserum specifically neutralizes virus
968
CONGDON AND KENNY
from infected colonies, indicating that virus detectable by PFU assay is fully assembled. However, cells from blebbed colonies resuspended in antiserum and diluted gave rise to blebbed colonies containing virus (unpublished data), suggesting intracellular protection or possibly a non-neutralizable viral genome. Therefore, a mechanism for vertical transfer of virus, as previously suggested (16), cannot be excluded. ACKNOWLEDGMENTS This study was supported in part by Public Health Service research grant AI-06720 and training grant AI-206 from the National Institute of Allergy and Infectious Diseases.
J. BACTERIOL. 10. 11. 12.
13. 14.
15.
LTERATURE CITED 1. Adams, M. H. 1959. Bacteriophages, p. 29 and 109. Interscience Publishers, New York. 2. Barksdale, L., and S. B. Arden. 1974. Persisting bacteriophage infections, lysogeny, and phage conversions. Annu. Rev. Microbiol. 28:265-299. 3. Clyde, W. A. 1974. Studies on the mycoplasmatales viruses and mycoplasma pathogenicity. INSERM 33: 109-116. 4. Davis, B. 1973. Bacterial physiology, p. 103. In B. Davis, R. Dulbecco, H. Eisen, H. Ginsberg, W. B. Wood, and M. McCarty (ed.), Microbiology. Harper and Row, Inc.,
16. 17. 18.
19.
Hagerstown, Md. 5. Garwes, D. J., B. V. Pike, S. G. Wyld, D. H. Pocock, and R. N. Gourlay. 1975. Characterization of Mycoplasmatales virus-laidlawii 3. J. Gen. Virol. 29:11-24. 6. Gourlay, R. N. 1970. Isolation of a virus infecting a strain
20.
of M. laidlawii. Nature (London) 225:1165. 7. Gourlay, R. N. 1971. Mycoplasmatales virus-laidlawii 2, a new virus isolated from Acholeplasma laidlawii. J. Gen. Virol. 12:65-67. 8. Gourlay, R. N., J. Bruce, and D. J. Garwes. 1971. Characterization of Mycoplasmatales virus-laidlawii 1. Nature (London) New Biol. 229:118-119. 9. Gourlay, R. N., D. J. Garwes, J. Bruce, and S. G. Wyld. 1973. Further studies on the morphology and
21. 22. 23.
composition of Mycoplasmatales virus-laidlawii 2. J. Gen. Virol. 18:127-133. Gourlay, R. N., and S. G. Wyld. 1972. Some biological characteristics of Mycoplasmatales virus-laidlawii 1. J. Gen. Virol. 14:15-23. Gourlay, R. N., and S. G. Wyld. 1973. Isolation of Mycoplasmatales virus-laidlawii 3, a new virus infecting Acholeplasma laidlawii. J. Gen. Virol. 19:279-283. Inosca, EL 1953. Genetique. Sur une propriete de BaciUus megaterium liee a la presence d'un prophage. C.R. Acad. Sci. 237:1794-1795. Kenny, G. E. 1971. Immunogenicity of Mycoplasma pneumoniae. Infect. Immun. 3:510-515. Kenny, G. E. 1973. Contamination of mammalian cells in culture with mycoplasmata, p. 107-129. In J. Fogh (ed.), Contamination in tissue culture. Academic Press Inc., New York. Knudson, D. L, and R. MacLeod. 1970. Mycoplasma pneumoniae and Mycoplasma salivarium: electron microscopy of colony growth in agar. J. Bacteriol. 101: 609-617. Liss, A. 1977. Acholepasma laidLawii infection by group 3 mycoplasmavirus. Virology 77:433-436. Liss, A., and J. Maniloff. 1973. Infection of Acholeplasma laidlawii by MV-LI virus. Virology 55:118-126. Maniloff, J., J. Das, and J. R. Christensen. 1977. Viruses of Mycoplasmas and Spiroplasmas. Adv. Virus Res. 21:343-380. Maniloff, J., and A. Lis. 1974. Comparative structure, chemistry and evolution of mycoplasmaviruses, p. 583604. In E. Kurstak and K. Maramorosch (ed.), Viruses, evolution and cancer. Academic Press Inc., New York. Milne, R. G., G. W. Thompson, and D. Taylor-Robinson. 1972. Electron microscope observations on Acholeplasma laidlawii viruses. Arch. Gesamte Virusforsch. 37:378-385. Putzrath, R. M., and J. Maniloff. 1977. Growth of an enveloped mycoplasmavirus and establishment of a carrier state. J. Virol. 22:308-314. Razin, S., and 0. Oliver. 1961. Morphogenesis of Mycoplasma and bacterial L-form colonies. J. Gen. Microbiol. 24:225-237. Tully, J. G. 1973. Biological and serological characteristics of the Acholeplasmas. Ann. N.Y. Acad. Sci. 225: 74-93.
Vol. 138, No. 3
Alteration of Colonial Morphology of Acholeplasma laidlawii and Acholeplasma modicum by Infection with Mycoplasmatales Viruses ALICE L. CONGDON AND GEORGE E. KENNY* Department of Pathobiology, SC-38, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 98195 Received for publication 16 April 1979
Morphologically aberrant colonies resulted from the infection of Acholeplasma laidlawii with two of its three known viruses and from Acholeplasma modicum cells naturally carrying virus. The patterns of colonial alteration differed between cells infected with the two A. laidlawii viruses. Colonies derived from single cells infected with the bullet-shaped virus MV-Li (Mycoplasmatales virus-laidlawii1) had a radial sectoring pattern of intracolonial swellings ("blebs"), whereas cells infected with the tailed icosahedral virus MV-U3 contained bubble-like blebs. Colonies from cells infected with the enveloped virus MV-L2 appeared identical to those obtained from uninfected cells. Aberrant colonies contained 106 colonyforming units of organisms and 106 plaque-forming units of virus serologically identical to the infecting type, indicating that both the virus and host organism were capable of simultaneous replication. Enumeration of virus by means of counting aberrant colonies was 30-fold more sensitive than infectious center assay for MV-Li and 1.2- to 2-fold higher for MV-L3. Furthermore, blebbed colonies were observed with A. modicum cells only at times of concurrent spontaneous plaquing with a new virus specific to A. modicum. Thus, blebbing in colonies provides a valuable marker for detection of the Mycoplasmatales viruses. The order Mycoplasmatales comprises membrane-bounded cells which are the smallest freeliving cells known. Three morphologically distinct viruses have been isolated recently from Acholeplasma laidlawii, one of the organisms in this order: the bullet-shaped MV-Li (MV-L, Mycoplasmatales virus-laidlawii-1; 6,8, 10, 17); the enveloped, spherical MV-L2 (7); and the icosahedral, tailed virus MV-L3 (11). These simple organisms, therefore, are capable of supporting the replication of radically diverse viruses, suggesting that other members of the Mycoplasmatales, including the pathogens, may also contain viruses. The three viruses were isolated from either spontaneous plaques or from washes of lawns of A. laidlawii (18), indicating that the viruses are carried in some cryptic form. As with animal viruses, the development of methods for detecting nonlytic viruses (i.e., those which do not form plaques) may be required for detection of these cryptic viruses. We have observed that strikingly altered and distinct colonies arise from A. laidlawii cells infected with MV-Li or MV-L3 and from cultures of Acholeplasma modicum naturally carrying a new virus MV-Mi (Congdon and Kenny, submitted for publication). Our study 962
focused on determining the virus content of infected colonies, the mechanisms by which they arise, and the numerical relationships of altered colonies to standard measures such as plaques and infectious centers. MATERIALS AND METHODS Viruses and acholeplasmata. MV-Li (7, 8) was obtained from J. Maniloff of the University of Rochester, Rochester, N.Y. MV-L2 (9) and MV-L3 (5) were kindly provided by R. N. Gourlay, Compton, England. Viruses were plaque purified three times. Plaque purification of MV-L2 was carried out in the presence of 1:20 dilution of rabbit antiserum to MV-Li having a kinetic neutralization value (1) of 67,000. A. laidlawii strain BN-1 (19) was obtained from J. Maniloff, and A. laidlawii strain M1353 (3) was from W. Clyde, University of North Carolina, Chapel Hill, N.C. Both strains were cloned three times in our laboratory; serially cultivated in the presence of 1:100 dilution of antisera to MV-LI, MV-L2 and MV-L3; and selected for their efficiency in plaquing the three viruses. A. modicum PG-49 was obtained from J. Tully (23), transferred twice in our laboratory, and frozen at -700C. Media and buffers. Soy peptone broth (14) for growth of cells and virus lysates contained 2% soy peptone (Humko Sheffield, Lyndhurst, N.Y.), 0.5% NaCl, and 20 mM TES [N-tris(hydroxymethyl)-
VOL. 138, 1979
VIRAL DISTORTION OF ACHOLEPLASMIC COLONIES
methyl-2-aminoethane-sulfonic acid], pH 8.0, and was supplemented with 5% fresh yeast dialysate (14), 5% agamma horse serum (North American Biologicals), 80 mM glucose, and 400 U of penicillin per ml. The composition of solid medium was similar, with the omission of both glucose and penicillin and the addition of 0.65% agarose. Agarose medium (12 ml) was placed into each petri dish (60 by 15 mm) and used for the determination of colony-forming units (CFU) and as bottom support for the overlay technique of the plaque assay. The medium for the overlay for plaque assays consisted of 2% tryptose (Difco), 0.5% NaCl, 20 mM TES (pH 8.0), 80 mM glucose, and 0.5% agarose per titer with 5% agamma horse serum. Virus dilutions and storage were carried out in buffer consisting of 0.8% NaCl, 20 mM TES (pH 7.4), and 0.1% gelatin; this buffer was used for all experiments unless otherwise specified. Plaque assay. Plaque-forming units (PFU) and infectious centers were assayed by a modification of the overlay technique (1). Frozen portions of A. laidlawii were thawed, diluted 1:10 in medium without glucose, incubated overnight at 370C in 2.5% C02, diluted 1:3 to 1:8, and incubated in a 37°C water bath with shaking for 5 to 7 h. A 0.1- to 0.2-ml portion of virus dilution was placed on the soy peptone support medium and mixed with 2 ml of overlay containing 0.4 ml of indicator organisms. Lawns of A. modicum were prepared with 2 ml of tryptose overlay medium and 1 ml of broth culture. Plates were incubated at 37°C in 2.5% CO2 for 36 to 48 h. A. laidlawii strain BN-1 was used as indicator for MV-Li, and strain M1353 was used for MV-L2 and MV-L3. Polylysine (molecular weight, 140,000) was added at a concentration of 100 Agg/ml for plaque enhancement with MV-L2. Infection of cells for colony observation. Log-
phase cultures of A. laidlawii strain BN-1 containing 108 to 2 x 108 cells per ml were obtained by 1:10 dilution of overnight cultures and incubation at 370C in a New Brunswick gyratory shaker water bath for several hours. Cultures were infected at a multiplicity of 5 to 10 (PFU/CFU) with either MV-Li, MV-L2, or MV-L3. After a l-min adsorption, cells were diluted in buffer (400), and portions (0.05 ml) were plated in duplicate on plates which were then incubated at 37°C in a 2.5% C02-air atmosphere. Infectious center and blebbing colony assay. Log-phase cells ofA. laidlawii BN-1 strain and M1353 strain at a concentration of 108 to 2 x 108 were exposed to indicated concentrations of MV-Li and MV-L3, respectively. After an adsorption period of 9 min at 370C in the gyratory water bath antiserum was added to the cell suspension at a concentration sufficient to neutralize 98.3 to 99.7% of any remaining free virus during the ensuing 10-min incubation period. For assays of CFU, cells were diluted at 00C, and 0.05-ml portions were plated in triplicate on prewarmed 370C plates, allowing for absorption of the fluid before the end of the latent period for virus release from infected cells, thus preventing the spread of virus. (We found the latent period to be 45 min for MV-Li and 60 min for MV-L3 with the strains of A. laidlawii used as the host.) Infectious centers and input virus were assayed by the overlay technlique described above. To assay for both viable cells and virus, individual colonies were
963
picked with Pasteur pipettes, resuspended in 1 ml of buffer with vigorous pipetting with a 1-ml pipette, diluted, and plated for CFU and PFU. Antisera. Immunogens were prepared by infecting log-phase A. laidlawii grown in broth containing 5% agamma rabbit serum with MV-Li, MV-L2, or MV-L3 at a multiplicity of infection of 0.001 to 0.01. At the end of 16 to 36 h of incubation at 370C in a gyratory water bath, cells were pelleted by centrifugation at 10,400 x g for 30 min. Virus was precipitated from the supernatant by addition of polyethylene glycol (molecular weight, 6,000) and NaCl to final concentrations of 8% and 0.5 M, respectively. Virus was precipitated overnight at 40C, collected by centrifugation at 8,000 x g for 15 min, and dialyzed overnight against buffer without gelatin. Precipitates of MV-Li were treated with 0.2% Nonidet P-40 (final concentration), layered over a 10-ml cushion of 30% sucrose, and centrifuged at 83,000 x g for 4 h in a Beckman SW 25.1 rotor. The resuspended pellet was brought to a density of 1.37 g/ ml with CsCl in 20 mM TES, pH 7.4, and centrifuged at 177,000 x g for 17 h in a Beckman type 65 rotor. The band with viral activity was centrifuged a second time in CsCl at a density of 1.37 g/ml and dialyzed overnight. Precipitated MV-L3 was chloroform extracted, pelleted through a 3-ml 30% sucrose cushion by centrifugation at 83,000 x g for 4.5 h, and banded twice in CsCl at a density of 1.48 g/ml by centrifugation in a Beckman 65 rotor for 18 h at 177,000 x g. The visible band with viral activity was dialyzed overnight. The immunogen for MV-L2 was prepared by dialyzing polyethylene glycol-precipitated virus because a suitable purification scheme had not yet been devised. The immunization scheme was as described previously for mycoplasmata (13). Immunogens contained 10'° to 10" PFU/ml. Sera were obtained within 7 days of the final injection. Neutralization of virus in colonies. Single blebbed colonies were resuspended on day 6 in 1 ml of buffer with vigorous pipetting, and 0.1 ml of the suspension was added to tubes containing 0.2 ml of a 1: 100 dilution of antiserum. Homologous kinetic neutralization values (1) of the antisera were 67,100 (MVLi), 257 (MV-L2), and 40,158 (MV-L3); cross-neutralization was not observed. After 10 min of incubation at 370C, dilutions were made in buffer at 00C and plated in duplicate for PFU. Inhibition of bleb formation. Agarose plates were pretreated with 0.2 ml of a 1:10 dilution of antiserum to MV-Li, MV-L2, or MV-L3. Cells were infected at 370C for 10 min with each of the three viruses, diluted, and plated in duplicate. Colonies (500 minimum) were observed for blebbing on day 8.
RESULTS Alteration of morphology of infected colonies. While enumerating CFU after infection with MV-Li, strikingly altered colonies were observed on plates incubated at 37°C for 5 days or longer. This phenomenon was investigated by infecting broth cultures of A. laidlawii with each of the three viruses (MV-Li, MV-L2, and MV-L3) and observing colonial growth at 24-h
964
CONGDON AND KENNY
intervals (Fig. 1). Colonies seen on days 1 and 2 (Fig. la and lb) resembled those from uninfected controls, but by day 3 (Fig. lc) some colonies were distorted with a blebbed appearance (presence of multiple intracolonial centers and swellings). By day 4 (Fig. ld), radial sectoring characteristic of MV-Ll-infected colonies was easily discernible, and on day 6 (Fig. le) the colonies appeared highly blebbed in a typical pattern of radial sectoring. Blebbing was most marked when colonial density was sparse (500 to 1,000
.
J. BACTERIOL.
colonies per 60-mm plate; Fig. 1). A similar result was obtained after plating cells infected with the icosahedral virus MV-L3. In that case, blebbed colonies again appeared after 72 h, but blebs were larger and developed in a random pattern rather than displaying sectoring (Fig. lf). In contrast, cells infected with MV-L2 gave rise to nonnal fried-egg-appearing colonies (Fig. lg) which were morphologically identical to colonies from uninfected cells (Fig. lh). Rare sectoring was occasionally observed in uninfected colonies
f
...
.:
::
::
..
..
.:
Si
::
::
..:
t
..
..
..
. w
*
.. ...
lS, *. t;. ....
..
*:
..: ... * ....:
FIG. 1. Log-phase A. laidlawii strain BN1 cells were infected at a multiplicity of I to 10 PFU/CFU with each of the three mycoplasmatales viruses. Colonies from cells infected with MV-Li were photographed on days 1 (a), 2 (b), 3 (c), 4 (d), and 6 (e). Colonies from cells infected with MV-L3 (f), MV-L2 (g), and uninfected (h) were photographed on day 6 postinfection. Bar = 0.5 mm.
VIRAL DISTORTION OF ACHOLEPLASMIC COLONIES
VOL. 138, 1979
(Fig. lh), which was easily distinguishable from the severe alteration induced by MV-Li infection (Fig. le). PFU and viable cells within aberrant colonies. Blebbed colonies were examined for virus by resuspending colonies on day 6 with vigorous pipetting in 1 ml of broth, diluting, and plating in agar overlays containing indicator A. laidlawii. Each blebbed colony contained from 105 to 106 PFU serologically identical to the infecting virus (Table 1). (Note that the neutralization constants for all three antisera allowed for greater than 99.9% neutralization of serologically related viruses.) All of 200 blebbed colonies examined contained high-titer virus. Rarely, smooth, normal-appearing colonies were observed in plates prepared from the infected broth cultures; however, all such colonies tested contained high-titer virus. However, colonies obtained from uninfected cultures did not contain virus demonstrable by this method. Colony blebbing was inhibited by pretreatment of plates with antiserum only to the infecting virus. Antisera to the two heterologous viruses had no effect on the blebbed colony formation. Furthermore, the smooth colonies appearing with the homologous antiserum did not contain PFU, whereas high titers of virus were present with heterologous antisera. The aberrant colonies which contained such large numbers of PFU appeared to be products of a single infected cell rather than aggregates of cells. Blebbing was observed in all colonies even when log-phase cells, infected with MV-Li at a multiplicity of 5, were filtered through a polycarbonate filter which decreased the recovery of CFU from 105 to 107.
Temporal sequence of virus appearance. To monitor the simultaneous growth of virus and cells, colonies infected with MV-Li were resuspended with vigorous pipetting and plated for both PFU and CFU (Table 2). Virus was clearly evident in colonies on day 1 with 4.4 x 105 PFU/colony, a value at least 10% of the total TABLE 1. Specific neutralization of virus from blebbed colonies PFU/colony of cells infected with: Treatment' None Nonimmune serum MV-LI antiserum MV-L2 antiserum MV-L3 antiserum
MV-Li 1.45 x 105 3.15 x 105
10'
MV-L2 2.19 x 106 2.98 x 106 6.57 x 106
1.85 x i05 3.05 x 105
4.69 x 106
1
x
1
x
10'
MV-L3 1.68 x 106 2.27 1.11 2.00 9
x x x
106 106
10W
x 10'
Antiserum was diluted 1:100, and neutralization was carried out for 10 min at 37'C. Neutralization constants (K values) were 67,100 (MV-Li), 257 (MV-L2), and 40,158 (MVL3). '
965
virus found in older colonies. Interestingly, blebbed colonies had more PFU than CFU until day 4, the day blebbing became most pronounced. Uninfected colonies contained more CFU initially and reached a higher maximum level of viable cells than infected colonies. Many of the resuspended cells from the blebbed colonies gave rise to colonies which again blebbed (Table 3). The number of aberrant progeny colonies from the original blebbed colony decreased as the age of the parental colony increased. On day 1, 87.5% of the resuspended cells gave rise to blebbed colonies, whereas the percentage dropped drastically to 3.0% by day 9, but all colonies contained virus regardless of their blebbed or smooth appearance. The presence of virus in these smooth progeny colonies was in direct contrast to the lack of virus in smooth colonies from among the original blebbed colonies of the primary infection. Also, at no time was virus isolated from uninfected control colonies. Blebbing in A. modicunL Spontaneous TABLE 2. Numbers of viruses and cells in blebbed colonies of A. laidlawii infected with MV-LIa CFUc Days of icubation
PFUb in MV-
Ll-infected cul- MV-Ll-intures
fected cultures trs
Unnfected cultures
4.4 x 105 1.73 x 105 2.3 x 105 1.53 x 106 8.8 x 105 2.5 x 106 3.64 x 106 2.5 x 106 3.8 x 106 6.2 x 106 2.36 x 106 3.92 x 106 9.2 x 105 5 2.4 X 106 6.9 x 106 6 8.9x105 5.7x106 1.1x106 7 1.16 x 106 3.4 X 106 4.9 x 106 9 5.1 x 105 2.2 x 106 1.1 X 106 aColonies were resuspended by vigorous pipetting, and PFU and CFU were enumerated. The results are the average of four colonies per datum point.
1 2 3 4
b
PFU/colony. Colonies from uninfected cultures
showed no PFU.
'CFU/colony. TABLE 3. Proportion of blebbing in progeny colonies of blebbed colonies Day colony dispersed 1 4 5 7 9
Blebbing" ( 87.5 53.2
Presence of virus' + + + + +
31.5 4.9 3.0 'Colonies were assayed for blebbing on day 7 after resuspension and plating. Three blebbed colonies were
for each time point. resuspended ' Both smooth and blebbed colonies had titers between 8 x i0 and 2 x 106 PFU.
966
CONGDON AND KENNY
J. BACTERIOL.
plaques were observed in overlay lawns of A. modicum PG49 during host range studies with MV-Li, MV-L2, and MV-L3. The plaquing agent was filterable at 0.1 ,um through polycarbonate filter, chloroform labile, and capable of plaquing only on A. modicum but not on A. laidlawii and was not neutralized by potent antisera to each of the three A. laidlawii viruses. This agent appeared to be a new virus which we have named Mycoplasmatales virus-modicum 1, MV-Mi (Congdon and Kenny, submitted for publication). Of particular interest, cultures which gave spontaneous plaques also produced blebbed colonies. All colonies from the frozen stock culture were blebbed, whereas colonies from cells which had been subcultured for several passages were smooth. Closer examination and enumeration revealed a marked transition in colony morphology concurrent with the loss of spontaneous plaques (Table 4). Initially blebbing was observed in 100% of the colonies, decreased to 80% by the day 2 of subculturing, and was followed by a drastic drop to 7 and 2% on days 3 and 4 of subculturing. Spontaneous plaques disappeared in lawns of these subcultures on day 4 and did not reappear upon further subculturing. This transition from blebbed colonies to smooth in cultures of A. modicum which appeared to be naturally carrying a virus and which were somehow induced to productive infection was strikingly similar to the transition seen in laboratory-infected A. laidlawii cells (Table 3). Comparison of virus assay by blebbing, infectious centers, and plaque formation. Since blebbed colonies arose from single infected cells, we compared the number of blebbed colonies with both infectious centers and input PFU
(Fig. 2) to determine the sensitivity of the blebbing assay for virus quantitation. Both the input plaque assay and the infectious center assay (simultaneously performed) for MV-Li were linear and gave nearly identical values (Fig. 2a). Most strikingly, the number of blebbed colonies greatly exceeded the number of PFU or infectious centers at all virus doses. As judged from the linear portion of the blebbed CFU curve (relative viruses doses: 1:8 through 1:64), the blebbing assay was 30-fold more sensitive than the infectious center assay. Therefore, the apparent multiplicity of infection was 3 when judged by blebbing rather than the 0.1 value measured by the PFU/CFU ratio. This greater multiplicity of infection might have influenced the 33% reduction in CFU observed at the two highest virus doses, although even at a multiplicity of 10 PFU/CFU, half of the CFU survived (data not shown). The results of MV-L3 differed from those with MV-Li (Fig. 2b). The PFU assay was linear over a broad range, but as expected at high multiplicities, infectious centers diverged from PFU because the number of cells had become a limiting component (a similar effect was observed with MV-Li at multiplicities of greater than 1 PFU/CFU). The substantial CFU reduction (Fig. 2b) probably contributed to reduced infectious centers. The number of blebbed CFU was always lower than total CFU in contrast to MV-Li, where nearly all colonies blebbed at the highest multiplicities tested. Except for the highest multiplicity, the number of blebbed colonies exceeded the number of infectious centers by a ratio of 1.2 to 2.2.
TABLE 4. Concurrent decrease of blebbing and spontaneous plaquing in A. modicum CFU"
Day
Total Total
% with ~~blebs(
Spontaneoush plaques
3.8 X 106 100 2.9 x 107 93.8 Sparse lawn + 1.02 x 108 79.7 + 9.68 x 10' 6.9 6.1 X 107 1.7 4 Frozen stock culture of A. modicum PG-49 was diluted 1:10 in soy peptone broth on day 0 and subcultured by serial 1:5 dilutions on days 2 through 5. bAppearance of plaques in overlay lawns made with 1-ml portions of the cultures used for the blebbing assay. Lawns were incubated for 36 to 48 h at 37°C. +, Many plaques; -, no plaques, but heavy lawn growth of A. modicum. ' Colonies were examined for blebbed morphology on days 7 to 9 after plating on soy peptone agarose. 0 1 2 3
RELATIVE VIRUS DOSE
RELATIVE VIRUS DOSE
FIG. 2. Comparison of virus enumeration by blebbed colony, PFU, and infectious center assays. Log-phase A. laidlawii cells strain BNJ (a) and strain M1353 (b) were exposed to varying dilutions of MV-LI (a) and MV-L3 (b) for 8 to 9 min at 37°C, diluted into antisera at 0°C for 5 to 8 min, and plated for infectious centers and CFU/ml. The maximum multiplicity of undiluted virus was 0.2 (PFU/CFU) for MV-LI and 1.0 for MV-L3. Blebbed colonies were scored on day 8.
VIRAL DISTORTION OF ACHOLEPLASMIC COLONIES
VOL. 138, 1979
DISCUSSION Results concerning striking colony alterations resulting from persistent infection of procaryotes with viruses are rare in the literature. Size decreases and smooth and rough appearance modifications have been noted with infected bacterial colonies (2), and more strikingly, colonies of Bacillus megaterium had finger-like projections with toothed appearance at the ends when infected with a lysogenic bacteriophage (12). Colonies of A. laidlawii infected with MV-Li have been described as cratered (20), but micrographs did not demonstrate the radical alteration reported here. Furthermore, in the present study, blebs appeared raised when colonies were examined after longitudinal sectioning (unpublished observations). Blebbing appears to result from the generation of new centers of growth in infected colonies, analogous to formation of the yolk portion in normal colonies (15, 22). The new centers could result from selective pressures stimulated by the large numbers of virus within the maturing colony (Tables 1 and 2). The predominant cell type in the colony changes with age, as illustrated by the decreased secondary blebbing of progeny cells resuspended from older infected colonies (Table 3). It is likely that these newly selected cells develop new centers of growth similar to the papillae or sectors in bacterial colonies which form as a result of mutation in aging colonies (4). Our results suggest that the infection of suspended A. laidlawii cells with its virus has several consequences: (i) in an appropriate lawn, plaques and infectious centers are fonned; (i) when plated on the surface of agar plates, colonies carrying virus are produced which may bleb (MV-Li, MV-L3, and MV-Mi) or not (MV-L2); and (iii) reduction in the number of CFU may be observed at high multiplicities. For MV-Li, the number of blebbed colonies exceeded the number of plaques and infectious centers by a factor of 30, suggesting that under the conditions studied, only 3% of the infected cells gave rise to infectious centers. This result might be ascribed to poor survival of infected cells in the agarose overlay when plated for infectious centers; to a delay of synthesis or release of virus resulting in fewer plaques due to overgrowth by indicator cells; or to an enhanced survival of infected cells when plated for CFU on an agarose surface. The close correspondence of blebbing with PFU and infectious centers for MV-L3-infected cells suggests that cells which bleb are also responsible for infectious centers. The use of antiserum in comparison of the infectious center and blebbing assay did not affect the results (Fig. 2) because
967
the PFU assay (necessarily done without antiserum) exactly corresponded to the infectious center assay, indicating that the incubation time at 37°C was sufficient to permit penetration. The increased sensitivity of the blebbing assay over the infectious center assay has important consequences for determination of numbers of functional virus particles. The degree of increased efficiency in enumeration may vary with the strain of A. laidlawii and sensitivity of the plaque assay in various laboratories. Nevertheless, the present data suggest that apparent virus yields per cell may be underestimated. Although particles which induce blebbing might be defective in plaque induction, it is noteworthy that both particle types were neutralized by antiserum and a large number of PFU were present in blebbed colonies. The survival of MV-L3-infected cells is of considerable interest because Liss (16) has shown killing of A. laidlawii by MV-L3, a conclusion in apparent contrast to our results. However, Liss shows paradoxically that cell death only occurred after 30 min of incubation with high (-5 PFU/CFU) multiplicities of MV-L3, whereas plating before 30 min results in colony formation. Our cells were infected, treated with antibody, and plated within 20 to 30 min (see Materials and Methods). Thus, the results are in agreement and suggest that A. laidlawii cells can survive infection with MV-L3 and grow while simultaneously propagating high titers of virus (Table 1), a result previously demonstrated for both MV-Li (17) and MV-L2 (21). We observed reduction in CFU by the highest multiplicities of MV-Li and MV-L3 tested (Fig. 2). The reduction was not proportional to virus dose, i.e., only 50% CFU reduction was observed at a multiplicity of 10 PFU/CFU for MV-Li, therefore cell killing, particularly by lysis from without, may not totally account for this reduction. Other factors such as cell aggregation by excess virus may also influence this result. Detection of blebbed colonies may be a powerful tool in recovering viruses in other Mycoplasma species. A significant example is our observation that A. modicum PG-49, which naturally carried virus, showed blebbing at the time when spontaneous plaquing was observed, but not when the culture had lost its ability to spontaneously plaque (Table 4). The changes in colonial morphology of infected A. laidlawii cells were similar (Table 3), suggesting that blebbing is directly correlated with active virus production. Lastly, the mechanism of viral transfer is unclear. Since antiserum inhibits bleb formation, horizontal transfer appears reasonable. Furthermore, antiserum specifically neutralizes virus
968
CONGDON AND KENNY
from infected colonies, indicating that virus detectable by PFU assay is fully assembled. However, cells from blebbed colonies resuspended in antiserum and diluted gave rise to blebbed colonies containing virus (unpublished data), suggesting intracellular protection or possibly a non-neutralizable viral genome. Therefore, a mechanism for vertical transfer of virus, as previously suggested (16), cannot be excluded. ACKNOWLEDGMENTS This study was supported in part by Public Health Service research grant AI-06720 and training grant AI-206 from the National Institute of Allergy and Infectious Diseases.
J. BACTERIOL. 10. 11. 12.
13. 14.
15.
LTERATURE CITED 1. Adams, M. H. 1959. Bacteriophages, p. 29 and 109. Interscience Publishers, New York. 2. Barksdale, L., and S. B. Arden. 1974. Persisting bacteriophage infections, lysogeny, and phage conversions. Annu. Rev. Microbiol. 28:265-299. 3. Clyde, W. A. 1974. Studies on the mycoplasmatales viruses and mycoplasma pathogenicity. INSERM 33: 109-116. 4. Davis, B. 1973. Bacterial physiology, p. 103. In B. Davis, R. Dulbecco, H. Eisen, H. Ginsberg, W. B. Wood, and M. McCarty (ed.), Microbiology. Harper and Row, Inc.,
16. 17. 18.
19.
Hagerstown, Md. 5. Garwes, D. J., B. V. Pike, S. G. Wyld, D. H. Pocock, and R. N. Gourlay. 1975. Characterization of Mycoplasmatales virus-laidlawii 3. J. Gen. Virol. 29:11-24. 6. Gourlay, R. N. 1970. Isolation of a virus infecting a strain
20.
of M. laidlawii. Nature (London) 225:1165. 7. Gourlay, R. N. 1971. Mycoplasmatales virus-laidlawii 2, a new virus isolated from Acholeplasma laidlawii. J. Gen. Virol. 12:65-67. 8. Gourlay, R. N., J. Bruce, and D. J. Garwes. 1971. Characterization of Mycoplasmatales virus-laidlawii 1. Nature (London) New Biol. 229:118-119. 9. Gourlay, R. N., D. J. Garwes, J. Bruce, and S. G. Wyld. 1973. Further studies on the morphology and
21. 22. 23.
composition of Mycoplasmatales virus-laidlawii 2. J. Gen. Virol. 18:127-133. Gourlay, R. N., and S. G. Wyld. 1972. Some biological characteristics of Mycoplasmatales virus-laidlawii 1. J. Gen. Virol. 14:15-23. Gourlay, R. N., and S. G. Wyld. 1973. Isolation of Mycoplasmatales virus-laidlawii 3, a new virus infecting Acholeplasma laidlawii. J. Gen. Virol. 19:279-283. Inosca, EL 1953. Genetique. Sur une propriete de BaciUus megaterium liee a la presence d'un prophage. C.R. Acad. Sci. 237:1794-1795. Kenny, G. E. 1971. Immunogenicity of Mycoplasma pneumoniae. Infect. Immun. 3:510-515. Kenny, G. E. 1973. Contamination of mammalian cells in culture with mycoplasmata, p. 107-129. In J. Fogh (ed.), Contamination in tissue culture. Academic Press Inc., New York. Knudson, D. L, and R. MacLeod. 1970. Mycoplasma pneumoniae and Mycoplasma salivarium: electron microscopy of colony growth in agar. J. Bacteriol. 101: 609-617. Liss, A. 1977. Acholepasma laidLawii infection by group 3 mycoplasmavirus. Virology 77:433-436. Liss, A., and J. Maniloff. 1973. Infection of Acholeplasma laidlawii by MV-LI virus. Virology 55:118-126. Maniloff, J., J. Das, and J. R. Christensen. 1977. Viruses of Mycoplasmas and Spiroplasmas. Adv. Virus Res. 21:343-380. Maniloff, J., and A. Lis. 1974. Comparative structure, chemistry and evolution of mycoplasmaviruses, p. 583604. In E. Kurstak and K. Maramorosch (ed.), Viruses, evolution and cancer. Academic Press Inc., New York. Milne, R. G., G. W. Thompson, and D. Taylor-Robinson. 1972. Electron microscope observations on Acholeplasma laidlawii viruses. Arch. Gesamte Virusforsch. 37:378-385. Putzrath, R. M., and J. Maniloff. 1977. Growth of an enveloped mycoplasmavirus and establishment of a carrier state. J. Virol. 22:308-314. Razin, S., and 0. Oliver. 1961. Morphogenesis of Mycoplasma and bacterial L-form colonies. J. Gen. Microbiol. 24:225-237. Tully, J. G. 1973. Biological and serological characteristics of the Acholeplasmas. Ann. N.Y. Acad. Sci. 225: 74-93.
