APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, p. 971-974 Copyright © 1977 American Society for Microbiology
Vol. 33, No. 4 Printed in U.S.A.
NOTES Simple, Effective Method for Purifying the AS-1 Cyanophage M. B. BARKLEY1 AND P. R. DESJARDINS* Department of Plant Pathology, University of California, Riverside, California 92521 Received for publication 24 September 1976
Highly infectious, purified AS-1 virus was obtained by treating virus-induced culture lysates with bentonite followed by rotary evaporation concentration and density gradient centrifugation.
Purification of the AS-1 cyanophage from its blue-green algal hosts is often complicated by the association of virions with cellular pigments and membranous and polysaccharidelike material in culture lysates. Safferman et al. (5) and Pearson et al. (4) reported purification procedures for this cyanophage. One method (5) involves concentration in the Sharples centrifuge, differential centrifugation, and density gradient centrifugation and often results in retention of pigmented membranous material that sediments with the virus in a sucrose gradient column. The other method (4) utilizes CsCl2 equilibrium density gradient centrifugation and results in marked losses of both infectivity and particle integrity. Both methods have the additional disadvantage of requiring 12 to 20 liters of crude lysate. In the present communication, a new method for purifying the AS-1 cyanophage is described that is simple, rapid, and requires only small volumes of crude lysate. Virus-induced lysates of Anacystis nidulans IU 625 were obtained by inoculating the host during its exponential phase of growth. Host cells were grown in 500-ml Erlenmeyer flasks containing 200 ml of modified Hughes medium (1), incubated at 30°C under 200 ft-c (ca. 2,152 lx) of fluorescent light, and aerated at 300 ml/ min. Continued aeration of the inoculated cell suspension after lysis resulted in frothing, which tended to remove cellular debris from the lysate suspension. Time of incubation for complete lysis of the culture depended on the multiplicity of infection. With multiplicities of infection of 0.01, 0.1 to 1, and >5, the times required for lysis were ca. 24 h, 10 to 12 h, and ca. 8 h, respectively. The untreated lysate usually had a minimum infectivity titer of the order of 109 to I Present address: "The Cottage," St. Thomas Road, Mulgoa via Penrith, New South Wales, 2750, Australia.
5 x 109 plaque-forming unit (PFU)/ml. Further clarification and purification of the virus is illustrated in the flow diagram in Fig. 1. Treatment of clarified lysate with unfractionated bentonite (City Chemical Corp., N. Y.) removed cellular debris and microscopic material without reduction in infectivity. Dialysis of clarified virus against 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.8, prior to concentration is critical if virus degradation, resulting from high salt concentration, is to be avoided. It is also critical that virus concentrated by rotary evaporation be given a low speed centrifugation and dialyzed against buffer immediately following the concentration step. Tris(hydroxymethyl)aminomethane-hydrochloride buffer (0.01 M, pH 7.8) has been found suitable for this dialysis step also, since infectivity of highly purified virus remains stable in this buffer for 2 months at either 4°C or room temperature. Infectivity titers at various steps in the procedure varied somewhat from one experiment to another. In a typical experiment when the titer of the bentonite-clarified virus was 2.9 x 109 PFU/ml, the titer of virus concentrated 10-fold by rotary evaporation was 2.4 x 1010 PFU/ml. When the virus, concentrated by rotary evaporation, is centrifuged through linear sucrose gradient columns (0.3 to 1.0 M sucrose), two ultraviolet-absorbing zones are observed (Fig. 2). The first broad zone occurs near the meniscus, as a second sharp zone bands midway down the column. Although some infectivity was found in the zone near the meniscus (104 to 106 PFU/ml), the major portion of the infectivity was found in the bottom zone. The titers in this zone varied from 3.8 x 1010 to 1 x 1011 PFU/ml. Electron microscopic examination of the top zone (Fig. 3A and B) revealed the presence of degraded virus (or unassembled virus precur971
972
APPL. ENVIRON. MICROBIOL.
NOTES
Lvsate from AS-1 Virus Inoculated Culture of Anacvstis nidulans IU 625 Primarv Clarification
(continued aeration)
I
Secondary Clarification 100 ml partially clarified lysate + 0.4 g sterilized bentonite; stir 30 min at room temp Purification
Centrifuge at 6500 rpj for 10 min (Sorvall GSA Rotor)
PelAet
Supernatant Filter through Whatman No. 2 filter paper
discard
Filtrate Dialyze against 0.01 M Tris-HCl
buffer, pH 7.C Concentration Rotary evaporate at medium speed at 34 ± 1 C
Centrifuge at 6500 rpm for 10 min (Sorvall SS34 Rotor) Pellet discard
Su2ernatant
Layer 1-2 ml onto preformed linear 0.3 M to 1.0 M sucrose in 0.01 M Tris-HCl buffer, pH 7.8, gradients; centrifuge at 20,000 rpm for 30-45 min in SW 27 rotor
I
Fractionate and collect bottom zone of highly ourified virus
Dialvze immediately against 0.01 M TrisHC1 bdffer, 'pH 7.8, to remove sucrose Centrifuge at 6500 rpm for 10 mmin (Sorvall SS34 Rotor) or
Filter through sterile 0.2 p cellutlose acetate membrane filter to remove contaminating microorganisms
FIG. 1. Flow diagram for purification ofAS-1 cyanophage from virus-induced lysate ofA. nidulans IU 625 cultivated in liquid medium. (Note: SS-34 rotor at 6,500 rpm = 5,100 x g; and GSA rotor at 6,500 rpm 6,900 x g.).
sors) and what appeared to be particulate bentonite (2, 3), which often appeared to be associated with exposed tailcores of virus particles with contracted sheaths (Fig. 3B). Since the zones were prepared for electron microscopy immediately after density gradient centrifugation, the association of degraded virus particles with particulate bentonite probably occurred during the clarification or concentration steps of the procedure. Presumably, the upper zone also contains viral and host nucleic acids (deoxyribonucleic acid), as some optical density was moved from this zone to the top of the gradient after treatment with deoxyribonuclease. Virus in the bottom zone (Fig. 3C) was free of cellular material and relatively homogeneous with respect to particle integrity. In comparison, virus purified by CsCl2 equilibrium centrifugation (4) appeared free of cellular material but was unsatisfactory, because of
E 5')
z 0
a. 0 co
co
-J/
RELATIVE DEPTH IN COLUMN
FIG. 2. Scanning profile of bentonite-treated, AS1 virus-induced lysate of A. nidulans IU 625 culture after 10-fold concentration by rotary evaporation and centrifugation through a linear sucrose gradient column. The major peak on the right is the intact virus zone. The arrow indicates a change to greater absorbance sensitivity of the scanning device.
VOL. 33, 1977
NOTES
973
FIG. 3. AS-1 virus purified by the method described and by CsC12 equilibrium centrifugation, negatively stained with uranyl acetate. (A, B) Top zone and (C) bottom zone of bentonite-treated virus after centrifugation through a linear sucrose gradient. (D) Virus purified by CsCI2 equilibrium centrifugation. Bar represents 0.2 ,um.
marked loss of titer (ca. 100 PFU/ml less than the crude lysate) and particle degradation (Fig. 3D). This method of purification has been found to
be simple, effective, and inexpensive. In addition, it permits rapid purification of AS-1 cyanophage from crude lysate. Only very small volumes (100 ml) of lysate are required to ob-
974
NOTES
APPL. ENVIRON. MICROBIOL.
tain relatively high yields (ca. 1 x 1011 PFU/ml) of highly infectious virus. The use of bentonite clarification in conjunction with density gradient centrifugation could be applied to the purification of other cyanophages, since some of these viruses undoubtedly share similar problems in clarification and purification.
Sciences Training Program, University of California, Riverside.
We thank R. S. Safferman for originally providing the host cultures and the AS-1 virus and J. G. Bald for his helpful suggestions. Also, we thank S. A. Swiecki and S. N. West for technical assistance and preparation of the illustrations. The research leading to this report was supported by the Office of Water Research and Technology, USDI, under the matching fund program of Public Law 88-379, as amended, and by the University of California, Water Resources Center, as part of Office of Water Research and Technology Project No. B-176-CAL and Water Resources Center Project UCAL-WRC-W-496. M. B. B. received support as a predoctoral trainee under Public Health Service training grant no. ES 00084-05, from the Division of Environmental Health
Proj. 49-Clay Mineral Standards, Prelim. Report No. 6, New York. 3. Jackson, M. L., W. Z. Mackie, and R. P. Pennington. 1946. Electron microscope applications in soils research. Soil Sci. Soc. Am. Proc. 11:57-63. 4. Pearson, N. J., E. A. Small, and M. M. Allen. 1975. Electron microscopic study of the infection of Anacystis nidulans by the cyanophage AS-1. Virology 65:469-479. 5. Safferman, R. S., T. 0. Diener, P. R. Desjardins, and M. E. Morris. 1972. Isolation and characterization of AS-1, a phycovirus infecting the blue-green algae, Anacystis nidulans and Synechococcus cedrorum. Virology 47:105-113.
LITERATURE CITED 1. Allen, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4:14. 2. Davis, D. W., T. G. Rochow, F. G. Rowe, M. L. Fuller, P. F. Kerr, and P. F. Hamilton. 1950. Electron micrographs of reference clay materials. In Am. Pet. Inst.
Vol. 33, No. 4 Printed in U.S.A.
NOTES Simple, Effective Method for Purifying the AS-1 Cyanophage M. B. BARKLEY1 AND P. R. DESJARDINS* Department of Plant Pathology, University of California, Riverside, California 92521 Received for publication 24 September 1976
Highly infectious, purified AS-1 virus was obtained by treating virus-induced culture lysates with bentonite followed by rotary evaporation concentration and density gradient centrifugation.
Purification of the AS-1 cyanophage from its blue-green algal hosts is often complicated by the association of virions with cellular pigments and membranous and polysaccharidelike material in culture lysates. Safferman et al. (5) and Pearson et al. (4) reported purification procedures for this cyanophage. One method (5) involves concentration in the Sharples centrifuge, differential centrifugation, and density gradient centrifugation and often results in retention of pigmented membranous material that sediments with the virus in a sucrose gradient column. The other method (4) utilizes CsCl2 equilibrium density gradient centrifugation and results in marked losses of both infectivity and particle integrity. Both methods have the additional disadvantage of requiring 12 to 20 liters of crude lysate. In the present communication, a new method for purifying the AS-1 cyanophage is described that is simple, rapid, and requires only small volumes of crude lysate. Virus-induced lysates of Anacystis nidulans IU 625 were obtained by inoculating the host during its exponential phase of growth. Host cells were grown in 500-ml Erlenmeyer flasks containing 200 ml of modified Hughes medium (1), incubated at 30°C under 200 ft-c (ca. 2,152 lx) of fluorescent light, and aerated at 300 ml/ min. Continued aeration of the inoculated cell suspension after lysis resulted in frothing, which tended to remove cellular debris from the lysate suspension. Time of incubation for complete lysis of the culture depended on the multiplicity of infection. With multiplicities of infection of 0.01, 0.1 to 1, and >5, the times required for lysis were ca. 24 h, 10 to 12 h, and ca. 8 h, respectively. The untreated lysate usually had a minimum infectivity titer of the order of 109 to I Present address: "The Cottage," St. Thomas Road, Mulgoa via Penrith, New South Wales, 2750, Australia.
5 x 109 plaque-forming unit (PFU)/ml. Further clarification and purification of the virus is illustrated in the flow diagram in Fig. 1. Treatment of clarified lysate with unfractionated bentonite (City Chemical Corp., N. Y.) removed cellular debris and microscopic material without reduction in infectivity. Dialysis of clarified virus against 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.8, prior to concentration is critical if virus degradation, resulting from high salt concentration, is to be avoided. It is also critical that virus concentrated by rotary evaporation be given a low speed centrifugation and dialyzed against buffer immediately following the concentration step. Tris(hydroxymethyl)aminomethane-hydrochloride buffer (0.01 M, pH 7.8) has been found suitable for this dialysis step also, since infectivity of highly purified virus remains stable in this buffer for 2 months at either 4°C or room temperature. Infectivity titers at various steps in the procedure varied somewhat from one experiment to another. In a typical experiment when the titer of the bentonite-clarified virus was 2.9 x 109 PFU/ml, the titer of virus concentrated 10-fold by rotary evaporation was 2.4 x 1010 PFU/ml. When the virus, concentrated by rotary evaporation, is centrifuged through linear sucrose gradient columns (0.3 to 1.0 M sucrose), two ultraviolet-absorbing zones are observed (Fig. 2). The first broad zone occurs near the meniscus, as a second sharp zone bands midway down the column. Although some infectivity was found in the zone near the meniscus (104 to 106 PFU/ml), the major portion of the infectivity was found in the bottom zone. The titers in this zone varied from 3.8 x 1010 to 1 x 1011 PFU/ml. Electron microscopic examination of the top zone (Fig. 3A and B) revealed the presence of degraded virus (or unassembled virus precur971
972
APPL. ENVIRON. MICROBIOL.
NOTES
Lvsate from AS-1 Virus Inoculated Culture of Anacvstis nidulans IU 625 Primarv Clarification
(continued aeration)
I
Secondary Clarification 100 ml partially clarified lysate + 0.4 g sterilized bentonite; stir 30 min at room temp Purification
Centrifuge at 6500 rpj for 10 min (Sorvall GSA Rotor)
PelAet
Supernatant Filter through Whatman No. 2 filter paper
discard
Filtrate Dialyze against 0.01 M Tris-HCl
buffer, pH 7.C Concentration Rotary evaporate at medium speed at 34 ± 1 C
Centrifuge at 6500 rpm for 10 min (Sorvall SS34 Rotor) Pellet discard
Su2ernatant
Layer 1-2 ml onto preformed linear 0.3 M to 1.0 M sucrose in 0.01 M Tris-HCl buffer, pH 7.8, gradients; centrifuge at 20,000 rpm for 30-45 min in SW 27 rotor
I
Fractionate and collect bottom zone of highly ourified virus
Dialvze immediately against 0.01 M TrisHC1 bdffer, 'pH 7.8, to remove sucrose Centrifuge at 6500 rpm for 10 mmin (Sorvall SS34 Rotor) or
Filter through sterile 0.2 p cellutlose acetate membrane filter to remove contaminating microorganisms
FIG. 1. Flow diagram for purification ofAS-1 cyanophage from virus-induced lysate ofA. nidulans IU 625 cultivated in liquid medium. (Note: SS-34 rotor at 6,500 rpm = 5,100 x g; and GSA rotor at 6,500 rpm 6,900 x g.).
sors) and what appeared to be particulate bentonite (2, 3), which often appeared to be associated with exposed tailcores of virus particles with contracted sheaths (Fig. 3B). Since the zones were prepared for electron microscopy immediately after density gradient centrifugation, the association of degraded virus particles with particulate bentonite probably occurred during the clarification or concentration steps of the procedure. Presumably, the upper zone also contains viral and host nucleic acids (deoxyribonucleic acid), as some optical density was moved from this zone to the top of the gradient after treatment with deoxyribonuclease. Virus in the bottom zone (Fig. 3C) was free of cellular material and relatively homogeneous with respect to particle integrity. In comparison, virus purified by CsCl2 equilibrium centrifugation (4) appeared free of cellular material but was unsatisfactory, because of
E 5')
z 0
a. 0 co
co
-J/
RELATIVE DEPTH IN COLUMN
FIG. 2. Scanning profile of bentonite-treated, AS1 virus-induced lysate of A. nidulans IU 625 culture after 10-fold concentration by rotary evaporation and centrifugation through a linear sucrose gradient column. The major peak on the right is the intact virus zone. The arrow indicates a change to greater absorbance sensitivity of the scanning device.
VOL. 33, 1977
NOTES
973
FIG. 3. AS-1 virus purified by the method described and by CsC12 equilibrium centrifugation, negatively stained with uranyl acetate. (A, B) Top zone and (C) bottom zone of bentonite-treated virus after centrifugation through a linear sucrose gradient. (D) Virus purified by CsCI2 equilibrium centrifugation. Bar represents 0.2 ,um.
marked loss of titer (ca. 100 PFU/ml less than the crude lysate) and particle degradation (Fig. 3D). This method of purification has been found to
be simple, effective, and inexpensive. In addition, it permits rapid purification of AS-1 cyanophage from crude lysate. Only very small volumes (100 ml) of lysate are required to ob-
974
NOTES
APPL. ENVIRON. MICROBIOL.
tain relatively high yields (ca. 1 x 1011 PFU/ml) of highly infectious virus. The use of bentonite clarification in conjunction with density gradient centrifugation could be applied to the purification of other cyanophages, since some of these viruses undoubtedly share similar problems in clarification and purification.
Sciences Training Program, University of California, Riverside.
We thank R. S. Safferman for originally providing the host cultures and the AS-1 virus and J. G. Bald for his helpful suggestions. Also, we thank S. A. Swiecki and S. N. West for technical assistance and preparation of the illustrations. The research leading to this report was supported by the Office of Water Research and Technology, USDI, under the matching fund program of Public Law 88-379, as amended, and by the University of California, Water Resources Center, as part of Office of Water Research and Technology Project No. B-176-CAL and Water Resources Center Project UCAL-WRC-W-496. M. B. B. received support as a predoctoral trainee under Public Health Service training grant no. ES 00084-05, from the Division of Environmental Health
Proj. 49-Clay Mineral Standards, Prelim. Report No. 6, New York. 3. Jackson, M. L., W. Z. Mackie, and R. P. Pennington. 1946. Electron microscope applications in soils research. Soil Sci. Soc. Am. Proc. 11:57-63. 4. Pearson, N. J., E. A. Small, and M. M. Allen. 1975. Electron microscopic study of the infection of Anacystis nidulans by the cyanophage AS-1. Virology 65:469-479. 5. Safferman, R. S., T. 0. Diener, P. R. Desjardins, and M. E. Morris. 1972. Isolation and characterization of AS-1, a phycovirus infecting the blue-green algae, Anacystis nidulans and Synechococcus cedrorum. Virology 47:105-113.
LITERATURE CITED 1. Allen, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4:14. 2. Davis, D. W., T. G. Rochow, F. G. Rowe, M. L. Fuller, P. F. Kerr, and P. F. Hamilton. 1950. Electron micrographs of reference clay materials. In Am. Pet. Inst.
