APPLuD MICROBIOLOGY, Feb. 1975, p. 234-239 Copyright 0 1975 American Society for Microbiology
Vol. 29, No. 2 Printed in U.SA.
Co-Evolution of a Virus-Alga System JOHN COWLISHAW* AND MARK MRSA Department of Biological Sciences, Oakland University, Rochester, Michigan 48063 Received for publication 18 July 1974
Plectonema boryanum, a filamentous blue-green alga, was cloned and then allowed to reach a steady state in a quasi-continuous culture in the presence of the algal virus, LPP-1. The culture was maintained for a 3.5-month period during which time at least four distinct culture lysings were evident. After the fourth lysis the culture reached a steady-state level which was identical in its algal concentration to the preinfection level. Upon testing the characteristics of the evolved alga and virus variants, the following was determined: cell variants resistant to both the original virus and the derived virus had evolved, and there was no evidence of lysogeny present among these cells. The evolved virus strains still grew on the parental algal strain, though with altered plaque morphology. Furthermore, they were antigenically similar to the parental virus, and showed no significant difference in adsorption rate or growth characteristics on parental cells. However, a low-grade chronic viral infection persisted in the culture. Rapid re-establishment of a dense, stable culture is apparently the normal laboratory response of a procaryotic cell-virus system. LPP-1 is the first discovered algal virus, isolated by R. Safferman in 1963 (19). It infects three closely related species of blue-green algae, which have been classified by F. Drouet into a single species, Schizothrix calcicola, the most prevalent blue-green alga on earth (22). Furthermore, LPP viral types are evidently a widely distributed virus group, having been isolated repeatedly in America (8, 22, 23), Israel (14), India (26), and Scotland (3), from algaerich environments such as waste-stabilization ponds as well as less eutrophic lakes and rivers. Furthermore, they represent a stable population inasmuch as they can be re-isolated over a period of many months at the same location (14, 22). Their presence is correlated with the extent of cultural pollution (24). Although the host species are ubiquitous in eutrophic waters, they are rarely found as the dominant alga (22). Furthermore, it is only with difficulty that these species can be found at all in environments containing established LPP-1 populations (14, 24). Thus, one might presume that LPP-type viruses are in dynamic equilibrium with their host in eutrophic waters, serving an important function as a natural algal control. Algal viruses therefore offer hope for both the understanding and the control of algal bloom populations (2, 15, 18). The infectious cycle (25), virion morphology (4, 5, 11, 12), and photosynthetic needs of LPP-1 (13) have been worked out in detail.
Furthermore, blue-green algal virus epidemics have been simulated (8, 20). However, no studies on the population dynamics of the virus and alga have been published. The present paper describes an experiment to simulate the natural interaction of LPP-1 and Plectonema boryanum. We have looked at the long-term characteristics pf a quasi-continuous, infected algal culture. Similar experiments have been reported for a bacteriophage-bacterium system (7, 17). Wide fluctuations in Escherichia coli concentration and either T3 or T4 concentration were followed after 20 to 40 h (T4) or 500 to 700 h (T3) by a stable phase in which bacterial concentration returned to the pre-infection value, but lowgrade, chronic viral growth continued. This stability did not end in 50 to 80 weeks. Similar results are found in our system.
234
MATERIALS AND METHODS Algal and viral strains. The alga, P. boryanum, and its virus, LPP-1, were originally obtained from R. Safferman, Environmental Protection Agency, National Environmental Research Center, Cincinnati, Ohio. The parental alga and virus were genetically purified by cloning on agar; and r+ type virus was chosen. Culture methods. Algal cultures were grown in a medium designed by Allen (1). It contains per liter: 1.5 g of NaNO,, 39 mg of KHPO4, 75 mg of MgSO4 * 7H2O, 20 mg of Na2CO,, 36 mg of CaCl2- 2H.O, 78 mg of Na2SiO, -9HO, 1 mg of EDTA,
VOL. 29, 1975
VIRUS-ALGA SYSTEM
6 mg of ferric citrate, 6 mg of citric acid, 2.86 mg of H,BO,, 1.81 mg of MnCl,*4HO, 0.222 mg of ZnSO4-7H2O, 0.178 mg of MoO,, 0.079 mg of CuSO4.5HO, and 0.0494 mg of Co(NO0)2.6H2O. Cultures were grown in 100-ml quantities in a 250-ml Erlenmeyer flask, magnetically stirred, aerated with purified, piped-in laboratory air (0.5% CO,) through a fritted glass disk, and lighted with one or two 20-watt cool-white fluorescent lamps giving about 150 footcandles incident on the culture. This culture was maintained as a quasi-continuous culture at about 8 x 107 cells/ml by replacing a portion of the culture with fresh growth medium daily. A second culture was allowed to grow more densely (10w to 2 x 108 cells/ml) and was used as the source of the indicator cells on plates for typical plaque assays. Culture temperature was about 25 C. Plaque assays. Virus titer was assayed and plaque morphology was studied by plating 0.5 ml of virus, 2.0 ml of indicator algae, and 2.5 ml of top agar on plates containing 20 ml of bottom agar. Both top and bottom agar contained Allen's medium and 1.5% agar (Difco). Plates were incubated under illumination of 175 footcandles for 3 to 5 days, at room temperature (approximately 22 C). Spectrophotometry. Spectrophotometric readings were taken on portions of the infected algal culture using a Beckman DBG Spectrophotometer at 540 and 632 nm. Algal colony assays. Algal filament titer was assayed and resulting colonies were picked and regrown by spreading 0.1 ml of an appropriate dilution of culture on 20 ml of bottom agar in a petri plate, and then incubating for 10 to 14 days under illumination. Microscopy. Filament concentration, cell concentration, and cell morphology were studied by light microscopy using a Neubauer-Hausser counting chamber. At a magnification of 450, the number and contour length of all the filaments in the field of view (20 to 100 filaments) were measured. At a magnification of 1,000, the number of cells in, and contour length of, several filaments were measured, yielding the average longitudinal length of a cell (2.5 Mtm). Finally, the cell concentration was calculated by: (filaments/milliliter) x (aver(cells per milliliter) = age contour length/filament) (average contour length/cell) Cells per filament was calculated from the same data. Serological procedures. LPP-1 antiserum was prepared by injection of rabbits with centrifugally purified and concentrated LPP-1 preparations. Neutralization tests were carried out at 22 C. Viral adsorption studies. Adsorption rates were calculated from the decrease of nonsedimentable plaque-forming units (PFU) as a function of time in an algal culture shaken at room temperature (22 C) on an Eberbach reciprocal shaker to which virus had been added. Usually, a 30-ml algal culture in a 250-ml Erlenmeyer flask was used with a multiplicity of infection much less than 1. One-step growth experiments. Virus was allowed to absorb for 60 min in a 10-ml algal culture in a
235
shaken flask (multiplicity of infection
Vol. 29, No. 2 Printed in U.SA.
Co-Evolution of a Virus-Alga System JOHN COWLISHAW* AND MARK MRSA Department of Biological Sciences, Oakland University, Rochester, Michigan 48063 Received for publication 18 July 1974
Plectonema boryanum, a filamentous blue-green alga, was cloned and then allowed to reach a steady state in a quasi-continuous culture in the presence of the algal virus, LPP-1. The culture was maintained for a 3.5-month period during which time at least four distinct culture lysings were evident. After the fourth lysis the culture reached a steady-state level which was identical in its algal concentration to the preinfection level. Upon testing the characteristics of the evolved alga and virus variants, the following was determined: cell variants resistant to both the original virus and the derived virus had evolved, and there was no evidence of lysogeny present among these cells. The evolved virus strains still grew on the parental algal strain, though with altered plaque morphology. Furthermore, they were antigenically similar to the parental virus, and showed no significant difference in adsorption rate or growth characteristics on parental cells. However, a low-grade chronic viral infection persisted in the culture. Rapid re-establishment of a dense, stable culture is apparently the normal laboratory response of a procaryotic cell-virus system. LPP-1 is the first discovered algal virus, isolated by R. Safferman in 1963 (19). It infects three closely related species of blue-green algae, which have been classified by F. Drouet into a single species, Schizothrix calcicola, the most prevalent blue-green alga on earth (22). Furthermore, LPP viral types are evidently a widely distributed virus group, having been isolated repeatedly in America (8, 22, 23), Israel (14), India (26), and Scotland (3), from algaerich environments such as waste-stabilization ponds as well as less eutrophic lakes and rivers. Furthermore, they represent a stable population inasmuch as they can be re-isolated over a period of many months at the same location (14, 22). Their presence is correlated with the extent of cultural pollution (24). Although the host species are ubiquitous in eutrophic waters, they are rarely found as the dominant alga (22). Furthermore, it is only with difficulty that these species can be found at all in environments containing established LPP-1 populations (14, 24). Thus, one might presume that LPP-type viruses are in dynamic equilibrium with their host in eutrophic waters, serving an important function as a natural algal control. Algal viruses therefore offer hope for both the understanding and the control of algal bloom populations (2, 15, 18). The infectious cycle (25), virion morphology (4, 5, 11, 12), and photosynthetic needs of LPP-1 (13) have been worked out in detail.
Furthermore, blue-green algal virus epidemics have been simulated (8, 20). However, no studies on the population dynamics of the virus and alga have been published. The present paper describes an experiment to simulate the natural interaction of LPP-1 and Plectonema boryanum. We have looked at the long-term characteristics pf a quasi-continuous, infected algal culture. Similar experiments have been reported for a bacteriophage-bacterium system (7, 17). Wide fluctuations in Escherichia coli concentration and either T3 or T4 concentration were followed after 20 to 40 h (T4) or 500 to 700 h (T3) by a stable phase in which bacterial concentration returned to the pre-infection value, but lowgrade, chronic viral growth continued. This stability did not end in 50 to 80 weeks. Similar results are found in our system.
234
MATERIALS AND METHODS Algal and viral strains. The alga, P. boryanum, and its virus, LPP-1, were originally obtained from R. Safferman, Environmental Protection Agency, National Environmental Research Center, Cincinnati, Ohio. The parental alga and virus were genetically purified by cloning on agar; and r+ type virus was chosen. Culture methods. Algal cultures were grown in a medium designed by Allen (1). It contains per liter: 1.5 g of NaNO,, 39 mg of KHPO4, 75 mg of MgSO4 * 7H2O, 20 mg of Na2CO,, 36 mg of CaCl2- 2H.O, 78 mg of Na2SiO, -9HO, 1 mg of EDTA,
VOL. 29, 1975
VIRUS-ALGA SYSTEM
6 mg of ferric citrate, 6 mg of citric acid, 2.86 mg of H,BO,, 1.81 mg of MnCl,*4HO, 0.222 mg of ZnSO4-7H2O, 0.178 mg of MoO,, 0.079 mg of CuSO4.5HO, and 0.0494 mg of Co(NO0)2.6H2O. Cultures were grown in 100-ml quantities in a 250-ml Erlenmeyer flask, magnetically stirred, aerated with purified, piped-in laboratory air (0.5% CO,) through a fritted glass disk, and lighted with one or two 20-watt cool-white fluorescent lamps giving about 150 footcandles incident on the culture. This culture was maintained as a quasi-continuous culture at about 8 x 107 cells/ml by replacing a portion of the culture with fresh growth medium daily. A second culture was allowed to grow more densely (10w to 2 x 108 cells/ml) and was used as the source of the indicator cells on plates for typical plaque assays. Culture temperature was about 25 C. Plaque assays. Virus titer was assayed and plaque morphology was studied by plating 0.5 ml of virus, 2.0 ml of indicator algae, and 2.5 ml of top agar on plates containing 20 ml of bottom agar. Both top and bottom agar contained Allen's medium and 1.5% agar (Difco). Plates were incubated under illumination of 175 footcandles for 3 to 5 days, at room temperature (approximately 22 C). Spectrophotometry. Spectrophotometric readings were taken on portions of the infected algal culture using a Beckman DBG Spectrophotometer at 540 and 632 nm. Algal colony assays. Algal filament titer was assayed and resulting colonies were picked and regrown by spreading 0.1 ml of an appropriate dilution of culture on 20 ml of bottom agar in a petri plate, and then incubating for 10 to 14 days under illumination. Microscopy. Filament concentration, cell concentration, and cell morphology were studied by light microscopy using a Neubauer-Hausser counting chamber. At a magnification of 450, the number and contour length of all the filaments in the field of view (20 to 100 filaments) were measured. At a magnification of 1,000, the number of cells in, and contour length of, several filaments were measured, yielding the average longitudinal length of a cell (2.5 Mtm). Finally, the cell concentration was calculated by: (filaments/milliliter) x (aver(cells per milliliter) = age contour length/filament) (average contour length/cell) Cells per filament was calculated from the same data. Serological procedures. LPP-1 antiserum was prepared by injection of rabbits with centrifugally purified and concentrated LPP-1 preparations. Neutralization tests were carried out at 22 C. Viral adsorption studies. Adsorption rates were calculated from the decrease of nonsedimentable plaque-forming units (PFU) as a function of time in an algal culture shaken at room temperature (22 C) on an Eberbach reciprocal shaker to which virus had been added. Usually, a 30-ml algal culture in a 250-ml Erlenmeyer flask was used with a multiplicity of infection much less than 1. One-step growth experiments. Virus was allowed to absorb for 60 min in a 10-ml algal culture in a
235
shaken flask (multiplicity of infection
