Resistance of blue-green algae to 60Co gamma radiation

Radiation Botany, 1969, Vol. 9, 481 to 489. Pergamon Press. Printed in Great Britain.

RESISTANCE OF B L U E - G R E E N ALGAE T O SOCo G A M M A RADIATION* MARJORIE P. KRAUS Department of Chemistry, University of Delaware, Newark, Delaware 19711, U.S.A.

(Received 19 May 1969)

Abstract--Many characteristics of blue green algae suggest that they should be excellent organisms for the investigation of biological interaction with radiation, particularly ionizing radiation. Therefore, the relative resistance to gamma radiation from a e°Co source has been measured for 23 cyanophycean algae. Since the organisms represent a wide range of cell types it was necessary to use dry weight as the end point for viability determination. Using the radiation resistant bacterium Micrococcus radiodurans as a primary standard and the bacterium Sarcina lutea as a secondary standard, the algae have been compared with one another and with S. lutea using a dry weight end point; S. lutea has also been compared with M. radiodurans on a colony count basis. The algae show a wide range of radiation resistance and have been grouped into three categories: (1) low resistance, with an LDg0 of less than 400 krads, for example, Microcystis aeruginosa, Anacysti~s nidulans and some species of Schizothrix calcicola; (2) moderate resistance, (equal or better than M. radioduraru) with an LDgoof more than 400 krads but less than 1200 krads, for example, OsciUatoria brevis, Anabaena variabilis and Scytonema hoffmannii; (3) high resistance, with an LDg0of greater than 1200 krads, for example, Plectonema boryanum, Lyngbya estuarii, Arostoc muscorum and Microeoleus vaginatus. R~smm~---Maints caract~res des algues bleues sugg~rent qu'elles doivent ~tre d'excellents organismes dam l'dtude des interactions biologiques des radiations, spdcialement les radiations ionisantes. C'est pourquoi, on a mesurd la rdsistanee relative de vingt-trois cyanophycdes aux rayons gamma d'une source de e°Co. Du fait que ces organismes prdsentent une grande variation de types cellulaires, il s'est av~rd ndcessaire d'utiliser le poids sec comme crit~re final en vue de ddterminer la viabilitC En utilisant la bact~rie rdsistante Microccus radiodurans comme premier dtalon et la bactdrie Sarchza lutea comme ~talon secondaire, les algues ont dtd compar~es entre elles et avec S. lutea sur la base du poids see comme critSre final. S. lutea a aussi ~t~ compard ~ M. radioduranssur la base d'un comptage de colonies. Les algues montrent une large gamme de rdsistance aux radiations. Elles ont dtd groupdes en trois categories: (1) celles de faible rfisistance avec une LDg0 plus faible que 400 krads par exemple Microqystis aeruginosa, Anacystis nidulans et diverses esp~ces de Schizothrix calcicola; (2) celles de r~sistanee moddrde (dgale ou sup~rieure il M. radiodurans) avec und Ln~0 de plus de 400 krads mais inf~rieure ~ 1200 krads, par exemple OsciUatoria brevis, Anabaena variabilis et Scytonema hoffmannii; (3) celles de forte r~sistance avee une LDg0 sup~rieure ~ 1200 krads par exemple Plectonema botyanum, Lyugbya estuarii, Nostoc muscorum et Microcoleus vaginatus. Z u s ~ m m e n f a s s u n g - - Z a h l r e i c h e Eigenschaften blaugrtiner Algen lassen vermuten, dass dieser Organismus ausgezeichnet geeignet ist ftir biologische Untersuchungen der Strab_lenwirkung, insbesondere mit ionisierender Strahlung. Die relative Resistenz gegen GammastrabAen aus einer s°Co-Strahlenquelle wurde daher fiir 23 Cyanophyceen bestimmt. Da eine *This research was supported in part by the U.S. AEC and this is document number NYO-3383-15. 481

MARJORIE P. KRAUS

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Reihe verschiedener Zelhypen bei diesen Organismen auftreten, wares notwendig, dasTrockengewicht als Endpunkt t'fir die Bestimmung der Lebensflihigkeit heranzuziehen. Unter Verwendung des strahlenresistenten Bakteriums Micrococau radiodurans als ersten Standard und des Bakteriums Sardna luUa als zweiten Standard, wurden die Algen miteinander und mit S. luUa verglichen, wobei das Trockengewicht als Endpunkt bentitzt wurde; S. LuUa wurde ebenfalls mit M. radiodurans verglichen, indem die Koloniezahl bestimmt wurde. Die Algen zeigen grosse Unterschiede in ihrer Strahlenresistenz und wurden in drei Gruppen unterteilt: (1) geringe Resistenz, mit einer LDg0 von weniger als 400 krad, z.B. Microcystis aeruginosa, Anacystis nidulans und einige Spezies yon Schizothrix calcicola; (2) mittlere Resistenz (gleieh oder besser als M, radiodurans) mit einer LDg0fiber 400 krad aber unter 1200 krad, z.B. Oscillatoria brevis, Anabaena variabilis und Scytonema hoffmannii; (3) hohe Resistenz, mit einer LDg0fiber 1200 krad, z.B. Pleaonema boryanum, Lyngbya estuarii, Nostoc mus¢orum und Mi~rocoleus vaginatus. INTRODUCTION

NORTH of Lake Superior in the Precambian formation known as the Gunflint Chert, have been found fossils which date back two billion years. The morphology of some of these are remarkably similar to Oscillatoria abundant today.(a) Other algal-like fossils with a much less well preserved morphology dating back nearly three billion years also have been found in South Africa.(z°) T h e ability of blue-green algae to pioneer on barren soil and to withstand dessication and extreme climatic conditions .is well known. Observations of these sorts make it logical to expect that the biophysical character of these organisms m a y reflect a capability for coping with radiation. I f we assume with BERNAL,(4) 'that by and large, the chemical processes found today are wide spread and do repeat, with necessary variations, reactions and substances which existed before the advent of totally organized organisms themselves', we might expect to find in such organisms as have progressed through the radiation flux of ancient atmospheres the structures and mechanisms for utilizing and/or surviving exposure to high doses of radiation. We report here, initially, the relative sensitivity to ionizing radiation of a small group of representative Cyanophyceae. The examination is currently being carried on with a larger group. In 1961, a group from Brookhaven National LaboratoriesC2S) measured the radiosensitivity of organisms cultured from soils at the Nevada testing site which had been subjected to large doses of g a m m a radiation. They found that three species, Synechococcus cedorum, Phonnidium tenuis and Microcoleus vaginatus survived a dose

of over a million rads received within a six hour interval. In 1962, GODWARD(z4) recorded an ultimate survival value for a species of Anabaena of 100-200 krads and intimated that some blue-green algae had remarkably high radiation resistance. The rapid strides now being made in elucidating processes of repair of D N A lesions in irradiated unicellular organisms(25) rflake the study of similar, or different, processes in bluegreen algae important not only for the contributions it m a y make to the mechanisms of radiation protection itself but also for the advances which will accrue in the understanding of the cellular organization and genetics of Cyanophycean forms.

MATERIALS A N D M E T H O D S The radiation source was a Gammacell 220, Atomic Energy of Canada, giving 64.5 krads/hr at the outset of the experiment. As the n°Co source decayed, longer periods of exposure were required for the same dosage. The algae, Anacystis nidulans IU625, Fremyella diplosiphon IU481, Plectonema boryanum IU594, P. calothicoides IU598 and Phormidium luridum IU426 were purchased from the Indiana collection.C3z) 'Texas 98', a Nostocacean alga, was supplied by Dr. T. Kantz and Tolypothrix tenuis was a gift fi'om Dr. E. Gantt. T h e rest, of our own isolation, have been identified by Dr. Francis Drouet, Academy of Natural Sciences, Philadelphia. T h e y were cultured in modified Chu medium(7) whose nitrate content was doubled in order to discourage the production of extracellular polysaccharide.01) Uniform,

RESISTANCE OF BLUE-GREEN ALGAE TO e°Co GAMMA RADIATION healthy cells in late log phase growth were generally used for irradiation. To assay radiation damage, it is necessary to count viable cells after exposure. In a heterogeneous group such as this consisting of unicellular, colonial, filamentous, branching and variously differentiated forms a single criterion for viability measurement is difficult to achieve since conventional procedures such as plating for colony counting or optical density measurements of liquid cultures are not applicable to the whole group. Arbitrarily, as a first approximation, the common denominator chosen was the amount of growth determined as dry weight produced by the irradiated cells after a suitable period. This criterion fixes the concentration at which the algae must be irradiated to yield a concentration of cells after irradiation which produces growth proportional to the number of viable cells. This interval, which varies with the alga, was obtained by trial runs varying both the concentration of the algal suspension and the point in log phase growth at which the cells were harvested. T h e algae were prepared for exposure by aseptically harvesting, by eentrifugation at 2000 g, a pellet of sufficient volume to inoculate both the samples to be irradiated and the required controls. The pellet was dispersed in ten times its volume of fresh medium from which aliquots were also taken for cell counting and dry weight determinations. In order to adequately manipulate filamentous algae it was necessary to homogenize them briefly using an A. H. T h o m a s tissue homogenizer. In distributing algae among the samples great care was taken to keep the algae well suspended. Loosely-capped vials containing 10 ml aliquots were placed in positions of equivalent radiation intensity in the sample holder of the Gammacell. Those samples not irradiated for the entire period were kept in the dark until the completion of the exposure when the contents of each vial was transferred to 65 ml fresh Chu medium and grown at 24°C for 15-21 days under continuous cool fluorescent light of 160 f.c. No regard was paid to the time of day when irradiation was initiated or to the establishment of any sort of absolute synchrony in the stages of cell development. At the end of the

483

growth period the collected pellet from each sample was dried to constant weight in a 69°C oven. As a standard against which to compare the algae, the radiation resistant bacterium, Micrococcus radiodurans, was obtained from Dr. A. V¢. Anderson of Oregon State College and cultured as described by BRuc~..Cs) Another bacterium, Sarcina lutea, of our own isolation and adapted both to the conditions under which the algae were grown and to those used for M. radiodurans, provided a means of comparison. S. lutea was grown overnight in Tryptocase Soy broth, collected by centrifugation, washed twice with Chu medium or with 0.067 M phosphate buffer, p H 7.0, and irradiated at a concentration of 0.15 mg dry wt/ml. T h e bacteria were also inoculated at the same concentration into vials containing the alga, Schizothrix calcicola, 'SF'. After irradiation, 0-05 ml of well suspended bacteria or algal bacterial mixture was spread uniformly over a T.S. agar plate and allowed to grow at room temperature. Colony counts for S. lutea were read on the second day. Alternatively, aliquots from the vials containing bacteria were transferred to 50 ml T.S. broth and allowed to develop for 36 hr. T h e cultures were then autoclaved, the bacteria collected by centrifugation, washed, dried and weighed. S. lutea and M. radiodurans were compared under vigorous agitation in 0-067 M phosphate buffer at p H 7.0. M. radiodurans for irradiation was collected from 8 hr shake cultures. Cells washed with buffer were centrifuged at 2000 g for 10 rain and resuspended in buffer at a concentration of 0.I ml pellet per 100ml buffer. Five ml of the bacterial suspension was introduced into each tube of the agitating apparatus which consisted of a chamber in which compressed air entered an opening in the centre of the upper plate of a lucite reservoir and escaped through a lower plate by means of capillaries introduced into the test tubes containing the suspension to be irradiated. T h e compressed air was bubbled through concentrated sulfuric acid, cotton filters and sterile phosphate buffer. Tests indicated no bacterial contamination. Serial dilutions were m a d e from the g a m m a rayirradiated samples and from controls at suit-

484

P. KRAUS

MARJORIE

able intervals. Time economy demanded that the serial dilutions be made at the end of each irradiation rather than at the end of the run. M. radiodurans was plated out using Kilburn's agar,(6) incubated at 33°C in the dark and read on the fourth day.

pending on the particular alga. The time of harvest for each irradiated algal species was chosen by visual inspection and allowed for any incipient growth at highest doses to become established. Harvesting was generally between the 17th and 21st day. Curves for -(-irradiated S. lutea and 3~I. radiodurans at a concentration of 6 × 107 eells/ml are plotted in Fig. 1 as dose in krads against the log of the number of cells giving rise to colonies. The curve for M. radiodurans is in reasonable agreement with that obtained by BRuce.C6) using X-rays. Since our exposure times were more than twice as long as his, some discrepancies might be expected. O u r survival curves for S. lutea consistently showed a greater resistance than those obtained by Bruce for the same organism and probably indicates that we were using different strains. I n Fig. 2 curves based on dry weight growth and plotted as per cent growth vs.-dose are shown for S. lutea, the alga Schizothrix calcicola 'SF', and a mixture of equal concentrations of alga and bacteria. The curve for S. lutea gave

RESULTS

Experiments to determine the optimum concentration at which to irradiate the algae by measuring, for a series of inoculant concentrations, the dry weight harvested at various stages of growth, showed that for the group as a whole, an inoculant of from 0.5 to 2.0 mg dry wt/75 ml medium gave growth proportional to the weight of cells inoculated. A larger inoculant than this exhausted the medium before all cells had attained maturity and actually resulted in a decrease in yield: the growth curve at the higher concentration crossed that at the lower. The most desirable time for harvest, in order to obtain the greatest weight in the proportionality region, was from the 15th to the 21st day, de-

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RESISTANCE OF BLUE-GREEN ALGAE TO '°Co GAMMA RADIATION

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Fro. 2. Comparative 'survival curves' for y-irradiated alga, Schizothrix calcicola 'SF', and bacterium, Sardna lutea, and for a mixture of alga and bacterium in terms of viability as determined by growth measured as dry weight. © Schizothrix "SF" (alga) × Sarcina lutea (bacterium) • Sarcina lutea + Schizothrix the same end point whether determined by dry weight or by colony count. T h e algal bacterial mixture illustrates the relative resistance of bacteria with respect to alga as well as a derogatory effect of bacteria on the algal growth. As the n u m b e r of bacteria in the mixture is reduced by higher irradiation doses, the growth of the algae returns to normal. The control is for bacteria-free growth. T h e average dry weight of M . radiodurans is about 131 m g per ml packed cells and of S. lutea about 128 mg/ml. O u r average cell count obtained by dilution plating of M . radiodurans was 1"3 × 1011 cells/g, a figure in fair agreement with Bruce's of 1 × 1011 cell/g.(8) Thus, the density of M . radiodurans as exposed to irradiation by Bruce was about 0.2 mg/ml, which is similar to the average density at which both our algal and bacterial cells have been irradiated. Cell counts for some filamentous algae can be obtained by sonication or, for those algae susceptible to lysozyme, by counting protoplasts after separating the individual cells of the trichome by the enzyme's action. Figure 3 illustrates the variations of LDg0 at different concentrations of irradiated algae. Curve I is drawn at the lower limiting concentration for suitably manipulating the algae;

curve I I approximates the concentrations normally irradiated; curve I I I is at the upper concentration limit beyond which satisfactory growth conditions are not possible. T h e curves shown are for data on Nostoc muscorum; identical curves were obtained for Nostoc linkia and 'Tex. 98' and similar ones for Plectonema botyanum and for other radiation resistant algae. T h e curves illustrate that a LDg0 figure is meaningful only when the concentration of cells is given. I t appears, though no definite data can be presented, that there is more difference in the range of LD'S with concentration in the case of resistant organisms than for those which are sensitive to radiation. An increase in the quantity of a sensitive alga irradiated does not increase its LD appreciably. Figure 4 is a composite plot for all the more resistant algae. It has been prepared as a semilog plot from the best curve drawn through the averages of the data for each alga, the n u m b e r of runs varying from one to five. I t is obvious that the experimental design is unable to provide information necessary to accurately describe the shape of the shoulder, and that the extrapolated LDg0 m a y be in error due to the fact that the cells with the lowest exposures m a y have reached a premature stationary state due

486

MARJORIE P. KRAUS

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Fro. 4. 'Survival curves' for y-irradiated blue-green algae compared with those for a sensitive (Sarcina lutea) and a radiation resistant (Mi, rococcus radiodumns) bacterium. 1. S. lutea; 2. M. radiodumns; 3. Scyton, ma hoffmannii; 4. Oscillatorla brevis; 5. Goccochloris aagnlna Spreng ; 6. Oscillatoria tenuL~; 7. Nostoc muacomm; 8. Nostoc llrdda ; 9. Plectonema calothricoides ; 10. P. boryanum; 11. mature, aged culture JVostoc nuscomm; 12. Lyngbya estua~i; 13. Microcoleus vaginatus.

RESISTANCE OF BLUE-GREEN ALGAE TO 6°Co GAMMA RADIATION to nutrition deficiency. Also they have had a longer period to undergo dark repair. While the general experimental error is high (and is indicated by the confidence limits shown on the previous graphs), the accuracy of the data is greatest at the lowest concentrations. The data, therefore, are capable of indicating the relative resistances among the members of the group. Knowing the concentration of algae irradiated, a conservative figure for an LDg0has been determined for an irradiated density for each alga of about 0" 15 mg/ml. In Table 1 the algae have been placed in categories according to the degree of radiation resistance they have displayed. These placements were arrived at both from the above

487

Microscopic examination of the cells after moderate exposures indicates no abnormalities of cell division resulting in abnormal growth. At the highest exposures endured, distortions of the cell do occur. Some of these distortions mimic those seen in the presence of antibiotic or viral attack and may give clues as to the structures and substances involved.

DISCUSSION

Although no Chamaesiphonaceae or Rivulariaceae have been included in this study, the data do not suggest any trends along taxonomic or morphological lines. This is not entirely surprising. SPARROW et a1.(2,~9,3°) in a

Table 1. Resistance to y-radiation of some blue-green algae*

LDg0 = < 400 krads Sensitive Anaeystis marina (K) Anaeystis nidulans (IU 625) Schizothrtx calcicola 'BB' (K) Microcystis aeruginosa (K) Coccochlorispeniocystis (K)

LOg0 = 400--1200 krads Moderately resistant Scytonema hoffmannii (K) Phormidium autumnale (K) Coccochloris stagnina Spreng (K) OsciUatoria brevis (K) Anabaena qariabilis (K) Fremyella'diploslphon (IU 481) Tolypothrix tenuis Anaeystis thermalis (K)

LDgo= > 1200 krads Highly resistant OsciUatoria tenuis (K) Phormidium luridum (IU 426) Plectonema ealothricoides (IU 598) Plectonema boopanum (IU 594) Sehizothrix calicola 'SF' (K) Nostoc linkia (K) aVostoc muscorum (K) aVostoc sp (TEX. 98) Lyngbya estuarii (K) Mitrocoleus vaginatus (K)

* IU: Algae from the Indiana Collection; K: Kraus isolates. measurements and by others in which the algae, at a density of 0.15 mg/ml, were irradiated with increasing doses. In these cases the end point was taken at the greatest exposure at which eventual permanent growth was obtained. For comparison with M . radiodurans, the categories are (1) low resistant or radiosensitive algae, (those with lower resistance than M . radiodurans) with an LDg0 of less than 400 krads, (2) moderately radiation resistant, (those similar in range to M . radiodurans) with an LDg0 of more than 400 krads and less than 1200 krads, and (3) highly resistant, (more resistant than M . radiodurans) with an LDg0 of over 1200 krads.

survey of a large number of plants and microorganisms, studied the correlations between DNA content, chromosome volume and radiation sensitivity and found that differences in sensitivity disappear when the data are examined in terms of energy absorbed per chromosome. The blue-green algae, with an arbitrary chromosome number of one (or perhaps with 'loose' DNA) are therefore an interesting group to examine further. Some green algae whose radiation sensitivities have been examined(aS,28, ~4) fall into our radiosensitive category. The green alga, Trebouxia, however, may be one which does not. Some amoebae(~°) and lichensO2) have

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MARJORIE P. KRAUS

remarkable resistance. It is interesting that for the lichens the criterion of viability was the greenness of the algal symbiont. The crustose lichens were found to be most resistant. While it is well known that the blue-green Nostocaceae and Scytonemataceae are common lichen symbionts, the alga associated most often in the literature with crustose forms appears to be Trebouxia. (x) In spite of the lack of precise information on the form of the shoulders shown in Fig. 4, there is no doubt that they are longer than those obtained for M. radiodurans. Experimentation on M. radiodurans has revealed(2°) an excision and repair mechanism for damaged DNA, involving an enzymatic system which is apparently not damaged by high exposure to radiation.ISS) Alexander and Lett have estimated that the absorption of energy equivalent to 600 eV of ionizing radiation can effect a main chain break in DNA. It has been reportedIa) that M. radiodurans can repair double strand breaks. Suggestions have been made that the nucleoprotein structure preserves the integrity of the DNA spacing, allowing repair. The finding of Cyanophycean protein moieties with high sulfhydryl content and with considerable opportunity for hydrophobic bonding(21) should encourage the examination of this aspect. Korogodin, and Lyman and Haynes (cited in reference 19) using heavy ionizing particles on yeast ceils, found that irradiated suspensions recovered when kept in water or minimal medium for one or two days after irradiation. In our own work we have noted that irradiated suspensions of resistant algae remaining in the irradiationdarkened vial for several days before reculturing improve in viability. It has not been possible, however, to increase the viability of the more sensitive algae a significant amount in this 1Tlanner.

In the alga, Plectonema bo~yanum, WERmN and RUpERT(82) have defined a photoreactivating enzyme capable of reversing nucleotide base dimerization lesions resulting from U V irradiation. For P. bo~yanum a virus has been discovered(~7) and virus resistant and possible lysogenic hosts are available. (17,~.s)Using various related strains of Schizothrix calcicola(9) charac-

terized, for example, by radiation sensitivity, chromatic adaptation, photoreactivity or resistance to antibiotics one may be able to carry out genetic studies giving insight into relationships between chloroplast and other replicating structures. In the y-irradiation of S. calcicola 'BB', which possesses the property of chromatic adaptation, it has been noted that a succession of substances (blue, pink, yellow and yellowgreen) have been leached into the medium as the algae succumb to irradiation. The synthetic pathways for the interplay ofphycocyanin and phycoerythrin (from which these colours probably come) have been studied by FUJITA and HATTORI(TM)while the ultra structures involved with phycobilin production are the subject of investigations by GANTT et al.Oa) Finally, there are the problems involving the roles of water. This field has been explored particularly well by POWERS et al.(2~) using irradiated spores. The resistant Nostocaceae in the heterocyst or akinete forms should be amenable to a similar type of study. The nature of y-radiation enables it to initiate fast reactions of electron transfer and to alter reactions responsible for accumulation of the stabilized products of photochemistryCtn) which occur before the more typically biochemical reactions. With their powerful fluorescences, the cyanophycean pigments could be used to monitor, by the use of fast spectral instrumentation, the reactions occurring during the interval between radiochemical and biochemical reactions. It is possibly in this manner that these photosynthetic organisms can best exhibit their potential as tools for research. Acknowledgements--The author is grateful to Dr. C. TRUMaOX~, Chemistry Department, UniversiW of Delaware, for many helpful discussions and expresses thanks to Dr. BANERJIand the Civil Engineerhag Department, University of Delaware, for laboratory space used in carrying out this research. REFERENCES

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RESISTANCE OF BLUE-GREEN ALGAE T O 6°Co G A M M A R A D I A T I O N

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