Arch. Microbiol. 103, 297--302 (1975) �9 by Springer-Verlag 1975

Photoreactivation of UV-Irradiated Blue-Green Algae and Algal Virus LPP-1

P. K. SINGH

Central Rice Research Institute, Cuttack, Orissa, India

Received November 4, 1974

Abstract. Ultraviolet (UV) sensitivity and photoreactivation of blue-green algae Cylindrosperrnum sp., Plectonema bory- anum, spores ofFischerella muscicola and algal virus (cyano- phage) LPP.1 were studied. The survival value after UV irradiation of filaments of Cylindrospermum sp. and Virus LPP-1 showed exponential trend and these were compara- tively sensitive towards UV than F. muscicola and P.bory- anum. Photoreactivation of UV-induced damage occurred in black, blue, green, yellow, red and white light in Cylindro- spermum sp., however only black, blue and white light were capable of photorepair of UV-induced damage in P. bory-

anum, spores of F.muscicota and virus LPP-1 in infected host alga. Pre-exposure to yellow and black light did not show photoprotection. The non-heterocystous and nitrogen fixation-less mutants of Cylindrospermum sp. were not induced by UV and their spontaneous mutation frequency was not affected after photoreactivation. The short tri- chome mutants of P. boryanum were more resistant towards UV.

The occurrence of photoreactivation of UV-induced killing in wide range of light in Cylindrospermum sp. is the first report in organisms.

Key words: Photoreactivation -- UV-Irradiation - Cylindrospermum - Plectonema boryanum - Fischerella muscicola -- Cyanophage LPP-1.

Photoreactivation of UV-induced damage has been extensively studied in bacteria and bacterial viruses (Rupert and Harm, 1966; Witkin, 1969a, b). Wu et al. (1967) observed photoreactivation of blue-green algal virus LPP-1 and its host alga Plectonema boryanum. Van Baalen (1968) reported photoreactivation in a marine coccoid blue-green alga AgmeneIlum quadruplicatum. The D N A photoreactivating enzyme was isolated fi-om blue-green algae P.boryanum (Werbin and Rupert, 1968) and Anacystis nidulans (Saito and Werbin, 1970). Author studied photoreactivation in detail in spores of blue-green alga Fischerelta muscicota and algal viruses LPP-1, 1)-2 and AR-1 (Singh, 1967; Singh and Singh, 1972). The present study deals with the photo- reactivation of UV-induced killing in blue-green" algae, algal virus LPP-1 and reports discovery of an alga capable of photoreactivation in wide range of visible light unlike other organisms.

Materials and Methods

The colony forming mutant of non colony forming alga Cylindrospermum sp. (Singh, 1974) was used for irradiation. It formed short trichomes of average 12 cells with terminal heterocysts at both ends and chain of subterminal spores. This mutant was similar in growth behaviour, UV sen sitivity,

photoreactivation to the parent alga and contained gas- vacuoles in vegetative cells, but filaments were devoid of fast movement on agar plates. The exponentially growing vegetative filaments of alga were used in experiments.

The vegetative cells ofuniseriate filaments ofF. muscicola became multiseriate at the time of sporulation due to forma- tion of septa in each cell. Thus, each parent cell was con- verted into a packet of endospores. The sporulation was almost synchronized and spores were easily separated from each other on account of the highly fragile nature of packets. The spores were thin-walled, mostly spherical, light brown in colour, highly refractile in nature and germinated in 24 hrs after inoculating on agar plates. Upon germination on agar plates, they formed discrete and spherical pin-head colonies. Only spores of the alga were used in experiments.

Exponentially growing filaments of P.boryanum after homogenization with glass beads and its short trichome mutants were used for UV-irradiation. The parent alga lacked the capacity to form distinct colonies on agar plates, but its short trichome mutants obtained after 5-bromouracil treatment (P. K. Singh, unpublished) formed spindle shaped colonies. The algal virus LPP-1 (Safferman and Morris, 1963) was multiplied on P. boryanum strain 594 of Indiana University Culture collection and filtered lysate with sintered glass filter was used for irradiation. This virus formed distinct clear plaques in lawn of P. boryanum in 3 or 4 days of plating.

Standard microbiological techniques were employed in isolation and culture of algae. The axenic cultures were incubated at 24:k1~ in a culture room and illuminated at

298 Arch. Microbiol., Vol. 103, No. 3 (1975)

approximately 250 ft. c. light intensity from day light fluorescent tubes for 14 hrs per day. Modified Chu-10 medium (Safferman and Morris, 1964) with trace elements (Allen and Arnon, 1955) was used for culturing of Cylindro- spermum sp. and P. boryanurn while F. muscicola was grown in Allen and Arnon's medium (1955) without any source of combined nitrogen (AA-N medium).

For UV irradiation, aliquots of 0.2 ml of growing cultures of Cylindrospermum sp. were diluted in 20.0 ml medium. The irradiation was performed in a 75 mm sterilized petridish containing 20.0 ml of filaments suspension and tiny sterilized iron needle. A General Electric germicidal lamp giving its main out put of 253.7 nm at a distance of 22.5 cm from the lamp (85 ergs/mm2/sec) was used for irradiation and suspension was continuously stirred during the irradiation period on a magnetic stirrer. Samples were pipetted out at various times and 0.1 ml aliquots were spread on agar plates which were prepared before 24 hrs of use with 0.8% Difco agar in Chu-10 medium. These agar plates were wrapped in black cloth for 22 hrs before expo- sure to light to avoid photoreactivation. Diluted spores of F. nuscicola and vegetative filaments of P. boryanum were irradiated and plated similarly except AA-N medium was used in agar plates for F. muscicola. The free virus particles of LPP-1 were irradiated in similar way and mixed with host alga P. boryanum in melted nutrient agar containing finally 0.5% Difco agar and its aliquots of 5.0 ml were spread uniformly on to each of the agar plates containing 1.0% agar by double agar layer technique (Adams, 1959). These were kept in dark for 12 hrs and then exposed to light. To prevent photoreactivation effects, all operations were done under a low intensity of yellow light. Colonies of Cylindrospermum sp. and F.muscicola were counted on 7th and llth day, respectively. Survival of P.boryanum in control and 30 sec of UV dose was determined on 3rd day and in rest of doses containing less population, counting of colonies was done on 6th day. The colonies of short tri- chome mutants were counted on 12th day and the plaques of LPP-1 were assayed on 4th day.

For photoreactivation studies, each pair of agar plates containing control and UV-irradiated samples were incu- bated for 2--22 hrs under one of the following conditions in culture room: 1. darkness; 2. black light; 3. blue light; 4. green light; 5. yellow light; 6. red light, and 7. white light. The virus LPP-1 with host was exposed for 6--10 hrs in different light and 11 hrs in darkness. Next, these plates were exposed to white light. Black light illumination was provided from a pair of 4 feet. General Electric "Black light" lamps mounted side by side and these lamps emitted maximum light of 355 rim. The yellow light illumination was provided from a single 4 feet General Electric lamp. The blue, green and red illuminations were provided through coloured celophane filteres of range 380--480, 480-560, and 620 700 nm respectively. Photoreactivating sector was calculated from the equation I-DI/D2, where D1 was the dose that gave without illumination, the same survival as did the UV dose D2 with maximum photoreactivation (Harm, 1968)o

The exponentially growing cultures in white light of Cylindrospermum sp. were exposed to 4 hrs under black and yellow light, diluted and irradiated with UV to observe photoprotection.

Acriftavine of concentrations 0.2--1.0 ~zg per ml was incorporated in agar plates to observe its effect on dark repair and photorepair,

ResLllts UV survival curve of vegetative filaments of colony forming mutant of Cylindrospermum sp. was exponent- ial showing one hit mechanism (Fig. l). The alga survived up to 45 sec of UV dose and the dose that inactivated 37 % of survival (D37) was 3 sec. The little variation in UV survival was observed from experi- ment to experiment. The period of 10--14 hrs in dark was not enough for inactivation and some photo- reactivation still occurred after exposure to light. Strong photoreactivation was observed in this alga in all the wavelengths of light used in experiments (Fig. 1). The UV-irradiated filaments up to 45 sec of dose, showed complete and striking photorecovery, if incubated immediately in white light. Exposure of 2--22 hrs under different wavelengths of light, i.e. black, blue, green, yellow and red showed strong photoreactiva- tion. The survival during photoreactivation was affected by the light intensity and time of exposure to light. There was no increase in UV survival when filaments were exposed to black and yellow light before UV irradiation. Therefore, photoprotection was not ob- served in the alga. The incorporation of 0.5 and 1.0 ~g per ml of acriflavine in agar plates showed strong killing of filaments in control itself. The above con- centrations of acriflavine ,showed reduction in UV survival in post-treatment, but further experiments will clarify the occurrence of dark repair in the alga. The slight inhibition of photoreactivation was also ob- served with acriflavine.

It was very easy to make bacteria-free cultures of Cylindrospermum sp., because UV-irradiated algal cells at higher doses showed high survival due to photo- reactivation in yellow and red light. However, bacterial cells lacked the photorepair in these wavelengths and did not show survival. Therefore, cultures irradiated to high doses of UV and exposed to the above wave- lengths of light were bacteria-free.

The spores of F.muscieola were comparatively resistant towards UV (Fig. 2). The UV survival curve was multihit, sigmoidal type and D37 was 2 rain. Photo- reactivation of UV-irradiated spores occurred in black, blue and white light only and it was not observed in green, yellow, and red light. Photoreactivating sector varied from 0.6--0.72. Photoreactivation was strong and almost similar in black, blue and white light.

The UV survival of filaments of P.boryanum was 0.4 % at 90 sec of dose (Fig. 3). There was small shoulder at lower doses of UV survival curve and D37 was 30 sec. The strong photoreactivation was observed in black, blue and white light (Fig.3), but no photo- reactivation occurred in green, yellow and red light. Photoreactivating sector varied from 0.6--0.75 and it

P. K. Singh: Photoreactivation after UV-Irradiation 299

100

0.1 L 0

10

o l t 9

1 0 0 [ ~ ~

1

o.ll 0 3o 60 1

UV dose (sec) UV dose ( min ) 1 2

0.1 I,, 0

Fig. i. UV-irradiated survival and photoreactivation of Cylindrospermum sp. e ~ o dark; 0

100

10

1 l I 1 I , 1 . . . . . . .

30 60 90 120 UV dose(sec)

3 0 whitelight; A ..... A btue

light; [] [] black light; zx ~ red light; II �9 green light; • • yellow light Fig. 2. UV-irradiated survival and photoreactivation of spores ofF. muscicola, e - - - o dark; O �9 white light; E ] - - � 9 blue

light; �9 �9 black light; • • red light; A - - A green light; zx zx yellow light Fig.3. UV-irradiated survival and photoreactivation of P.boryanum. e ~ e dark; � 9 white light; Jl, blue

light; �9 �9 black light; z~--zx yellow light

was same in black, blue and white light at 30, 60 and 90 sec of UV doses. The short trichome mutant clones 3 and 5 were more resistant towards UV and Da7 was 1.6 rain for clone-3 (Fig.4). Photoreactivation in these mutants was observed in blue and white light and it was less in black light. Photoreactivation did not occur in yellow, green and red light in mutants also. The mutant clone 5 showed less photoreactivability in white light than clone-3.

The UV survival curve of virus LPP-1 was exponen- tial type and Dz7 was 3 sec (Fig. 5). Photoreactivation occurred in black, blue and white light and photo- reactivating sector varied from 0.52--0.8. The exposure of 6--10 hrs of UV-irradiated infected virus towards black light showed photorepair (Fig. 5). The incorpora- tion of 0.2 ~zg per ml of acriflavine in both bottom and top layers showed more photodynamic inactivation of virus in white and blue light than other wavelengths of light used in experiments. The UV survival of virus was reduced in post treatment with acriflavine in green light, red light and dark and reduction of survival was more at higher doses of UV (Fig. 5). Thus, showed the presence of host cell reactivation. Photoreactiva- tion was comparatively more inhibited in white and blue light in presence of acriflavine (Fig. 5).

The number of non-heterocystous and non-nitrogen fixing spontaneously occurring mutants of Cylindro- spermum sp. increased with the number of transfers of the cultures in nitrate containing medium (Fig. 6) and their numbers decreased when cultures were transferred in nitrogen-free medium. These spontaneously occur- ring mutants were not observed when cultures were grown regularly in nitrogen-free medium. There was no induction of these mutants with UV and spontaneous mutation frequency was unchanged after photo- reactivation of UV-irradiated filaments in different wavelengths of light (Fig. 7).

Discussion

The high UV sensitivity of blue-green alga Cylindro- sperrnum sp. might be due to lack or low efficiency of excision repair and post replication or recombinational repair mechanisms. The strains of bacterium E.coli differed by a factor of more than a thousand in their sensitivity to UV, but only because they differed either in the ability of repair pyrimidine dimers or in the ability to tolerate unrepaired dimers or both (Witkin, 1969a, b). There Was also no induction of non-nitrogen fixing mutants (Singh and Singh, 1968; Singh et al.,

300 Arch. Microbiol., Vol. 103, No. 3 (1975)

10 t . . . . . . . . 1 ...... I

60 90 120 UV dose (sec)

4

0 15 30 45 60 UV dose ( sec )

5

162

lO ~ E r

o = -

:~10 -t

2 Z~ 6 8 10 12 N u m ber of transfers (days)

6

Fig. 4. UV-irradiated survival and photoreactivation of short trichome mutant of P. boryanum, x - - • dark; O O white light; �9 �9 blue light; ~ zx black light; I �9 red light; [] [] yellow light

Fig. 5. UV-irradiated survival and photoreactivation of algal virus LPP-1 on host alga P. boryanum and effect of 0.2 Fg per ml of acriflavine (AF) on survival in post treatment. D [] dark; O O white light; | | blue light; • - - • black light; a zx red light; ~ ~ green light; ~ A dark 4- AF; �9 �9 white light -1- AF; • 2 1 5 blue light + AF;

I �9 black light + AF; �9 �9 red light + AF; [] ..... [] green light + AF Fig. 6. Spontaneous mutation frequency of non-heterocystous, long filament forming and non-nitrogen fixing mutants of

Cylindrosperrnurn sp. in number of cultures in nitrate containing medium

1972) in the alga after UV-treatment and spontaneous mutation frequency was not affected when UV- irradiated cultures were exposed to photoreactiving light. The absence of mutation induction by UV in bacteria was atributed due to lack of dark repair mechanisms and therefore, none of the strains carrying

10-2

i #

o

- \ 16 , , 0 15 30 45

uv dose (sec)

Fig. 7. Effect of photoreactivation of UV irradiated culture on spontaneous non-heterocystous mutation frequency. �9 �9 dark; �9 O white light; zx-.---zx blue light;

D red light; A A yellow light; �9 �9 green light

the excision repair less (exr-) allele derived from strain Bsl or Bs2 produced UV-induced mutations of any kind (Witkin, 1969a, b). Similarly, the alga is highly sensitive to UV and its mutagenic effect appears to be entirely eliminated.

Mostly two types of photoreactivation are observed in bacteria (1) photoenzymatic repair is the basic mechanism responsible for photoreactivation (Harm, 1968). It involves a light activated enzymatic reactiva- tion by a photoreactivating enzyme, which is activated by light of wavelength about 400 nm (Wutff and Rupert, t962). (2) Indirect photoreactivation was obtained in photoreactivating less (phr-) strain of E. coli B by post illumination of UV-irradiated cells with wavelength of 334 nm which is the optimal range for photoprotection (Jagger and Stafford, 1965). While 405 nm, being effective for enzymatic photoreactivation, but ineffec- tive for photoprotection, failed to produce indirect photoreactivation in bacteria (Rupert and Harm, 1966). In the present experiments, photoreactivation was very efficient in green, yellow and red light in Cylindrospermum sp. However, pre-exposure of black and yellow light did not show photoprotection. This was the unique property of this alga, because there was

P. K. Singh: Photoreactivation after UV-Irradiation 301

no report on photoreactivation in yellow and red light in any microorganisms or plants. Photoreactivation occurred almost to the same extent in blue and black light which were the action spectra for photoenzymatic repair in other blue-green algae (Saito and Werbin, 1970). Jagger and Stafford (1965) have concluded in phr- strains of E. coti B that indirect photoreactivation operated through a photoreactivating light induced delay in growth and division but, delay in growth and division were not observed in the present experiments by photoreactivating light, i.e. green, yellow and red. Therefore, the possibility of indirect photorepair is likely to be absent in the alga. Witkin (t969a) men- tioned that photoreversal of UV-induced killing is an example of direct enzymatic photoreactivation and thus present observations of high photoreversal in wavelengths ranging from 355--700 nm of UV- induced killing was due to enzymatic photorepair. It is likely that occurrence of high mutation frequency towards various markers unlike many blue-green algae (P. K. Singh, unpublished) in this alga is some how linked to the efficient wavelength independent photo- repair mechanisms. The filaments of P. boryanum were comparatively resistant towards UV than Cylindro- spermum sp. Photoreactivating enzyme(s) has been isolated from P.boryanum and tested by the Herno- philus influenzae transforming assay (Werbin and Rupert, 1968). The survival data was presented and photoreactivation was observed in this alga in the present study in black, blue and white light but, it was absent in green, yellow, and red light. The UV resistance of short trichome mutants of P. boryanum might be due to their slow growth than parent alga, where chances of repair was more.

The UV-irradiated spores of F.muscicola showed high photoreactivation only in black, blue and white light as observed in P. boryanum. Singh (1967) and Singh and Singh (1972) made detail study on UV irradiation and various repair mechanisms, where very efficient photoreactivation was observed in blue and white light. In addition, it was reported in this study in black light.

Photoreactivation of UV-irradiated virus LPP-1 in infected host alga P. boryanum was studied (Wu et aL, 1967; Singh, 1967; Singh and Singh, 1972). Photo- reactivation of UV-irradiated LPP-1 was maximum after 6 hrs of exposure to white light and minimum exposure time was 1 hr (Singh, 1967; Singh and Singh, 1972). Wu et al. (1967) failed to observe photoreactiva- tion of LPP-1 in black light. However, photoreactiva- tlon was observed in the present study when infected host alga was exposed to 8 or 10 hrs of black light and exposure to longer periods of black light had inactivat- ing effect. This might be one of the reason of absence of

photoreactivation in Wu et at. (1967) experiments. The UV survival of LPP-1 was less in red and yellow light than the dark. Similar decrease in UV survival in yellow light was also reported in alga Synechocystis aquatitis (Zhevner and Shestakov, 1972).

The acriflavine is known to produce photodynamic inactivation, protection against UV inactivation in pre-treatment and inhibitor of dark repair in post treatment in blue-green algae and algal viruses (Singh, 1967, 1968; Singh and Singh, 1972). The reduction of UV survival in presence of acriflavine in different colours of light might be due to inhibition of host cell reactivation. The occurrence of UV reactivation after infection of UV-irradiated virus in UV irradiated host alga P. boryanum was reported earlier (Singh and Singh, 1972).

It has been invariably observed in this study that UV survival in green, yellow and red light was less than dark except Cylindrospermum sp. Therefore, it is suggestive that UV survival of algae and algal viruses capable of photorepair in black and blue light only, should be determined in yellow or red light and not in dark. The keeping in dark is inhibitory for growth and therefore, dark UV survival might be similar to the liquid holding recovery in bacteria (Rupert and Harm, 1966).

Acknowledgements. I am grateful to Prof. Dr. R.N. Singh, Head of Botany Department, Banaras Hindu University, for valuable suggestions and interest in this work. I am also grateful to Dr. S. Y. Padmanabhan, Director, and Dr. S. Patnaik, Head of Crops & Soils Division, for providing facilities and useful criticism of this work.

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Dr. P. K. Singh Central Rice Research Institute Cuttack-753006, Orissa, India