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Protistology 1 (2), 76-81 (1999) July, 1999
Protistology
Correlation between virus-sensitivity and isoenzyme spectrum in symbiotic Chlorella-like algae
B. Linz a, A. Linz 1b, A.V. Migunova and K.V. Kvitko
Laboratory of Microbiology and 1 Laboratory of Plant Genetics, Biological Research Institute of St.Petersburg State University, Russia (a Dept, of Biomolecular Sciences, University of Manchester, Institute of Sciencs and Technology; UK;b Bundesanstalt fur Zuchtungsforschung, Institut fur Zuchtungsmethodik, Germany)
Summary
The collection of virus-sensitivity Chlorella-like algae, symbionts of Paramecium bursaria and same others resistant to viruses Chlorella strains is described. Specific agglutination by antiserums according to the groups of virus-sensitivity algae is observed. The difference of the isoenzyme patterns of two virus-sensitive Chlorella strains groups from European and American populations are shown. Into gropes this patterns were identical. This characteristics of each free-living , resistant to virus Chlorella strains are individual. The correlation between virus-sensitivity, isoenzyme spectrum and serological characterization of Chlorella strains are exposed.
Key words: zoochlorellae, PBCV-virus, symbionts, virus-sensitivity, isoenzyme, serological characterization, agglutination, surface antigen specificity
Introduction
Symbiotic Chlorella-like algae, zoochlorella are a very specific (in the ecological sense) group of eukaryotic organisms. The sensitivity of zoochlorella cells to viruses was found as a frequent situation for Hydra viridis (Meints et al., 1981, Van Etten et al., 1981, 1991) and Paramecium bursaria symbionts (Kawakami and Kawakami, 1978; Van Etten et al., 1981, 1983, 1991; Kvitko and Gromov, 1984; Reisser et al., 1986, 1991; Yamada et al., 1991). In both host organisms the symbiotic algae cells are defended against virus attack in individual, host derived “perialgaT vacuoles and become defenseless after distraction of the host cells. The natural selection among zoochlorellae should be working in the direction of forming clones of specialized forms with very stable combinations of characters. We consider this correlation between virus-sensitivity and a zoochlorella status as an empirical rule, the application sphere for this rule we have to investigate.
We studied Chlorella strains isolated from Paramecium bursaria and specific to them viruses (Phycodnaviridae). The variability of such Chlorella markers as isoenzyme spectrum, surface antigen specificity and sensitivity for two known ecotypes of viruses is the subject of this paper.
Material and Methods
Chlorella strains and culture conditions. The Chlorella strains studied were: a) Isolates from European populations of Paramecium bursaria: Ch. spec. 241.80 (from the Sammlung of Algenkulturen at Gottingen (SAG), FRG; cf. Schlosser,1982); Ch. spec. strains Pbi, PbAm and PbBS (all from W.Reisser, Gottingen; cf. Reisser et al.,1988). Ch. spec. strain OCh (isolated from P bursaria at lake Cherlivoye in Karelia, Russia, by E.S.Krayeva; cf. Kvitko et al.,1988). Isolates from American populations of P. bursaria, known as Ch. spec. 211-6 (= “Ch. paramecii” Loefer, from SAG; cf. Schlosser,1982); Ch. spec. strain NC64A (from J.Van Etten, Lincoln, Nebraska; cf. Van Etten et al.,1983). b) Mutants of strain NC64A (Migunova et al.,1992): As-21-skb-1 - resistant to streptomycin and two toxic aminoacids: kanavanin and b-Alanin; As-21-2Du - resistant to diuron, an inhibitor of photosynthesis; Az4 (k) - fast growth phenotype. c) Type strains of free-living Chlorella serving for comparison: Ch. protothecoides Kruger 211-7a (SAG); Ch. vulgaris Beijerinck 211-11b (SAG); and Ch. sorokiniana 211-8k (SAG, all are obtained from the Collection of Algae of St.Petersburg State University; cf. Gromov and Titova,1988). Chlorella strains OCh, PbAm, PbBS, and 241.80 were cultured in FES medium as described by
© 1999 by Russia, Protistology.
Table 1. Isoenzyme visualization
Enzymes Abbreviation Gel concentration (%) Visualisation methods (references)
Aspartataminotransferase AAT 7,5 Braun et al., 1978
Aconitase ACO 6,0 Wehling.1986
Diaphorase DIA 6,0 Wehling.1986
Malatdehydrogenase MDH 5,0 Adams, Joly,1980
NAD-dependent Aromatic
Alcohol Dehydrogenase AADH-NAD 6,0 Jaaska,1978
NADP-dependent Aromatic
Alcohol Dehydrogenase AADH-NADP 6,0 Schmidt et al., 1984
Superoxide Dismutase SOD 7,5 Beauchamp, Fridovich,1971
Reisser (Reisser,1984). All the other strains studied were cultured in a modified Bold’s basal medium (MBBM) as described by Van Etten (Van Etten et al.,1983).
Test for virus-sensitivity. 200 ml of mid-log phase growing algae at a concentration of 1x108 to 2x108 cells per ml were mixed with 3 ml of 0,75% soft agar MBBM and overlaid on Petri plates containing 15 ml of MBBM plus 1,5% agar. Algal lawns were inoculated by stabbing a needle with diluted suspensions of the NC64A-virus PBCV-1 (kindly given by J.Van Etten) and the Pbi-virus 101G-2, isolated by the plaque assay method described by Van Etten (Van Etten et al.,1983) from a pond near St.Petersburg in 1991. Algal plates were incubated at 23°C in continuous light and observed after 3 days.
Serological characterization of Chlorella strains. Antisera were raised against Chlorella strains OCh, 241.80, and NC64A, respectively, and assayed by the agglutination test as described (Migunova et al.,1992).
Analysis of isoenzymes. Chlorella cells were disrupted in test tubes with glass beads (0,65 mm in diameter) in a 5mM Na-phosphate buffer, pH 6.0, containing 10mM NaCl, 50mM Na2S2O3, 2mM dithiothreitol, and 15% sucrose. Homogenization using a Vortex mechanical homogenizer was carried out on ice. The crude cell extract was centrifuged (5000g for 25min) and the supernatant was used for isoenzyme analysis. The samples were layered onto a vertical polyacrylamide gel of various concentration (Table 1) and electrophoresed under native conditions. Isoenzymes were visualized by enzyme-specific reactions (Table 1) using corresponding dyes (see references).
Results
Sensitivity of Chlorella strains to viruses Studied Chlorella strains can be divided into 3 groups by sensitivity to viruses (Table2): 1) sensitive to Pbi-vi-ruses, resistant to NC64A-viruses; 2) sensitive to NC64A-viruses, resistant to Pbi-viruses; 3) resistant both to Pbi-viruses and NC64A-viruses. To the first group belong Chlorella strains OCh, PbAm, PbBS, and 241.80, to
the second group belong Chlorella strain 211-6, strain NC64A and its mutants Az4 (k), As-21-skb2-1 and As-21-2Du. The type strains of free-living Chlorella studied represent the third group. All virus-sensitive Chlorella strains were isolated from European (OCh, PbAm, PbBS, 241.80) and American (211-6 and NC64A) strains of Paramecium bursaria, respectively, or are mutants (subclones) of Chlorella strain NC64A.
Serological characterization of Chlorella strains
Specific agglutination of algal cells depending on Chlorella strain and used antiserum is observed in analysis of antiserum titer with the agglutination method. The Chlorella strains sensitive to Pbi-viruses show a high inverted titre (28) of agglutination reaction with antisera raised against Chlorella strains OCh and 241.80, respectively (Table 2), but they do not show any reaction with the antiserum raised against strain NC64A. Conversely, Chlorella strain NC64A and its mutants, and strain 211-6 show a high inverted titre of agglutination with the antiserum raised against Chlorella NC64A and do not agglutinate with any antiserum against European Chlorella strains.
Chlorella strains, resistant to both the Pbi-virus and NC64A-virus agglutinate very poor with all used antisera, or do not show any reaction (Table 2). Thus, specific agglutination according to the groups of virus-sensitivity is observed.
Isoenzyme analysis
The used antibodies bind to antigenes located on the surface of the Chlorella cells. In our next experiment we analyzed several isoenzymes which are involved in the metabolism in the cell. We adapted methods that are successfully used for the visualization of isoenzymes in higher plants (Table 1). We have got patterns for 7 isoenzymes (see below).
We began our study with comparing isoenzyme patterns of the analyzed virus-sensitive strains (Fig. 2). The European virus-sensitive strains Och, 241.80, PbAm and PbBS showed identical electrophoretic mobility of the isoenzymes NAD-dependent Aromatic Alcohol Dehydrogenase (AADH-NAD), NADP-dependent Aromatic
Table 2. Sensitivity of Chlorella strains to viruses and serological characterisation of the strains using the agglutination method
Chlorella strains Sensitivity to Pbi-virus NC64A-virus 101G-2 PBCV-1 Inverted antiserum titers* Antisera raised against strains Och 241.80 NC64A
European zoochlorella
Och + - 2 8 2 8 2 0
241.80 + - 2 8 2 8 2 0
PbBS + - 2 8 2 8 2 0
PbAm + - 2 8 2 8 2 0
American zoochlorella
211-6 — + 2 1 2 0 2 8
NC64A — + 2 1 2 1 2 8
As-21-skp-1 - + 2 0 2 0 2 8
As-21-2Du - + 2 1 2 1 2 7
Az 4 (k) - + 2 0 2 0 2 8
Type strains
Ch. protothecoides
211-7a - - 2 1 2 1 2 0
Ch. sorokiniana
211-8k - - 2 2 2 3 2 3
Ch. vulgaris
211-11b — - 2 1 2 0 2 3
* Inverted antiserum titers were identified using the agglutination method in microtiter plates.
Alcohol Dehydrogenase (AADH-NADP) and Aconitase (ACO). We have obtained similar patterns for AADH-NAD and ACO between the American Chlorella strains 211-6 and NC64A and the mutants of NC64A. These patterns are different to the patterns of the European Chlorella strains. We did not find any patterns of AADH-NADP for the American isolates.
To address the question of taxonomic grouping of virus-sensitive Chlorella strains we included cell extracts of the 3 type strains Chlorella protothecoides 211-7a, Chlorella sorokiniana 211-8k and Chlorella vulgaris 211-11b for the analyses of the isoenzymes Diaphorase (DIA), Aspartataminotransferase (AAT), Superoxide Dismutase (SOD) and Malatdehydrogenase (MDH). Again, the European virus-sensitive Chlorella strains OCh, 241.80, PbAm, and PbBS show identical electrophoretic mobility of isozymes of all analysed isoenzymes (Fig. 1). The patterns are different to the isoenzyme patterns of both the American virus-sensitive Chlorella strains and type strains. However, the AAT isoenzyme patterns of Chlorella sp. strains OCh, 241.80, PbAm, and PbBS and Ch. vulgaris
211-11b are identical. Similarity of patterns of all analysed isoenzymes between Chlorella strains 211-6 and NC64A, and NC64A subclones As-21-skb-1, As-21-2Du and Az4 (k), is shown in Fig. 1. Isoenzyme patterns are specific for these strains and are not similar to the patterns of any other Chlorella strain. However, the Diaphorase patterns of Chlorella protothecoides 211-7a are almost similar to these of the American strains. It should be stressed, unlike strain NC64A, its mutants show lower activity or absence of some patterns, and also formation of isoenzyme patterns de novo. It is known that every Chlorella mutant can show some minor changes in its own specific intensity and quantity of isoenzyme patterns (Bers et al., 1971). Moreover, forms of isoenzymes can change when cells are exposed to the effect of protein synthesis inhibitors (Bers, 1973). We suppose that insignificant changes in isoenzyme spectrum of strain As-21-skb-1 are caused by the effect of protein synthesis inhibitors during the selection of this strain.
Thus, Chlorella-like algae strains OCh, 241.80, PbAm, and PbBS isolated from Paramecium bursaria of various places in Europe are sensitive to one group of viruses (Pbi-
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AADH-NADP
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surface antigenes and show similar patterns of all analysed isoenzymes, with the exception of mutants As-21-skb-1 and As-21-2Du showing minor changes for some of the enzymes.
Based on these results we suppose a correlation between Chlorella surface antigenes including the receptor for viruses and isoenzyme patterns in virus-sensitive Chlo-rella strains. In order to check our supposition we isolated Chlorella viruses which should confirm or refute our grouping of Chlorella strains by virus-sensitivity, surface antigenes, and isoenzyme spectrums by attacking Chlo-rella strains with different isoenzyme patterns.
Viruses that infect Chlorella NC64A we found in water collected in Tadzhikistan (Voitsekhovsky et al.,1994). Plaque forming viruses of Chlorella Pbi-type were found in water samples collected near St.Petersburg. However, several attempts to find plaque forming virus attacking both Chlorella NC64A and Chlorella Pbi have been unsuccessful. Thus, isolated by us viruses did not change our grouping of symbiotic Chlorella strains.
Discussion
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Fig. 1. The electrophoretic mobility of the isoenzymes (A -AADH-NAD, B - AADH-NADP, C - ACO) of the Chlorella strains. The European virus-sensitive Chlorella strains (Och, 241-80, PbBS, PbAm), the American Chlorella strains (2116, NC64A, As-21-skb-1, Az4(k), As-21-Du). The difference of the isoenzime patterns of two virus-sensitive Chlorella strains groops from European and American populations are shown. Into groops this patterns were identical.
viruses), they have got similar surface antigenes and they are characterized by identical isoenzyme patterns of analysed isoenzymes: aconitase (ACO),
aspartataminotransferase (AAT), diaphorase (DIA), malat dehydrogenase (MDH), NAD-dependent aromatic alcohol dehydrogenase (AADH-NAD), NADP-dependent aromatic alcohol dehydrogenase (AADH-NADP), and superoxide dismutase (SOD). On the other hand, American Chlorella strains 211-6 and NC64A isolated from Paramecium bursaria of various geographical origin, and the analysed subclones of strain NC64A: As-21-skb2-1, As-21-2Du, Az4 (k) are sensitive to an other group of viruses (NC64A-viruses), they are characterized by similar
We have analysed virus-sensitivity, surface antigenes of living cells and spectrums of 7 isoenzymes of 6 symbiotic Chlorella strains, 3 subclones of symbiotic Chlorella strain NC64A, and 3 type strains Ch. vulgaris, Ch. sorokiniana, and Ch. protothecoides. It is now evident, that virus-sensitive Chlorella strains, former endosym-bionts of the protozoan Paramecium bursaria isolated in Europe (strains OCh, 241.SC, PbAm, and PbBS) show identical isoenzyme patterns. Based on morphological, physiological and biochemical studies Reisser (19S4; Reisser et al.,19SS) suggests that strains PbAm, PbBS, and 241.SC are closely related to the Chlorella vulgaris group to which belong Ch. vulgaris, Ch. sorokiniana and Ch. lobophora. Our finding that aspartataminotransferase isoenzyme patterns of Pbi-virus sensitive strains and type strain Ch. vulgaris 211-11b are identical, supports this suggestion. On the other hand, the Diaphorase patterns of the American strains and type strain Chlorella protothecoides 211-7a are almost similar. However, it was not found any similarity between isoenzyme patterns of American virus-sensitive Chlorella strains, former endo-symbionts of P bursaria, and investigated type strains Chlorella vulgaris 211-11b and Chlorella sorokiniana 211-Sk, although strain NC64A is closely related to free-living Chlorella vulgaris (Reisser et al.,19SS) by morphological and physiological features, and strain 211-6 belongs to the Chlorella vulgaris group (Huss et al.,19S9). Analysis of G+C mol % contents and deoxyribinucleic acid reassociation (Huss et al., 19S9) revealed that strain 2116 is closely related to European virus-sensitive Chlorella strain Pbi (99 % homology) although they are members of different systems Paramecium bursaria - Chlorella - vi-
Figs 2-3. Isoenzyme patterns (A - DIA; B - AAT; C - SOD; D - MDH) are specific for the European Chlorella strains (Och, 241-S0, PbBS, PbAm), for the American strains (211-6, NC64A, As-21-skb-1, Az4(k), As-21-Du) and are not similar to the patterns of other virus-resistant Chlorella strain (Chlorella protothecoides 211-7a, Chlorella sorokiniana 211-Sk and Chlorella vulgaris 211-11b).
rus, but strain 241.80 shows less DNA homology (65%) with strain Pbi although they belong to the same system.
Thus, taxonomy of symbiotic Chlorella is a complex problem to solve which we currently have begun electrocaryotyping of Chlorella chromosomes using pulsed field gel electrophoresis.
Acknowledgements
We are thank Prof. W. Reisser (Gottingen, FRG) for kindly providing strains Pbi, PbAm and PbBS and Prof. J. Van Etten (Lincoln, Nebraska, USA) for strain NC64A and virus PBCV-1.
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Address for correspondence: Prof. Konstantin V. Kvitko, Laboratory of Microbiology, Biological Institute of St. Petersburg State University, Oranienbaumskoye sh. 2, Stary Peterhof, St.Petersburg, 198004, Russia. E-mail: [email protected]
The manuscript is presented by S.I.Fokin
