Phylogenetic significances of the photoreactivable sectors of species of Haitsenula

A. SARACMEK AND ROSALEE I R E L A N D Wichita State Ut~iversity, Ifficl~itn, Kansa.~

Accepted June 9, 1971

SARACHEK, A, , and R. IRELAND. 1971. Phylogenetic significances of the photorcactivable sectors of species of Hanser~ula. Can. J. Microbiol. 17: 1217-1221.

Each species of the genus Hanser~~ila has becn tested for the extent to which cellular inactivation by ~~ l t r av~o le t radiation is mitigated by photoreactivation. The values obtained conform to a pattern con- sistent with the phylogenetic relationships bctwcen the species proposed by Wickerham. These findings corroborate previous indications that (i) fixation of the capacity for photoreactivation is an important element in the evolution of yeasts and that (ii) opportunity for environmental exposure of yeasts to solar ultraviolet radiation is not the determining factor in natural selection for thcir abilities to photo- reactivate.

The term photoreactivation refers to recovery by biological systems from the adverse effects of ultraviolet radiation (uv.) through postirradia- tion treatment with visible light (8). Though two different mechanisms for such recovery exist, the critical feature of both is the elimination of pyrimidine dimers induced in DNA by uv. (9). Direct photoreactivation results from a inono- merization of the dimers catalyzed by a single light-activated enzyme (19). Indirect photore- activation ensues from the non-enzymatic light- induced inhibition of cell growth which prolongs the time during which dimers can be eliminated by dark repair processes before the onset of DNA replication (9, 10). Indirect photoreactiva- tion appears to be of limited biological impor- tance, having been demonstrated positively in a few strains of microorganisms (1, 9). In contrast, direct photoreactivation is exhibited by a wide variety of higher animal (3) and plant (15) cells, as well as both eucaryotic and procaryotic microorganisms (8). Moreover, in higher animals and plants, the cellular contents of photore- activating enzyme vary with particular stages of ontogeny and are tissue specific in mature in- dividuals (3, 15). Both the broad distribution of photoreactivating enzyme in nature and the changes in its occurrence during differentiation of multicellular organisms impute a far-reaching biological significance either to the photorepair process or to some yet to be uncovered, non- radiobiological function of the photoreactivating enzyme.

A previous survey of 83 species of yeasts dis- tributed among 12 perfect and 10 imperfect

Received February 12, 1971

genera revealed that the ability of uv.-killed cells to undergo direct photoreactivation is genus dependent and, presumably, a fi~nda- mental feature of the evolutionary divergence of yeasts (16). Though the findings also s~~ggested that capability for photoreactivatioil may be an absolute property of each genus and, therefore, a useful taxonomic criterion, the n~unber of species examined within individual genera was insufficient to establish that point unequivocally. All scven, arbitrarily selected species of Hail- senula included in that study were found to photoreactivate, but to markedly different de- grees. The present paper reports the proportion of lethal uv.-induced damage subject to direct photorepair (i.e., the photoreactivable sector) in each of the 25 species currently accepted in the genus Hansenula. Wickerhain (24) has arranged these species according to a phylo- genetic scheme which constitutes the most sub- stantial statement of probable evolutionary re- latio~lships within a major yeast genus. Our observations were made to determine (i) whether ability to photoreactivate is universal among the Hansetiulas and (ii) whether the magnitudes of the photoreactivable sectors for individual species correlate with the systematic placement of the species in Wickerham's scheme.

Materials and Methods The test organisms are listed in Table 1 ; ATCC strains

were purchased from the American Type Culture Col- lection, Rockville, Maryland, U.S.A. and NRRL strains were provided by Dr. Lynferd J. Wickerham or Dr. C. P. Kurtzman of the Northern Regional Research Labo- ratory, Peoria, Illinois, U.S.A.

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1218 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 17, 1971

Yeasts were cultured before or after irradiation at 25°C on the glucose-peptone-yeast extract agar of Busbee and Sarachek (2). Cells to be irradiated were harvested from 2-day-old slants. Asci were observed in cultures of H. glrrcozytnn, H. snt~rnzrrs var. sntlrrnus, H. satrrrtlrrs var. srrbsrrficietzs, and H . silvicola; however, in no case did the proportion of asci exceed 0.5% of the total cell population. Preparation of cells for irradiation as well as

Line 1A Line 1B

H. sntrrrrrrrs var. sotrrrtfrrs

-H. mtrri.tira var. I srrbsrficicns

Line 3 A

I H. beckii

I

procedures for irradiation and photoreactivation were described in detail by Busbee and Sarachek (2). Cell survivals were assessed by colony counts made, depend- ing upon the strain, 3 to 5 days after treatment. For each of the photoreactivable species, visible light reduced the inactivational effectiveness of uv. by a constant pro- portion (i.e., dose-reduction factor (DRF)) to the 95% level of kill, at least. DRF's for individual species were

Line 5

b Line 2 Line 4

var.

var.

HOMOTHALLIC LINES I

Y More primitive species

than now known

HETEROTHALLIC LINES

FIG. 1. Phylogenetic relationships between species of Harlsenula as proposed by Wickerham (24). Line lA, homothallic species evolving toward the free-living state. Line lB, homothallic species evolving to dependence upon warm-blooded animals? Line 2, heterothallic species evolving to free-living state. Line 3, homothallic species evolving to greater dependence upon coniferous trees. Line 4, heterothallic species evolving to greater dependence upon coniferous trees. Line 5, homothallic species, various habitats. NOIT: Cnndida crtilis is identified as the permanently diploid, asporogenous derivative of H. jadinii (20, 23).

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SARACHEK AND IRELAND: PHOTOREACTIVATION, HANSENULA 1219

determined from the regression of the uv. dosages with- out photoreactivation on those with photoreactivation required to produce equivalent kills (13). As defined by Dulbecco (5), the photoreactivable sector, o r segment of the uv. inactivational cross section of an organism elimi- nated by photoreactivation, is expressed by the value I -DRF.

As an enzymatic process, direct photoreactivation is markedly temperature-dependent whereas non-enzymatic, indirect photoreactivation is not (9). Our standard pro- cedure for photoreactivation involved exposure of ir- radiated cells to visible light for 1 h at 25°C. When tested at the 99% level of uv. inactivation, all photoreactivable test organisms were found to exhibit maximal recovery after 20 to 30min illumination and to show no photo- inactivation over the full I-h exposure period. More- over, at the same level of inactivation, recoveries by these organisms when illuminated at 5°C for 15 min were less than 15% of those obtained with the same exposure time at 25°C. Thus, under our standard experimental con- ditions, all strains were fully photoreactivated and the pronounced dependency of their recoveries on temper- ature during illumination indicates that the photo- reactivation observed is of the direct, enzymatic sort.

Results and Discussion Figure 1 illustrates the phylogeny of the genus

Hansenula as outlined by Wickerham (24). The species of the genus have been grouped into five major and one subsidiary lines of descent based on habitat and homothallism vs. heterothallism. The most primitive species are haploid, are usually associated with trees, ferment few or no sugars, produce extracellular phosphomannans, and have vitamin requirements. Progressive evolution is taken to be associated with a shift to a diploid growth form, losses of vitamin re- quirements and the capacity for phosphomannan formation, acquisition of ability to ferment several sugars, and a tendency to greater or lesser ecological dependency upon trees. Since species of Kluyverornyces (7), Sacclzaro~nyces (22, 26), and Sclzizosaccharomyces (12) are known to undergo interconversions between homothallic and heterothallic states on the basis of single- gene mutations, acceptance of completely in- dependent homothallic and heterothallic lines of descent in Hansenula might require closer scrutiny. Nevertheless, the Wickerham scheme does express a generally cogent and coherent phylogeny for Hansenula founded upon reason- ably systematic criteria.

Table 1 records values for the photoreactivable sector of each species indicated in Fig. 1. It is seen that four of the species, H. bimundalis, H. capsulata, H. petersonii, and H. ntickerhamii, do

not photoreactivate at all; the four species com- prising line 5 of Wickerham's scheme together with H. nonfermentans and H. henricii of line 3 photoreactivate poorly with photoreactivable sectors of 0.1. The remaining species photo- reactivate well with photoreactivable sectors ranging from 0.3 to 0.7. These findings bear the following phylogenetic implications.

1. Photoreactivation is a very prominent though not absolute characteristic of Hansenula and, therefore, is not a positive diagnostic criterion for the genus.

2. The non-photoreactivating species of Hall- senula are primitive ones. The absence of photo- reactivation in H. petersonii suggests that the subsidiary line 1B (Fig. 1) might more properly emerge from line 1A between H. capsulata and H. silvicola, one "notch" lower than its presently assumed origin. On the bases of biochemical

TABLE 1

Photoreactivable (PR) sectors of the species of Harzsenlrta indicated in Fig. 1

Species PR sector

Hansenlrta mlonzata ATCC 580 H, beckii NRRL Y-1482 H. begerirzckii NRRL YB-4312 H. biinlmdatis var. arnericana NRRL

Y-2156 H. birnlrndalis var, mnericmla NRRL

Y-2157 H. binzlmdatis var. bimluzdalis NRRL Y-5343 - - - .-

H. califorrzica NRRL Y-1680 H. conadensis NRRL Y-1880 H. capsrrlata NRRL Y-1842 H. ciferrii NRRL Y-1031 H. dir?zerzrzae NRRL Yl3-3239 H. fabiarzii NRRL Y-1871 H. >lucozyma NRRL YB-2185 H. heraicii NRRL YB-2194 H. holsrii NRRL Y-2155 H. iadinii ATCC 18201 H.mirzuta NRRL Y-411 H. rnrakii NRRL Y-1364 H. ?zonfernzeiztans NRRL YB-2203 0.1 H. oetersonii NRRL Yl3-3808 0 .0 H. >latypodis NRRL Y-6732 0.1 H. polynzorpfra (H. arzgusta) ATCC 14754 0.1 H. saturnus var. satlrrtzrrs ATCC 18119 0.7 H. saturnus var. subsrrficiens NRRL

YB-1657 H. silvicola ATCC 16764 H. silvicola NRRL Y-1678 0.4 H. subpelliculosa NRRL Y-1683 0.5 H. wickerhamii NRRL -4943 0.0 H. lvirrgei NRRL Y-1987 0.3 Candida ~rtilis* ATCC 9950 0.5

*Imperfect form of H. jadinii (20, 23).

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1220 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 17, 1971

and ecological considerations, Wickerham and Burton (25) proposed several years ago that the genus Pichia must have evolved from primitive Hansen~rla species. We have reported (16) that the species of Piclzia recognized at the time of that proposal by the treatise of Lodder and Kreger-van Rij (11) do not photoreactivate. Thus, our photoreactivation findings are con- sonant with the placement of primitive species in Wickerhain's phylogenetic scheme and with the presumption that the genus Piclzia arose from such species.

3. Comparisons of the unique cell wall mannan structures of Haiiseti~rla species have prompted Spencer et al. (21) to suggest that H. nonfernzentans and H. henricii should be removed from line 3 and transferred to line 5. Our observation that these two species together with the four species of line 5 have the same small photoreactivable sector of 0.1, clearly distin- guishable from the photoreactivable sectors of all other Hansei~ulas, substantiates that sug- gestion.

4. The magnitudes of the photoreactivable sectors of the strongly photoreactivating yeasts do not correlate with either the level of evolu- tionary development of those species nor their assignments to the various lines of descent.

5. Pyrimidine dimers constitute the critical uv.-induced DNA lesions responsible for cellular inactivation. A substantial body of evidence indicates that non-genetic forms of uv. damage also can cause inactivation of yeasts (4, 6, 18). Though the sites of these damages have not been identified positively, the protein-synthesizing apparatus (1 6, 17, 18), the cytokinetic mechanism (16), and the cellular membranes (6) have been proposed as particularly likely candidates. Cleavage of pyrimidine dimers is the only known function of photoreactivating enzyme (19). Thus, dissimilarities in photoreactivable sectors for species of Hansenula suggest that the species differ either in the proportion of their pyrimidine dimers accessible to photoreactivating enzyme or in the relative contributions of genetic and non-genetic damages to their inactivation. In these terms, the fact that species with small photoreactivable sectors form a separate phylo- genetic grouping from those with large ones implies that systematic differentiation occurs within the genus Haizseizula either for some radiobiologically significant feature of nuclear

organization or for the relative proportions of cellular genetic and 11011-genetic uv.-sensitive target materials.

6. The absence of photoreactivatioi~ in prim- itive species and its occurrence in advanced ones could be explained in one of two ways. First, the gene for photoreactivating enzyme might enter the genus through intergeneric malings involving Hatzsetiula species of intermediate levels of evolution. Since the yeast genera (with the possible exception of Hansenula) are largely taxonomic agglomerates without well-substanti- ated systematic definitions, it is possible that there might be organisms currently consigned to other genera with sufficient natural affinities to Hanseiiuln to conduct the gene(s) for photore- activating enzyme introgressively into that genus. However, such an explanation is unlikely since it would require that the gene(s) be introduced independently into the five lines of descent. Alternatively, it is possible that all species of Haiisen~rla bear a gene for photoreactivating enzyme, but that the genetic constitution of each primitive species is such that either the gene exists in a fully repressed state or the activity of the formed enzyme is completely inhibited. In these terms, acquisition of photoreactivating activity with progressive evolution of the genus would be associated with genetic changes which release the repression at the gene or enzyme level. The fact that photoreactivating enzyme activity in animal (3) or plant (15) cells appears and disappears at different stages of develop- ment lends credence to these possibilities.

Sunlight transmitted through the earth's atmosphere contains ultraviolet radiation of wavelengths suitable for induction of pyrimidine dimers in DNA as well as longer wavelengths of light capable of activating photoreactivating enzymes (14). It would be reasonable to assume that positive selectioll for the capacity for photo- reactivation is favored in organisms which naturally experience appreciable exposures to sunlight. Our earlier observation of the genus dependency of enzymatic photoreactivation in yeasts (16) was interpreted to mean that strong selective pressures for the retention or loss of the genetic potential for photoreactivation are in- volved in the evolution of these organisms. However, since a great many species in photo- reactivating and in uon-photoreactivating genera occupy equivalent ecological niches, it was

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SARACHEK AND IRELAND: PHO TOREACTIVATION. HANSENULA 1221

argued that selection for photoreactivability does not relate primarily to the need to ameliorate the detrimental cellular effects of sunlight. The present study establishes clear correlations between the intrageneric, phylogenetic status of species of Hanse~zzlla and their qualitative and quantitative capabilities for photoreactivation. Though the ecological predilections of photo- reactivating and non-photoreactivating species are different, the habitats of both groups offer comparable possibilities for contact with sun- light. These findings, therefore, reinforce the views that (i) selection for or against the ability to photoreactivate is an inlportant feature of the evolution of yeasts but that (ii) such selection is exerted upon some as yet unknown function of the photoreactivating enzyme other than its capacity for repair of pyrimidine dimers induced by solar ultraviolet. radiation.

Acknowledgments

These studies were aided in part by a contract AT(l1-1)-1772 with the U.S. Atomic Energy Commission.

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24. WICKERHAM, L. J. 1969. New homothallic taxa of Hansetl~rla. Mycopathol. Mycol. Appl. 37 : 15-32.

25. WICKERHAM. L. J., and K. A. BURTON. 1962. Phy- logeny and biochemistry of the genus Hnnsetnrln. Bacteriol. Rev. 26: 382-397.

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