Trans. Br. mycol. Soc. 91 (3), 439-442

[ 439 ]

Printed in Great Britain

CHANGE FROM HOMOTHALLISM TO HETEROTHALLISM INSCHIZOSACCHAROMYCES MALIDEVORANS:

BIOCHEMICAL AND TAXONOMIC CONSEQUENCES

By P. D. BRIDGECAB International Mycological Institute, Ferry Lane, Kew, Surrey TW9 ]AF, U.K.

AND J. W. MAYDepartment of Microbiology, Monash University, Clayton, Victoria ]168, Australia

Whereas a homothallic line of Schizosaccharomyces malidevorans assimilates maltose,heterothallic (asporogenous) variants which fall into two mating-type groups (h ' and h-)interfertile with S. pombe all fail to assimilate maltose. These results explain discrepancies inpublished descriptions of the species and support the synonomy of S. malidevorans withS. pombe.

The fission yeast Schizosaccharomyces malidevoranswas first described on the basis of a single isolatefrom Listan grapes grown in Spain. The primordialline of this monotypic species deposited at theAustralian Wine Research Institute (AWRI) washomothallic (ascospores being produced abund-antly) and assimilated maltose, being distinguishedfrom S. pombe by the rough appearance of itsascospores and its failure to ferment maltose(Rankine & Fornachon, 1964). Subsequently, itwas reported that ascospores of S. pombe frequentlypossessed a rough outer wall (Slooff, 1970), leavingmaltose as the only clear-cut distinguishing char-acter. However, another line of S. malidevoransmaintained at the Centraalbureau voor Schim-melcultures (CBS), Delft, The Netherlands wasreported not to assimilate maltose, although it wasreported to produce ascospores (Slooff, 1970;Barnett et al., 1983; Yarrow, 1984). On the otherhand, auxotrophic mutants derived from the CBSline exhibited gene recombination preferentiallywith the h mating type of S. pombe, indicating thatthe CBS line was heterothallic and corresponded tothe h" mating type of S. pombe (Sipiczki et al.,1982). In view of these discrepancies between thereported properties of S. malidevorans, we havenow re-examined subcultures of the AWRI andCBS lines for their ability to sporulate and toassimilate maltose.

MATERIALS AND METHODS

Cultures

Lines of S. malidevorans were obtained fromAWRI (AWRI 158) and CBS (CBS 5557). The

reference mating types of S. pombe were obtainedfrom Prof. J. M. Mitchison, Department ofZoology, University of Edinburgh (L972, h) andfrom Prof. U. Leupold, Institute of General Micro-biology, University of Bern (L975, h"), Cultureswere grown at 25°C and maintained on slopes ofOxoid Malt Extract agar (MEA) at 4°.

Physiological and biochemical properties

Assimilation of maltose was tested as the ability togrow within 14 don EMM2A(G-o) agar containing1 % (wIv) maltose as described by Bridge & May(1984). Fermentation of maltose was tested as theability to produce gas within 28 d in liquidEMM2A(G-o) containing 1 % (w/v) maltose asdescribed by Bridge & May (1984). After 14 dincubation at 25° on MEA, sporulating andnon-sporulating colonies were distinguished byexposure to iodine vapour (Leupold, 1970),sporulating colonies staining blue (iodine-positive)and non-sporulating colonies appearing lightyellow (iodine-negative). The presence or absenceof ascospores was confirmed microscopically.

Interfertility and mating-type determination

Asporogenous lines of S. malidevorans were testedfor interfertility with other asporogenous lines ofS. malidevorans and with the h and h ' matingtypes of S. pombe by 'cross-patching' on MEA.After incubation for 28 d at 25°, the formation ofascospores was determined by exposure to iodinevapour (as above). In order to eliminate anyvariation in environmental conditions which could

440 Change from homothallism to heterothallism

Table 1. Half matrix showing interfertility and designated or inferred mating type of asporogenous lines ofS. malidevorans and mating-type reference strains of S. pombe

S. malidevorans S. pombe

Group 1 Group 2 L972 L975 Mating type

S. malidevorans AWRI 158Group 1 0 A 0 A h-Group 2 0 A 0 h+

S. malidevorans CBS 5557 A 0 h+S. pombe

L972 0 A h-L975 ° h+

0, no ascospores produced; A, ascospores produced.

induce sporulation, cell lines were tested togetherwith S. pombe controls, as patches on one plate. Toeliminate any chemical effects from other colonies,subcultures from the CBS line were re-testedagainst S. pombe in isolation.

RESUL TS

Properties of 'wild-type' lines of S. malidevorans

Cultures of S. malidevorans AWRI 158 were (i)iodine-positive and produced abundant ascosporeson MEA, and (ii) assimilated maltose but failed toferment it. Thus this line is self-fertile and can beinferred to be homothallic. On the other hand,cultures of S. malideuorans CBS 5557 were (i)iodine-negative and failed to produce any asco-spores on MEA, and (ii) failed to either assimilateor ferment maltose. Although this line is not self-fertile, it is fertile with S. pombe L972 (h) but notwith L975 (h"), and thus may be inferred to belongto the h" mating type of S. pombe (Table 1).

Isolation and properties of asporogenous lines ofS. malidevorans AWRI 158

Examination of iodine-treated colonies of S.malideoorans AWRI 158 revealed the presence ofiodine-negative papillae. Lines, each derived froma single papilla, could be divided into two groups:group 1 (2 isolates) and group 2 (6 isolates).Members of a particular group were not interfertilebut were fertile with members of the other group.Furthermore, members of group 1 were fertilewith S. pombe L975 (h ") but not L972 (h'), andhence they were mating type h"; whereas membersof group 2 were fertile with L972 but not L975,and hence they were mating type h" (Table 1).None of the lines in either group was able toassimilate or ferment maltose.

DISCUSSION

The occurrence of heterothallism in subcultures ofthe CBS line of S. malideoorans and the inter-fertility of these with S. pombe has previously beendemonstrated by Sipiczki et al. (1982) and ourresults show that the ability of S. malidevorans toassimilate maltose is dependent upon expression ofhomothallism, heterothallic lines (either h" or h-)lacking this capacity. Homothallism depends uponthe rapid ability to switch expression of twoclosely-linked mating-type genes so that both h 'and h cells are present in a clonal culture, whereasheterothallism is caused by mutations in themating-type region or in unlinked genes regulatingswitching, resulting in a culture of predominantlyeither h ' or h- cells (Gutz et al., 1974).

The nature of the relationship between thesimultaneous shifts from homothallism to hetero-thallism and from maltose assimilation to non-assimilation are not known. Sipiczki et al, (1982)found that a gene involved in maltose assimilation(mal") was probably closely linked to leu-a onchromosome I, whereas the mating-type regionhas been mapped on chromosome II (Gygax &Thuriaux, 1984). While this might suggest aprocess involving a regulatory mechanism, at leastone gene involved in switching (swi 2) has beenallocated to chromosome I, although its positionhas not been precisely defined (Gygax & Thuriaux,1984).

With respect to homothallism and ability toassimilate maltose, our results for S. malidevoransAWRI 158 agree with those of Rankine & For-nachon (1964) and our results for S. malidevoransCBS 5557 agree with those of Sipiczki et al. (1982)but differ from those of Slooff (1970), Barnett et al.(1983) and Yarrow (1984). These results, togetherwith such results for assimilation of glycerol andgrowth at 37° as are available, are shown in Table

P. D. Bridge and J. W. May

Table 2. Summary of reported properties of two lines of S. malidevorans

Assimilation ofGrowth

Source of information Line Homothallism Maltose Glycerol at 37 DC

Rankine & Fornachon (1964) AWRI 158 + +Bridge & May (1984) AWRI158 + + +Vasiliadis et al. (1987) AWRI158 +Bridge & May (1984) CBS 5557Vasiliadis et al. (1987) CBS 5557 +Sipiczki et al. (1982) CBS 5557 -*Sloof (1970) CBS 5557 + +Barnett et al. (1983) CBS 5557 + +Yarrow (1984) CBS 5557 + +

+, positive; -, negative; ., not recorded by authors.* Result obtained with auxotrophic mutant.

441

2. Clearly the line maintained at CBS differssignificantly from the primordial line maintainedat AWRI. Furthermore, since heterothallism andassimilation of maltose have not been observedtogether in any of the present lines this combinationof characters (see Slooff, 1970; Barnett et al., 1983;Yarrow, 1984) is probably a consequence of a lossof homothallism in the line at CBS sometimefollowing acquisition.

In regard to the taxonomic status of S. malideoo-rans, the proposal of Sipiczki et al. (1982) to reduceit to a variety of S. pombe is consistent withgroupings based on chemotaxonomy (Yamadaet al., 1973; Stewart & Widanapatirana, 1974;Claisse & Simon-Becam, 1978), numerical tax-onomy (Bridge & May, 1984) and glycerol assimila-tion (Vasiliadis et al., 1987). However, thedescription given by Sipiczki et al. (1982) forS. pornbe var. malidevorans differs significantlyfrom the original description and from otherproperties which we have ascertained for theprimordial line maintained at AWRI (see Table 2).Since other proposed differential properties (viz.rough spore surface, decomposition of malic acid,production of H

2S,growth at 37° and assimilation

of glycerol) have been found to occur in both taxa(Slooff, 1970; Bridge & May, 1984; Vasiliadis exal., 1987), the only reliable difference between theprimordial line of S. rnalidevorans and typical S.pornbe is the inability of the former to fermentmaltose. Consequently, as the difference in a singlecharacter - viz. maltose fermentation - has alreadybeen rejected as sufficient basis for the separationof S. slooffiae and S. octosporus (Yarrow, 1984) (seealso Sigler & Hawksworth, 1987), there is nojustifiable alternative to the reduction of S.rnalidevorans to synonomy with S. pornbe.

This work was supported in part by a MonashUniversity Special Research Grant.

REFERENCES

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BRIDGE, P. D. & MAY, J. W. (1984). A numericalclassification of fission yeasts of the genus Schizo-saccharomyces Lindner. Journal ofGeneral Microbiology130, 1921-1932.

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GUTZ, H., HESLOT, H., LEUPOLD, U. & LOPRIENO, N.(1974). Schizosaccharomyces pombe. In Handbook ofGenetics, 1 Bacteria, Bacteriophages and Fungi (ed.R. C. King), pp. 395-446. New York: Plenum Press.

GYGAX, A. & THURIAUX, P. (1984). A revised chromosomemap of the fission yeast Schizosaccharomyces pombe.Current Genetics 8, 85-92.

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RANKINE, B. C. & FORNACHON, J. C. M. (1964). Schizo-saccharomyces malidevorans sp. n., a yeast decomposingt-malic acid. Antonie van Leeuwenhoek 30, 73-75.

SIGLER, L. & HAWKSWORTH, D. L. (1987). InternationalCommission in the Taxonomy of Fungi (ICTF). Codeof practice for systematic mycologists. Mycopathologia99,3-7·

SIPICZKI, M., KUCSERA, J., ULASZEWSKI, S. & ZSOLT, J.(1982). Hybridization studies by crossing and proto-plast fusion within the genus Schizosaccharomyces.Journal of General Microbiology 128, 1989-2000.

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VASILIADIS, G. E., SLOAN, J., MARSHALL, J. H. & MAY,J. W. (1987). Glycerol and dihydroxyacetone metabo-lizing enzymes in fission yeasts of the genus Schizo-saccharomyces. Archives of Microbiology 147,263-267.

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(Received for publication 5 January 1988)