Dipodascus Capitatus, Dipodascus Spicifer and Geotrichum Clavatum: Genomic Characterization
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Antonie van Leeuwenhoek 74: 229–235, 1998. 229 © 1998 Kluwer Academic Publishers. Printed in the Netherlands. Dipodascus capitatus, Dipodascus spicifer and Geotrichum clavatum: Genomic characterization Maudy Th. Smith∗ & G.A. Poot Centraalbureau voor Schimmelcultures, Yeast Division, Julianalaan 67, 2628 BC Delft, The Netherlands (∗ Author for correspondence) Received 16 July 1998; accepted 20 July 1998 Key words: Dipodascus capitatus, D.spicifer, Geotrichum clavatum, yeast, taxonomy, DNA heterogeneity Abstract The G+C contents of 25 strains of Dipodascus capitatus, Dipodascus spicifer and Geotrichum clavatum were found to be heterogeneous on basis of derivative graphs of the melting profiles. Strains showing similar derivative graphs of the melting curve exhibited high levels of DNA homology (80-100%); strains showing dissimilar derivative graphs exhibited low levels of DNA homology (5 to 45%). Being considered separate taxa on basis of these parameters, D. capitatus, D. spicifer and G. clavatum could be identified by a combination of the key characteristics growth on xylose, cellobiose, salicin and arbutin. Introduction (1986). Geotrichum capitatum is frequently isolated from clinical specimens from man and cattle (Hellman In their revision of the ascomycetous genus Geotrichum & Raethel, 1964; Gemeinhardt, 1965; Saëz & Rinjard, Link:Fr. and its teleomorphs, de Hoog et al. (1986) 1973; Baird et al., 1985; Polachek et al., 1992; Aal- introduced five new species, among which three baek et al., 1994). In view of the clinical importance closely related taxa, Dipodascus capitatus, D. spicifer of G. capitatum as well as its morphological and phys- and Geotrichum clavatum. Species of Geotrichum iological similarities with D. spicifer and G. clavatum, and its teleomorphs are characterized morphologically Guého et al. (1987) re-evaluated this complex in more by formation of septate hyphae, disarticulating into detail examining a total of 11 strains. These authors arthroconidia, and by absence of budding yeast cells. recognized the existence of two distinct, closely re- Only, D. capitatus and D. spicifer produce sympo- lated human-pathogenic species, G. capitatum and G. dial rachids, in addition. Physiologically D. capitatus, clavatum, and a third group, represented by a single D. spicifer and G. clavatum are very similar, vary- strain isolated from rotten cacti and identified as D. ing in the capacity to utilize D-xylose, cellobiose, spicifer. salicin and arbutin. The most common of these three In 1997, Phaff et al. described a new Dipodascus species is the heterothallic D. capitatus. Its anamorph, species, D. starmeri, also occurring in cactus necroses. still erroneously referred to as Trichosporon capita- By analyzing the phylogenetic relationship of this tum (Arnold et al., 1981; Barbor et al., 1995), is in species and other members of the genera Dipodascus the present classification known as Geotrichum cap- and Geotrichum on basis of large subunit ribosomal itatum (Diddens et Lodder) von Arx (von Arx et al., DNA nucleotide sequence divergence, these authors 1977; Weijman, 1979; de Hoog et al., 1986). The concluded that D. starmeri is close to D. ingens.They anamorph species known as Blastoschizomyces capi- also pointed out some apparent conspecificities among tatus (Diddens & Lodder) Salkin, Gordon, Samsonoff species of these genera, in particular between D. cap- & Rieder (Salkin et al., 1985; Pagano et al., 1996; itatus, D. spicifer and G. clavatum.Inviewofthe Perfect & Schell, 1996; Plum et al., 1996) was con- conflicting conclusions of Guého et al. (1987) and sidered a synonym of G. capitatum by de Hoog et al. Phaff et al. (1997) on the taxonomic status of the MENNEN/SCHRIKS:DISK/CP: Pips Nr.:185891; Ordernr.:233762-mc (antokap:bio2fam) v.1.1 anto1012.tex; 7/12/1998; 11:13; p.1 230 three taxa based on DNA similarity data and rRNA Results sequence analyses, respectively, we present our obser- vations on detailed analyses of DNA melting profiles Physiological and cultural characteristics of 25 strains, genomic DNA reassociations performed with a selection of strains as well as nutritional char- Among 157 ascomycetous arthroconidial yeast iso- acteristics. lates examined physiologically, only 6 strains showed the same nutritional profile as the type strain of G. clavatum, 19 strains resembled the type strain of D. Materials and methods capitatus, whereas not a single strain was similar to the type culture of D. spicifer (Table 2). Characters to Cultures examined distinguish the 3 groups from one another are growth on D-xylose, cellobiose, salicin and arbutin. The range A total of 157 arthroconidial ascomycetous yeastiso- of growth rates on GPYA medium of the three groups lates was examined physiologically. Only those strains was similar, being 5-10 mm/7days for the D. capita- showing physiological similarities either to D. capita- tus group, 8-9 mm/7days for the type culture of D. tus, D. spicifer or G. clavatum, are listed in Table 1 spicifer and 5-8 mm/7days for the G. clavatum group with indication of their origin. (Table 2). Physiology and cultural characteristics Nuclear DNA base composition Abilities to grow on carbon compounds were tested The G+C contents of the 25 strains calculated from according to the methods described by van der Walt & the Tm values of the melting graphs directly, as well Yarrow (1984). Cultures were grown in liquid media in as from the derivatives of these graphs are presented in Table 1. All strains showed two peaks in their deriva- test-tubes for four weeks at 25 ◦C and shaken contin- uously at 30 rpm. Utilization of nitrogen compounds tive graphs (Figure 1 A and B). The type strain of D. was examined by the auxanographic method after one spicifer has the lowest GC value at 28.6 mol% and the week. highest at 38.5 mol%. For the G. clavatum group, rep- For measuring growth, a heavy inoculum of a 1–7 resented by 7 strains (the type strain included) the low- days old culture was streaked onto 4% glucose-0.5% est value ranged from 25.0–27.3 mol% and the highest peptone-0.5% yeast autolysate-2% agar (GPYA) in from 37.5–39.3 mol%; for the D. capitatus group rep- resented by 20 strains (the type strains of D.capitatus Petri dishes, and incubated for 7 days at 25 ◦C. The growth measurements were taken from the center to and Blastoschizomyces pseudotrichosporon included) the edge of the streak. these values were 27.0–30.0 mol% and 37.0–39.4 mol%, respectively. The distance between the tops of Genomic DNA analysis the two peaks is 9.7 mol% for D. spicifer, 9.4–10.7 mol% for the D. capitatus group and 11.4–12.1 mol% For DNA extraction, strains were grown for 2 days for the G. clavatum group. at 25 ◦C on a rotary shaker at 125 rpm in 2L YM broth (Difco Laboratories, Detroit, Mich.) using 1 L Infraspecific DNA relatedness flat-bottom flasks. The procedures for isolation and purification of DNA, and the methods of determining Strains used in infraspecific reassociations are marked DNA base composition were performed according to in Table 1. Some of the infraspecific reassociation Smith et al. (1995). DNA hybridizations were done combinations are shown in Table 3. Three combina- according to Seidler and Mandel (1971), modified by tions of four selected strains of G. clavatum, the type Kurtzman et al. (1980). The optimal reassociation included, gave reassociation values from 82–96%, temperature of 55 ◦C was determined following the clearly indicating their conspecificity. Fourteen com- procedure of the latter authors. Reassociation experi- binations of eleven selected strains of D. capitatus, ments were performed at least twice. Strains used in including the type and the mating types, reassociated reassociation tests are marked in Table 1. at 90–100%. anto1012.tex; 7/12/1998; 11:13; p.2 231 Table 1. Origins and other informations of strains examined, with their mol% G+C calculated from DNA melting curves and from derivative graphs Strain Origin and/or other information G+C content (mol%) as G+C content (mol%) as determined determined by Tm methoda by first derivates Value(s) Difference Geotrichum clavatum CBS 425.71Tc human lung tissue, USA 32.2(31.1d ) 26.5 38.6(40.0e) 12.1 CBS 576.82 human patient with asthma, 35.1(33.2d ) 27.1 39.0(41.0e) 11.8 CBS 758.85c cactus rot, USA 33.0(29.5d ) 26.7 38.6(38.7e) 11.8 CBS 969.87 man, France 33.0 26.6 38.5 11.9 CBS 970.87c condensation droplets in 34.4 27.2 38.6 11.4 brewery, Belgium CBS 489.88 liver of man, France 30.8 25.0 37.5 12.5 IP 95662c man, France 33.2 27.3 39.3 12.0 G. clavatum 26.6 0.7 38.6 0.5 11.9 0.3 ± ± ± Dipodascus capitatus CBS 197.35c wood pulp, Sweden, MT A 32.6(36.3d ) 30.0 39.4(40.2e)9.4 CBS 311.76 sputum of man, Germany CBS 312.76c sputum of man, Germany 31.8 27.0 37.0 9.9 CBS 162.80c bovine mastitis milk, UK 33.8 27.1 37.8 10.7 CBS 571.82Tc wood pulp, Sweden, LT of 31.8(33.7d ) 27.6 37.8(40.1e) 10.2 Trichosporon capitatum CBS 572.82 wood pulp, Sweden 33.9 29.3 39.6 10.3 CBS 573.82c yeast cake, 34.6 28.6 38.3 9.7 CBS 574.82 sputum of man, Norway 33.2 28.8 38.5 9.7 CBS 575.82c man, South Africa 32.5 27.5 37.7 10.2 CBS 577.82c sputum man, Germany 33.1(36.1d ) 28.2 37.9(40.5e)9.7 CBS 578.82 AUT of G.