J. Basic Microbiol. 44 (2004) 1, 36–41 DOI: 10.1002/jobm.200310272

© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0233-111X/04/0102-0036

(Department of General and Environmental Microbiology, Faculty of Sciences, University of Pécs, Pécs, P.O. Box 266, H-7624, Hungary; 1Department of Microbiology, Faculty of Sciences, University of Szeged, P.O. Box 533, H-6701 Szeged, Hungary)

Electrophoretic karyotype of two Micromucor species

ÁGNES NAGY, MIKLÓS PESTI, LÁSZLÓ GALGÓCZY1 and CSABA VÁGVÖLGYI1*

(Received 12 February 2003/Accepted 12 August 2003)

Electrophoretic karyotype analysis was applied to obtain information on the organisation and intrageneric variability of the nuclear genome in three Micromucor isolates of two different species (M. isabellina and M. ramanniana). A protoplast formation protocol, conditions for the preparation of highly-intact chromosome-size DNA molecules and for the separation of DNA molecules were established. The chromosomal banding patterns revealed substantial variability among the isolates: 11 to 14 chromosomal mobility groups were resolved. The DNA in the Micromucor chromosomes were rather small; their estimated sizes were calculated to be between 2.60 and 0.4 Mb. Using Saccha-romyces cerevisiae and Schizosaccharomyces pombe as size standard, the minimum total genome sizes were estimated to be between 24.19 and 24.9 Mb.

The Mortierella and Micromucor species belong to the most common saprophytic soil fungi. Though the classical work of GAMS (1977) gave a key and description for the genus Mortierella harbouring boths the members of the “subgenus Mortierella” and the “subgenus Micromucor” (“the Mortierella isabellina-group”) the need for a major taxonomic revision was clearly emphasized. Recent studies provided substantial evidence to handle these two taxa as separate genera, e.g. comparative studies using extracellular polysaccharides (DE RUITER et al. 1993) and sterols (WEETE and GANDHI 1999) revealed that the fungi in the genus Mortierella and the genus Micromucor are essentially different. Phylogenetic studies based on nuclear ribosomal-DNA, actin and translation elongation factor EF-1a gene se-quence data identified the Micromucor (with Umbelopsis) forming a basal sister group to all other Mucorales (VOIGT et al. 1999, O’DONNELL et al. 2001, VOIGT and WÖSTEMEYER 2001) a highly different position from Mortierella. These new data also create an interest to learn more about the genetic background of these fungi of special taxonomic position. Electrophoretic karyotype analysis using different approaches of pulsed field gel electro-phoresis (PFGE) has led to significant progress in fungal genetics. Various PFGE tech-niques allowed the separation of DNA molecules even larger than 10 megabase pairs (Mb) in size (ZOLAN 1975); as a result, the physical karyotypes of numerous previously geneti-cally uncharacterised fungal species have been established. Though there are few reports also on the genome analyses of zygomycetes fungi (KAYSER and WÖSTEMEYER 1991, NAGY et al. 1994, DÍAZ-MÍNGUEZ et al. 1999, NAGY et al. 2000, BURMESTER and WÖSTEMEYER 1994) these are rare in comparison with other fungal groups. In the present study, orthogonal field alternation gel electrophoresis (OFAGE) and the contour clamped homogeneous electric field (CHEF) technique were applied to obtain information on the organisation and intrageneric variability of the nuclear genome in Micromucor isabellina and Micromucor ramanniana. This data will be used as a first step toward a better understanding of the size and structure of the genome of Micromucor spe-cies.

* Corresponding author: Dr. C. VÁGVÖLGYI; e-mail: csaba@bio.u-szeged.hu

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Materials and methods

Strains: Chromosomal DNA was prepared from the strains Micromucor isabellina P19 (Fungal Reference Centre, Friedrich Schiller University, Jena, Germany), M. ramanniana NRRL 1296 and M. ramanniana NRRL 5844 (USDA, Agricultural Research Service, Peoria, Illinois, USA) strains. They were maintained on malt extract agar (0.5% malt extract, 0.5% yeast extract, 0.5% glucose, 1.0% KH2PO4 and 1.5% agar) slants at 4 °C. Saccharomyces cerevisiae YNN295 (BIO-RAD) and Schizo-saccharomyces pombe 972 h– (BIO-RAD) were applied as chromosomal DNA standards. Protoplast formation: Sporangiospore suspensions were harvested from cultures grown on malt-extract agar for 7 days. Small drops of this suspension were applied to cellophane membranes laid on the surface of malt extract agar plates. After incubation for 16 h at 25 °C, the cellophane sheets with the young colonies were transferred into the protoplasting solution containing 1.5% (w/v) Helix pomatia gastric juice and 0.025% Trichoderma lysing enzyme (SIGMA) in 0.6 M sorbitol and kept at 25 °C with occassional shaking for 2 h. (H. pomatia gastric juice was extracted from the digestive tract (gut) of snails as a viscous fluid. It could be stored as a freeze-dried powder at 4–8 °C for several years with unchanged protoplast-forming activity.) The Micromucor protoplasts were filtered through a loose prewetted cotton wool plug to separate them from the mycelial remnants. Chromosomal sample preparation: For the preparation of chromosomal DNA, the protoplasts were pelleted by centrifugation for 10 min at 3500 rpm, and washed twice by resuspending them in 0.6 M sorbitol. The suspension of 1–5 × 108 protoplasts/ml was then mixed with an equal volume of 1.4% low-gelling-temperature agarose (SIGMA Type VII) in 0.6 M sorbitol. The solidified plugs were incubated in 0.5 M EDTA/0.1 M Tris-HCl/1% sodium laurylsarcosine (pH 9.5) with 1 mg/ml Proteinase K at 52 °C for 24 h. The agarose plugs were subsequently washed twice in 50 mM EDTA (pH 8.0) and stored at 4 °C. Electrophoresis conditions: The PFGE experiments were performed by using a Pulsaphor (PHAR- MACIA-LKB) system with a hexagonal electrode array, and a CHEF DR II apparatus (BIO-RAD). The plugs containing chromosomal DNA were inserted into the slots of a 0.9% chromosomal-grade agarose gel slab in 0.5 × TBE (45 mM Tris/borate/5 mM EDTA, pH 8). The running buffer was 0.5 × TBE, with continuous recirculation at 10 °C. After electrophoresis, the gels were stained for 15–30 min in 0.5 µg/ml ethidium bromide and destained overnight in distilled water.

Results

To determine the best conditions for Micromucor genomic DNA separation, several OFAGE and CHEF parameters were tested. A CHEF experiment optimised for the separa-tion of S. pombe chromosomal DNAs revealed that Micromucor chromosomal DNAs are rather small; they are not comparable in size to those detected in the genome of S. pombe (Fig. 1). The optimum conditions for Micromucor genomic DNA separation were found for both the CHEF and the OFAGE system. However, the main advantage of the OFAGE sys-tem, was its relatively short separation time in comparison with CHEF. The separations could preferentially resolved either the small or the large chromosomal DNAs under one set of conditions. Resolution of the chromosomes below 2.2 Mb was achieved by using a short switching time (125 s), relatively high voltages (180 V) and a short running time (40 h). Such parameters did not satisfactorily separate chromosomes larger than 2.2 Mb. A longer switching time (600 s), lower voltages (75 V) and an extended separation time (96 h) proved excellent for the separation of chromosomes over 2.2 Mb. With OFAGE, the best separation (Fig. 2) was achieved by changing the switching time and the voltages in the course of one electrophoretic run (75 V, for 72 h with a switching time of 600 s, then 180 V for 12 h with a switching time of 125 s). Practically the same resolution could be achieved with CHEF, but with a significantly longer running time (results not shown).

38 Á. Nagy et al.

© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Fig. 1 Separation of Micromucor chromosomal DNAs by CHEF optimized for the resolution of S. pombechromosomal DNAs. Lane 1–3: chromosomal DNA from M. isabellina P19, M. ramanniana NRRL 1296 and M. ramanniana NRRL 5844, respectively; Lane 4: S. cerevisiae YNN295; Lane 5: S. pombe 972 h–. Electrophoretic conditions (CHEF): 74 V, for 72 h with a switching time of 5000 s, then 48 V for 72 h with a switching time of 3000 s, then 48 V for 72 h with a switching time of 2100 s. Sizes of S. pombechromosomal DNAs are indicated at the right margin

Fig. 2 Optimized separation of chromosomal DNA mole-cules from Micromucor strains by OFAGE. Lane 1: S. cerevisiae YNN295; Lane 2–4: chromosomal DNA from M. isabellina P19, M. ramanniana NRRL 1296 and M. ramanniana NRRL 5844, respectively. Elec-trophoretic conditions: 75 V, for 72 h with a switching time of 600 s, then 180 V for 12 h with a switching time of 125 s. Sizes of some S. cerevisiae chromoso-mal DNAs are indicated at the left margin

Micromucor karyotype 39

© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The estimated chromosomal numbers and sizes of the chromosomal DNAs for these Mi-cromucor strains are listed in Table 1. These data were confirmed with a comparison of results obtained from slightly different running conditions. None of the strains gave the same electrophoretic pattern; 12 chromosomal mobility groups were resolved for M. isabellina, and 14 or 11 for M. ramanniana. The intensities of some bands were higher, suggesting that they contain multiple unresolved chromosomes (Fig. 2 and Table 1). Accordingly, the most probable chromosomal numbers were 16 for M. isabellina and 16 or 14 for M. ramanniana. The estimated total minimum genome sizes for Micromucor (24.19–24.90 Mb), are larger than those reported for other yeast genomes, but smaller than those observed for the majority of filamentous fungi.

Discussion

In this study the first details have been gained about the genome organisation in fungi belonging to the genus Micromucor. Based on the application of OFAGE and CHEF gel electrophoresis, we have determined the genome sizes, the numbers and of sizes of chromo-somal DNAs in the two most abundant Micromucor species (M. ramanniana and M. isabellina). All these strains have distinct chromosomal banding patterns which, at least in the case of M. ramanniana when two strains have been studied, also provides a prelimi-nary indication of intraspecific chromosomal length polymorphism. The most striking result of these experiments was that the Micromucor chromosomal DNAs are surprisingly small; most of them are comparable in size to those detected in the genome of Saccharomyces (e.g. S. cerevisiae YNN295: 245 kb–2200 kb). All the zygomy-cetes genomes investigated until now by electrophoretic karyotyping revealed larger chro-mosomal DNAs and consequently larger genome sizes (Table 2). At the same time, there are some (mainly phytopathogenic) fungi when small or relatively small chromoso- mal DNAs have been observed. Examples known as e.g, Ustilago hordei (0.17–3.15 Mb, Table 1 Estimated chromosomal DNA and minimum total genome sizes of Micromucor strains. Possible chromosomal comigrations indicated by bolder bars

Chromosome mobility group

Strain

M. isabellina P19

M. ramanniana NRRL 1296

M. ramanniana NRRL 5844

S. cerevisiae

1 2.60 2.60 2.60 2.20 2 2.40 2.50 2.50 1.60 3 2.00 1.70 2.15 1.125 4 1.70 1.60 2.10 1.02 5 1.65 1.55 1.95 0.945 6 1.60 1.50 1.70 0.85 7 1.40 1.43 1.60 0.80 8 1.30 1.35 1.40 0.77 9 1.20 1.30 1.30 0.70 10 1.04 1.25 1.22 0.63 11 0.95 1.20 0.47 0.58 12 0.42 1.06 0.46 13 0.85 0.37 14 0.40 0.29 15 0.245

Genome size (Mb) 24.90 24.19 24.41 13.71

40 Á. Nagy et al.

© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Table 2 Estimated values for chromosomal DNAs and genome sizes for zygomycetes based on electrophoretic karyotyping experiments

Organism Calculated genome size (Mb)

Chromosome size range (Mb)

No. of chromosomes

References(s)

Absidia glauca 33.3–42 1.22–7 11–12 KAYSER and WÖSTEMEYER (1991)

Micromucor isabellina

24.9 0.42–2.6 16 Recent study

Micromucor ramanniana

24.19–24.41 0.4–2.6 14–16 Recent study

Mucor bainieri 42.6 5–8.4 6 NAGY et al. (2000) Mucor circinelloides 38.7–39

36–40.62 2.3–8.1 1.9–9.1

9–10 8–9

NAGY et al. (1994) DÍAZ-MÍNGUEZ et al. (1999)

Mucor mucedo 30.2 2.6–5.2 9 NAGY et al. (2000) Mucor plumbeus 39.8–40.7 2.5–8.4 7 NAGY et al. (2000) Parasitella parasitica n.d. 2–6.5 n.d. BURMESTER and

WÖSTEMEYER (1994)

n.d.: not determined MCCLUSKEY and MILLS 1990), Septoria nodorum (0.5–3.5 Mb, COOLEY and CATEN 1991) and Alternaria alternata (0.4–5.7 Mb, AKAMATSU et al. 1999). Some phytopathogenic filamentous fungi also contain small supernumerary chromosome(s): these are dispensable genetic elements which are not found in all the representatives of the species but some of them carry functional genes (the best known example is the supernumerary chromosome set of Nectria hematococca mating population (MP) VI; COVERT 1998). In case of the investi-gated Micromucor species the presence of very small, distinct chromosomal DNA mole-cules – with slightly variable size (0.4–0.47 Mb) among the isolates – has also been de-tected. However, this band was present in all (though only limited number) of the isolates tested until now so the supernumerary therme is not applicable in this stage of the study. At the same time, it is worth to mention that these are among the smallest intact chromosomal DNAs detected in filamentous fungi. Phylogenetic studies (VOIGT et al. 1999, O’DONNELL et al. 2001) suggested that the Mi-cromucor-Umbelopsis group is sufficiently distinct from the Mucoraceae and unrelated to Mortierella. A recent work (MEYER and GAMS 2003) also convincingly showed that the taxa belonging to the Micromucor (except single known isolate of M. longicollis) are unre-lated to Mortierella. Furthermore, based on the results of restriction fragment length poly-morphism (RFLP) and ITS1 sequence analysis the new family Umbelopsidaceae (Mu-corales) with the single genus Umbelopsis and the new combinations U. isabellina, U. ramanniana, and U. autotrophica are proposed. Though the electrophoretic karyotyping is not the method of choice to solve phylogenetic problems it is worth to mention that the strikingly different genom organisation of the inves-tigated Micromucor isolates from all other zygomycetes studied until now has an indirect support of the results of the new phylogenetic studies which suggest their unique position inside the Mucorales. In addition, the separation of chromosomal DNAs by pulsed field gel electrophoresis could allow the efficient investigation of the genetic makeup in these fungi where classical genetic analysis is unavailable.

Acknowledgements

This work was supported in part by Hungarian Scientific Research Fund (OTKA) grants D2911 and T 032738.

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References

AKAMATSU, H., TAGA, M., KODAMA, M., JOHNSON, R., OTANI, H. and KOHMOTO, K., 1999. Molecular karyotypes for Alternaria plant pathogens known to produce host-specific toxins. Curr. Genet., 35, 647–656.

BURMESTER, A. and WÖSTEMEYER, J., 1994. Variability in genome organization of the zygomycete Parasitella parasitica. Curr. Genet., 26, 456–460.

COOLEY, R. N. and CATEN, C. E., 1991. Variation in electrophoretic karyotype between strains of Septoria nodorum. Mol. Gen. Genet., 228, 17–23.

COVERT, S. F., 1998. Supernumerary chromosomes in filamentous fungi. Curr. Genet., 33, 311–319. DÍAZ-MÍNGUEZ, J. M., LÓPEZ-MATAS, M. A. and ESLAVA, A. P., 1999. Complementary mating types of

Mucor circinelloides show electrophoretic karyotype heterogeneity. Curr. Genet., 36, 383–389. GAMS, W., 1977. A key to the species of Mortierella. Persoonia, 9, 381–391. KAYSER, T. and WÖSTEMEYER, J., 1991. Electrophoretic karyotype of the zygomycete Absidia glauca:

evidence for differences between mating types. Curr. Genet., 19, 279–284. MCCLUSKEY, K. and MILLS, D., 1990. Identification and characterization of chromosome-length

polymorphisms among strains representing the fourteen races of Ustilago hordei. Mol. Plant Microb. Interact., 3, 366–373.

MEYER, W. and GAMS, W., 2003. Delimitation of Umbelopsis (Mucorales, Umbelopsidaceae fam. nov.) based on ITS sequence and RFLP data. Mycol. Res., 107, 339–350.

NAGY, Á., VÁGVÖLGYI, CS., BALLA, É. and FERENCZY, L., 1994. Electrophoretic karyotype of Mucor circinelloides. Curr. Genet., 26, 45–48.

NAGY, Á., PALÁGYI, ZS., VASTAG, M., FERENCZY, L. and VÁGVÖLGYI, CS., 2000. Electrophoretic karyotypes of some related Mucor species. Antonie van Leeuwenhoek, 78, 33–37.

O’DONNELL, K., LUTZONI, F. M., WARD, T. J. and BENNY, G. L., 2001. Evolutionary relationships among mucoralean fungi (Zygomycota): Evidence for family polyphyly on a large scale. Mycolo-gia, 93, 286–296.

DE RUITER, G. A., VAN BRUGGEN-VAN DER LUGT, A. W., ROMBOUTS, F. M. and GAMS, W., 1993. Approaches to the classification of the Mortierella isabellina group (Mucorales). Taxonomic value of extracellular polysaccharides. Mycological Research, 97, 690–696.

VOIGT, K., CIGELNIK, E. and O’DONNELL, K., 1999. Phylogeny and PCR identification of clinically important zygomycetes based on nuclear ribosomal-DNA sequence data. J. Clin. Microbiol., 37, 3957–3964.

VOIGT, K. and WÖSTEMEYER, J., 2001. Phylogeny and origin of 82 zygomycetes from all 54 genera of the Mucorales and Mortierellales based on combined analysis of actin and translation elongation factor EF-1α genes. Gene, 270, 113–120.

WEETE, J. D. and GANDHI, S. R., 1999. Sterols and fatty acids of the Mortierellaceae: taconomic impli-cations. Mycologia, 91, 642–649.

ZOLAN, M. E., 1995. Chromosome-length polymorphism in fungi. Microbiol. Rev., 59, 686–698. Mailing address: Dr. CSABA VÁGVÖLGYI, Department of Microbiology, Faculty of Sciences, Univer-sity of Szeged, P.O. Box 533, H-6701, Szeged, Hungary Tel: (36) 62 544 516; Fax: (36) 62 544 823 e-mail: csaba@bio.u-szeged.hu