SCANNING OF GEOTRICHUM CANDIDUM GENOME BY RFLP-PCR OF RDNA AND RAPD

Michał Piegza, Wojciech Barszczewski, Danuta Witkowska, Regina Stempniewicz, Małgorzata Robak
Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

In the present work we focused on the comparison of genome diversity of 23 strains of Geotrichum candidum isolated from different sources. The analysis of restriction fragment length polymorphism after amplification of rDNA cluster with NS3-ITS4 primers (RFLP-PCR rDNA) and randomly amplified polymorphic DNA (RAPD) analysis with four primers [(GTG)₅, (GAC)₅, (GACA)₄ and M13] were applied for scanning the genome. In RFLP-PCR rDNA with Msp I the physiological classification at species level was confirmed, however two types of pattern were obtained with Hae III. The diversity among some strains was observed in RAPD. Microsatellite primer (GACA)₄ was the most discriminating one, only one species specific product was observed. For 20 strains, primer (GTG)₅ gave very similar pattern (>80%) allowing the determination of three species specific products (920, 720 and 510 bp). Species specific products were also found with (GAC)₅ and M13 primers. The microsatellite overall dendrogram allowed the individual recognition of five strains of G. candidum, for which the patterns similarity was <70%. RAPD patterns similarity of 80% or more, obtained for the rest of strains, reflected rather low genome diversity of G. candidum.

Key words: Geotrichum candidum, RFLP-PCR, RAPD-PCR.

INTRODUCTION

Geotrichum candidum belongs to the ascomycetous yeast. However, it is a microorganism showing cell morphological characteristics typical for both yeasts and moulds [6, 7]. Historically, the synonyms for G. candidum are: Oidium lactis, Oospora lactis, Oidium nubilum, Oidium humi. According to Guarro et al [8], the teleomorphic form of this yeast is known as Galactomyces geotrichum and, recently, de Hoogh and Smith [4], based on data concerning ribosomal RNA genes, have separated the genus of Galactomyces into six species. One of these species is described as Galactomyces geotrichum (syn. Endomyces geotrichum, Dipodascus geotrichum) without a described anamorphic form, and another one as Galactomyces candidus with Geotrichum candidum as anamorphic state. Most of the collection isolates of G. candidum, Oidium lactis, Oospora lactis belong to the last group. The confusion in the nomenclature of species and genus create a need to study more isolates coming from different sources and to compare their morphology, physiology and genome organization to precisely define the type of the strain. According to Gente et al. [6], strains showing moulds-like and intermediate colony morphology have larger chromosomes than the yeast-like ones. Their genome size varies from 10,9 Mb to 16,9 Mb and the number of chromosomes from 5 to 8. Such simple molecular techniques as RFLP-PCR rDNA (Restriction Fragments Length Polymorphism of rDNA) and microsatellite RAPD (Randomly Amplified Polymorphic DNA), developed recently for some other yeast [2, 12, 14], may be useful for the study of G. candidum genome. Both techniques allow the study of size and sequence variability of amplified genomic DNA from different strains without sequencing of any genome fragment and the rigorous use of the same protocols allows good reproducibility.

G. candidum is mainly isolated from traditional produced cheese [9, 12], but it has been well documented that strains of that species could be useful for biological barley protection [3, 11]. The use of strains isolated from different sources (milk, cheese, fruits) could not only protect, but also be helpful in changing the barley grain structure by the action of secreted enzymes [5].

Trying to discriminate between 23 strains of G. candidum, we decided to characterize the genetic variability of isolates from milk cream, malt, different types of cheese and from chicken feathers. The genome similarity was studied by RFLP- PCR rDNA and RAPD analyses.

MATERIALS AND METHOD

Microorganisms and culture conditions:
Twenty three strains isolated from four sources and identified by API 32C-BioMerieux as Geotrichum candidum were studied. The origin, number of line and strain symbols used in the electrophoretic patterns were presented in Table 1.

DNA extraction:
The DNA template extraction was performed according to Al-Rawi and Kavanagh [1] with a minor modification. One ml of 21 hours culture (YM) was centrifuged at 15000 g for 10 minutes (Sigma centrifuge 3-16K). The biomass was washed with PSB (pH=7.4) and centrifuged. The washed cells were suspended in 200 µl of 0.06 M Tris-HCl lysing buffer (pH=6.8) containing 2% SDS, 5% b-mercaptoethanol and 10% glycol. The suspension was vortexed for 1 hour at 50°C. The 200 µl mixture of phenol:chloroform:isoamyl alcohol (25:24:1) and 0.3g of glass beads were added to each tube and the tubes were vortexed for 10 minutes. The 200 µl of TE-buffer were added and preparations were centrifuged for 5 minutes at 15000 g. Then 200 µl of aqueous phase were transferred into 400 µl of ice-cold 96 % ethanol. After 30 minutes at –20°C the mixture was centrifuged and the precipitate was dried. Finally, 50 µl TE-buffer were added. 5 µl of prepared DNA were added to the PCR tubes.

Table 1. Description of the strains

Origin	Strain name	Symbols used in figures
Brie cheese	G. candidum KB5 G. candidum KB6	X5 X6
Camembert cheese	G. candidum KC3 G. candidum Sc12	X3 Sc12
Malt	G.candidum MSK10	10
	G.candidum MSK14	14
	G.candidum MSK13	1-3
	G.candidum MSK31	3-1
	G.candidum MSK310	3-10
	G.candidum MSK311	3-11
	G.candidum MSK33	3-3
	G.candidum MSK34	3-4
	G.candidum MSK35	3-5
	G.candidum MSK39	3-9
	G.candidum MSK411	4-11
	G.candidum MSK412	4-12
	G.candidum MSK48	4-8
	G.candidum SS220K3	K3
	G.candidum SS228K2	K2
	G.candidum SS32B1	B1
	G.candidum SS47D2	D2
Cream	G.candidum SmP1	SP
Chicken feathers	G.candidum PH1	PH

The species identification was confirmed by API 32C – BioMerieux. All isolates were stored on YM slants at 4°C.

RFLP-PCR rDNA analysis:
Primers NS3-ITS4, which with Saccharomyces cerevisiae S288C DNA template could amplify rDNA fragment of 2059 bp (size calculated on the “Saccharomyces Genome Database” rDNA sequence), were chosen for RFLP-PCR. PCR and restriction conditions were the same as described previously [2]. Reaction mixture contained 0.3 µl of Taq polymerase, 3.0 µl of PCR buffer, 3.0 m l of 25 mM MgCl₂, 0.3 µl of 10 mM dNTP mix, 0.1 µl of each primer, 5.0 µl of template DNA, and 18.2 µl sterile distilled water. The amplification was made in Tpersonal thermocycler (Biometra) under the following conditions: 5 min denaturation at 95°C, 35 cycles at 95°C for 30 s, 61.5°C for 30 s, 72°C for 3 min, and final extension at 72°C for 5 min.

After amplification 4 µl of PCR products, 0.2 µl of restriction enzymes each (Hae III, ScrF I, Msp I), 1 µl of appropriate buffer and 4.8 µl of sterile water were mixed. The reaction mixtures were incubated at 37°C for 2 hours. After the incubation time, the mixtures were mixed with 2 µl of 6 X Loading Dye Solution (Fermentas) and loaded on agarose gel 1% in 0.5 TBE-buffer containing 5 µl of ethidium bromide (10mg/mL). Restriction products were separated by electrophoresis at 120V during 2 hours. GeneRuler 100 bp DNA Ladder Plus (Fermentas) was used as molecular weight DNA standard. DNA fragments were visualized in UV light, electrophoretic patterns were photographed in Vilber Lourmat system and analysed with BioCapt software. The obtained profiles were analyzed with BioGene software. Dendograms were obtained by means of the Unweighted Pair Group Method using Arithmetic Average (UPGMA) clustering analysis on the base of Dice Coefficient [10] and the overall dendrogram were calculated with WEPA as described earlier for Candida sake strains [14].

RAPD-PCR analysis:
Primers (GAC)₅, (GTG)₅, (GACA)₄ and core sequence of phage M13 were used for RAPD analysis. All reaction mixtures contained 0.3 µl of Taq polymerase, 3.0 µl of 10xPCR-Buffer, 3.0 µl of 25 mM MgCl₂, 0.3 µl of 10 mM dNTP mix, 0.1 µl of chosen primer, 18.3 of µl distilled water and 5.0 µl of matrix DNA. The reaction was performed in Tpersonal thermocycler under the following conditions: denaturation at 94°C for 3 min., 40 cycles at 94°C for 20 s, 50°C for 1 min, 72°C for 20 s, and the final extension at 72°C for 6 min. Electrophoresis, gel documentation and numerical analyses were the same as described for RFLP-PCR analysis.

RESULTS

After amplification with NS3-ITS4, the primers we were supposed to get only one product similar to S. cerevisiae S288C rDNA fragment. Unfortunately, after the electrophoretic separation, we recovered two PCR products for all G. candidum strains. One of them was 1700 bp and the second was smaller, 640 bp (Fig. 1).

Figure 1. Size of NS3-ITS4 rDNA amplification products of five Geotrichum candidum strains from, the left: Marker, 3-11, PH, D2 X5, X6

Digestion of the amplified regions with Hae III, Msp I and ScrF I restriction enzymes allowed the differentiation of some isolates, but very similar patters were obtained for almost all the studied strains (Fig. 2). The strains were separated into two groups using Hae III enzyme. PCR product for one of the group was about 1540, and for the other one two dominant products were detected: 680 and 470 bp (Fig. 2A). There was no clear difference in the profiles after restriction with Msp I enzyme (Fig. 2C). For most of the strains, on electrophoresis profiles, three products of 630, 535 and 495 bp were obtained. Only two profiles were a little bit different. For the strain MSK₄12 no digestion product was observed and for the strain SS228K₂ a product of 960 bp was detected. The most significant difference in the profiles was observed after restriction of PCR products with ScrF I enzyme, because for a few number of strains the degradation of amplicons was not completed (Fig 2B). However, in the electrophoretic patterns obtained for most of them, similar products were detected (990, 716 and 660 bp).

Figure 2. Restriction profiles of NS3-ITS4 rDNA fragment of Geotrichum candidum strains obtained with three endonucleases: Hae III (A), Scr F1 (B), Msp I (C)

RAPD analysis with three microsatellite primers and M13 core sequence allowed the differentiation of some strains isolated from different sources (Fig. 3). A detailed study revealed that (GTG)₅ primers patterns profiles indicated three dominant products: 970, 720 and 510 bp (Fig. 4B). For (GAC)₅ primer two to elven products were observed, but the most important were two products sized of 510 and 460 bp. For sixteen of 23 strains, products of 670 bp and 736 bp were also observed with this primer (Fig. 3A). Using (GACA)₄ primer we found one very intensive product for all of strains, about 480 bp and a few products in smaller quantities. For five strains: MSK₃11, MSK₃5, MSK₄12, PH1 and SP1 we were able to recognize characteristic products (Fig. 3C). M13 primer amplification showed three characteristic products in electrophorograms: 1100, 997 and 900 bp (Fig. 3D). For strains MSK14 and SP1 an additional one, very different product was observed.

The most important is that dendrograms obtained for (GTG)₅ primer showed 100% of similarity between 15 strains, and 65% of resemblance for all of them (Fig. 4, 5). With (GACA)₄ primers, the strains show 20% of product size similarity, but we found three groups with 80% similarity. In four cases the strains profiles were identical: for X6 and 14, for 3-1 and 3-9, for 4-11 and K2, for 3-3 and D2. Moreover, amplification with primer M13 divided all isolates into three groups with 80% profile similarity. 50% of patterns of the strains showed resemblance. We observed a more important difference for (GAC)₅ primer, especially that the strain D2 had no similarity with the other strains. In the overall dendrogram, which was made for all four primers, we observed that, taken together, G. candidum 23 strains had 50% of similarity and a few pairs were grouped with 90% (Fig. 5).

Figure 3. RAPD-PCR profiles of Geotrichum candidum strains obtained for four primers: (GAC)5 (A), (GTG)5 (B), (GACA)4 (C), M13 (D)

Figure 4. Dendrogram of RAPD profiles for Geotrichum candidum

Figure 5. Dendrogram of RAPD profiles for Geotrichum candidum for all four primers

DISCUSSION

The presence of two products of G. candidum DNA amplification with NS3-ITS4 primers was unexpected, but obtained in all repetition. During the parallel amplification of the same rDNA region, only one product was obtained for Debaryomyces hansenii, Candida spp, Yarrowia lipolytica (results not shown). For G. candidum, both obtained fragments were smaller than the theoretical one calculated on the basis of rDNA sequence of S. cerevisiae [SGD]. The theoretical amplification of S. cerevisiae S288C rDNA with NS3-ITS4 primers would give a fragment of 2059 bp. So, the structure of rDNA cluster must be different in G. candidum (smaller expected products). The rDNA non-coding region could be specially affected. For example, a large fragment of nucleotides in the intergenic spacer of rDNA region could be deleted. There is little published data showing rDNA sequence for Galactomyces / Geotrichum. Six sequences of 18S rRNA were deposited in GenBank and for the intergenic regions only the sequences composed of ITS1 (sized from 72 to 104 bp), ITS2 (sized from 106 to 420 bp) and 5,8S (sized from 104 to108 bp) regions were described in detail [4]. In case of ITS2 region, a very important size variation was observed (up to 4 times). The variation could explained the presence of smaller products, specially of 1700 bp.

The second consequence of ITS2 length variability could be related to rDNA polymorphism and the presence of two amplicons. However, another possible explanation of the detection of the other products could concern the presence of an extranuclear genetic material or mitochondrial rDNA. To date there are no publications on G. candidum mitochondrial DNA or plasmids. It is worth noticing that one of the used primers, NS3, in pair with NS4 amplified mitochondrial rDNA as well, giving about 365 pb product in S. cerevisiae [15].

Vasdinyei and Deak [13] have used NS1-ITS2 primers to amplify a fragment of rDNA and Hae III and Msp1 in restriction analysis. However, the amplified region of rDNA was smaller than in this research. In restriction analyses with Hae III, we found four fragments (520, 500, 270, 150bp) close in size to the products obtained by Vasdinyei and Deak [13] with the same endonuclease. So, Hae III was cutting in the region laying between sequences complementary to NS3 and ITS2. The size of Msp I restriction fragments, compared for both researches (mentioned above and ours), was considerably different.

In this RAPD study using M13 primer, the obtained amplification products were almost of identical size to those described by Vasdinyei and Deak [13]. Those products could be considered as characteristic for species. Also, after the DNA amplification with (GAC)₅ we found two products which could be considered as characteristic for the species.

Gente et al. [7] analyzed G. candidum strains using 14 microsatellite primers. They show 80% of molecular similarity between 60 strains, despite the fact that they were isolated from different sources, just like in the present study. According to the same authors, strains showing 83% of similarity in PCR product profiles have to be considered identical. Also, Prillinger et al. [12], in the study of 25 isolates of G. candidum obtained 0-10% of patterns diversity in RAPD with decamer 2 (5- TGCCGAACTG-3). So, it is not surprising that only five of our isolates – SP, X5, D2, 4-8 and X3 – were individually identified. The rest of them were separated into two branches, not corresponding to the origin of isolates.

Summarizing, the scanned genome variability of twenty three isolated strains of G. candidum yeasts was observed, but we supposed that all isolates belonged to a single species. RAPD analysis allowed individual recognition of five out of 23 strains. Probably, there are conserved regions in their respective genomes. These regions seem to be common not only for the strains isolated in the same environment (e.g. milk and dairy products) but also among the strains coming from different isolation sources (milk, cream and dairy products, malt, chicken feathers). Unexpected, but very interesting was the presence of two products of genomic DNA amplification with NS3-ITS4 primers for all studied G. candidum strains. It has to be explained.

REFERENCES

1. Al-Rawi N., Kavanagh K., 1998, A rapid method for the extraction of whole cell proteins from Candida species, Journal of Microbiological Methods, 34, 107-112.

2. Barszczewski W., Robak M., 2004, Differentiation of contaminating yeasts in brewery by PCR-based techniques, Food Microbiology, 21 (2), 227-231.

3. Boivin P., Malanda M., 1997, Improvement of malt quality and safety by adding starter culture during the malting process, Technical Quarterty, 34 (2), 96-101.

4. de Hoog G.S., Smith M.Th., 2004, Ribosomal gene phylogeny and species delimitation in Geotrichum and its teleomorphs, Studies in Mycology, 50, 489-515.

5. Dziuba E., Wojtatowicz M., Stempniewicz R., Foszczyńska B., 2000, The use of Geotrichum candidum starter cultures in malting of brewery barley, in Food Biotechnology, Progress in Biotechnology, edited by Bielecki S. at all, vol 17, Elsevier , Amsterdam, 311-316.

6. Gente S., Desmasures N., Jacopin C., Plessis G., Beliard M., Panoff J.M., Gueguen M., 2002, Intra species chromosome-length polymorphism in Geotrichum candidum revealed by pulsed field gel electrophoresis, International Journal of Food Microbiology, 76, 127-134.

7. Gente S., Desmasures N., Panoff J.M., Gueguen M., 2002, Genetic diversity among Geotrichum candidum strains from various substrates studied using RAM and RAPD-PCR, Journal of Applied Microbiology, 92, 491-501.

8. Guarro J., Gene J., Stchigel A.M., 1999, Developments in Fungal Taxonomy, American Society for Microbiology, 12 (3), 454-500.

9. Marcellino N., Beuvier E., Grappin R., Gueguen M., Benson D.R., 2001, Diversity of Geotrichum candidum strains isolated from traditional cheesemaking fabrications in France, Applied and Environmental Microbiology, 67 (10), 4752-4759.

10. Nei M., Li W-H., 1979, Mathematical model for studying genetic variation in terms of restriction endonucleases, Proc Natl Acad Sci. USA, 76, 5269-5273.

11. Piegza M., Witkowska D., Stempniewicz R., Rywińska A., 2005, Geotrichum hydrolytic activity in milled malt and barley medium, EJPAU Biotechnology, vol 8, issue 1.

12. Prillinger H., Molnar O., Eliskases-Lechner F., Lopandic K., 1999, Phenotypic and genotypic identification of yeasts from cheese, Antonie van Leewehoek, 75, 267-283.

13. Vasdinyei R., Deak T., 2003, Characterization of yeast isolates originating from Hungarian dairy products using traditional and molecular identification techniques, International Journal of Food Microbiology, 86, 123-130.

14. Walczak E., Czaplińska A., Barszczewski W., Wilgosz M., Wojtatowicz M., Robak M., 2007, RAPD with microsatellite as a tool for differentiation of Candida genus yeasts isolated in brewing, Food Microbiology – in press.

15. White T. J., Bruns T., Lee S., Taylor J. 1990, Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, in: Innis M. A., Gelfand D. H., Sninsky J. J. and. White T. J, “PCR Protocols – A Guide to Methods and Applications”, London. Academic Press , 315-322.

ACKNOWLEDGEMENTS

This research was supported by a grant from State Committee for Scientific Research within the project no. 2PO6T 052 26.

 

Accepted for print: 27.09.2006

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Michał Piegza
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
phone +48 71 320 7737
fax +48 71 320 7794
51-630 Wrocław
Poland
email: michal.piegza@upwr.edu.pl

Wojciech Barszczewski
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Norwida 25, 50-373 Wrocław, Poland
Phone: +48 71 3205463
email: wojciech.barszczewski@up.wroc.pl

Danuta Witkowska
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
Poland
email: wit@ozi.ar.wroc.pl

Regina Stempniewicz
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Norwida 25, 50-373 Wrocław, Poland
Fax. 4871- 3284124
Phone: 48-71-3205116

Małgorzata Robak
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
Poland
email: malgorzata.robak@up.wroc.pl

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