Saccharomyces species assignment by long range ribotyping

Robert Messner 1), Hansjörg Prillinger 1), Cletus P Kurtzman 2), and Elena Slavikova 3)

1) Institut für Angewandte Mikrobiologie
Universität für Bodenkultur,Nußdorfer Lände 11 A- 1190 Wien

2)National Center for Agricultural Utilization Research,
Ill 61604 Peoria, USA

3)Slovak Academy of Sciences,
Institute of Chemistry, CS-84238 Bratislava, Slovakia

Abstract

Type strains of 10 genotypically distinct Saccharomyces species are differentiated by ribosomal DNA restriction fragment analysis (ribotyping). The full length of the chromosomal ribosomal repeat was amplified in two parts, the 18SrDNA including both ITS regions (2600 bp) and the 25SrDNA (3300 bp). Restriction fragments generated by 9 enzymes from these two products yield characteristic patterns, by which unknown Saccharomyces isolates are assigned to the type strains. For convenient separation and detection only fragments longer than 200 bp were monitored. In contrast to molecular differentiation methods of highest resolution as RAPD-PCR or fingerprinting, the results from ribotyping are absolutely reproducible and thereby suitable for databases.

The phylogeny computed from the discrete character matrix for presence / absence of fragments by the PHYLIP program package is in complete accordance to the phylogeny derived from ribosomal RNA sequence analysis. By this the field of application of the long range ribotyping can be regarded basically as equal to DNA sequence analysis of the same locus. Because distant relationships are recognized, missidentified genera were detected upon the species assignment. This cannot be done by methods of higher resolution like RAPD-PCR or fingerprinting.

Introduction

A recent shift from phenotypic to genotypic characteristics was accompanied by severe changes within the taxonomy of the genera Saccharomyces and Kluyveromyces. For example, species like S. aceti and S. diastaticus described by physiology are joined with the species S. cerevisiae upon molecular analysis with RAPD-PCR (cf. Poster of Molnár et al.).

The ribotyping may fill the gap between the resolution power of the RAPD analysis and the database compatibility of DNA sequencing. Molina and coworkers have shown the lack of variation within species S. bayanus, S.cerevisiae and S. kluyveri by restriction polymorphisms of the 18SrDNA (Molina et al., 1992a) and of the ITS (internal transcribed spacer) regions (Molina; et al., 1992b)

Here the ribotyping of 10 Saccharomyces type strains is presented, which had been tested by RAPD analysis from a field of 64 strains.

Table 1: Saccharomyces strains investigated. All strains represent the type strains of the respective species.


     Strain                               Strain designations
                                          HA    CBS	 Origin
S. bayanus (Saccardo)	                  389    380    DGVPG 6171
S. castelli (Capriotti)		          408   4309      CBS 4309
S. cerevisiae (Meyen ex Hansen)           227   1171      DSM 70449
S. dairensis (Naganishi)                   56    421     NRRL Y-12639
S. exiguus (Rees ex Hansen                  8    379     NRRL Y-12640
S. kluyveri (Phaff, Miller et Shifrine)    57   3082     NRRL Y-12651
S. paradoxus (Batschinskaya)	          405    432      CBS 432
S. pastorianus (Hansen)			  452   1538      CCY 21-6-7
S. servazzii (Capriotti)                   55   4311     NRRL Y-12661
S. unisporus (Jörgensen)                   75    398      IFG 1201

Ribotyping patterns of the Saccharomyces species S. bayanus, S. cerevisiae, S. paradoxus, S. pastorianus, S. castelli, S. dairensis, S. exiguus, S. kluyveri, S. servazzii, S. unisporus and the outgroup Torulaspora (Lanes 1 to 11). Combinations of PCR products and restriction enzymes were: 18SrDNA and ITS regions by HphI (Panel A) and by CfoI (Panel B), 25SrDNA by HphI (Panel C) and by HinfI (Panel D).

Table 3: Discrete character matrix of presence (1) and absence (0) of restriction fragments. Conserved fragments are present in all Saccharomyces strains investigated and are indicated by "C".

18S rDNA + ITS :

S. bayanus     11010001000 111011000 00010001000101 1000000101010 110000010100 00110010010 100010000010 1000101 100100010
S. crevisiae   11010001000 111011000 00010000010101 0100001101110 100000110110 00110010001 100010000010 1000101 100100010
S. paradoxus   11010001000 111011000 00010000010101 0100001101110 100000110101 10000010001 100010000010 1000101 100100010
S. pastorianus 11010001000 111011000 00010001000101 1000000101010 110000010100 00110010010 100010000010 1000101 100100010
S. castelli    11010000100 110101010 10001000000100 1000010000111 101000010100 00100110010 010010001001 1010001 010001010
S. dairensis   11010000010 110010100 10010000000100 1000010001110 101000010100 00100011010 000110010000 1001001 010001010
S. exiguus     11010000001 111000101 01000100000100 1001010000010 100010010100 01000010010 010010001001 1000001 001101010
S. kluyveri    11010000000 110001001 00001000011100 1000100100001 100001010100 00101010010 100010000000 1000011 010001010
S. servazzii   11100111000 110001010 00010000100110 1010000000010 100100010100 10000000110 001011000101 1000001 000001111
S. unisporus   11101111000 110101010 00100010000100 1010000010010 100100010100 10000000110 001011000101 1000011 000011011
Torulaspora    11010000000 110010100 10001000000100 1000001110010 100000011100 10000010010 100010100000 1100001 010001010
               CC          CC                   C                 C      C C                   C        C     C        C
Restricted by    AluI        CfoI      HaeIII         HinfI         HphI         MspI        MvaI         RsaI    TaqI

25S rDNA :

S. bayanus     001011100100 000111110 010111001010 1110011011 011001111 0111000010001 11000 1010000111 10010111
S. crevisiae   100010100100 100101010 010111001010 1110011011 011001111 0111000010001 11000 1010000111 10010111
S. paradoxus   001000100101 100101010 010101011010 1110011011 011001111 0111000010001 11000 1010000111 10010111
S. pastorianus 001011100100 000111110 010111001010 1110011011 011001111 0111000010001 11000 1010000111 10010111
S. castelli    100010100100 000111100 010111001010 1100011111 011001111 0010111000010 11000 0011001111 10011101
S. dairensis   001110100100 000111100 011011001011 1100011111 011001111 0010111000010 11000 0110000111 10011101
S. exiguus     001100100110 011101001 100111100011 1011011010 011001111 0010111001010 10011 1010100111 10011101
S. kluyveri    001001101100 101001000 011011001110 1100111110 100111001 0010111010000 11000 0011011110 10110101
S. servazzii   001100100110 000111100 100101110011 1110011011 011011001 0000111101110 11000 0110000111 01010110
S. unisporus   001100100101 010101100 100101110011 1110011011 011011001 0110100000110 11000 0110000111 01010110
Torulaspora    010001010101 000111100 010111001010 1100011111 011001111 1010100000010 10100 0011001111 10011101
                     C  C        C         C    C  C    CC C       C  C               C       C    CC     C C
Restricted by    AluI         CfoI      HaeIII       HinfI      HphI      MspI         MvaI   RsaI       TaqI

Conclusion

This ribotyping method has been developed to offer a convenient tool for diffentiation of numerous strains at the species level. The longer the fragments which can be amplified without by-products, the less tedious and expensive the separation of small restriction fragments required. The enlarged ribotyping is able to fill the gap between the convenient RAPD analysis for highest resolution and the sequencing of rDNA for phylogeny above the genus level.

Acknowledgements

For kindly providing strains we are thankful to Dr. A. Vaughan-Martini (DBVPG), Dr. A. Martini (DBVPG), Dr. G.I. Naumov, Dr. K. Scheide, and Prof. U. Stahl.

This work was supported by the Austrian Fond zur Förderung der Wissenschaftlichen Forschung, Project P9255.

References

Molina FI, Inoue T, Jong S-C (1992a) Ribosomal DNA restriction analysis reveals genetic heterogeneity in Saccharomyces cerevisiae Meyen ex Hansen
Int. J. Syst. Bacteriol. 42:499-502

Molina FI, Inoue T, Jong S-C (1992b) Restriction polymorphisms in the internal transcribed spacers and 5.8S rDNA of Saccharomyces Curr.
Microbiol. 25:251-255

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03/20/95 C by IAM