Biomedical Engineering Reference
In-Depth Information
5.4.1
Culture-Dependent Approaches
5.4.1.1
Yeasts
Identification is the localization of individuals in a classification scheme by means
of diagnostic characteristics resulting in the assignment of names. The diagnostic
characteristics to be used are provided in the species descriptions and, in the case of
yeasts, are collected in the monograph “The yeasts: a taxonomic study,” currently in
its fourth edition [
1
], with the fifth edition about to be released [
204
] . While the
fourth edition still included instructions for the phenotypic identification of yeasts
[
199
], the fifth edition reformulates them as phenotypic “characterization” instead
[
205
]. This is a consequence of the need to use DNA-based methods to recognize
the since 1998 twofold increased number of yeast species. Nevertheless, the accu-
rate description of fermentation and assimilation abilities as well as other pheno-
typic characters of yeasts continues to be of interest in technological, ecological and
taxonomic frameworks.
Among the DNA-based methods currently applied to yeast identification the
partial sequencing of the large subunit (LSU) ribosomal ribonucleic acid genes
occupies a key position [
206
]. The DNA sequences of the variable regions D1 and
D2 located at the 5' end of the LSU of virtually all known yeast species are docu-
mented in the public databases of the International Nucleotide Sequence Database
Collaboration (INSDC, including GenBank, the European Molecular Biology
Laboratory, and the DNA Data Bank of Japan). The entries of the three submis-
sion hubs are bundled, daily updated, and made available for searches by the
NCBI (
www.ncbi.nlm.nih.gov/nucleotide/
). Few distinct yeast species show no
or low sequence divergence in the D1/D2 LSU rDNA, can therefore not reliably
be distinguished, and require complementary analyses such as
Saccharomyces
bayanus
and
S. pastorianus
[
206
] ,
Hanseniaspora meyeri
and
Hanseniaspora
clermontia; Hanseniaspora guilliermondii
and
Hanseniaspora opuntiae
[
207
] ,
Meyerozyma guilliermondii
and
Meyerozyma caribbica; Trichomonascus ciferrii
and
Candida mucifera; K. marxianus
and
Kluyveromyces lactis
[
55
] ,
Debaryomyces hansenii, Debaryomyces fabryi
and
Debaryomyces subglobosus
[
208
]. Genetic regions that show in most cases larger divergence than the D1/D2
LSU rDNA and for which substantial sequence records have been accumulated
include the ITS region of the ribosomal gene cluster. This region is favored by
mycologists as the barcoding locus of fungi, although no common threshold
value to distinguish intraspecific from interspecific variation can be defined
[
209,
210
]. No systematic evaluation of ITS sequence variation to answer this
question for ascomycetous yeast species exists to date. In comparison to sequenc-
ing, RFLP analysis of ITS sequences offers simplified access to partial DNA
sequence information. The accessed information is determined by the recogni-
tion sites of the applied restriction enzymes and typically includes only a few
nucleotides, necessitating a range of restriction enzymes to reliably distinguish
the species in question.
