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Microbiology, Vol. 69, No. 2, 2000, pp. 229–233. Translated from Mikrobiologiya, Vol. 69, No. 2, 2000, pp. 280–285.
Original Russian Text Copyright © 2000 by Naumov, Tokareva, Naumova, Bab’eva.
The generic and species taxonomy of saturn-spored
soil yeasts is often revised. The genus
with the type species
1925 based on the ability to form saturn-shaped
ascospores and assimilate nitrates. Later, this genus
was divided into two genera,
with the affiliation of
This genus also comprised some new sat-
urn-spored species, including
1977, the genus
Genomic hybridization studies performed in our labo-
ratory proved the validity of the reestablishment of the
 and allowed the discrimination of
five sibling species,
W. beijerinckii, W. mrakii, W. sat-
urnus, W. suaveolens
Later, the sixth sibling species
However, the classification of saturn-spored yeasts
was revised again, in our opinion, without reasonable
grounds [9, 10]. For instance, the species
43–56% DNA homology with the species
were reclassified as
showed DNA homology levels
(68 and 72%, respectively) that were sufficient to con-
sider them varieties. DNA homology between
It should be noted that such reclassification of spe-
cies of the genus
does not agree well with the
results of genomic hybridization studies  and the
was first described in
to the genus
was reestablished .
, showing merely
varieties. Only the pair
and the pair
was from 79%  to
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results of the hybridization of mitochondrial DNA .
Indeed, the dot hybridization of the mitochondrial
ORF1 probe of
CBS 1707 with the mitochon-
drial DNA of
CBS 255 and CBS 1670, and
6342 was very high, while the level of hybridization
strains was either weak (strains NRRL
YB-3985, NRRL YB-4818, and NRRL YB-838) or
even zero (strains CBS 5761 and NRRL YB-3285).
Furthermore, hybridization with the mitochondrial
CBS 5763 was also absent.
Traditionally, yeasts are classified based on their
morphological and physiological characteristics,
including the pattern of utilizable carbon and nitrogen
sources. Because of the considerable variability of
nutritional requirements, physiological tests do not
always lead to reliable taxonomic affiliation. Moreover,
these tests are time-consuming. On the other hand,
progress in the field of molecular biology gave rise to
new methods of yeast identification based on the com-
parison of nucleic acids and proteins. First of all, these
are various types of the polymerase chain reaction
method, including RAPD-PCR (random amplified
polymorphic DNA analysis), AP-PCR (arbitrarily
primed PCR), and UP-PCR (PCR with universal prim-
ers) [13, 14], as well as the methods of DNA reassoci-
ation and ribosomal gene sequencing [15–17]. To the
best of our knowledge, PCR has not yet been applied to
the classification of yeasts of the genus
In the present work, we used UP-PCR to investigate
a large collection of
soils in various regions of Russia and other countries.
strains isolated from
Discrimination between the Soil Yeast Species
with the Universal Primer N21
by the Polymerase Chain Reaction
G. I. Naumov*
*State Research Institute of Genetics and Selection of Industrial Microorganisms,
Pervyi Dorozhnyi Proezd 1, Moscow, 113545 Russia
**Department of Soil Biology, Faculty of Soil Science, Moscow State University, Vorob’evy gory, Moscow, 119899 Russia
Received June 29, 1999; in final form, September 6, 1999
, N. G. Tokareva*
**, E. S. Naumova*, and I. P. Bab’eva**
universal primer N21, were found to belong to two sibling species,
iation of the strains studied agrees well with the results of genetic and physiological investigations.
—Thirty-five yeast strains of the genus
, analyzed by the polymerase chain reaction with the
Williopsis saturnus, Williopsis suaveolens,
biological species, UP-PCR
MATERIALS AND METHODS
The strains of the genus
are listed in the table.
Isolation of DNA.
24 h on complete YPD medium of the following com-
position (g/l): glucose, 20; peptone, 10; yeast extract,
10; agar, 20. A loopful of yeast cells was suspended in
l of 50 mM Tris–HCl buffer (pH 7.8) containing
50 mM NaCl, 500 mM
roylsarcosine, and 500
g/ml proteinase K. The sus-
pension was incubated at 65
lysis, and the concentration of NaCl in the lysate was
increased to 1 M. The lysate was mixed with an equal
volume of a chloroform–isoamyl alcohol (24 : 1) mix-
ture and shaken for 15 min. After centrifugation at
for 5 min, the upper phase was transferred to
another tube, and DNA was precipitated by adding
0.6 volume of isopropanol and centrifugating for
15 min. The DNA precipitate was washed with 70%
ethanol and dissolved in 50
l of TE buffer (1 mM Tris–HCl
with 0.1 mM EDTA, pH 7.8). The final concentration of
DNA in TE buffer was about 200 ng/
Polymerase chain reaction analysis.
cation with the universal primer N21 (5'-GGATC-
CGAGGGTGGCGGTTCT) was carried out in a mix-
l) containing 2.5 mM MgCl
dNTP, 0.2 mM primer, 0.05 U/
(Dynazyme II), and 20–200 ng of DNA. PCR amplifi-
cation (30 cycles) was performed in a PHC-3 thermal
used in this study
Strains were grown at 28
C for 1 h to induce cell
, 0.4 mM of each
l DNA polymerase
cycler (Techne Inc.) with the following steps: 50-s
DNA denaturation at 94
C, 80-s primer annealing at
C, and 60-s DNA synthesis at 70
noted that primer N21 was already successfully used
for the discrimination of three sibling species in the
PCR amplification with the microsatellite primer
was carried out in a mixture (20
1.5 mM MgCl
, 0.4 mM of each dNTP, 0.25 mM
primer, 0.1 U/
l SuperTaq DNA polymerase, and
200 ng of DNA. PCR amplification (30 cycles) was
performed in the same thermal cycler with the follow-
ing steps: 30-s DNA denaturation at 94
annealing at 50
C, and 60-s DNA synthesis at 72
PCR products were separated by electrophoresis
through 1.2% agarose gel run at 60 V in 1
(90 mM Tris, 20 mM EDTA, and 90 mM boric acid) for
4–5 h. After electrophoresis, the gel was stained with
ethidium bromide and photographed under UV light.
The homology of DNA samples
amplified with primer N21 was determined by hybrid-
izing them with the PCR products of strain CBS 255.
The PCR products were transferred to a Hybond N
membrane (Amersham, United Kingdom) according to
the recommendation of the manufacturer. DNA was
fixed on the membrane by annealing at 80
labeled with digoxigenin-11-dUTP (Boehringer Man-
nheim, Germany). The amplified DNA of strain CBS
255 purified on a QIAGEN column (Germany) was
C. It should be
C, 30-s primer
C for 2 h and
Some properties of
strains used in this study
on L-rhamnose on citric acidon mannitol
CBS 254 (type strain)
VKM Y: 1635, 1636 1637, 1638
CBS 255 (type strain)
1116, 1117, 1118, 1119, 1121, 1134
1951, 1952, 1955, 1957, 1958, 1959,
1961, 1962, 1964, 1966, 1967, 1970
The Slovenian Alps
Russia, Krasnodar krai
Russia, Smolensk oblast
Russia, Tver oblast
Russia, Novgorod oblast
Russia, Tula oblast
Russia, Rostov oblast
Russia, Tula oblast
Russia, Rostov oblast
Note: CBS, Centraalbureau voor Schimmelcultures (Delft, the Netherlands); KBP, the Collection of Yeasts of the Department of Soil Bio-
logy, Faculty of Biology, Moscow State University; VKM, All-Russia Collection of Microorganisms; CCY, Culture Collection of
Yeasts, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia.
DISCRIMINATION BETWEEN THE SOIL YEAST SPECIES231
used as a DNA probe. DNA hybridization and the
detection of hybridization products were carried out
according to the recommendations of Boehringer Man-
RESULTS AND DISCUSSION
The 35 strains used in this study were isolated from
soils in European Russia, Denmark, the United States,
Slovenia, the Netherlands, and the Himalayas (see
table). In contrast to the earlier studies, where the non-
CBS 5761 and
CBS 1670 were used, in the present work, we investi-
gated the type strains
254 can assimilate L-rhamnose, citric acid, and manni-
tol [7, 10, 19]. Based on the ability to assimilate these
carbon sources, we divided all the strains studied into
two groups (table). The first group included nine strains
(CBS 112, CBS 258, CBS 1994, CBS 5761, VKM Y-1635,
VKM Y-1636, VKM Y-1637, VKM Y-1638, and KBP
3655), which, similarly to
able to utilize L-rhamnose, citric acid, and mannitol.
The second group included the remaining 25 strains,
which, similarly to
unable to assimilate these carbon sources.
The UP-PCR analysis of strains with the use of
primer N21 (Figs. 1 and 2) showed that the molecular
weight of PCR products varied from 0.4 to 3.0 kb,
depending on the particular strain. In the similarity of
their PCR product patterns, strains again fell into two
groups. All ten strains of group I had identical PCR prod-
uct patterns characterized by the presence of three major
DNA fragments 0.6, 0.8, and 1.1 kb in size (Fig. 1) and
exhibited the range of utilizable carbon sources typical
On the other hand, the remaining 25 strains com-
prised group II with the PCR product patterns charac-
terized by the presence of a major DNA fragment
0.4 kb in size (Fig. 2) and the range of utilizable carbon
sources typical of
two subgroups could be distinguished, one subgroup char-
acterized by the presence of a second major 1.0-kb DNA
fragment in the PCR product pattern (Fig. 2, lanes
and the other subgroup, by a 1.8-kb DNA fragment
(Fig. 2, lanes
). The PCR product patterns inside
the subgroups differed only in the presence or absence
of some minor bands. At the same time, strain KBP
1117 (Fig. 2, lane
) differed from the strains of both
subgroups by exhibiting two additional major DNA
fragments about 0.8 and 2.5 kb in size. The dot hybrid-
ization analysis of the PCR products of the strains of
both subgroups and the type strain CBS 255 confirmed
that they all belong to the species
are not presented).
In our opinion, the minor differences in the PCR
product patterns of strains comprising the subgroups of
CBS 254 and
CBS 254, were
CBS 255, were
CBS 255. Inside group II,
group II are due to intraspecies polymorphism. The
hybridization species specificity of the UP-PCR prod-
ucts, which manifests itself in the absence of cross-
hybridization of DNA fragments from different PCR
product patterns, has been shown for many species of
yeasts and mycelial fungi [13, 14]. By comparison, we
performed the PCR-based analysis of strains compris-
ing group II with the use of the microsatellite primer
. Again, these strains fell into two subgroups,
although the differences in the PCR product patterns of
these subgroups were not so pronounced as in the case
of analysis with primer N21. In addition to the DNA
fragments typical of the strains of the second subgroup,
the strains of the first subgroup exhibited a 0.65-kb
DNA fragment (data are not presented).
We were not able to reveal a correlation between the
intraspecies polymorphism of
and the sites from which they were isolated, since the
strains of subgroup I were isolated from soils in the
Netherlands, the United States, and the Smolensk,
Tver, and Novgorod oblasts of Russia, and the strains
of subgroup II, from soils in the Tula, Rostov, and Tver
oblasts of Russia.
Based on the results of UP-PCR analysis and phys-
iological tests (see table), we transferred strain CCY
38-4-1 from the species W. saturnus to the species
The natural habitats of W. saturnus and W. suaveo-
lens species are alluvial boggy soils in the river flood-
plains of the temperate zone . All the strains iso-
lated in the central part of European Russia were found
to belong to W. suaveolens. On the other hand, only
four of the 26 strains isolated from Russian soils could
be assigned to W. saturnus (see table). All four of these
strains were isolated from the chernozem-like soil of a
virgin meadow in a warm rice-growing region of Kras-
nodar krai. The abundance rate of W. saturnus in the
soils of this region is more than 50% . Therefore,
there is a high fidelity of W. suaveolens to the soils of
1 2 3 4 5 6 7 8 9 10M
Fig. 1. Electrophoresis of the UP-PCR products of W. satur-
nus strains. Lanes: 1, CBS 254; 2, CBS 5761; 3, CBS 112;
4, CBS 1994; 5, CBS 258; 6, KBP 3655; 7, VKM Y-1635;
8, VKM Y-1636; 9, VKM Y-1637; 10, VKM Y-1638. M
denotes molecular weight markers (kb).
MICROBIOLOGY Vol. 69 No. 2 2000
NAUMOV et al.
the central part of European Russia, whereas the spe-
cies W. saturnus is predominant in the soils of the
southern part of European Russia. Unfortunately, we
could not study the divergent strain W. saturnus KBP
2708 from the Far Eastern population of soil yeasts ,
since it has been lost.
The species W. saturnus and W. suaveolens exhibit a
high level of DNA homology, are indistinguishable in
the 18S and 26S rRNA gene sequences, and differ only
slightly in less conservative ITS sequences [15–17]. As
already noted, there are two points of view on the taxo-
nomic status of W. saturnus and W. suaveolens: some
authors treat them as distinct species , and others
consider W. suaveolens to be a variety of W. saturnus
[9, 10]. The results of UP-PCR analysis presented in
this paper and the genetic data obtained earlier [5, 7]
prove that W. saturnus and W. suaveolens are distinct
Earlier, the four strains of saturn-spored soil yeasts,
CBS 1670, KBP 1955, KBP 2706, and KBP 2709, were
assigned to the species W. suaveolens . Such an affil-
iation of these species is consistent with the results of
their UP-PCR analysis with the use of primer N21
(Fig. 2). It should be emphasized that the PCR-based
method has some advantages over the genetic hybrid-
ization method. Indeed, the latter method enables the
study of only sporulating fertile cultures, whereas the
PCR method is applicable to all cultures and allows a
great number of strains to be analyzed within a short
time period. In particular, the PCR method employing
the universal primer N21 is appropriate for discrimi-
nation of the sibling species W. saturnus and W. sua-
We are grateful to D. Yarrow (Centraalbureau voor
Schimmelcultures, the Netherlands) for providing
yeast strains and to V.I. Golubev for fruitful discussion.
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11 12 13 14 15 16 17 18 19M 20 21 22 23 24 25
Fig. 2. Electrophoresis of the UP-PCR products of W. suaveolens strains. Lanes: 1, CBS 5761; 2, CBS 255; 3, CBS 1670; 4, KBP
1103; 5, KBP 1104; 6, KBP 1116; 7, KBP 1951; 8, KBP 1952; 9, KBP 1955; 10, KBP 1957; 11, KBP 1958; 12, KBP 1959; 13, KBP
1961; 14, KBP 1962; 15, KBP 1964; 16, KBP 1966; 17, KBP 1967; 18, KBP 1970; 19, KBP 1118; 20, KBP 1119; 21, KBP 1121;
22, KBP 1134; 23, KBP 2706; 24, KBP 2709; 25, KBP 1117. M denotes molecular weight markers (kb).
MICROBIOLOGY Vol. 69 No. 2 2000 Download full-text
DISCRIMINATION BETWEEN THE SOIL YEAST SPECIES233
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