Download Saccharomyces boulardii Using Intraspecific Protoplast Fusion

Journal of Applied Sciences Research, 3(3): 209-217, 2007
© 2007, INSInet Publication
Genetic Construction of Potentially Probiotic Saccharomyces boulardii Yeast Strains
Using Intraspecific Protoplast Fusion
1
Nivien A. Abosereh, 1 Hassan Abdel-Latif A. Mohamed and 2Azzat B. Abd El-Chalk
1
Microbial Genetics Dept. and 2Dairy Dept., National Research Centre, Cairo, Egypt.
Abstract: Highly efficient bio-therapeutic yeast Saccharomyces boulardii strains, were obtained by intraspecific protoplasts fusion. The selected fusants F1 (S.b.M1 X S.b.M 2) (ad - X lys -) and F2 (S.b.M2 X
S.b.M3) (lys -´arg -) and their parents; S.b.M1(ad -), S.b.M2 (lys -) and S.b.M 3 (arg -) as well as the wild type
strain (S. boulardii ) were examined by Electron microscope and tested for their anti-microbial activities,
tolerance to low pH and 3% bile salt. Results showed that the number of intact cells were about 3000,
4500 and 5500 cells / ml in the protoplast suspensions of S.b.M1, S.b.M2 and S.b.M3, respectively. On
the other hand, the protoplast formation percentage of S.b.M1, S.b.M2 and S.b.M3, were 1.91, 3.91 and
4.82% , respectively. The regeneration percentage of S. S.b.M1, S.b.M2 and S.b.M3, were 0.9364, 1.9273
and 2.3455%, respectively. Mixed protoplasts; (S.b.M1 X S.b.M2), (S.b.M1 X S.b.M3) and (S.b.M 2 X
S.b.M3) produced hybrid fusants colonies reached to 215, 345 and 410, respectively. Results revealed that
the fusion between S.b.M1 and S.b.M 2 had higher regeneration percentage (10.42%) than the other
fusions (S.b.M1) X (S.b.M 3) and (S.b.M2) X (S.b.M3) which showed regeneration percentage of 8.15%
and 7.94%, respectively. The produced regenerated fusants showed colonies with larger cell size than the
parental strains. Electron micrographs of cell collected during generation of protoplast fusion showed that
the outer membrane has peeled away from the inner membrane or no longer visible. The fusion between
cells that still retain some outer membrane and thus would technically be considered spheroplasts rather
than true protoplasts. Some fusants remnants of the outer membrane, thus, it is not necessary to remove
the entire outer membrane in order to achieve fusion. No antimicrobial effect was scored with the tested
S. boulardii strains on E. coli. The highest clear zone diameter was achieved by fusant F2 (S.b.M 2 X
S.b.M3) against B. cereus, while the lowest one was achieved by S. boulardii (W .T ) against S. aurues.
At low pH 3 and in the presence of 3% Bile salt, the fusant F2 (S.b.M2 X S.b.M3) gave the highest
level of growth among the other tested yeast strains. This indicate that the produced intra-specific fusants
of S. boulardii, have highly antagonistic effect on some pathogenic bacteria and also highly tolerate to
low pH 3 and 3% Bile salts in comparison to their parents that may be due to the regulatory effect of
the ploidy state. These fusants are, therefore, recommended for various medical applications.
Key words: Protoplast fusion, Electron microscope, Auxotrophic mutants, Bile salts, Anti-microbial
agent for the treatment of microbes associated with
diarrhea and colitis[6 ].
Saccharomyces boulardii is safe, non-toxic, nonpathogenic, thermophilic yeast and its treatment alone
reduced bacterial translocation. It is useful as biotherapeutic agent in combination with standard
antibiotic for the treatment of and recurrent Clostridiuni
difficile diarrhea and colitis. Diet supplementations with
probiotics (Lactobacilli and Saccharomyces boulardii)
were also helped to reduce some effects of aging [2 ,1 3 ,1 8 ].
In order to obtain strains showing more suitable
properties, genetic manipulation methods have been
used. Genetic improvement of Saccharomyces and other
yeast strains has traditionally relied on random
mutagenesis [3 8 ]. However, due to the aneuploid, diploid
or polyploid nature of most strains used in beer and
INTRODUCTION
Several studies have been reported the use of
yeasts (Saccharomyces boulardii or S. cerevisiae) as a
potential bio-therapeutic agent (probiotic) for the
treatment of microbes associated with diarrhea and
colitis. Viable yeast cells were recently used to
improve the resistance of the intestinal ecosystem to
bacterial infection. The role of probiotics in preventing
or curing gastrointestinal illnesses have been reported [1 9 ]
and many yeast species showed potential benefits of
the host, so that they could be used as bio-therapeutic
agents [3 7 ].
S. boulardii is a rich source of B vitamins and
chromium, so that it was used with S. cerevisiae (a
commercial baker 's yeast) as a potential bio-therapeutic
Corresponding Author:
Nivien A. Abosereh, Microbial Genetics Dept, National Research Centre, Cairo, Egypt
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J. Appl. Sci. Res., 3(3): 209-217, 2007
wine making, traditional crossing techniques have not
been very successful. Thus, the use of new techniques
such as protoplast fusion and genetic transformation is
necessary. New genotypes were obtained by protoplast
fusion, which showed recombinant features, while the
transformed strains showed heterologous genes [2 7 ].
Protoplast fusion is versatile technique for inducing
genetic recombination in a variety of prokaryotic and
eukaryotic microorganisms. Protoplast is induced to
fuse and form transient hybrids. During this hybrid
state, the genomes may re-assort and genetic
recombination can occur [2 1 ]. So far, an increasing
number of recombinant strains have been formed.
The aim of this research study is to obtain highly
efficient Saccharomyces boulardii biotherapeutic strains
through protoplast fusion techniques.
harvested by centrifugation at 3000 rpm for 20 mins.
The cell pellets were washed three times with distilled
water and resuspended in hypertonic buffer (0.1 M
Tris–HCl. pH 7.5, 2 mM EDTA, 50 mM 2Mercaptoethanol and 0.45 M KCl) and incubated at
37 o C for 10 mins with gentle agitation. The cells were
again centrifuged and resuspended in lytic buffer (0.7
M KCl, 1% 2-Mercaptoethanol and 0.1% snail enzyme)
and incubated at 37 o C for 17 hrs. Using microscope,
the cells were periodically checked for the formation of
protoplast with iodine solution (2 g potassium iodine,
1 g iodine, up to 300 ml distilled water). Protoplast
was then collected by centrifugation at 3000 rpm for
10 mins, washed at least three times with the washing
buffer (0.1M phosphate buffer, pH 7.5 and 0.8 M
sorbitol and resuspended in the same buffer.
M ATERIALS AND M ETHODS
Protoplast fusion: Protoplast suspension of both
parental yeast strains were mixed in 1: 1 ratio,
centrifuged at 3000 rpm for 10 minutes. The pellets
were resuspended in fusion buffer (10 mM Tris-HCl,
pH 8.1, 10 mM CaCl2 and 35% PEG with M.W .
4000).
The suspensions were incubated at 37 o C for 30
mins. 0.1 ml of the suspension was mixed with 10 ml
of melted regeneration minimal medium (MMR1) and
poured into plates containing a thin bottom layer of the
same medium supplemented with (nutritional
requirement).
Plates were incubated at 37 o C for 5 days. For
fusion control, parental protoplasts were submitted to
the same treatment [1 2 ].
M icrobial strains and culture condition: Table (1)
illustrates the microbial strains used in this
investigation; three auxotroph mutants (S. b. M1, S. b.
M2 and S. b. M 3) were produced after irradiation
treatment to the wild type Saccharomyces boulardii by
gamma radiation [1 ]. Four bacterial strains (Bacillus
cereus, Escherichia coli, Pseudomonas aeruginosa and
Staphylococcus aureus) were used as test strains in
antimicrobial bioassay.
M edia: Complete medium (CM ) [9 ]: The yeast strains
were maintained as slant of YEP agar medium as (CM )
and stored at 5 O C. W hen required, the yeast culture
was activated by transferring to YEP plates, and
incubated at 37 O C for 48 hrs.
Nutrient broth (NB) or Nutrient agar (NA)
media [2 9 ]: The tester bacterial strains were grown at
37 0 C in NB medium or on NA medium with vigorous
shaking.
Minimal medium (MM) [3 3 ]: W as used to test the
yeast mutants, i.e., auxotrophic cells and the induced
fusants
Minimal medium for regeneration (MMR) [3 1 ]: It
consists of ; 0.67% yeast nitrogen base (YNB), 3%
sorbitol, 0.1% glucose, 0.6% KCl and 3% agar.
Regeneration minimal medium (Top layer medium)
(MMR1): It is similar to MMR but without KCl and
agar was reduced to 1.8%. This medium was used for
the stabilization of fusion products.
Electron M icroscopy (EM ): Sample of the generated
fusants were centrifuged and fixed in 2% glutaraldhyde
in 50 mM sodium cacoclylate buffer, pH 7.2 at 4 o C
overnight. The pellets were then washed 3 times in the
same buffer, post fixed in 1% osmium tetroxide in
sodium cacodylate buffer for 1h at room temperature,
and immersed in 1% urangl acetate for 2 hrs. The
fixed samples were dehydrated with acetone and
embedded in supurr's epoxy resin. Thin sections
(60 ìm thick) were collected on formuar-coated EM
grids and stained with uranyl acetate and lead citrate.
Images were obtained on Philips CM 1 0 (FE 1 Co,
Hillsboro, OR) transmission electron microscope
operating at 80KV with Gatan (Pleasanton, CA)
Bioscan digital camera.
Induction of yeast protoplasts: Yeast cells were
converted to protoplasts according to Farahank et al.[1 2 ]
method with a slight modification. Yeast strains were
aerobically grown to early stationary phase in 25 ml
Erlenmeyer flasks containing 10 ml aliquots of CM
medium for 20 hrs, at 37 o C, then the cells were
Antimicrobial activity: The antimicrobial activity of
the cell-free supernatants for the fusant strains against
the tester organisms was determined using agar
diffusion well assay[3 6 ].
Tolerance to low pH: The cells of the tested yeast
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J. Appl. Sci. Res., 3(3): 209-217, 2007
strains were harvested from fresh culture by
centrifugation (4000 rpm, 10 min. at 4 o C). The yeast
cells were washed three times with sterile saline
(0.85% NaCl) and inoculated at 1% in YEP broth
acidified to pH (3.0) by 10% HCl according to Chou
and W einer[8 ].
The aim of the present study is to use protoplast
fusion technique to improve some probiotic properties
(i.e. antimicrobial activity and resistant to high
concentration of Bile Salt) of the S. boulardii strains.
Bile Salt Tolerance: Cultures of S. boulardii were
evaluated for viability and growth in YEP broth
medium supplemented with 0.2% sodium thioglycolate
and 3% Oxgell bile salt (Oxoid) according to El-Shafai
et al.[1 1 ].
Protoplast Formation and Fusion: Biologically,
species were defined as "a group of organisms that can
interbreed with one another" [5 ]. However, for many
microorganisms, and particularly a number of groups of
yeasts and fungi, no sexual stage could be discovered
RESULTS AND DISCUSSIONS
Table 1: List of the m icrobial strains used
M icrobial Strain
Phenotype
Genotype
Source
S. boulardii
Prototrophic
W ild type
NRC 1
----------------------------------------------------------------------------------------------------------------------------S.b.M 1*
Auxotroph
ad S.b.M 2*
lys S.b.M 3*
arg --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bacillus cereus (ATC C33018)**
Prototrophic
M IRCEN 2
E. coli (ATC C 69337)**
S. aurues (ATC C20231)**
----------------------------------------------------------------------------------------------------------------P. aeruginosa (NRRL B-272.A)**
NRRL 3
* (S.b.M ) is referring to S. boulardii m utants that obtained after irradiating the wild type Saccharom yces boulardii by gam m a radiation in
previous article.
** Test strains used in antim icrobial bioassay.
1
(N RC) K indly obtained from N ational Research Centre (N RC), D okki, Giza, Egypt.
2
(M IRCEN ) Kindly obtained from Egyptian M icrobial Culture Collection (EM CC) at Cairo, M icrobiological Resources C entre (M IRCEN ),
Fac. of A gric., Ain Sham s U niv., Egypt.
3
(NRRL) Kindly obtained from National Centre for Agricultural Utilization Research, USA.
in their life cycles, so the biological definition of a
species proved unworkable. The availability of facile
methods to sequence first proteins and then genes led
to the rapid growth of molecular phylogenetics and the
use of sequence comparisons to both define and
identify species [2 3 ,2 2 ,2 6 ,3 5 ]. W hereas, S. boulardii is a
wild-type strain that is rep o rted ly unable to
sporulate [2 4 ,2 3 ], but its ploidy and mating type have not
been published [3 9 ], so that, the protoplast fusion
technique was done to obtain S. boulardii intraspecific
fusants. The mutants; ( Sb.M1) and (S.b.M2) adenine
and lysine auxotrophs, respectively and another mutant
(S.b.M3) argenine auxotroph were used in this study
(Figure 1).
In figure (1) we expect that the initial colonies
formed on MMR medium
resulted from fused
protoplasts containing genomic DNA from two parent.
It appears that if
fusion occurs between two
auxotrophic parents , complementation between the two
parental genomes can support growth on MM medium
even if recombination has not occurred to combine
wild type alleles into a single chromosome. During
growth of the initial colonies on either M M R or M .M.,
the recombination
can
occur to generate
true
prototrophs. Alternatively, the parental genomes may
Fig. 1: M odel for the example of heterodiploid cells
obtained by fusion of protoplasts of two
auxotrophic parent strains of S. boulardii.
segregate during cell division without recombination,
or recombination may occur in such a way that wild
type alleles are not combined. In the latter cases, the
resulting strains will be auxotrophs, but they will
retain the enzymes that were present in the biparental
progenitor. In the absence of significant protein
turnover , the concentration of these enzymes will be
reduced by one-half during each cell division , so that
there is likely to be enough enzyme to allow synthesis
of leucine, adenine or argenine for several cell
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Table 2: Protoplast form ation
Parent strains
N um ber of survivals cells / m l
Intact yeast cells*
Protoplast form ation**
------------------------------------------------------------------------------------------N um ber / m l
%
N um ber / m l
%
S.b.M 1 (ad -)
1.1´10 8
3000
0.003
2.1x10 6
1.91
-----------------------------------------------------------------------------------------------------6
S.b.M 2 ( lys )
4500
0.004
4.3 x10
3.91
-----------------------------------------------------------------------------------------------------S.b.M 3 (arg -)
5500
0.005
5.3 x10 6
4.82
*growing on M M R m edium supplem ented with adenine or lysine or argenine.
**According to the m icroscopic exam ination of the protoplast suspensions.
(A)
(B)
(C)
Fig. 2: Electron micrographs of cell collected during generation of protoplast.
generation. Such heterogeneity has been well
documented in previous studies of protoplast fusion [1 7 ]
(D)
into M MR medium supplemented with adenine or
lysine or argenine to determine the number of intact
yeast cells.
Table (2) showed that the number of intact S.b.M1
cells was about 3000 cells / ml of protoplast
suspension while it was about 4500 and 5500 cells /
ml for S.b.M2 and S.b.M3, respectively. On the other
hand, the protoplast formation percentage of S.b.M1
was 1.91% while it was 3.91 and 4.82% for S.b.M2
and S.b.M3, respectively.
The regeneration percentage of the induced
protoplasts was determined by mixing 0.1 ml of
protoplast suspension with the top layer medium
(M MR1) and poured onto plates containing a thin
bottom layer of the regeneration medium (MMR)
supplemented with 20 µg / ml of adenine or lysine or
Protoplast Induction and Efficiency: An-equal
number of cells (15 x 10 7 cells / ml) of S.b.M1 or
S.b.M 2 or S.b.M3 were used in the protoplast induction
according to Farahank et al. [1 2 ] method. After protoplast
induction, protoplast formation may depend on the
sensitivity of the yeast strains to lytic enzymes and on
the reaction of different cells of the same culture as
well as the walls of older cells being highly
resistant[2 5 ]. The results indicated that the protoplasts
were efficiently induced as the microscopic examination
of the protoplast suspension showed that almost all the
cells were converted into protoplasts within . 20 hrs.
A sample of the protoplast suspensions were separated
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argenine (Table 3). The results showed that after
incubation at 37 o C for 4 days, the regeneration
percentage of S.b.M1 was 0.9364% while it were
1.9273 and 2.3455% for S.b.M2 and S.b.M3,
respectively.
The efficiency of fusion was expressed as a
percentage of reverting protoplasts. Mixture of
complementing protoplasts but untreated with fusion
buffer were used as a control. Protoplast suspensions
(4.5 ml) of both yeast strains were mixed, centrifuged
and resuspended in 2 ml protoplasting buffer. Two
hundreds micro-liters of the mixture were mixed with
the top layer and poured onto the selective regeneration
medium MMR without any supplementation of adenine
or lysine or argenine.
Table (4) showed that after 7 days of incubation at
37 o C, 215, 345 and 410 hybrid fusants colonies were
found per mixed protoplasts, (S.b.M1 X S.b.M2),
(S.b.M1 X S.b.M 3) and (S.b.M 2 X S.b.M 3),
respectively. The fusion between (S.b.M1) X (S.b.M2)
had higher regeneration percentage (10.42%) when
grown on M MR medium than the other fusions
(S.b.M1) X (S.b.M3) and (S.b.M2) X (S.b.M3) that
showed regeneration percentage of 8.15 and 7.94%,
respectively. These results are in agreement with the
results of Stobinska et al.[3 2 ]. The use of PEG as a
spheroplast fusionic agent has a number of limitations,
which have been discussed in detail by [4 1 ].
Data
presented in this study showed that regeneration of
protoplasts produced large sized cell colonies than the
parental strains (Figure 2). T his may due to the effect
of hybrid vigour. These results are in agreement with
the results of Dzuiba and Chmeilewska [1 0 ]. Results of
intergeneric protoplast fusion between Saccharomyces
cerevisiae and Kluveromyces lactis induced by PEG
were reported by Borun and Jinke [4 ]. They stated that
the cell volume of fusant was about the sum of the
cells of two parents. DNA contents of most fusant
were about two to three time higher than that of the
parents. On the other hand, Selebane et al.[3 0 ] found
that fusants of Candida shehatae and Pichia stipitis
showed only marginal in cell DNA content when
compared with their parents.
Figures (2-A and 2-B) showed electron
micrographs of cell collected during generation of the
fused protoplasts. For most of the cells in the field, the
outer membrane has peeled away from the inner
membrane or no longer visible. W e have observed
fusion between cells that still retain some outer
membrane and thus would technically be considered
spheroplasts rather than true protoplasts [4 0 ]. W hile as,
Figures (2-C and 2-D) show the cells which contain
the nucleuses of the parents cells after complete fusion
occurred.
Based upon the results reported here, we suggest
that the efficiency of protoplast fusion is critically
dependent upon both technical and organismic factors.
W e note that some fusants remnants of the outer
membrane, thus, it is not necessary to remove the
entire outer membrane in order to achieve fusion.
Two of the produced fusants were isolated, named
F1 (resulted after fusion between S.b.M1 and S.b.M2)
and F2 (resulted after fusion between S.b.M2 and
S.b.M3) and subjected to further studies.
Antimicrobial Activity: The ability of cell-free of five
yeast strains; S. boulardii (W .T.) strain and three
mutants; (S.b.M 1), (S.b.M2) and (S.b.M3) the adenine,
lysine and argenine auxotrophs, respectively as well as
two fusants; F1 (S.b.M1 X S.b.M2) (ad -´lys -) and F2
(S.b.M2 X S.b.M3) (lys -´arg -) to retard or suppress the
gro w th o f so m e ha rm ful ind ica to r b a c teria
(Staphylococcus aureus, Bacillus cereus, Pseudomonas
aeruginosa and Escherichia coli) is illustrated in Table
(5) and Figure (3).
Table (5) revealed that in case of E. coli, negative
results of the antimicrobial effects of the tested yeast
strains were found. It also showed that S. boulardii
wild type strain was not able to give any antimicrobial
effect on E. coli and P. aeruginosa as reported by
Kuhle et al. [2 0 ]. The diameter of clear zone was
achieved by fusant F2 (S.b.M2 X S.b.M3) against B.
cereus (11 mm) was larger than their parents; S.b.M2
and S.b.M3 and the wild type strain (S. boulardii) that
showed (0.0, 0.0 and 8 mm, respectively). W hile the
smallest clear zone diameter was achieved by fusant F1
(S.b.M1 X S.b.M2) and their parent S.b.M1 against P.
aeruginosa (8 mm).as well as the wild type strain (S.
boulardii) and the other parent S.b.M2 that showed (0.0
and 10 mm, respectively).
Figure (3) shows the clear zone apparent when the
two fusants; F1 (S.b.M1 X S.b.M2) and F2 (S.b.M2 X
S.b.M3) were used as antagonistic effecter on
Pseudomonas aeruginosa comparison with the wild
type strain (S. boulardii).
Tolerance of Low pH: The ability to grow (tolerance)
at pH 3 was varied among the two fusant strains and
their parents as well as the original strain S. boulardii
(W .T) (Table 6). Data showed clearly that the
logarithmic scale of colony-forming units(log CFU) of
survival percentage at pH 3. Results indicated that
fusant F2 (S.b.M2 X S.b.M3) gave the highest level of
growth being (5.0 log CFU ) and the other fusant F1
(S.b.M1 X S.b.M2) showed (3.8 log CFU). W hile as
their parents, (S.b.M1), (S.b.M2) and (S.b.M3) as well
as the wild type strain (S. boulardii ) that showed
level of growth being (3.9, 4.5, 3.2 and 4.7 log CFU,
respectively).
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J. Appl. Sci. Res., 3(3): 209-217, 2007
Table 3: Regenerated colonies on M M R
Parent strains
N um ber of survivals colonies / m l*
S.b.M 1 (ad -)
1.1´10 8
S.b.M 2 ( lys -)
Num ber of regenerated colonies / m l**
% Regeneration
5.12´10 6
0.9364
------------------------------------------------------------------------6.83´10 6
1.9273
------------------------------------------------------------------------6.08´10 6
2.3455
S.b.M 3 (arg -)
*Grow ing on CM .
**Growing on M M R m edium supplem ented with adenine ,lysine, or argenine.
Table 4: Form ation of hybrid fusants colonies.
Y east hybrids
N um ber of Protoplast form ation / m l*
N um ber of hybrid fusants colonies / m l**
% Protoplast fusion
S.b.M 1 X S.b.M 2
2063
215
10.42
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------S.b.M 1 X S.b.M 3
4233
345
8.15
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------S.b.M 2 X S.b.M 3
5163
410
7.94
*According to the m icroscopic exam ination of the protoplast suspensions.
**Appeared on M M R m edium without any supplem entation.
Table 5: The antim icrobial effect of cell-free Saccharom yces boulardii fusants and their parents on various indicator bacteria.
Y east strains
D iam eter of inhibition zone (m m ) of indicator bacteria
------------------------------------------------------------------------------------------------------------------------------------------B. cereus
E. coli
P. aeruginosa
S. aureus
S. boulardii (W .T.)
8
0.0
0.0
5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------S.b.M 1 (ad -)
0.0
0.0
8
8
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------S.b.M 2 (lys )
0.0
0.0
10
0.0
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------S.b.M 3 (arg -)
0.0
0.0
8
6
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------F1 (S.b.M 1XS.b.M 2)*
0.0
0.0
8
10
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------F2 (S.b.M 2XS.b.M 3)*
11
0.0
9
0.0
* Fusants
Table 6: Survival (as Percentage of log CFU ) of Saccharom yces boulardii fusants and their parents at the low pH 3.
S. boulardii strain
Control
pH 3
--------------------------------------------------------------------------------------------------------------------Percentage of logarithm ic colony-form ing units(% Log CFU )
S. boulardii (W . T.)
7.2
4.7
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------S.b.M 1 (ad -)
7.6
3.9
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------S.b.M 2 (lys -)
7.9
4.5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------S.b.M 3 (arg -)
7.1
3.2
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------F1 (S.b.M 1XS.b.M 2) *
7.8
3.8
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------F2 (S.b.M 2XS.b.M 3) *
7.9
5.0
*Fusants
Table 7: Survival (as Percentage of log CFU ) of Saccharom yces boulardii fusants and their parents in the presence of 3% bile salt.
S. boulardii strain
Control
3 % bile salt
-------------------------------------------------------------------------------------------------------------------Percentage of logarithm ic colony-form ing units(% log CFU ))
S. boulardii (W T)
8.0
7.7
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------S.b.M 1 (ad )
7.6
6.5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------S.b.M 2 (lys -)
7.2
7.3
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------S.b.M 3 (arg -)
7.9
6.5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------F1 (S.b.M 1XS.b.M 2)*
8.1
6.1
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------F2 (S.b.M 2XS.b.M 3) *
8.5
8.3
* Fusants
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Fig. 3: Pseudomonas aeruginosa inhibition by S. boulardii fusants.
Bile Salt Tolerance: The ability to grow (tolerance) in
the presence of 3% Bile salt was varied among the two
fusant strains and their parents as well as the original
strain S. boulardii (W .T) (Table 7). Data showed
clearly that the logarithmic scale of colony-forming
units( log CFU ) of survival percentage in the presence
of 3% Bile salt. Results indicated that fusant F2
(S.b.M2 X S.b.M3) gave the highest level of growth
being (8.3 log CFU) and the other fusant F1 (S.b.M 1
X S.b.M2) showed (6.1 log CFU). W hile as their
parents, (S.b.M 1), (S.b.M2) and (S.b.M3) as well as
the wild type strain (S. boulardii ) showed level of
growth being (6.5, 7.3, 6.5 and 7.7 log CFU,
respectively). Although the Bile salt concentration of
the human G1 tract varies, the mean intestinal Bile
concentration is believed to be 0.3% , [1 5 ]. This
concentration of Bile has consequently been used in
most studies screening for Bile resistant strains [1 6 ,7 ].
This property may provide these strains with an
advantage in vivo. These results are generally in
harmony with those reported by Kuhle et al. [2 0 ] who
indicated that Saccharomyces boulardii displayed good
resistance to 0.3% Bile salt .
The efficiency of genome shuffling is a function of
the efficiencies of generation and fusion of protoplasts,
recovery of protoplasts after fusion, recombination
between heterogeneous chromosomes in the diploid or
multiploid cells obtained after fusion, and the eventual
segregation of true proto-fusants containing only one
genome [2 8 ]. Genetic improvement of Saccharomyces and
other yeast strains has traditionally relied on random
mutagenesis or classical breeding and genetic crossing
(i.e. protoplast fusion) of two strains followed by
screening for mutant or fusants exhibiting enhanced
properties of interest[3 8 ,3 4 ].
The Appearance of characters in the fusants
progeny that does not exist in their parents may be due
to the regulatory effects of the ploidy state. Galitski et
al. [1 4 ] reported that microarray-based gene expression
analysis identified genes showing ploidy-dependent
expression in isogenic Saccharomyces cerevisiae strains
that varied in ploidy from haploid to tetraploid. These
genes were induced or repressed in proportion to the
number of chromosome sets, regardless of the mating
type. Ploidy-dependent repression of some G 1 cyclins
could explain the enlarged cell size associated with
higher ploidies, and suggests ploidy-dependent
modifications of cell cycle progression. Moreover,
ploidy regulation of the FLO11 gene had direct
consequences for yeast development. Also, Andalis et
al.[3 ] noted that they cannot eliminate the possibility
that one or more of the genes whose regulation differs
in tetraploids is responsible for their failure to survive
in stationary phase.
From the present results, it is concluded that the
produced intraspecific fusants of S. boulardii,-that have
a highly antagonistic effect on some pathogenic
bacteria and also have a highly tolerate to low pH
degree and high bile salts concentration than their
parents may be due to the regulatory effect of the
ploidy state - are recommended for various medical
applications.
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