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Department of Environmental Health Sciences, University of California, Los Angeles, CA 90095–1772
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • boron • growth • dose response • yeast • Saccharomyces cerevisiae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and fish (Eckhert 1998
, Rowe et al. 1998
, Rowe and Eckhert 1999
), but the molecular
basis for essentiality has not been determined for either. There is
also strong evidence that boron is probably required for embryonic
development in frogs and mice (Fort et al. 1998
,
Lanoue et al. 1998
). The use of yeast
(Saccharomyces cerevesiae) has proven a powerful tool in
advancing our knowledge of the molecular trafficking of copper, zinc
and iron in eukaryotes (Eide 1998
, Pufahl et al. 1997
). This unicellular eukaryotic organism shares
significant genetic and protein homologies with mammals and to a lesser
extent with plants. Yeast have the advantage of growing rapidly in
liquid culture and being genomically characterized, so that the effects
of elemental manipulations can be evaluated at the genetic level. There
are two widely used media for yeast (Fiechter et al. 1987
). Only one is supplemented with boric acid. The objective
of this investigation was to assess whether yeast growth is stimulated
by boron and could provide a suitable model with which to study the
molecular biology of boron. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Media preparation.
Powder stocks of amino acids, salts, vitamins/trace elements, and
carbon/nitrogen sources were prepared and stored at room temperature in
moisture- and light-insulated polyethylene containers. To eliminate
trace contamination of boron, ultrapure reagents were used whenever
possible. The media were prepared from concentrated
mixes.2
,3
,4
,5
Liquid medium was prepared by mixing 2.48 g amino acid stock,
25 g carbon/nitrogen source stock, 1.7 g salt stock, 34 mg
vitamin/trace element mix with 1 L of ultrapure (18 M
) (<0.0925
µmol B/L) water as previously described
(Eckhert 1998
) in an autoclaved polycarbonate bottle
(Fisher Scientific). Mixing was performed for 2 h at room
temperature with a plastic-coated magnetic stir bar.
Boron removal.
Final boron removal was accomplished by filtering the media through a column of borate anion–specific Amberlite (Rohm and Has Company, Philadelphia, PA). In a TFE 2-L separation column, 30 mL of Amberlite resin (per L of media) (Sigma IRA-743) was treated, in sequence, with the following: 150 mL 3 mol/L ammonia hydroxide, 600 mL distilled water, 300 mL of 1 mol/L hydrochloric acid, 150 mL distilled water, 300 mL of 0.16 mol/L nitric acid, 600 mL distilled water followed by 1 L of media. All liquid was allowed to filter through the resin at an effluent rate of ~2 drops/s. Media were collected in a sterile polycarbonate filter apparatus, filter-sterilized and stored at 4°C in sterile polycarbonate bottles until used. The plasticware was obtained from Fisher Scientific.
Yeast growth and counting.
An overnight culture was prepared with 50 mL of boron-free medium in a 500-mL sterile baffled polycarbonate culture flask that was inoculated with a single colony from a YPD agar plate (Difco, Detroit, MI) streaked 2 d previously with Sacchromyces cerevisiae. All streaking and inoculations were carried out with a 2-mm platinum/iridium inoculation loop. The culture was then incubated at 30°C in a dry air shaker at 360 rpm for 24 h. The overnight culture (8 mL) was used to inoculate 400 mL of boron-free medium in a 2-L sterile baffled polycarbonate culture flask. The culture was then incubated under the same conditions as the overnight culture. At early log phase, the sample was mixed thoroughly and divided evenly between two 2-L sterile baffled polycarbonate culture flasks. One of the cultures (+B) was supplemented with 185 µmol/L boric acid (Fisher, 99.8% purity); the other culture (nonboron, NB) was supplemented with a comparable volume (4 mL/L of culture) of ultrapure water. This did not change the pH of the culture. At 6, 9, 12, 15 and 24 h, the culture was counted by diluting 10 µL well-mixed culture with 900 µL sterile water in a sterile microcentrifuge tube and loading an improved Neimbauer hemocytometer with 10 µL of the diluted culture. The number of yeast cells in 10 areas of 1 mm2 was counted. Dividing cells were assessed as two separate cells if a septum was apparent. Cell density was determined by direct counting rather than by turbidity because of an apparent difference in cell size between B+ and B- treatments.
Preparation and analysis of samples for boron content.
At 9.17 h, 5-mL aliquots were taken in triplicate from each
culture (+B and NB) and placed in a 15-mL polyethylene tube. The cells
were pelleted by centrifugation at 2000 x g, room
temperature, for 10 min. The supernatant was then transferred to a new
polyethylene tube and stored at -70°C until analyzed. The pellet was
washed twice with 10 mL sterile boron-free medium, snap frozen in
liquid nitrogen and stored at -70°C until analyzed. Analysis of
boron content was carried out using inductively coupled plasma-mass
spectrometry as previously described (Eckhert 1998
).
Analysis of ethanol production.
Cultures supplemented with 0, 4.6, 23.1, 92.5 and 185 µmol/L boric acid were prepared. After 24 h, the yeast were pelleted by centrifugation, and the media fractions were analyzed for ethanol content using the Sigma Diagnostics Alcohol Reagent kit (based on the amount of NAD+ produced by alcohol dehydrogenase as measured by a spectrophotometric shift at 340 nm).
Statistical analysis.
Data were analyzed with a statistical software package (SigmaStat for
Windows, Jandel Scientific Software, San Rafael, CA). The effect of
boron was evaluated using the Kruskal-Wallis two-way ANOVA on ranks
and Dunn’s multiple comparison test (Dixon and Massey 1983
). Data are presented as means ± SEM A
value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows the results of three independent experiments with separate
cultures averaged together. At 9 h (early log phase growth), the
addition of 185 µmol B/L to the media resulted in an
increase in the growth rate compared with the culture supplemented with
H2O. This increase in growth rate was significant
(P < 0.05) at 3 h. The difference in growth rate
increased in magnitude until the cultures reached stationary phase (24
h). At 24 h, 4 h into the stationary phase, the cell density
of the B-supplemented cultures was > 50% greater than that
of the nonsupplemented cultures. Part of this increase was due to
continued growth during the stationary stage. Figure 2
gives the dose-response curve. Maximum boron stimulation occurred
at concentrations <0.8 µmol B/L. Ethanol production at
24 h was independent of boron concentration of the culture media
(Fig. 3
). Cellular boron concentrations became significantly reduced over the
course of the experiment (Table 1
). Yeast cells in both the boron-supplemented and nonsupplemented
media lost >95% of their boron. There was no difference in the
cellular concentrations between groups at either the time of
supplementation (9.17 h) or at 24 h.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Boron is an ingredient of some but not
all widely used yeast media (Fiechter et al. 1987
); until this report
it had not been shown to be essential or to stimulate growth. In 1946,
Wickerhan published the composition of the growth medium used by the
USDA at the Northern Regional Research Laboratory in Peoria, IL in
their yeast strain classification program (Wickerhan 1946
). That medium became the basis of several different media
sold by Difco for the classification of yeast (Difco Manual 1999
). However, when Olson and Johnson (1949)
evaluated the constituents of growth media in 1949, to
develop one that provided growth yields equal to those of natural
media, they found that boron was not among those that stimulated
growth. Using glass flasks and distilled water to prepare media, these
investigators were able to show that Na, Mg, K, P, Fe, Cu and Zn were
required by yeast, but not B, Mn, Co, Sn or I. Their basal levels of
boron were probably too high to see growth stimulation because
distillation in metal or glass contributes to the boron concentration
of the water. This is because boron is added to glass to increase its
expansion properties, and tin distillation storage tanks contain boron.
Since that report, two reviews of the nutritional requirements of yeast
have been published; boron is not included as a growth stimulator or
nutrient in either report (Rose 1987
, Suomalainen and Oura 1971
).
The production of ethanol by yeast can achieve sufficiently high
concentrations to inhibit growth. Boron has been shown to inhibit yeast
alcohol dehydrogenase (Smith and Johnson 1976
) in a
cell-free system and to reduce the amount of ethanol produced.
Ethanol production was therefore measured in this study to determine
whether the difference in growth rate in the deceleration and plateau
phase (15–24 h) was related to differences in its production. The
ethanol concentrations at 24 h were found to be independent of
boron concentration of the culture media (Fig. 3)
. Therefore, we
conclude that the differences in growth rate and carrying capacity of
the media were due to boron rather than to a secondary effect of
ethanol production.
Although boron stimulated yeast growth, we did not detect significant
differences in cellular boron concentrations at 24 h. The cellular
boron concentrations were significantly reduced after a long period of
cell replication (Table 1)
. NB culture cells continued to retain a
significant amount of boron despite the fact that they had been grown
in media with <1 µmol B/L for 48 h (24 h in the
overnight culture + 24 h in the experimental culture),
demonstrating that S. cerevisiae vigorously maintains
cellular boron content. This ability contrasts with the zebrafish
embryos that become rapidly depleted in boron when incubated under low
boron conditions (Rowe and Eckhert 1999
). The difference
between the ability of the two species in retaining boron may explain
why boron-deficient environments lead to only a depression of
growth in yeast, whereas zebrafish embryos die under similar
conditions.
S. cerevisiae has previously been shown to be a good model for studying the molecular trafficking of copper, iron and zinc. This study suggests that boron can be added to that list. In contrast to these metals, boron does not have a radioactive isotope with a half-life longer than 0.8 s, 8B; thus, trafficking experiments will have to use indirect techniques to co-localize the element with cellular proteins.
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FOOTNOTES |
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3 The 10X-concentrated carbon/nitrogen source stock contained: 200 g dextrose (Cat. # BP350–1, purity 99.8%,
FisherBiotech, Fisher Scientific, Fair Lawn, NJ) and 50 g ammonium sulfate (Cat. # A-5132, Sigma Chemical, St. Louis, MO).
4 The 100X-concentrated salt stock contained (purity 98–99.8%, where appropriate, all purchased from Sigma
Chemical, St. Louis, MO): 85 g KH2PO4 (Cat. # P-0662), 15 g K2HPO4 (Cat. #
P-3786), 50 g MgSO4 · 7H2O (Cat. # M-5921), 10 g NaCl (Cat. # S-7653), and 10 g
CaCl2 · 2H2O(Cat. # C-5080).
5 The 1000X-concentrated vitamin/trace element stock contained (purity 98–99.5%, where appropriate, all purchased
from Sigma Chemical, St. Louis, MO): 20 mg D-biotin (Cat. # B-4501), 2 g D-calcium pantothenate (Cat. # P-5710), 2
mg folic acid (Cat. # B-F-7876), 10 g myo-inositol (Cat. # I5125), 400 mg nicotinic acid (Cat. # N-4126), 200 mg
p-aminobenzoic acid (Cat. # A-9878), 800 mg pyridoxine (PN)-HCl (Cat. # P-9755), 400 mg riboflavin (Cat. # R-4500), 400 mg
thiamine-HCl (Cat. # T-4625), 40 mg CuSO4 (Cat. # C-1297), 100 mg KI (Cat. # P-2963), 200 mg FeCl3 (Cat. #
F-7134,), 400 mg MnSO4 · H2O (Cat. # M-7634), 200 mg Na2MoO4 · 2H2O (Cat. #
M-1003) and 400 mg ZnSO4 · 7H2O zinc sulfate.
Manuscript received May 12, 1999.
Initial review completed July 7, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1. Difco Manual of Dehydrated Culture Media and Reagents for Microbiological and Clinical Laboratory Procedures 11th ed. 1999 Becton Dickinson and Company Franklin Lakes, NJ.
2. Dixon W. J., Massey F. J., Jr Introduction to Statistical Analysis 4th ed. 1983 McGraw-Hill New York, NY.
3.
Eckhert C. D. Boron stimulates embryonic trout growth. J. Nutr. 1998;128:2488-2493
4. Eide D. J. The molecular biology of metal ion transport in Saccharomyces cerevisiae. Annu. Rev. Nutr. 1998;18:441-469[Medline]
5. Fiechter A., Kappeli O., Meussdoerffer F. Batch and continuous culture. Rose A. H. Harrison J. S. eds. The Yeasts 1987;vol. 2:99-128 Academic Press Orlando, FL.
6. Fort D. J., Propst T. L., Stover E. L., Strong P. L., Murray F. J. Adverse reproductive and developmental effects in Xenopus from insufficient boron. Biol. Trace Elem. Res. 1998;66:237-259[Medline]
7. Lanoue L., Taubeneck M. W., Muniz J., Hanna L. A., Strong P. L., Murray F. J., Nielsen F. H., Hunt C. D., Keen C. L. Assessing the effects of low boron diets on embryonic and fetal development in rodents using in vitro and in vivo model systems. Biol. Trace Elem. Res. 1998;66:271-298[Medline]
8.
Olson B. H., Johnson M. J. Factors producing high yeast yields in synthetic media. J. Bacteriol. 1949;57:235-246
9.
Pufahl R. A., Singer C. P., Peariso K. L., Lin S., Schmidt P. J., Fahrni C. J., Culotta V. C., Penner-Hahn J. E., Halloran T. V. Metal Ion Chaperone Function of the Soluble Cu (1) Receptor Atx1. Science (Washington, DC) 1997;278:853-856
10. Rose A. H. Responses to the chemical environment. Rose A. H. Harrison J. S. eds. The Yeasts 1987;vol. 2:6-40 Academic Press Orlando, FL.
11. Rowe R. I., Bouzan C., Nabili S., Eckhert C. D. The response of trout and zebrafish embryos to low and high boron concentrations is U-shaped. Biol. Trace Elem. Res. 1998;66:262-270
12. Rowe R. I., Eckhert C. D. Boron is required for zebrafish embryogenesis. J. Exp. Biol. 1999;202:1649-1654[Abstract]
13. Smith K.W., Johnson S. L. Borate inhibition of yeast alcohol dehydrogenase. Biochemistry 1976;5:560-565
14. Suomalainen H., Oura E. Yeast nutrition and solute uptake. Rose A. H. Harrison J. S. eds. The Yeasts 1971;vol. 2:3-74 Academic Press New York, NY.
15. Warington K. The effect of boric acid and borax on the broadbean and certain other plants. Ann. Bot. 1923;37:629-672
16.
Wickerhan L. J. A critical evaluation of the nitrogen assimilation tests commonly used in the classification of yeasts. J. Bacteriol. 1946;52:293-302
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