DIVISION OF ECOLOGY

DIVISION OF ECOLOGY
Head
František Nerud, PhD.
A major focus in the environmental area in the Division of Ecology is on the study of
the relationships among the diversity, structure and function of the anthropogenically
damaged soil microbial ecosystem (fungi, bacteria) and the interactions with biotic and
abiotic components of their environment that regulate and affect microbial community activities. Another research line concerns the use of the most important bioremediation methods
like mycoremediation, composting and phytoremediation, for biodegradation of persistent
aromatic pollutants (PAH, PCB, endocrine disruptors). Bacterial aerobic/anaerobic degradation, rhisosphere microbial communities and ligninolytic fingi are investigated in this respect.
Elucidation of the bioavailability phenomenon and its influence on bioremediation, identification of degradation metabolites and monitoring of ecotoxicity during bioremediation are other
research tasks. Besides this the substantial importance of humic substances, as a key environmental factor determining the composition of soil microbial community, for ectomycorrhizal
fungi has been confirmed. Mechanisms of biological chlorination of soil organic matter and
the effect of mineral chloride on microflora have been followed.
The Division maintains the Collection of Basidiomycetes (see http://www.
biomed.cas.cz/CCBAS/fungi.htm) and part of this Collection significant for
agriculture is now incorporated in the National Program of Protection and Utilization of Genetic
Resources of Economically Significant Plants and Microorganisms (see http://www.
vurv.cz/collections/vurv.exe/search). This program was established to
professionally and financially secure genetic resources of plants and microorganisms important for agricultural production and research.
Division members have been active in mentoring graduate, undergraduate and high
school students in research projects. Furthermore, the Division coordinates the Center of
Environmental Microbiology and participates in the Center of Functional Cell Organization.
In addition, staff members also cooperate with civic organizations in the project concerning
the development and protection of the environment in the Pardubice region.
The research efforts are described in detail in the project summaries by each of the
principal investigators.
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Laboratory 141
ENVIRONMENTAL MICROBIOLOGY
(formerly BIOCHEMISTRY OF WOOD-ROTTING FUNGI)
Head
Petr Baldrian, PhD.
Scientific staff
Petra Dobiášová, MSc.
Ivana Eichlerová, PhD.
Jiří Gabriel, PhD., DSc.
Jana Herinková, MSc.
Ladislav Homolka, PhD.
Ludmila Lisá, MSc.
Věra Merhautová, MSc.
František Nerud, PhD.
Mirka Petránková, MSc.
Martina Štursová, MSc.
Josef Trögl, PhD.
Šárka Veselá, PhD.
Technical staff
Romana Kubecová
Doctoral students
Valášková, MSc.
Jaroslav Šnajdr, MSc., Michaela Urbanová, MSc., Vendula
Undergraduate students Jiří Dvořák, BSc., Leo Argote Vivar, BSc.,
Tomáš Větrovský, BSc., Jana Voříšková, BSc.
Research field and principal results
The research in the laboratory covers the ecology, physiology and biochemistry of
saprotrophic fungi, ecology of microorganisms in the soil environment and the technological
use of basidiomycetes and their extracellular enzymes along with the development of methods
for the preservation of fungal cultures. The laboratory worked under the name “Laboratory of
Biochemistry of Wood-Rotting Fungi” until the autumn of 2008.
Litter transformation by saprotrophic basidiomycetes and their role in forest soil
The production of lignin- and polysaccharide-degrading enzymes and changes of the
chemical composition of litter were studied with three isolates from a Quercus petraea forest.
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These isolates were capable of fresh litter degradation and were identified as Gymnopus sp.,
Hypholoma fasciculare and Rhodocollybia butyracea. Within 12 weeks of incubation, H. fasciculare decomposed 23 %, R. butyracea 32 % and Gymnopus sp. 38 % of the substrate dry
mass. All fungi produced laccase and Mn-peroxidase (MnP) and none of them produced
lignin peroxidase or other Mn-independent peroxidases. All fungi decreased the C/N ratio of
the litter from 24 to 15-19 and Gymnopus sp. also caused a substantial decrease in the lignin
content. Analytical pyrolysis mass spectrometry of litter decomposed by this fungus showed
changes in the litter composition similar to those caused by white-rot fungi during wood
decay. All fungi also changed the mean masses of humic acid and fulvic acid fractions
isolated from degraded litter. Compared to the decomposition by saprotrophic basidiomycetes, litter degradation in situ on the site of fungal isolation resulted in the relative enrichment
of lignin probably due to the participation of non-basidiomycetous fungi and bacteria during
natural litter decomposition.
Phylogenetic composition and properties of bacteria coexisting
with the fungus Hypholoma fasciculare in decaying wood
White-rot fungi are major degraders of woody materials in terrestrial environments
because of their ability to decompose lignin. However, little is known about the possible
associations of white-rot fungi with other microorganisms during wood decay. We investigated the numbers, community composition and functional traits of bacteria present in
natural wood samples under advanced decay by the white-rot basidiomycete Hypholoma fasciculare. The wood samples contained high numbers of cultivable bacteria (0.2–8 × 109
colony forming units [CFU] g–1 dry wood). Most cultivable bacteria belonged to Proteobacteria and Acidobacteria (75 and 23 % of sequences, respectively). The same phyla were
also found to be dominant (59 and 23 %, respectively) using a non-culturable quantification
technique, namely direct cloning and sequencing of 16SrRNA genes extracted from wood.
Bacteria that could be subcultured consisted of acid-tolerant strains that appeared to rely on
substrates released by lignocellulolytic enzyme activities of the fungus. There were no indications for antagonism (antibiosis) of the bacteria against the fungus.
Microorganisms inhabiting hardwood forest soil and their enzymatic activity
Spatial variability and vertical distribution (0–8 cm) of the ligninolytic enzymes laccase
and Mn-peroxidase and several polysaccharide hydrolases were studied in a Quercus petraea
forest soil profile (Fig. 27). Activities of all tested enzymes exhibited high spatial variability
in the L and H horizons. Acid phosphatase and 1,4-β-N-acetylglucosaminidase exhibited low
variability in both horizons, while the variability of Mn-peroxidase activity in the L horizon,
and endoxylanase and cellobiohydrolase activities in the H horizon were very high. The
L horizon contained 4 × more microbial biomass (based on PLFA) and 7 × more fungal
biomass (based on ergosterol content) than the H horizon. The L horizon also contained
relatively more fungi-specific and less actinomycete-specific PLFA. The soil profile showed
a gradient of pH, content of organic carbon and humic compounds, microbial biomass and
enzyme activities, all decreasing with soil depth. Ligninolytic enzymes showed preferential
localization in the upper part of the H horizon. Differences in enzyme activities were accompanied by differences in the microbial community composition where the relative amount of
fungal biomass decreased and actinomycete biomass increased with soil depth. The results
also showed that the vertical gradients occur at a small (1 cm) scale.
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Fig. 27. Xaverov forest Natural Reserve near Prague, Czech Republic dominated by Quercus petraea
is the site of most studies in the ecology of soil and soil microorganisms.
Microbiology of primary succession on postmining deposits
Changes in the activity of extracellular enzymes and the changes in microbial
community and abiotic properties in the topsoil layer, as well as soil abiotic properties during
primary succession were investigated in a brown coal mine deposit area near Sokolov, Czech
Republic (Fig. 28). The study considered the chronosequence of 4 post-mining plots, 4-, 12-,
21- and 45-year old. The 4-year old site had no vegetation cover. Herbs and grasses (mainly
Calamagrostis epigeios) were present on the 12-year old plot, shrubs (Salix caprea) occurred
on the 21-year old plot and tree cover (Betula spp. and Populus tremuloides) developed on the
45-year old plot. Soil pH gradually decreased with site age, while the content of P, K, C and
N peaked in the 21-year old site. Succession age was the most important factor affecting the
total and bacterial PLFA contents, followed by the effects of soil layer and season while for
the fungal biomass content-related properties (ergosterol, fungal PLFA and the fungal/bacterial PLFA ratio), season was the most important. Activities of individual enzymes in the topsoil (0–5 cm depth) were significantly affected by both site age and season.
Maintenance and cryopreservation of fungi
A part of the basidiomycete cultures from CCBAS collection, which is significant for
agriculture, is incorporated in the National Program of Protection and Utilization of Genetic
Resources of Economically Significant Plants and Microorganisms (see a web page
http://www.vurv.cz/collections/vurv.exe/search). The replacement of the method of subculturing on agar media by storing on perlite in cryovials was successfully tested. Cultures of 33
basidiomycete strains out of 35 tested were viable with unchanged characteristics after four
years of maintenance on perlite in cryovials. These cultures can be a good substitute for agar
cultures in long-term maintenance of fungi. Storage under oil was tested for comparison, but
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it turned out to be unsuitable for the majority of our cultures. Enzyme profiles of the tested
brown rot basidiomycetes differ from those of white rot basidiomycetes. Enzyme profiles of
the tested white rot basidiomycetes are similar regardless of the laccase activity. Oak wood is
Fig. 28. Successional changes of vegetation in postmining sites in the Sokolov region, Czech Republic, are accompanied by dramatic changes in soil microbial community and its functioning. Sites
undergoing primary succession for 4, 12, 21 and 45 years were studied.
more resistant to fungal attack than pine and spruce wood, which is the most sensitive. Characterization of enzyme activities in our cultures using the Apizyme method continued and
characterization of carbohydrate metabolism using the Api 50 CH method was started in
2008. Stability of the cultures stored in liquid nitrogen was tested using DNA fingerprinting.
The results indicate that cryopreservation is a safe method for the long-term maintenance of
fungi.
Maintenance and cryopreservation of fungi
A part of the basidiomycete cultures from CCBAS collection, which is significant for
agriculture, is incorporated in the National Program of Protection and Utilization of Genetic
Resources of Economically Significant Plants and Microorganisms (see a web page
http://www.vurv.cz/collections/vurv.exe/search). The replacement of the method of subculturing on agar media by storing on perlite in cryovials was successfully tested. Cultures of 33
basidiomycete strains out of 35 tested were viable with unchanged characteristics after four
years of maintenance on perlite in cryovials. These cultures can be a good substitute for agar
cultures in long-term maintenance of fungi. Storage under oil was tested for comparison, but
it turned out to be unsuitable for the majority of our cultures. Enzyme profiles of the tested
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brown rot basidiomycetes differ from those of white rot basidiomycetes. Enzyme profiles of
the tested white rot basidiomycetes are similar regardless of the laccase activity. Oak wood is
more resistant to fungal attack than pine and spruce wood, which is the most sensitive.
Characterization of enzyme activities in our cultures using the Apizyme method continued
and characterization of carbohydrate metabolism using the Api 50 CH method was started in
2008. Stability of the cultures stored in liquid nitrogen was tested using DNA fingerprinting.
The results indicate that cryopreservation is a safe method for the long-term maintenance of
fungi.
Publications
Baldrian P., Trögl J., Frouz J., Šnajdr J., Valášková V., Merhautová V., Cajthaml T., Herinková J.: Enzyme
activities and microbial biomass in topsoil layer during spontaneous succession in spoil heaps after brown
coal mining. Soil Biol. Biochem. 40, 2107-2115 (2008).
Baldrian P., Valášková V.: Degradation of cellulose by basidiomycetous fungi. FEMS Microbiol. Rev. 32, 501521 (2008).
Baldrian P.: Ectomycorrhizal fungi and their enzymes in soils: is there enough evidence for their role as facultative soil saprotrophs? Oecologia 161, 657-660 (2009).
Baldrian P.: Effect of heavy metals on saprotrophic soil fungi. In: Sherameti I., Varma A. (eds): Soil Heavy
Metals. Soil Biology Series, vol. 19, Springer Verlag, Berlin-Heidelberg, in press.
Baldrian P.: Enzymes of Saprotrophic Basidiomycetes. In: Ecology of Saprotrophic Basidiomycetes (Boddy,
Watkinson and van West, eds.), Academic Press, USA, pp. 19-41, 2008.
Baldrian P.: Microbial Enzyme-Catalyzed Processes in Soils and their Analysis. Plant, Soil, and Environment, in
press.
Baldrian P. (2008) Wood-inhabiting ligninolytic basidiomycetes in soils: Ecology and constraints for applicability in bioremediation. Fungal Ecology 1, 4-12.
Baldrian, P.; Merhautová, V.; Cajthaml, T.; Nerud, F.; Stopka, P.; Gorbacheva, O.; Hrubý, M.; Beneš, M.J.
(2008) Synthesis of zirconia-immobilized copper chelates for catalytic decomposition of hydrogen peroxide
and the oxidation of polycyclic aromatic hydrocarbons. Chemosphere 72, 1721-1726.
Byss M., Elhottová D., Tříska J., Baldrian P.: Fungal bioremediation of the creosote-contaminated soil:
Influence of Pleurotus ostreatus and Irpex lacteus on polycyclic aromatic hydrocarbons removal and soil
microbial community composition in the laboratory-scale study. Chemosphere 73, 1518-1523 (2008).
Eichlerová I., Homolka L., Benada O., Kofroňová O., Hubálek T., Nerud F.: Decolorization of Orange G and
Remazol Briliant Blue R by the white rot fungus Dichomitus squalens: Toxicological evaluation and
morphological study. Chemosphere 69, 795-802 (2007).
Eichlerová I., Homolka L., Nerud F.: Decolorization of high concenrations of synthetic dyes, by the white rot
fungus Bjerkandera adusta. Dyes Pigm. 75, 38-44 (2007).
Gryndler M., Krofta K., Gryndlerová H., Soukupová L., Hršelová H., Gabriel J.: Potentially dangerous fusarioid
microorganisms associated with rot of hop (Humulus lupulus L.) plants in field culture. Plant Soil Environ.
54, 149-154 (2008).
Homolka L., Lisá L., Nerud F.: Basidiomycete cultures on perlite survive successfully repeated freezing and
thawing in cryovials without subculturing. J. Microbiol. Meth. 69, 529-532 (2007).
Homolka L., Lisá L.: Long-term maintenance of fungal cultures on perlite in cryovials - an alternative for agar
slants. Folia Microbiol. 53, 534-536 (2008).
Homolka, L. Lisá L., Kubátová A., Váňová M., Janderová B., Nerud F.: Cryopreservation of filamentous
micromycetes and yeasts using perlite. Folia Microbiol. 51, 153-157 (2007).
Kokol V., Doliška A., Eichlerová I., Baldrian P., Nerud F.: Decolorization of textile dyes by whole cultures of
Ischnoderma resinosum and by purified laccase and Mn-peroxidase. Enzyme Microb. Technol. 40, 16731677 (2007).
Řezáčová V., Baldrian P., Hršelová H., Larsen J., Gryndler M.: Influence of mineral and organic fertilization on
soil fungi, enzyme activities and humic substances in a long-term field experiment. Folia Microbiol. 52, 415422 (2007).
Šnajdr J., Baldrian P.: Temperature affects the production, activity and stability of ligninolytic enzymes in Pleurotus ostreatus and Trametes versicolor. Folia Microbiol. 52, 498-502 (2007).
Šnajdr J., Valášková V., Merhautová V., Cajthaml T., Baldrian P.: Activity and spatial distribution of lignocellulose-degrading enzymes during forest soil colonization by saprotrophic basidiomycetes. Enzyme Microb.
Technol. 43, 186-192 (2008).
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Šnajdr J., Valášková V., Merhautová V., Herinková J., Cajthaml T., Baldrian P.: Spatial variability of enzyme
activities and microbial biomass in the upper layers of Quercus petraea forest soil. Soil Biol. Biochem. 40,
2068-2075 (2008).
Soukupová L., Hršelová H., Gryndlerová H., Merhautová V., Gryndler M.: Alkali-extractable soil organic
matter: An important factor affecting the mycelial growth of ectomycorrhizal fungi. Appl. Soil Ecol. 40, 3743 (2008).
Steffen K.T., Cajthaml T., Šnajdr J., Baldrian P.: Differential degradation of oak (Quercus petraea) leaf litter by
litter-decomposing basidiomycetes. Res. Microbiol. 158, 447-455 (2007).
Tomšovský M., Popelářová P., Baldrian P.: Production and regulation of lignocellulose-degrading enzymes of
Poria-like wood-inhabiting basidiomycetes. Folia Microbiol. 54, 74-80 (2009).
Uhnáková B., Petříková A., Biedermann D., Homolka L., Vejvoda V., Bednář P., Papoušková B., Šulc M.,
Martínková, L.: Biodegradation of brominated aromatics by cultures and laccase of Trametes versicolor.
Chemosphere 76, 826-832 (2009).
Valášková V., Baldrian P.: Denaturing gradient gel electrophoresis as a fingerprinting method for the analysis of
soil microbial communities. Plant, Soil, and Environment, in press.
Valášková V., de Boer W., Klein Gunnewiek P., Pospíšek M., Baldrian P.: Phylogenetic composition and
properties of bacteria coexisting with the fungus Hypholoma fasciculare in decaying wood. ISME Journal 3,
1218-1221 (2009).
Valášková V., Šnajdr J., Bittner B., Cajthaml T., Merhautová V., Hofrichter M., Baldrian P.: Production of
lignocellulose-degrading enzymes and degradation of leaf litter by saprotrophic basidiomycetes isolated from
a Quercus petraea forest. Soil Biol. Biochem. 39, 2651-2660 (2007).
Žižka Z., Gabriel J.: Autofluorescence of the fruiting body of the fungus Macrolepiota rhacodes. Folia Microbiol. 53, 537-539 (2008).
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Laboratory 142
ENVIRONMENTAL BIOTECHNOLOGY
(formerly EXPERIMENTAL MYCOLOGY)
Head
Tomáš Cajthaml, PhD.
Scientific staff
Karel Novák, PhD.
Čeněk Novotný, PhD.
Kateřina Svobodová, PhD.
Part-time scientists
Václav Šašek, PhD.
Technical staff
Pavla Erbanová, MSc.
Ludmila Klanicová
Zdena Křesinová, MSc.
Doctoral students
Martin Šušla, MSc., Zuzana Čechová, MSc., Stefano Covino, MSc.
Undergraduate students
Lenka Kožená, Milan Muzikář, Monika Čvančarová, Michaela Fišarová,
Martina Plačková, Lucie Svobodová, Alena Filipová, Tatiana Stella,
Petr Šmejkal, Martin Ezechiáš
Research field and principal results
The research is mainly focused on the study of fate and degradation and organic pollutants and endocrine disrupting compounds by ligninolytic fungi, composting and bacteria;
on the study of mechanisms of degradation and practical utilization of microbial bioremediation technologies.
The plant determinants of symbiosis between nodule bacteria and host plants are studied
in the model system Pisum sativum–Rhizobium leguminosarum bv. viciae.
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Estimation of estrogenic and androgenic activity of compounds
with two in vitro yeast assays
Recently, investigations on environmental pollution by endocrine disrupting (ED)
chemicals, studies for their toxicity and biodegradability are now in progress. Sensitivity of
two bioassays towards several ED compounds, including a synthetic estrogen 17α-ethynylestradiol, bisphenol A and a fungicide triclosane, were compared. An androgenic potential of
Delor 103, a commercial mixture of various PCB congeners, was demonstrated using a bioluminescent yeast screen of androgenic compounds. Based on comparison of EC50 values,
Delor 103 was shown to be two orders less potent than the standard androgen testosterone. On
the other hand, chlorobenzoic acids, representing potential PCB degradation metabolites,
showed no androgenic activity but were slightly estrogenic. Their estrogenicity varied with
their chemical structure. An estrogen-induced β-galactosidase expression system in S. cerevisiae proved to be more sensitive towards PCBs than a bioluminescent estrogen screen. With
the β-galactosidase screen a slight estrogen-like activity of Delor103 was measured, whereas
no activity was detected using a bioluminiscent assay. However, the sensitivities of the two
assays towards chlorobenzoic acids were comparable. In addition, both yeast based bioassays
were tested for evaluation of ED activity of compounds yielding from biodegradation
experiments.
Biodegradation of endocrine-disrupting compounds and suppression
of estrogenic activity by ligninolytic fungi
Endocrine-disrupting compounds (EDCs) represent a large group of substances of natural and anthropogenic origin. They are widely distributed in the environment and can pose
serious risks to aquatic organisms and to public health. In this study, 4-n-nonylphenol, technical 4-nonylphenol, bisphenol A, 17α-ethinylestradiol, and triclosan were biodegraded by
eight ligninolytic fungal strains (Irpex lacteus 617/93, Bjerkandera adusta 606/93, Phanerochaete chrysosporium ME 446, Phanerochaete magnoliae CCBAS 134/I, Pleurotus ostreatus
3004 CCBAS 278, Trametes versicolor 167/93, Pycnoporus cinnabarinus CCBAS 595,
Dichomitus squalens CCBAS 750). The results show that under the used conditions the fungi
were able to degrade the EDCs within 14 d of cultivation with exception of B. adusta and
P. chrysosporium in the case of triclosane and bisphenol A, respectively. I. lacteus and
P. ostreatus were found to be most efficient EDC degraders with their degradation efficiency
exceeding 90 % or 80 %, respectively, in 7 d. Both fungi degraded technical 4-nonylphenol,
bisphenol-A, and 17α-ethinylestradiol below the detection limit within first 3 d of cultivation.
In general, estrogenic activities assayed with a recombinant yeast test decreased with advanced degradation. However, in case of I. lacteus, P. ostreatus, and P. chrysosporium the yeast
assay showed a residual estrogenic activity (28–85 % of initial) in 17α-ethinylestradiol
cultures. Estrogenic activity in B. adusta cultures temporally increased during degradation of
technical 4-nonylphenol, suggesting a production of endocrine-active intermediates. Attention
was paid also to the effects of EDCs on the ligninolytic enzyme activities of the different
fungi strains to evaluate their possible stimulation or suppression of activities during the
biodegradation processes.
PAH desorption from river floodplain soils using supercritical fluid extraction
Supercritical fluid extraction (SFE) with pure carbon dioxide was used to obtain desorption curves of Polycyclic aromatic hydrocarbons (PAHs) from river floodplain soils
which contain coal and coal-derived particles. Original soils, soils’ light fractions (ρ < 2 g
cm−3), and <63 μm fractions were studied for PAHs’ desorption kinetics. The desorption
curves were fitted with a simple two-site model to determine the rapidly released fraction (F)
representing bioavailability of PAHs. Desorption rate constants were one order of magnitude
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lower than those of “slow” and “very slow” desorption rates from other studies. This suggests
very slow and extremely slow desorption. Estimated time scales releasing 99 % of total
extractable contaminants ranged from decades for 2–4-ring PAHs and hundreds of years for
5–6-ring PAHs. We demonstrate that, despite high soil PAH concentrations which are due to
coal and coal-derived particles, the general environmental risk is reduced by the very slow
and extremely slow desorption rates.
A novel aspect of PAH biodegradation by ligninolytic fungus Irpex lacteus
The ligninolytic fungus Irpex lacteus was shown as an efficient degrader of oligocyclic
aromatic hydrocarbons (PAHs; ‘polycyclic aromatic hydrocarbons’) possessing 3–6 aromatic
rings in complex liquid media. The strain produced mainly Mn-dependent peroxidase in
media without pollutants. Activity of ligninolytic enzymes was higher in a N-limited medium.
However, after contamination with PAHs (especially pyrene) the values increased and
significant activity of Mn-independent peroxidase appeared in the complex medium. Other
factors (such as the increase in nitrogen concentration or the presence of solvent(s) for
dissolution of PAHs) had no effect. Cytochrome P-450 was detected in the microsomal
fraction of biomass grown in the complex medium. The rate of PAH degradation was also
affected by the presence of various combinations of PAHs. However, independently of the
enzyme activities, anthracene was shown to have a positive influence on degradation of
pyrene and fluoranthene.
Biodegradation of industrial dyes by ligninolytic enzymes
The isoenzymes of manganese-dependent peroxidase (MnP) and laccase produced in
cultures of Dichomitus squalens immobilized on polyurethane foam (PUF) and lignocellulose
were isolated, purified and characterized and their implication in decolorization of synthetic
dyes was proven, demonstrating also a synergistic cooperation of the isolated enzymes.
Similarly, MnP isoenzymes of another fungus, Irpex lacteus, growing in PUF-immobilized
cultures, were isolated and characterized, and their ability to decolorize azo-, anthraquinoneand triphenyl methane dyes demonstrated. In contrast to D. squalens, laccase of I. lacteus
was found to be tightly-associated with fungal mycelium and the biochemical characteristic
and dye-decolorization ability of its bound form was established. Degradation products of the
azo dye Reactive Orange 16 (RO16) obtained by treatment in PUF-immobilized I. lacteus
cultures were identified by LC-MS analysis and a pathway for RO16 degradation was
suggested. The research contributed to better understanding of the phenomenon of recurrent
colorization and the role of the individual enzymes in it. PUF-immobilized fungal cultures
were able to decolorize recalcitrant textile dyes in 5 sequential cycles at 2 g dye per liter and
>95% efficiency showing thus promising long term application characteristics. Commercial
carriers such as Filtren TM30, polyamide or luffa sponge were found to be suitable materials
for immobilization of high-degradation fungal strains in trickle-bed reactors.
Visualization of symbiotic tissue in intact root nodules of Vicia tetrasperma
using GFP-marked Rhizobium leguminosarum bv. Viciae
In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules
of indeterminate type is localized to the basal part of the nodule where the symbiotic zones
contain infected cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia.
Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics.
Tagging of Rhizobium leguminosarum bv. Viciae strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with
the advantages of the tiny nodules formed by Vicia tetrasperma (L.) SCHREB. allowed for
vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-
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shifted background channel and digital image stacking along z-axis enabled us to construct
a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal
microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the
nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This
signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous
detection of bacteria and secondary metabolites can be used for monitoring changes to intact
nodule physiology in the model legumes. The advantage of V. tetrasperma as a suggested
laboratory model for pea cross-inoculation group has been demonstrated.
Publications
Baldrian P., Merhautova V., Cajthaml T., Nerud F., Stopka P., Gorbacheva O., Hruby M., Benes M.J.: Synthesis
of zirconia-immobilized copper chelates for catalytic decomposition of hydrogen peroxide and the oxidation
of polycyclic aromatic hydrocarbons. Chemosphere 72(11), 1721-1726 (2008).
Baldrian P., Trogl J., Frouz J., Snajdr J., Valaskova V., Merhautova V., Cajthaml T., Herinkova J.: Enzyme
activities and microbial biomass in topsoil layer during spontaneous succession in spoil heaps after brown
coal mining. Soil Biol. Biochem. 40(9), 2107-2115 (2008).
Bartkova H., Kluson P., Bartek L., Drobek M., Cajthaml T., Krysa J.: Photoelectrochemical and photocatalytic
properties of titanium (IV) oxide nanoparticulate layers. 1st Internat. Symp.Transparent Conducting Oxides,
October 23-25, 2006 Crete, Greece, Thin Solid Films 515(24), 8455-8460 (2007).
Cajthaml T., Erbanova P., Kollmann A., Novotny C., Sasek V., Mougin C.: Degradation of PAHs by ligninolytic enzymes of Irpex lacteus. Folia Microbiol. 53(4), 289-294 (2008).
Cajthaml T., Kresinova Z., Svobodova K., Moder M.: Biodegradation of endocrine-disrupting compounds and
suppression of estrogenic activity by ligninolytic fungi. Chemosphere 75(6), 745-750 (2009).
Cajthaml T., Křesinová Z., Svobodová K., Sigler K., Řezanka T.: Microbial transformation of synthetic estrogen
17α-ethinylestradiol. Environ. Pollut. 157(12), 3325-3335 (2009).
Casieri L., Varese G.C., Anastasi A., Prigione V., Svobodova K., Marchisio V.F., Novotny C.: Decolorization
and detoxication of reactive industrial dyes by immobilized fungi Trametes pubescens and Pleurotus
ostreatus. Folia Microbiol. 53(1), 44-52 (2008).
Cerna I., Kluson P., Drobek M., Cajthaml T., Bartek L.: Ionic liquids – some reflections on their use in asymmetric homogeneous catalysis. Chemické Listy 101(12), 994-1001 (2007).
Chovanec P., Hovorka O., Novak K.: Visualization of symbiotic tissue in intact root nodules of Vicia tetrasperma using GFP-marked Rhizobium leguminosarum bv. viciae. Folia Microbiol. 53(2),139-146 (2008).
Gryndler M., Hrselova H., Cajthaml T., Havrankova M., Rezacova V., Gryndlerova H., Larsen J.: Influence of
soil organic matter decomposition on arbuscular mycorrhizal fungi in terms of asymbiotic hyphal growth
and root colonization. Mycorrhiza 19(4), 255-266 (2009).
Hubalek T., Vosahlova S., Mateju V., Kovacova N., Novotny C.: Ecotoxicity monitoring of hydrocarboncontaminated soil during bioremediation: A case study. Arch. Environ. Contamination Toxicol. 52(1),1-7
(2007).
Kluson P., Drobek M., Krejcikova S., Krysa J., Kalaji A., Cajthaml T., Rakusan J.: Molecular structure effects
in photodegradation of phenol and its chlorinated derivatives with phthalocyanines. Appl. Catal. B-Environ.
80(3-4), 321-326 (2008).
Kluson P., Luskova H., Solcova O., Matejova L., Cajthaml T.: Lamellar micelles-mediated synthesis of nanoscale thick sheets of titania. Materials Lett. 61(14-15), 2931-2934 (2007).
Kresinova Z, Svobodova K, Cajthaml T: Microbial degradation of endocrine disruptors. Chemické Listy 103(3),
200-207 (2009).
Leonardi V., Sasek V., Petruccioli M., D'Annibale A., Erbanova P., Cajthaml T.: Bioavailability modification
and fungal biodegradation of PAHs in aged industrial soils. Internat. Biodeterioration Biodegrad. 60(3),
165-170 (2007).
Novak K., Biedermannova E., Vondrys J.: Symbiotic and growth performance of supernodulating forage pea
lines. Crop Sci. 49(4), 1227-1234 (2009).
Olsovska J., Kamenik Z., Cajthaml T.: Hyphenated ultra high-performance liquid chromatography-Nano
Quantity Analyte Detector technique for determination of compounds with low UV absorption. J. Chromatogr. A 1216(30), 5774-5778 (2009).
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Pavko A, Novotny C.: Induction of ligninolytic enzyme production by Dichomitus squalens on various types of
immobilization support. Acta Chim. Sloven. 55(3), 648-652 (2008).
Pocedic J., Hasal P., Novotny C.: Decolorization of organic dyes by Irpex lacteus in a laboratory trickle-bed
biofilter using various mycelium supports. J. Chem. Technol. Biotechnol. 84(7), 1031-1042 (2009).
Prokopova I., Vickova E., Sasek V., Nahlik J., Soukupova-Chaloupkova V., Skolil J.: Aromatic–aliphatic
copolyesters based on waste poly(ethylene terephthalate) and their biodegradability. E-Polymers, Article No
052 (2008).
Rezanka T., Nedbalova L., Elster J., Cajthaml T., Sigler K.: Very-long-chain iso and anteiso branched fatty
acids in N-acylphosphatidylethanolamines from a natural cyanobacterial mat of Calothrix sp. Phytochemistry 70(5), 655-663 (2009).
Sampedro I., Cajthaml T., Marinari S., Marinari S., Stazi S.R., Grego S., Petruccioli M., Federici F., D’Annibale
A.: Immobilized inocula of white-rot fungi accelerate both detoxification and organic matter transformation
in two-phase dry olive-mill residue. J. Agricult.Food Chem. 57(12), 5452-5460 (2009).
Sampedro I., Cajthaml T., Marinari S., Petruccioli M., Grego S., D’Annibale A.: Organic matter transformation
and detoxification in dry olive mill residue by the saprophytic fungus Paecilomyces farinosus. Process
Biochem. 44(2), 216-225 (2009).
Snajdr J, Valaskova V, Merhautova V, Herinkova J, Cajthaml T, Baldrian P.: Spatial variability of enzyme
activities and microbial biomass in the upper layers of Quercus petraea forest soil. Soil Biol. Biochem.
40(9), 2068-2075 (2008).
Snajdr J., Valaskova V., Merhautova V., Cajthaml T., Baldrian P.: Activity and spatial distribution of lignocellulose-degrading enzymes during forest soil colonization by saprotrophic basidiomycetes. 10th Internatl.
Congr. Biotechnol. Pulp and Paper, June 10-15, 2007 Madison, WI, USA. Enzyme Microb. Technol. 43(2),
186-192 (2008).
Steffen K.T., Cajthaml T., Snajdr J., Baldrian P.: Differential degradation of oak (Quercus petraea) leaf litter by
litter-decomposing basidiomycetes. Res. Microbiol. 158(5), 447-455 (2007).
Susla M., Novotny C., Erbanova P., Svobodova K.: Implication of Dichomitus squalens manganese-dependent
peroxidase in dye decolorization and cooperation of the enzyme with laccase. Folia Microbiol. 53(6), 479485 (2008).
Susla M., Novotny C., Svobodova K.: The implication of Dichomitus squalens laccase isoenzymes in dye
decolorization by immobilized fungal cultures. Bioresource Technol. 98(11), 2109-2115 (2007).
Svobodova K., Majcherczyk A., Novotny C., Kues U.: Implication of mycelium-associated laccase from Irpex
lacteus in the decolorization of synthetic dyes. Bioresource Technol. 99(3), 463-471 (2008).
Svobodová K., Plačková M., Novotná V., Cajthaml T.: Estrogenic and androgenic activity of PCBs, their
chlorinated metabolites and other endocrine disruptors estimated with two in vitro yeast assays. Sci. Total
Environment 407(22), 5921-5925 (2009).
Svobodova K., Senholdt M., Novotny C., Rehorek A.: Mechanism of Reactive Orange 16 degradation with the
white rot fungus Irpex lacteus. Process Biochem. 42(9), 1279-1284 (2007).
Valaskova V., Snajdr J., Bittner B., et al.: Production of lignocellulose-degrading enzymes and degradation of
leaf litter by saprotrophic basidiomycetes isolated from a Quercus petraea forest. Soil Biol. Biochem.
39(10), 2651-2660 (2007).
Vosahlikova-Kolarova M., Krejcik Z., Cajthaml T., Demnerova K., Pazlarova J.: Biodegradation of methyl tertbutyl ether using bacterial strains. Folia Microbiol. 53(5), 411-416 (2008).
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Laboratory 143
FUNGAL BIOLOGY
Head
Milan Gryndler,
Assoc. Prof., PhD.
Scientific staff
Hana Hršelová, PhD.
Veronika Řezáčová, PhD.
Lucie Soukupová, MSc.
Part-time scientists
Hana Gryndlerová, MSc.
Zdeněk Přikryl, MSc.
Technical staff
Hana Máchová
Doctoral students
Lucie Soukupová, MSc., Marie Havránková, MSc.
Undergraduate students
Jitka Görnerová, BSc.
Research field and principal results
Interactions between ectomycorrhizal fungi and soil organic matter
We confirmed the substantial importance of humic substances for ectomycorrhizal
fungi: these substances act as a key environmental factor determining the composition of soil
microbial community. This can be seen from their strong and specific response to the
presence of humic substances (fulvic acid and humic acid) in the nutrition media. We started
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field observations focused on interactions of different soil parameters including humus quality
on ectomycorrhizal fungal communities in a forest ecosystem.
At the same time, positive effects of humic-like substances extracted from sewage
sludge have been observed and its possible practical use in large scale cultivation is being
evaluated. In spite of their origin in sewage sludge, these substances cannot represent a menace for human health and may replace humus components ordinarily extracted from the soil
not only in the cultures of ectomycorrhizal fungi but also as additives to plant fertilizers.
Interactions between arbuscular mycorrhizal fungi and soil organic matter
We observed significant association of hyphal growth of arbuscular mycorrhizal fungi
with some components of soil organic matter detected by using pyrolysis-GC-MS approach.
Our results indicate that mycelia of arbuscular mycorrhizal fungi are influenced by organic
matter decomposition both via compounds released during the decomposition process and
also by secondary metabolites produced by microorganisms involved in organic matter decomposition.
Interactions of soil chlorine with microbial communities
We continued the works on the mechanisms of biological chlorination of soil organic
matter in soil and the effects of mineral chloride on microflora possibly involved in chlorination or dehalogenation processes. We observed strong effects of chloride on the composition of soil microbial community and discovered a hitherto unknown group of eukaryotic
organisms (probably a basal fungal lineage) with exotic rDNA cassette sequence responding
positively to increased chloride concentration. The research is continued and the exotic
microbial lineage is further studied. We developed a specific primer selectively amplifying
the members of this microbial group and established trap cultures attempting to detect its
members in different ecosystems. The taxonomic position of this microbial group is examined
in detail.
Studies of fungi associated with plant cultures
We developed a molecular tool suitable for the detection of an uncultured fungus
causing a rot of hops wounded by inconsiderate soil management. This phytopathogenic
Fig. 29. Micropropagated seedlings of Prunus
domestica experimentally inoculated with the fungus
Entoloma clypeatum, a suspect root pathogen.
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fungus (Gibberella pulicaris) cannot be easily isolated from the wounded tissues but may
now be efficiently and selectively detected in field collected samples. This may help to trace
the sources of the infection agent in the field and modify management practices to reduce
plant damage.
We further finalized the development of the method of detection of potentially pathogenic fungi, Entoloma spp. (Fig. 29), damaging the roots of rosaceous woody plants (i.e. fruit
trees). The method is based on newly developed group-specific DNA primers allowing
selective partial amplification of fungal LSU-rRNA gene in roots, soil and cultivation substrates. The method is ready for field applications.
We essayed to exploit selected artificial substrate (miocene clay in mine spoil banks)
for production of high-biomass crops (reed canarygrass) and flax with application of arbuscular mycorrhizal fungal inocula. According to our results a significant part of organic fertilization of the substrate can be replaced by the inoculation with mycorrhizal fungi which may
enable economic biomass production of the mentioned plants.
Publications
Borovička J., Dunn C.E., Mihaljevic M., Gryndler M., Jelínek E., Rohovec J., Rohošková M, Řanda Z.:
Bioaccumulation of gold in macrofungi and ectomycorrhizae from the vicinity of the Mokrsko gold
deposit, Czech Republic. Soil Biol. Biochem. (2009), in press.
Clarke N., Fuksová K., Gryndler M., Lachmanová Z., Liste H.-H., Rohlenová J., Schroll R., Schröder P.,
Matucha M.: The formation and fate of chlorinated organic substances in temperate and boreal forest
soils. Environ. Sci. Pollut. Res. 16, 127-143 (2009).
Götz C., Fekete A., Gebefuegi I., Forczek S.T., Fuksová K., Li X., Englmann M., Gryndler M., Hartmann A.,
Matucha M., Schmitt-Kopplin P., Schröder P.: Uptake, degradation and chiral discrimination of N-acylD/L-homoserine lactones by barley (Hordeum vulgare) and yam bean (Pachyrhizus erosus) plants. Anal.
Bioanal. Chemistry 389, 1447–1475 (2007).
Gryndler M., Borovička J., Gryndlerová H., Gryndler E.: Fructification of Langermannia gigantea in artificially inoculated field soil. Czech Mycol. 60, 231–242 (2008).
Gryndler M., Egertová Z., Soukupová L., Gryndlerová H., Borovička J., Hršelová H.: Molecular detection of
Entoloma spp. associated with roots of rosaceous woody plants. Mycol. Prog., online first, DOI
10.1007/s11557-009-0615-3 (2009).
Gryndler M., Hršelová H., Cajthaml T., Havránková M., Řezáčová V., Gryndlerová H., Larsen J.: Influence of
soil organic matter decomposition on arbuscular mycorrhizal fungi in terms of asymbiotic hyphal growth
and root colonization. Mycorrhiza 19, 255-266 (2009).
Gryndler M., Krofta K., Gryndlerová H., Soukupová L., Hršelová H., Gabriel J.: Potentially dangerous fusarioid
microorganisms associated with rot of hop (Humulus lupulus L.) plants in field culture. Plant, Soil
Environ. 54, 149–154 (2008).
Gryndler M., Rohlenová J., Kopecký J., Matucha M.: Chloride concentration affects soil microbial community.
Chemosphere 71, 1401–1408 (2008).
Gryndler M., Sudová R., Püschel D., Rydlová J., Janoušková M., Vosátka M.: Cultivation of high-biomass crops
on coal mine spoil banks: can microbial inoculation compensate for high doses of organic matter? Bioresource Technol. 99, 6391–6399 (2008).
Gryndler M.: Mycorrhizal symbiosis – an indispensable component of the plant culture, pp. 177-200 in R.C. Ray,
O.P. Ward (Eds.): Microbial Biotechnology in Horticulture, Vol. 2. Science Publishers, Enfield, Jersey,
Plymouth 2008.
Hršelová H., Soukupová L., Gryndler M.: Humic acid-like material from sewage sludge stimulates culture
growth of ectomycorrhizal fungi in vitro. Folia Microbiol. 52, 627–630 (2007).
Matucha M., Gryndler M., Forczek S.T., Schröder P., Bastviken D., Rohlenová J., Uhlířová H., Fuksová K.:
A chlorine-36 and carbon-14 study of the role of chlorine in the forest ecosystem. J. Label. Comp.
Radiopharm. 50, 437–439 (2007).
Matucha M., Gryndler M., Schröder P., Forczek S.T., Uhlířová H., Fuksová K., Rohlenová J.: Chloroacetic
acids – degradation intermediates of organic matter in forest soil. Soil Biol. Biochem. 39, 382–385
(2007).
Püschel D., Rydlová J., Sudová R., Gryndler M.: Cultivation of flax in spoil-bank clay: Mycorrhizal inoculation
vs. high organic amendments. J. Plant Nutr. Soil Sci. 171, 872-877 (2008).
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Rohlenová J., Gryndler M., Forczek S.T., Fuksová K., Handová V., Matucha M.: Microbial chlorination of
organic matter in forest soil: investigation using 36Cl-chloride and its methodology. Environ. Sci. Technol. 43, 3652-3655 (2009).
Řezáčová V., Baldrian P., Hršelová H., Larsen J., Gryndler M.: Influence of mineral and organic fertilization on
soil fungi, enzyme activities and humic substances in a long-term field experiment. Folia Microbiol. 52,
415-421 (2007).
Soukupová L., Hršelová H., Gryndlerová H., Merhautová V., Gryndler M.: Alkali-extractable soil organic matter: an important factor affecting the mycelial growth of ectomycorrhizal fungi. Appl. Soil Ecol. 40, 37–
43 (2008).
Vohník M., Fendrych M., Kolařík M., Gryndler M., Hršelová H., Albrechtová J., Vosátka M.: The ascomycete
Meliniomyces variabilis isolated from a sporocarp of Hydnotrya tulasnei (Pezizales) intracellularly colonises roots of ecto- and ericoid mycorrhizal host plants. Czech Mycol. 59, 215–226 (2007).
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Laboratory 144
FUNCTIONAL CYTOLOGY
Head
Pavla Binarová,
Assoc. Prof., PhD.
Scientific staff
Jindřich Volc, PhD.
Ondřej Plíhal PhD.
Technical staff
Doctoral students
Jana Chumová, PhD.
Gabriela Kočárová, BSc.
Anna Doskočilová, MSc., Lucie Kohoutová, MSc.
Research field and principal results
The recent focus of the group is centred on the mechanisms underlying organization of
microtubule nucleation independent of centrosomes.
Multiple forms and protein interactions of γ-tubulin in acentrosomal cells suggest
functions other than microtubule nucleation
γ-Tubulin is required for the formation of microtubules at centrosomes but its function
in noncentrosomal microtubule nucleation is not understood. We showed that GFP γ-tubulin
is in vivo dynamically localized in cytoplasm, associated with membranes and microtubules,
and in nuclei (Fig. 30). We identified γ-tubulin complexes bound to membranes. To analyze
the role of γ-tubulin in acentrosomal plant cells further, we conditionally downregulated
γ-tubulin by inducible expression of RNAi constructs in Arabidopsis plants. Our data showed
that γ-tubulin is essential for microtubule nucleation from dispersed sites in acentrosomal
plant cells and suggested that the sites of γ-tubulin localization observed with membranes,
microtubules and nuclei are likely the sites of non-centrosomal nucleation and organization.
Moreover, the RNAi seedlings showed a disturbed organization of cell files, stomata
clustering and impaired development of highly polar cells such as root hairs. These data
suggest that some functions of γ-tubulin important for cytokinesis, cell specification and polar
growth are microtubule independent.
Protein complexes of γ-tubulin were purified from extracts of Arabidopsis and characterized. The γ-tubulin complex proteins (GCPs) are core components of γ-tubulin small and
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large complexes that are indispensable for microtubule nucleation from centrosomes. We
found complexes of γ-tubulin with Arabidopsis homologues of GCP2 and GCP3 and GCP4 in
cytoplasm and associated with membranes. Besides GCPs, proteins involved in cell cycle
signalling, stress signalling and cell wall metabolism were identified to interact with γ-tubulin. A recent goal of our research is to elucidate the physiological meaning of the interactions.
Fig. 30. Cell culture of Arabidopsis expressing N-terminal GFP γ-tubulin. Fusion with GFP provides
a tool for studying γ-tubulin dynamics in cells in vivo. (A) GFP γ-tubulin. (B) Differential interphase
contrast. Cytokinetic apparatus phragmoplast (arrow) with γ-tubulin-GFP accumulated with phragmoplast microtubules.
Enzymes involved in redox mechanisms of fungal degradation of lignocellulose
The sugar oxidoreductase pyranose dehydrogenase (PDH) purified from mycelial cultures of the mushroom Agaricus meleagris exhibited a broad substrate tolerance, oxidizing
a number of aldopyranoses to the corresponding C-2 or C-2,3 (di)dehydro sugars. The enzyme catalyzed in up to 95% yields the regiospecific conversion of D-galactose to 2-dehydroD-galactose, an intermediate in a possible biotechnologically interesting process for redox
isomerisation of D-galactose to the prebiotic sugar D-tagatose. Molecular and catalytic properties of this enzyme were compared with the data found for PDH from A. xanthoderma and
A. bisporus.
Three genes (pdh1, pdh2 and pdh3) encoding pyranose dehydrogenase from A. meleagris were cloned and analysed. It was found that they displayed a conserved structure and
organization. The respective cDNAs contained ORFs translating into plypeptides of 602 or
600 amino acids. pdh2 and pdh3 were essentially transcribed constitutively, whereas pdh1
expression was upregulated upon exhaustion of carbon source.
PDHs from different sources were also tested for their possible application in amperometric biosensors for detection of sugars based on the electrical wiring of the proteins with
osmium redox polymer on graphite electrodes.
A novel alcohol oxidase (AOX) purified from the brown rot basidiomycete Gloeophyllum trabeum was characterized as a homooctameric nonglycosylated protein containing
noncovalently bonded FAD. The observed extracellular targeting of AOX, compared with
AOXs peroxisomal localization in yeast, was traced to a unique C-terminal sequence of the
G. trabeum enzyme. This is relevant for the proposed role of this enzyme in lignocellulose
degradation.
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Publications
Binarová P. Cenklová V., Pochylová Z., Dráberová E., Dráber P.: The forms of gamma tubulin in higher plants,
pp. 23–43 in The Plant Cytoskeleton: Genomic and Bioinformatic Tools for Biotechnology and
Agriculture. (V.W. Baird, Y.B. Blume, D. Breviario, A.I. Yemets, Eds). Springer, Netherlands (2008).
Daniel G., Volc J., Filonova L., Plíhal O., Kubátová E., Halada P.: Characteristics of Gloeophyllum trabeum
alcohol oxidase, an extracellular source of H2O2 in brown rot decay of wood. Appl. Environ. Microbiol.
17, 6241–6253 (2007).
Kittl R., Sygmund C., Halada P., Volc J., Divne C., Haltrich D., Peterbauer C.K.: Molecular cloning of three
pyranose dehydrogenase-encoding genes from Agaricus meleagris and analysis of their expression by
real-time RT-PCR. Curr. Genet. 55, 117–127 (2008).
Kujawa M., Volc J., Halada P., Sedmera P., Divne C., Sygmund C., Leitner C., Peterbauer C., Haltrich D.:
Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma.
FEBS J. 274, 879–894 (2007).
Spíchal L., Kryštof V., Paprskářová M., Lenobel R., Stýskala J., Binarová P., Cenklová V., De Veylder L.,
Fischer P.M., Schmülling T., Strnad M.: Clasical anticytokinins do not interact with cytokinin receptors,
but inhibit cyclin-dependent kinases. J. Biol. Chem. 282, 14356–14363 (2007).
Sygmund C., Kittl R., Volc J., Halada P., Kubátová E., Haltrich D., Peterbauer C.: Characterization of pyranose
dehydrogenase from Agaricus meleagris and its application in the C-2 specific conversion of D-galactose.
J. Biotechnol. 133, 334–342 (2008).
Tasca F., Timur S., Ludwig R., Haltrich D., Volc J., Antiochia R., Gorton L.: Amperometric biosensors for detection of sugars based on the electrical wiring of different pyranose oxidases and pyranose dehydrogenases with osmium redox polymer on graphite electrodes. Electroanal. 19, 294–302 (2007).
Gemperlová L., Cvikrová M., Fischerová L., Binarová P., Fischer L., Eder J.: (Polyamine metabolism during the
cell cycle of synchronized tobacco BY-2 cell line. Plant Physiol. Biochem. 47(7), 584-91 (2009).
Pisanelli I., Kujawa M., Spadiut O., Kittl R., Halada P., Volc J., Michael D., Mozuch M.D., Kersten P., Haltrich
D., Peterbauer C.: Pyranose 2-oxidase from Phanerochaete chrysosporium – expression in E. coli and
biochemical characterization. J. Biotechnol. 142(2), 97–106 (2009).
Peterbauer C. K., Volc J.: Pyranose dehydrogenases: biochemical features and perspectives of technological
applications. Appl. Microbiol. Biotechnol., 2009 Sept 19 published on line.