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The luminescent system of the luminous fungus Neonothopanus nambi

Authors:
  • Institure of Biophysics, Siberian Branch of Russian Academy of Sciences, Krasnoayrsk, Russia
ISSN 16076729, Doklady Biochemistry and Biophysics, 2011, Vol. 438, pp. 138–140. © Pleiades Publishing, Ltd., 2011.
Original Russian Text © V.S. Bondar, A.P. Puzyr, K.V. Purtov, S.E. Medvedeva, E.K. Rodicheva, J.I. Gitelson, 2011, published in Doklady Akademii Nauk, 2011, Vol. 438, No. 5,
pp. 705–707.
138
The ability to emit visible light is characteristic of
many higher fungi. Currently, approximately 80 lumi
nous fungal species are known, which were found in
different regions of the globe (North and South
America, Europe, Asia (including Siberia), Australia,
Africa, and Brazil) [1–3]. However, the mechanisms
of luminescence of higher fungi remain obscure, and
there is no single opinion about the molecular organi
zation of the luminescent systems [2, 4, 5]. Two alter
native ideas are discussed: (1) the bioluminescence of
fungi is ensured by luciferase–luciferin a classical
enzyme–substrate system, and (2) the luminescence
is caused by oxidation of organic substrates by oxi
dases without a specialized enzyme. The existence of
a fungal luciferase, therefore, is debated or even
denied. The study of these aspects is not only of fun
damental importance for broadening the notion
about bioluminescence as a natural phenomenon but
also of practical value for using luminous fungi as
biomarkers that significantly enhance the capabilities
of bioluminescent analysis. Only a few studies on
application of luminescent fungi for biological testing
are known [6, 7].
We have investigated the luminescent system of the
luminous fungus
Neonothopanus nambi
, which was
found in the tropical forests of South Vietnam and was
initially described as species
Omphalotus af. illudent
[8].
This study was performed with a culture of the fun
gus
N. nambi
and its fruiting bodies, which was kindly
provided by Vietnamese researcher Dao Thi Van (pri
vate collection of strains BIOLUMI Co., Ltd., Ho
Chi Minh City, Vietnam). Mycelium was grown in a
liquid nutrient potato–sucrose medium (200 g potato,
20 g sucrose, 1 L of distilled water) in the dark at
26
°
С
for 8–10 days. In experiments we also used dry myce
lium samples and fruiting bodies of fungi obtained
after drying at
30–35
°
С
to a constant weight.
The level of light production by
N. nambi
mycelium
was assessed with a BLM 8801 luminometer (SKTB
Nauka, Krasnoyarsk, Russia), which was calibrated
using the Hastings–Weber radioactive standard (emis
sion of 10
8
photons in 1 s was taken as one fluorescent
unit). Luminescent signals were recorded with an
LKB 2210 recorder (LKB, Sweden). The fungal lumi
nescence spectra were recorded spectrofluorometri
cally using an AmincoBowman Series 2 luminescent
spectrometer (Thermo Spectronic, United States).
Mycelium was disintegrated by the following pro
cedures: (1) grinding at
4
°
С
in water and buffer sys
tems with neutral pH value, (2) preliminary freezing at
–20
°
С
in a freezer and at
–170
o
С
in liquid nitrogen
with subsequent grinding in a ceramic mortar, (3) dis
integration with a homogenizer in glass–glass system,
and (4) solubilization of samples in 5% Triton X100.
We have shown experimentally that freshly grown
mycelium of
N. nambi
is capable of longterm lumi
nescence (Fig. 1). It can be seen that mycelium sam
ples placed in the luminometer cell initially had a low
level of luminescence. Their luminescence signifi
cantly increased with time and could markedly exceed
the initial level. The time required for mycelium sam
ples to reach the maximum luminescence level varied
from 40 min to 5 h. Then, the level of light production
decreased and reached the steadystate level that was
retained during at least 10–12 h. Possibly, the observed
significant rise in light production was associated with
regenerative processes occurring in the mycelium after
the mechanical damage at the time of sampling for
analysis. The samples of freshly grown mycelium
placed in distilled water also retained the ability to
luminesce for a long time (for more than two weeks).
We found that the luminescence spectrum of
N. nambi
is located in the visible region of the spec
trum (wavelength range, 480–700 nm) with a maxi
mum at 527–535 nm (Fig. 2). These data agree well
The Luminescent System of the Luminous Fungus
Neonothopanus nambi
V. S. Bondar
a
,
b
, A. P. Puzyr
a
, K. V. Purtov
a
, S. E. Medvedeva
a
, E. K. Rodicheva
a
,
and
Academician
J. I. Gitelson
a
,
b
Received February 3, 2011
DOI:
10.1134/S1607672911030082
a
Institute of Biophysics, Siberian Branch,
Russian Academy of Sciences, Akademgorodok,
Krasnoyarsk, 660036 Russia
b
Siberian Federal University, Krasnoyarsk, 660041 Russia
BIOCHEMISTRY, BIOPHYSICS
AND MOLECULAR BIOLOGY
DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol. 438 2011
THE LUMINESCENT SYSTEM OF THE LUMINOUS FUNGUS
NEONOTHOPANUS NAMBI
139
with the results of several authors [4, 5, 9] obtained for
other species of luminous fungi. On the one hand, this
suggests a similarity, if not identity, of the molecular
basis of luminous fungal species, which have the same
maximum of light emission (at least those whose emis
sion spectrum was investigated). However, possibly
only the terminal luminescence emitters are similar in
different fungal species as it is observed in the case of
the green fluorescent protein, a common terminal
emitter discovered by the authors of [10] that is wide
spread in animals with different bioluminescent sys
tems.
The luminescent system of
N. nambi
is oxygen
dependent. Elimination of oxygen with sodium
dithionite or its replacement with gaseous nitrogen
makes luminescence drop to zero. Subsequent resto
ration of oxygen access entails the restoration of lumi
nescence.
The Fenton’s reagent system used in this study
(
Fe
2+
and
Н
2
О
2
) had no stimulatory effect on the fun
gal luminescent system. Simultaneous additions of
Fe
2+
(1 and 4 mM) and
Н
2
О
2
(20 mM) to the reaction
mixture did not increase the level of luminescence.
Light emission was not increased either after the addi
tion of sodium diethyldithiocarbamate (1 mM) as an
inhibitor of superoxide dismutase. On the basis of
these facts, it can be assumed that the studied lumines
cent system probably functions without the involve
ment of superoxide anion radical. However, it should
be noted that the enhancement of luminescence of
luminous fungi
P. stipticus, A. mellea, M. citricolor,
P. japonicus, O. olearius
, and
M. luxcoeli
by superoxide
anion radical was shown in [5, 11].
We found that luminescence of the
N. nambi
myce
lium was stimulated by addition of only hydrogen per
oxide (concentration range, 1–20 mM), which may
indicate the involvement of peroxidase (or several per
oxidases) in the mechanism of light emission by this
fungal species. This assumption seems reasonable
because it is known that many higher fungi contain
actively functioning ligninolytic enzyme complexes
that contain several enzymes exhibiting peroxidase
activity [12, 13].
We found that some bivalent metal ions affect the
luminescent system of
N. nambi
. The luminescence
intensity of the mycelium increased 1.5–3 times after
the addition of Mn
2+
ions (10–40 mM). Ca
2+
ions (5–
80 mM) had almost no effect on the light emission by
the mycelium, whereas Mg
2+
ions (20 mM) reduced
the level of luminescence by 15–25%. Ni
2+
and Co
2+
ions at a concentration of 10 mM reduced the level of
luminescence by 25–30% and 50–60%, respectively.
The addition of EDTA (10–20 mM) significantly
(2
3 times) increased the light emission by the myce
lium, apparently, due to the binding of bivalent metal
ions that may inhibit the fluorescence system.
The mechanism underlying the discovered stimu
lation of luminescence by manganese ions can be
explained taking into account the above hypothesis
and assuming that the ligninolytic enzyme complex of
the fungus
N. nambi
contains Mnperoxidase(s). It is
known that the peroxidase complex in higher fungi
may contain Mnperoxidase and a hybrid Mnperoxi
dase [14, 15]. This assumption requires further inves
tigation.
The mechanical disintegration of
N. nambi
myce
lium leads to an irreversible loss of the ability of the
fungus to luminesce. It was found that the moment of
disintegration of the mycelium is not accompanied by
light emission. It was also shown that the lumines
cence of homogenates obtained by different methods
of mycelium disintegration cannot be restored in the
presence of oxygen, by addition of manganese ions,
hydrogen peroxide, EDTA, or by replacement of
buffer systems. After freezing the mycelium samples in
water at
–20
°
С
and subsequent thawing them at room
80
0 200 1200
60
40
20
1000800600400
Bioluminescence, rel. units
Tim e, min
0
400 700
3
2
1
600500
Light emission, rel. units
Wavelength, nm
Fig. 1.
Time dependence of bioluminescence of
N. nambi
mycelium obtained by culturing in a liquid nutrient
potato–sucrose medium.
Fig. 2.
Bioluminescence spectrum of
N. nambi
mycelium
in vivo.
140
DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol. 438 2011
BONDAR et al.
temperature, the lightemitting ability of the fungus
was completely lost.
Similar results were also obtained with the myce
lium samples and fruiting bodies of fungi that were
previously dried to a constant weight at mild heating
conditions (at
30–35
°
С
). It was found that the dry
mycelium samples and fruiting bodies placed in water
or a buffer system with a neutral pH value did not
recover the light emitting capability. These results
indicate that the luminescent system of
N. nambi
is
located on the cell membrane or in other cellular
structures.
Our experimental data showed that the extracts
obtained after the disintegration of the
N. nambi
mycelium and removal of debris by centrifugation at
16000
g
for 10 min (centrifuge 5415R, Eppendorf,
Germany) at
10
°
С
contained a luminescence activa
tor. The addition of an aliquot of this extract to sam
ples of luminous mycelium enhanced light emission by
them by 1.5 or two orders of magnitude or more. Heat
ing of the extract containing the activator in a boiling
water bath for 1–3 min did not abolish its stimulatory
effect. The pretreatment of extracts with detonation
nanodiamond particles (a polyfunctional adsorbent
that binds protein and peptide components) also did
not abolish the stimulatory effect. These data suggest
that the revealed component is, most likely, a low
molecularweight heatresistant compound that may
be an emitter or controller of fluorescent systems in
the studied fungal species. Further research will focus
on the isolation of the revealed activator and study its
physical and chemical properties.
Thus, in this study we have obtained primary data
on the structural and functional organization and
physicochemical properties of the luminescence sys
tem of the luminous tropical higher fungus
N. nambi
.
It is shown that grown mycelium samples are capable
of longterm light emission and that the studied lumi
nescence system is oxygendependent and is probably
located in the cell membrane or in other structures of
mycelium. The stimulation of luminescence by Mn
2+
ions suggests the presence of Mnperoxidase(s) in the
ligninolytic enzyme complex of
N. nambi
. We detected
a lowmolecularweight heatstable component in the
fungal biomass that significantly (by orders of magni
tude) increased light emission by mycelium. Isolation
of this compound in its pure form, study of its physic
ochemical properties and functions in the reaction of
light emission by the fungus
N. nambi
is one of the pri
orities of our further studies.
ACKNOWLEDGMENTS
This work was supported by the Federal Agency for
Science and Innovation within the Federal Special
Purpose Program (state contract no. 02.740.11.0766),
the Russian Foundation for Basic Research (project
no. 080490307V’et_a), and the Russian–Vietnam
ese Tropical Research and Development Center
(project Ekolan E1.2).
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Luminescent fungi spontaneously emit light during certain stages of their life cycles. Most of them are luminous during a part of their mycelial stage, but not many of them are luminous when they form fruiting bodies. In the case of Panellus stipticus, both the mycelium and the fruiting body can be luminous, and the emission of light takes place when its luciferin is aerobically oxidized in the presence of the superoxide anion (O2) and a cationic surfactant. It is highly likely that the luminescence reactions of all kinds of luminous fungi are basically the same as that of P. stipticus. In order to determine the factor that makes a fungus luminous or non-luminous, we studied the relations between the light emission of fungi at various growth stages and the contents of luciferin, its precursor, superoxide dismutase (SOD), and catalase, on six species of luminescent fungi: Armillariella mellea, Mycena citricolor, Mycena lux-coeli, Omphlotus olearious, Panellus stipticus, and Pleurotus japonicus. The analysis of the data suggested that the fungi generally contain the components necessary for light emission, but also contain very large amounts of SOD which destroy O2−. If an appreciable amount of SOD is distributed at the site of light emission, the luminescence reaction is prevented. For the reaction to take place, it is essential that the SOD activity at the site is sufficiently low or inhibited, despite the high content of SOD in the whole tissue. Thus, the level of SOD activity at the site of light emission appears to be a limiting factor in regulating the luminescence of fungi.
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The total radiant flux from a solution containing a radioactive compound, hexadecane-l-C14, plus an appropriate scintillator has been determined. The procedure used involves comparison of its luminescent emission with the light scattered by glycogen illuminated with a monochromatic homogeneous light beam of known photon flux. From the result obtained the scintillation quantum yield of a β- disintegration from carbon 14 has been determined to be 793 photons; thus 5.25% of the energy appears as light.
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Metal cation toxicity to basidiomycete fungi is poorly understood, despite its well-known importance in terrestrial ecosystems. Moreover, there is no reported methodology for the routine evaluation of metal toxicity to basidiomycetes. In the present study, we describe the development of a procedure to assess the acute toxicity of metal cations (Na(+), K(+), Li(+), Ca(2+),Mg(2+), Co(2+), Zn(2+), Ni(2+), Mn(2+), Cd(2+), and Cu(2+)) to the bioluminescent basidiomycete fungus Gerronema viridilucens. The method is based on the decrease in the intensity of bioluminescence resulting from injuries sustained by the fungus mycelium exposed to either essential or nonessential metal toxicants. The assay described herein enables us to propose a metal toxicity series to Gerronema viridilucens based on data obtained from the bioluminescence intensity (median effective concentration [EC50] values) versus metal concentration: Cd(2+) > Cu(2+) > Mn(2+) approximately Ni(2+) approximately Co(2+) > Zn(2+) > Mg(2+) > Li(+) > K(+) approximately Na(+) > Ca(2+), and to shed some light on the mechanism of toxic action of metal cations to basidiomycete fungi.