Cell cycle arrest and apoptosis, two alternative mechanisms for PMKT2 killer activity
Antonio Santos, Alejandro Alonso, Ignacio Belda, Domingo Marquina⇑
Department of Microbiology, Faculty of Biology, Complutense University of Madrid, 28040 Madrid, Spain
a r t i c l e i n f o
Received 23 July 2012
Accepted 25 October 2012
Available online 5 November 2012
a b s t r a c t
Pichia membranifaciens CYC 1086 secretes a unique 30 kDa killer toxin (PMKT2) that inhibits a variety of
spoilage yeasts and fungi of agronomical interest. The cytocidal effect of PMKT2 on Saccharomyces
cerevisiae cells was studied. Metabolic events associated with the loss of S. cerevisiae viability caused
by PMKT2 were qualitatively identical to those reported for K28 killer toxin activity, but different to those
reported for PMKT.
At higher doses, none of the cellular events accounting for the action of PMKT, the killer toxin secreted
by P. membranifaciens CYC 1106, was observed for PMKT2. Potassium leakage, sodium influx and the
decrease of intracellular pH were not among the primary effects of PMKT2. We report here that this pro-
tein is unable to form ion-permeable channels in liposome membranes, suggesting that channel forma-
tion is not the mechanism of cytotoxic action of PMKT2. Nevertheless, flow cytometry studies have
revealed a cell cycle arrest at an early S-phase with an immature bud and pre-replicated 1n DNA content.
By testing the sensitivity of cells arrested at different stages in the cell cycle, we hoped to identify the
execution point for lethality more precisely. Cells arrested at the G1-phase by a-factor or arrested at
G2-phase by the spindle poison methyl benzimidazol-2-yl-carbamate (MBC) were protected against
the toxin. Cells released from the arrest in both cases were killed by PMKT2 at a similar rate. Neverthe-
less, cells released from MBC-arrest were able to grow for a short time, and then viability dropped rapidly.
These findings suggest that cells released from G2-phase are initially able to divide, but die in the pres-
ence of PMKT2 after initiating the S-phase in a new cycle, adopting a terminal phenotype within that
By contrast, low doses of PMKT and PMKT2 were able to generate the same cellular response. The evi-
dence presented here shows that treating yeast with low doses of PMKT2 leads to the typical membra-
nous, cytoplasmic, mitochondrial and nuclear markers of apoptosis, namely, the production of reactive
oxygen species, DNA strand breaks, metacaspase activation and cytochrome c release.
? 2012 Elsevier Inc. All rights reserved.
Yeast strains often achieve competitive advantage by producing
killer toxins, which kill off competing sensitive cells belonging to
either the same or a different species (Young, 1987; Ciani and
Fatichenti, 2001). The production of killer toxins is typically asso-
ciated with the secretion of protein toxins that kill susceptible
yeast cells in a two-step receptor-mediated fashion. The most thor-
oughly studied examples are the Saccharomyces cerevisiae toxins
K1, K2 and K28. Producers of these toxins are able to kill each other
but are immune to killer toxins of their own class.
antimicrobial activity that are currently known,including killer tox-
ins, only a minor proportion of them have been studied in detail in
relation to their mechanism of action (López-García et al., 2010).
Detailed knowledge of the mode of action of killer toxins is critical
forsupporting their potential application to biotechnological,thera-
peutic or agronomic fields. Killer toxins are a widespread phenome-
mode of action of killer toxins also being found. Several killer toxins
seem to be pore-forming related toxins (Breinig et al., 2002; Santos
and Marquina, 2004b), others have been found to block completion
of the cell cycle (Schmitt et al., 1996) and, finally, additional mecha-
nisms have been proposed for other killer toxins. For example,
HM-1,fromHansenulamrakii, kills sensitivecellsby interferingwith
b-(1 ? 3)-glucan synthesis (Takasuka et al., 1995), etc. Concerning
cell cycle interference by killer toxins, several examples have been
reported. K28 toxin, of S. cerevisiae, interferes with DNA synthesis
and Wingea robertsiae toxins provoke S-phase arrest and concomi-
tant activation of the intra-S-phase DNA damage checkpoint
(Klassen et al., 2004), or Kluyveromyces lactis zymocin, which is
responsible for cell cycle arrest in G1 (Tokunaga et al., 1989; Butler
et al., 1991, 1994; Studte et al., 2008). Nevertheless, recent evidence
1087-1845/$ - see front matter ? 2012 Elsevier Inc. All rights reserved.
⇑Corresponding author. Address: Department of Microbiology III, Faculty of
Biology, Complutense University of Madrid, Calle José Antonio Nováis, 28040
Madrid, Spain. Fax: +34 913 944 964.
E-mail address: firstname.lastname@example.org (D. Marquina).
Fungal Genetics and Biology 50 (2013) 44–54
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variable/alternative mechanismsdepending on toxin concentration.
Ionophoric mechanisms – those that disrupt the cytoplasmic mem-
brane function – have beencorrelated with high killer toxinconcen-
trations, even though small concentrations have been correlated
with apoptosis (Klassen and Meinhardt, 2005; Schmitt and Reiter,
2008; Santos and Marquina, 2011). These moderate-to-low concen-
trations seem to be related to conditions usually found in natural
environments, where the toxin concentration is presumably low
and killer yeast eliminates competitor sensitive yeasts through the
induction of apoptosis.
Within the genus Pichia, which is recognizably heterogeneous
and Robnett, 1998), the following produce different killer toxins: P.
acacie (McCracken et al., 1994; Klassen et al., 2008), P. anomala
(Comitini et al., 2004;Wang et al., 2007; Izgü et al., 2007), P. farinosa
(Suzuki and Nikkuni, 1994), P. inositovora (Klassen and Meinhardt,
2003), P. kluyveri (Middelbeek et al., 1979) and Pichia membranifac-
iens (Santos et al., 2000, 2009). In the same way, killer toxins from
the genus Pichia have heterogeneous activity mechanisms (Izgü
et al., 2007; Klassen et al., 2008; Middelbeek et al., 1979; Pfeiffer
and Radler, 1984; Santos and Marquina, 2004a, 2011).
In previous work, Marquina et al. (1992) found that P. membra-
nifaciens, the dominant yeast species isolated from spontaneously
fermenting olive brines, had a particularly strong, broad-spectrum
killer activity. P. membranifaciens produces two different killer tox-
ins (PMKT and PMKT2) that are active on spoilage yeasts and fungi
(Santos and Marquina, 2004a, 2011; Santos et al., 2005, 2009). Pre-
vious biochemical studies indicate that PMKT and PMKT2 are,
respectively, 18-kDa and 30-kDa proteins that interact with the cell
wall. PMKT was found to have affinity for (1 ? 6)-b-D-glucans,
whereas PMKT2 was adsorbed by cell wall mannoproteins, suggest-
ing the presence of two different primary receptors for these toxins
(Santos et al., 2000, 2009, 2007). We now know that PMKT acts by
disrupting plasma membrane electrochemical gradients leading to
the death of sensitive cells (Santos and Marquina, 2004b). By
exploring the transcriptional responses of S. cerevisiae to high doses
of PMKT, it was corroborated that the cellular response is related to
changes in ionic homeostasis with an activation of the High
Osmolarity Glycerol (HOG) signaling pathway (Santos et al., 2005;
Lelandais and Devaux, 2010). By contrast, low PMKT doses lead to
a cell death process accompanied by cytological and biochemical
indicators of apoptotic programmed cell death, and the global gene
expression response during that stimulus indicates that genes re-
lated to an oxidative stress response were induced, showing that
the coordinated transcriptional response is not consistent with that
obtained when ionophoric concentrations of PMKT are used.
On the other hand, despite all this knowledge concerning the
cellular mechanisms underlying the killing action of PMKT, nothing
is so far known about the mechanism of action of PMKT2. In light of
the above, the aim of this study was to gain insight into the molec-
ular events underpinning the killing action of PMKT2 and compare
them with the ionophoric events caused by PMKT – the most stud-
ied killer toxin from P. membranifaciens. We describe how the kill-
ing of S. cerevisiae cells may be expressed as two independent
mechanisms depending on toxin concentration. High PMKT2 doses
were observed to induce the cell cycle arrest of sensitive cells. By
contrast, low doses of PMKT2, as those described for PMKT, lead
to a cell death process in S. cerevisiae accompanied by cytological
and biochemical indicators of apoptotic programmed cell death.
2. Experimental procedure
2.1. Strains and general media
CYC 1086 (Complutense Yeast Collection, Complutense University
of Madrid, Spain), originally isolated from olive brines (Marquina
et al., 1992) and identified according to conventional methods
used in yeast taxonomy (Kurtzman and Fell, 1998). The killer toxin
from P. membranifaciens CYC 1086 (named PMKT2) is compared
here with PMKT obtained from P. membranifaciens CYC 1106
wild-type strain used in this study was S. cerevisiae Hansen
BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0). The strains were
maintained on agar slants containing YMA at 20 ?C (Santos and
For killer toxin production, P. membranifaciens CYC 1086 was
cultured in a buffered YMB medium (YMA without agar). The med-
ium was buffered to pH 4.5 with 0.1 M sodium citrate/phosphate.
Killer activity was determined using purified PMKT2 extracts by
a diffusion test (Woods and Bevan, 1968) on YMAMB plates
(Llorente et al., 1997) seeded with S. cerevisiae Hansen BY4741.
Following the incubation of the YMAMB plates for 3–4 days at
20 ?C, the diameter of the inhibition zone was considered to reflect
yeast killer activity under the conditions tested. Killer toxin activ-
ity was expressed in arbitrary units (AUs) (Santos et al., 2000).
2.2. PMKT2 production and purification
PMKT2 production and purification were performed according
to Santos et al., 2009, with slight modifications. Killer yeast was
cultivated for 4 days at 20 ?C, 150 rpm in 2-l Erlenmeyer flasks
with 1 l of buffered YMB. After centrifugation (5000g, 5 min, 4 ?C)
the supernatant was adjusted to a final glycerol concentration of
15% (vol/vol) and concentrated to a volume of approximately
100 ml bytangential ultrafiltration
membrane (Minisette membrane cassette, Omega-type, Filtron
Technology Corporation). Ice-cold ethanol was slowly added to a
final concentration of 45% (v/v) and, following 20 min incubation
at 0–4 ?C, centrifuged (8000g, 10 min, 0 ?C). The protein precipitate
was discarded and the proteins remaining in the solution were
subsequently precipitated by further addition of ice-cold ethanol
to a final concentration of 75% (v/v). The resulting pellet was dis-
solved in 1 mM sodium citrate/phosphate buffer (pH 4.0), and
the solution was used as killer toxin concentrate. All the steps for
subsequent purification were followed according to Santos et al.
(2009). Killer activity was determined as above and protein was
determined by the Bradford method.
2.3. Measurement of cell death
Cells of S. cerevisiae BY4741 were grown to logarithmic phase in
buffered YMB medium, collected, and resuspended in the same
medium containing killer activity (200 and 2000 AU/ml) to reach
a final cell concentration of around 2 ? 106cells/ml. A control with
inactivated killer toxin (5 min, 75 ?C) was run in parallel. Aliquots
were taken periodically, and further 10-fold dilutions were made
serially to a final dilution of 10?4. Volumes of 50 ll were used
for plating on YMA medium. The resulting colonies were counted
after 48 h incubation at 30 ?C (Fig. 1). Experiments were done by
2.4. Determination of plasma membrane integrity
Propidium iodide was used to determine membrane integrity.
Sensitive cells of S. cerevisiae were grown for 12 h at 20 ?C in buf-
fered YMB medium. The cells obtained were then resuspended in
the same medium to obtain a final cellular concentration of around
2 ? 106cells/ml in the presence of PMKT2 (200 and 2000 AU/ml) or
inactive killer toxin. Samples of 0.5 ml were taken at 1 h intervals
and 50 ll of a propidium iodide (PI) stock solution (50 lg/ml) was
added. After 1 min incubation, cells were measured in a counting
A. Santos et al./Fungal Genetics and Biology 50 (2013) 44–54
chamber (Fig. 1). An Olympus BX61 epifluorescence microscope
was used with a 12.5 megapixel cooled CCD camera (Olympus
DP71), using software for image archival and management (CellD
2.8, Olympus Software Imaging Solutions). Results were represen-
tative of three independent experiments.
2.5. Measurement of intracellular pH (pHi)
A BD FACSCalibur flow cytometer apparatus was used for flow
cytometry. A pH-sensitive fluorochrome SNARF-1-AM (semina-
phtorhodafluor-1-acetoxymethylester) (invitrogen) was used to
measure the internal pH changes (Fig. 2) of sensitive cells by flow
cytometry in the presence of 2000 AU/ml PMKT2 or with heat inac-
tivated killer toxin (Santos and Marquina, 2004b). Results were
representative of three independent experiments.
2.6. Determination of intracellular potassium and sodium
The amount of potassium and sodium was measured according
to the method developed by Middelbeek et al. (1979). 2000 AU/ml
of PMKT2, suspended in buffered YMB medium, was added to a cell
suspension (2 ? 106cells/ml), grown in buffered YMB medium.
Cells were incubated at 20 ?C and samples were taken at 1 h inter-
vals. After incubation, 2 ml samples were filtered by suction on
0.45 lm Millipore filters. The filters were washed twice with ice-
cold distilled water and frozen in liquid nitrogen for 5 min. They
were then placed in 2 ml boiling distilled water (heated for 30 s
at 100 ?C). Debris was removed by centrifugation (10,000g, 4 ?C)
and the sodium and potassium content was determined with a
Perking Elmer 3100 flame spectrophotometer (emission wave-
lengths: kNa+589 nm; kK+766.5 nm). Experiments were done by
triplicate (Fig. 2).
2.7. Patch-clamp techniques on liposomes
Liposomes and PMKT2-containing liposomes were obtained by
ultrasonication and then freeze–thawing in a lipid/water mixture,
as described by Santos and Marquina (2004b). Conventional
patch-clamp techniques were used for the determination of con-
ductivity (Hamill et al., 1981; Martinac et al., 1990). Recordings
of killer toxin activity were obtained from inside-out membrane
patches from PMKT2-incorporated liposomes. To maintain PMKT2
activity, the bath solution was constantly adjusted to pH 4.5 by
MES (Sigma) and the pipette solution was adjusted to pH 7.0 with
HEPES (Sigma). Digitalization, storage and analysis were as de-
scribed (Santos and Marquina, 2004b). A storage oscilloscope was
used to monitor membrane capacitance and obtain single-channel
recordings (Fig. 3).
2.8. Measurement of cellular DNA contents and cell cycle experiments
Samples of cells (2 ? 106cells/ml) from control (inactive toxin)
and toxin-treated cultures (2000 and 200 AU/ml) were fixed in 70%
(v/v) ethanol (?20 ?C) and stored at ?20 ?C. Each sample was
washed once with PBS, centrifuged, and then resuspended in
1 ml 50 mM Tris–HCl, pH 6.8, containing 1 mg/ml boiled RNAase
A (Sigma). After 2 h of incubation at 37 ?C, cells were centrifuged,
washed with PBS, and then resuspended in 200 ll of the same buf-
fer containing 50 lg/ml propidium iodide (PI). Data were collected
on a linear scale, and the fraction of single cells was determined by
forward-scatter and FL2 fluorescence. Under these conditions, fluo-
rescence is considered proportional to DNA content (Figs. 4 and 5).
Cell cycle experiments were conducted to map more precisely
the point in the cell cycle at which PMKT2 toxin has its lethal ef-
fect. Due to the temperature sensitivity of PMKT2, we were unable
to use temperature-sensitive cdc mutants, so we used metabolic
inhibitors instead. A culture grown overnight in buffered YMB
was refreshed to an OD600of 0.5 with pre-warmed buffered YMB
medium and grown for 2 h at 20 ?C in 250 ml Erlenmeyer flasks,
each containing 100 ml medium. This initial culture was divided
into six flasks. The cells growing in three flasks were arrested at
START by treatment with a-factor (Nova Biochem, Switzerland),
which was added to a concentration of 1 lM. The other three flasks
were treated to arrest cells at the G2-phase by addition of the drug
methyl benzimidazol-2-yl-carbamate (MBC) at 50 lg/ml.
After 3 h incubation, the medium and a-factor/MBC were re-
moved from two flasks by washing the cells with fresh buffered
YMB after centrifuging twice at 5000 rpm for 5 min. Finally, the
cells were resuspended in 10 ml fresh buffered YMB and then inoc-
ulated to 90 ml in pre-warmed buffered YMB to let them grow
without a-factor/MBC arrest (Fig.
(Fig. 4d) were not centrifuged, so a-factor/MBC were not removed,
and after 3 h they were supplemented with PMKT2 (2000 AU/ml).
After 3 h, the last two flasks, were washed by centrifugation to re-
move a-factor or MBC, and immediately supplemented with
2000 AU/ml PMKT2 (Fig. 4e). Aliquots were taken periodically
throughout all the experiments, and further 10-fold dilutions were
Fig. 1. Effect of PMKT2 on the viability of sensitive cells. Killer toxin-treated cells were exposed to 2000 AU/ml (A) and 200 AU/ml (B) of PMKT2 and viability (-j-) and
permeability to propidium iodide (-s-) were determined. Results were representative of three independent experiments.
A. Santos et al./Fungal Genetics and Biology 50 (2013) 44–54
made serially to a final dilution of 10?4. Volumes of 50 ll were
used for plating on YMA medium (pH 6.7). The resulting colonies
were counted after 48 h incubation at 30 ?C, with these conditions
rapidly inactivating any free toxin or toxin bound to the cell walls.
Experiments were done by triplicate.
2.9. Determination of apoptosis by TUNEL
S. cerevisiae BY4741 cells were grown at 20 ?C in buffered YMB
medium, collected, and subsequently resuspended and adjusted
to 2 ? 106cells/ml in the same medium containing PMKT2 (200
and 2000 AU/ml). A control with heat inactivated PMKT2 was run
in parallel. Aliquots were taken at appropriate intervals for a total
periodof 180 min. The proportionsof apoptoticcells in treatedpop-
ulations were ascertained by TUNEL (Terminal Deoxynucleotidyl
Transferase-mediated dUTP Nick End Labeling) assays, essentially
as previously published (Santos and Marquina, 2011). Sensitive
yeast cells were fixed with 3.7% (v/v) formaldehyde, and the cell
wall was digested with lyticase (Ludovico et al., 2001). Cells were
applied to poly-L-lysine coated slides, which were then rinsed with
PBS, incubated in a permeabilization solution (0.1% (v/v) Triton
X-100 and 0.1% (w/v) sodium citrate) for 2 min in ice, rinsed twice
with PBS and incubated with a 10 ll TUNEL reaction mixture
(terminal deoxynucleotidyl transferase 200 U/ml, FITC-labeled
dUTP 10 mM, 25 mM Tris–HCl, 200 mM sodium cacodylate and
5 mM cobalt chloride) for 60 min at 37 ?C. Finally the slides were
rinsed three times with PBS, and a coverslip was mounted with a
drop of Vectashield anti-fading agent (Vector Laboratories). Micro-
scope and image acquisitions were performed under an Olympus
BX61 epifluorescence microscope and digital images were acquired
with a 12.5 megapixel cooled CCD camera (Olympus DP71), using
software for image archival and management (CellD 2.8, Olympus
Software Imaging Solutions). Results were representative of three
2.10. DNA ladder assay. Pulsed field gel electrophoresis
The methods used to prepare cells and agarose plugs were fol-
lowed according to Ribeiro et al. (2006). Control cells (inactive tox-
in) and cells treated for 1–4 h with PMKT2 (2000 and 200 AU/ml)
were analyzed (Fig. 6).
Fig. 2. Physiological effects of high doses (2000 AU/ml) of PMKT2 on S. cerevisiae BY4741 cells. (A) Changes induced on pHiin PMKT2 treated cells (-j-) and cells treated with
inactivated PMKT2 (-h-). (B) Evolution of intracellular concentrations of sodium in treated (-s-) and untreated cells (-.-). (C) Evolution of intracellular concentrations of
potassium in treated (-j-) and untreated cells (-h-). Results were representative of three independent experiments.
Fig. 3. Conductance fluctuations of patches from liposomes due to the presence of
PMKT2. Current activity observed in the presence of inactive PMKT2-incorporating
liposomes (A) or active PMKT2-incorporating liposomes (B). The same was observed
in the absence of killer toxin. Results were representative of three independent
A. Santos et al./Fungal Genetics and Biology 50 (2013) 44–54
2.11. Determination of superoxide production by dihydroethidium
We monitored the red-DHE-derived fluorescence, which signals
the hydroxyethidium forthcoming from the reaction with the
superoxide produced by yeast cells (Peshavariya et al., 2007). The
protocol was developed as described by Santos and Marquina
(2011). Initially, sensitive cells were loaded with 10 lg/ml of
DHE for 60 min at 28 ?C. After loading with the probe, cells were
transferred to buffered YMB with PMKT2 (200 and 2000 AU/ml).
Fig. 4. High doses of PMKT2 induced cell cycle arrest at G1/S. Flow cytometry analysis of DNA content of S. cerevisiae 4741 revealed that cells treated for different time periods
with PMKT2 (A. 2000 AU/ml, B. Control) induced cell cycle arrest at an early S phase with a small-sized bud. In order to map more precisely the point in the cell cycle at which
PMKT2 toxin exhibits its lethal effect, we used sensitive cells that had been arrested with different drugs. A culture of exponentially growing cells at 20 ?C in buffered YMB
was divided into six at zero time and treated for 3 h with two different cell cycle inhibitors, a-factor (-j-) or methyl benzimidazol-2-yl-carbamate (MBC, -s-). (C) As
expected, cells arrested by a-factor or MBC were unable to growth until drugs were removed by washing. (D) Cells exposed to a-factor and MBC remained fully viable after
exposure to PMKT2 (2000 AU/ml). (E) Arrested cells recovered their sensitivity to PMKT2 when the cell cycle continued by washing out cell cycle drugs. MBC arrested cells
were able to grow, but only for a short time, the cells then died at the same rate as a-factor-treated cells.
Fig. 5. Apoptosis in S. cerevisiae BY4741 after different PMKT2 treatments. (A) Change in percentage of TUNEL positive cells over time after treatment with PMKT2: 200 (-s-)
and 2000 AU/ml (-j-). High PMKT2 doses did not cause the accumulation of TUNEL positive cells. Cells were treated with PMKT2 (B. 200 AU/ml) and the DNA content was
analyzed by flow cytometry after incubation for 2 h. The presence of low PMKT2 concentrations induced apoptosis that was observed by the accumulation of sub-G1 DNA
contents in cells. The control cells were treated with inactivated killer toxin (C). Results were representative of three independent experiments.
A. Santos et al./Fungal Genetics and Biology 50 (2013) 44–54
ROS production was monitored by epifluorescence microscopy.
Control cells were treated with heat-inactivated-PMKT2. Results
were representative of three independent experiments (Fig. 7b).
2.12. Annexin V staining
We monitored the exposed phosphatidylserine of S. cerevisiae
BY4741 cells (treated with 200 and 2000 AU/ml PMKT2) following
the annexin V labeling-green1derived fluorescence (FITC Annexin V
Apoptosis Detection Kit II, BD Pharmingen), according to the manu-
facturer’sprocedure. Control cells
inactivated-PMKT2. Fluorescence was monitored by epifluorescence
microscopy. Results were representative of three independent
experiments (Fig. 7c).
were treated withheat-
2.13. In vivo detection of metacaspase activation
Activated metacaspases in S. cerevisiae BY4741 cells (treated
with 200 and 2000 AU/ml PMKT2) were detected by epifluores-
cence microscopy using a FAM-FLICA™ apoptosis detection kit
(Poly Caspases Assay Kit, Immunochemistry Technologies), accord-
ing to the manufacturer’s instructions. Control cells were treated
with heat-inactivated-PMKT2. Fluorescence was monitored by epi-
fluorescence microscopy. Results were representative of three
independent experiments (Fig. 7d).
2.14. Cell fractionation and western blotting for cytochrome c
Cell homogenates, and mitochondrial and cytosolic fractions
were isolated from PMKT2 treated (200 and 2000 AU/ml) or un-
treated (heat-inactivated-PMKT2) cells, basically as previously de-
scribed (Santos and Marquina, 2011). Proteins were isolated from
S. cerevisiae BY4741 strain grown in buffered YMB medium after
0, 10, 20, 30 and 45 min of exposure to PMKT2. Cells were incu-
bated with zymolyase 20T to obtain spheroplasts. Spheroplasts
were homogenized on ice by 30 strokes in a tight-fitting Dounce
homogenizer. The homogenate was centrifuged, the supernatant
was stored and the pellet was re-homogenized and centrifuged.
Both supernatants were combined and mitochondria were precip-
itated. The resulting supernatant was used as cytosolic fraction.
The pellet was carefully resuspended, and the suspension was cen-
trifuged to remove residual cell debris. Mitochondria were resus-
pended in 0.6 M mannitol, 10 mM Tris–HCl, pH 7.4, to give an
approximate final concentration of 10 mg of protein/ml.
Thirty micrograms of either cytosolic or mitochondrial proteins
were resolved by using a 12% PAGE–SDS gel, and transferred to a
nitrocellulose membrane that was probed with two different anti-
bodies: polyclonal anti-cytochrome c (Pharmingen, San Diego, CA)
and monoclonal anti-phosphoglycerate kinase (Pgk1p) (invitro-
gen). Immunoblot analysis was performed as described by Bobba
et al. (1999). Densitometric values for immunoreactive bands were
quantified using a GS-800 Imaging Densitometer (Bio-Rad). Protein
normalization was based on the amount of Pgk1p for cytosolic and
mitochondrial fractions in each lane on the same membrane
(Fig. 8). Experiments were done by triplicate.
3. Results and discussion
3.1. Kinetic study of killing
This study shows that treatment with PMKT2 caused cell death
in different ways depending on the PMKT2 concentration. The
binding of toxin to cell wall sites is the first event (1–5 min)
in the action of a killer toxin against whole sensitive cells
(Kurzweilová and Sigler, 1994). In previous work (Santos et al.,
2009), the binding of PMKT2 to its cell wall receptor, mannopro-
teins, occurred in the first 2–3 min after toxin addition. Once the
toxin became bound, the number of viable cells decreased quickly
when 2000 AU/ml were used, with a mortality of approximately
80% being recorded after 5 h, with a cell death rate of 0.17 h?1
(Fig. 1a). Simultaneously, membrane permeability, indicative of
cellular necrosis, was followed by determining the fluorescence
of PMKT2-treated cells labeled with propidium iodide. Interest-
ingly, the increase in the number of cells with plasma membrane
permeabilization was observed only until after more than 4 h of
incubation. By contrast, lower PMKT2 concentrations (200 AU/ml)
were unable to kill sensitive yeast cells during the initial 5 h of
incubation, and plasma membrane permeabilization was observed
in parallel with cell death (Fig. 1b). As indicated earlier, the highest
concentration caused rapid cell death, whereas necrosis was
apparent only after 4–5 h (Fig. 1a). These results showed that
PMKT2 could exert a different mechanism of action depending
on toxin concentration.
3.2. Changes in cellular homeostasis caused by PMKT2
Different studies were conducted to determine the cytocidal ef-
fect of the killer toxin on S. cerevisiae BY4741. Following this ap-
proach, sensitive yeast was treated with high doses (2000 AU/ml)
of PMKT2, as done previously for PMKT (Santos and Marquina,
2004b), and several features were studied to identify a mechanism
related to channel formation and cellular homeostasis disruption.
The effect of intracellular pH on sensitive cells of active and
inactive killer toxin preparations was measured by flow cytometry
Fig. 6. DNA fragmentation assay. Genomic DNA analyzed by PFGE from viable cells
exposed to 200 or 2000 AU/ml PMKT2 for the indicated time intervals. Lanes
marked as control (C) were loaded with DNA isolated from exponentially growing
cells without further treatment. DNA laddering was observed after staining with
GelRed?solution and exposure to UV light. Results were representative of three
1For interpretation of color in Fig. 7, the reader is referred to the web version of
A. Santos et al./Fungal Genetics and Biology 50 (2013) 44–54
(Fig. 2a). The pHiof inactive toxin-treated cells, as well as of those
treated with PMKT2 (2000 AU/ml), remained constant in the 6.3–
6.6 range for 5 h. Thereafter, the pHiof PMKT2-treated cells de-
creased in parallel to the loss of cellular permeability (Fig. 1a). It
should be stressed that the described effect is observed 5 h after
adding the toxin, when the viability of the cells has decreased by
around 85% (Fig. 1a).
Similar results were observed for the intracellular pools of so-
dium and potassium. Actively growing cells took up K+from the
medium and maintained an intracellular pool of the ion. Initially,
the addition of PMKT2 to sensitive cells did not induce a leakage
of K+from the cells (Fig. 2b), recording an intracellular concentra-
tion of potassium of around 175 mM, similar to the figure for con-
trol cells. When sensitive cells were exposed to PMKT2, a marked
leakage of potassium ions out of the cells started 5 h after the addi-
tion of killer toxin, probably indicating that this is not a primary ef-
fect of the killer toxin. The leakage of potassium ran in parallel to
the permeabilization of the cells, but it was not directly associated
with cell death.
We also studied the changes in plasma membrane permeability
to Na+due to the action of PMKT2. The sodium concentration in
cytoplasm was approximately 5 mM in the initial incubation with
PMKT2 or in the control cells. When sensitive cells were exposed to
the killer toxin, a marked flow of sodium ions began 4–5 h after the
addition of the killer toxin (Fig. 2b). In the same way as K+ions, the
rapid intake of sodium ions was directly associated with the in-
creased permeability of the plasma membrane, but not with kill-
ing. Cells treated with inactivated killer toxin (control) showed
no changes in the intracellular pools of sodium and potassium dur-
Fig. 1a shows the changes in permeability to propidium iodide
in the presence of 2000 AU/ml of the active killer toxin. In the ini-
tial steps of toxin action, the physical integrity of the plasma mem-
brane was not affected by the addition of killer toxin. The presence
of changes in cell integrity (permeability to propidium iodide) was
detected only when the killing process was clearly advanced in
time. According to these results, these changes were probably a
late consequence of the cell death induced by PMKT2.
In conclusion, these findings indicated that PMKT2 has a mech-
anism that is not related to other ionophoric toxins (Martinac et al.,
1990; Kagan, 1983). These authors discovered that the ion chan-
nels formed in yeast spheroplasts and artificial liposomes by S.
cerevisiae K1 killer toxin do not discriminate between K+and Na+.
It has been reported that killer toxins induce the formation of
ion-permeable channels in lipid bilayer membranes (Kagan,
1983). According to the results shown in Figs. 1 and 2, these events
(pH acidification and ion movements) would not be due to the di-
rect effects of the killer toxin in the plasma membrane and are
probably a late consequence of the cell death induced by PMKT2.
At this stage in the research, it was obvious that PMKT2 and PMKT
were different in their mechanism of action, so an additional
experimental approach, based on patch-clamped liposomes, was
conducted to show that both toxins are unrelated as regard their
killing mechanism. The conductance of PMKT2 containing lipo-
somes was studied by the patch-clamp technique. One hundred
patches were tested in each experiment. We observed that purified
PMKT2 did not cause ionic leakage or channel formation at concen-
trations comparable to those at which it exerts antimycotic effects.
‘Staircase laddering’, a characteristic of channels induced by larger
microbicidal proteins, such as colicins and PMKT, was not observed
(Kagan, 1983; Santos and Marquina, 2004b). Liposomes, to which
PMKT2 had been incorporated at acidic pH, yielded patches with-
out a characteristic channel activity (Fig. 3a). Additionally, none
of the patches excised from liposomes with incorporated heat-
inactivated-killer toxin or without killer toxin was found to have
channel activity (Fig. 3b). These observations were reinforced by
the fact the presence of PMKT2 in the bath solution (pH 4.5) was
also unable to induce conductance on non-incorporated-PMKT2
liposomes. These results were different to those reported for PMKT
which is able to form non-selective channels in liposome mem-
branes (Santos and Marquina, 2004b). These channels are freely
permeable to common physiological ions suggesting that channel
formation is the cytotoxic mechanism of action of P. membranifac-
iens killer toxin through a discharge of cellular membrane potential
and changes in ionic homeostasis.
All together, these results indicate that PMKT2 has a mechanism
of action that is not related to the interference of the plasma mem-
brane and cellular homeostasis. The enhancement of membrane
permeability to sodium, potassium and protons after 4–5 h incuba-
tion could be considered indirect effects of PMKT2 activity, as these
effects did not occur prior to or concomitantly with cell death, indi-
cating that they were not primary effects. A plausible explanation
could be that when a cell is finally de-energized, by different mech-
anisms, any metabolite or ion accumulated in the cell against its
concentration gradient will tend towards equilibrium, and a flow
would be observed (i.e., K+, Na+, H+) (de la Peña et al., 1980; Suzuki
and Shimma, 1999). This is consistent with the notion that the late
intracellular acidification, potassium and sodium fluxes on sensi-
tive cells were concomitantly observed 4–5 h after toxin addition,
rather than permeability to propidium iodide. The reason so many
dead cells remain nonpermeabilized for a long time in the presence
of PMKT2 was determined after the cell cycle studies were con-
ducted. There might be a period of time during which the cell cycle
arrest initiated by the killer toxin does not cause plasma mem-
brane damage. After a prolonged time, the arrested cells were ren-
dered unviable and permeabilization occurred. In conclusion,
PMKT2 is not related to the action of other killer toxins described
previously, such as K1 from S. cerevisiae (de la Peña et al., 1981;
Martinac et al., 1990; Skipper and Bussey, 1977), P. kluyveri
(Kagan, 1983) or even PMKT, which is characterized by the forma-
tion of non-selective and poorly regulated channels (Santos and
Furthermore, effects on cellular morphology were detected
when sensitive cells were observed under a microscope or when
light scatter (forward scatter and side scatter) was analyzed using
flow cytometry. The addition of 2000 AU/ml of PMKT2 generated
the accumulation of unbudded cells or cells with a small bud,
indicating that PMKT2 was probably responsible for the cell cycle
arrest (Fig. 4). The following investigations were conducted to con-
firm this possibility.
3.3. Mechanism of action at high doses of PMKT2: Cell cycle inhibition
Some killer toxins exert their killing action by blocking the cell
cycle of sensitive cells (i.e., K. lactis killer toxin and K28). In previ-
ous investigations, we concluded that the killer toxin from P.
membranifaciens CYC 1106 (PMKT) does not lead to an accumula-
tion of cells with unreplicated DNA. As stated above, PMKT2 seems
to be different, so we therefore measured DNA content in toxin-
treated and untreated sensitive cells by flow cytometry analysis
of cells stained with propidium iodide (Fig. 4). Control cells with-
out toxin recorded the two peaks characteristic of a mixture of
pre-S-phase cells with a single DNA content (1n) and post-S-phase
cells with a DNA content of 2n, with the saddle representing cells
in S (Fig. 4b). This pattern changed slightly in the exponential
growth phase (0–12 h of culture growth). Only one population
(pre-S phase peak) was observed in the stationary phase of growth
after 24 h incubation (not shown). In contrast, cells treated with
toxin for 7 h lacked the second (2n) DNA peak, indicating that
PMKT2 toxin leads to an accumulation of yeast cells with unrepli-
cated chromosomal DNA (Fig. 4a). This result could indicate that if
PMKT2 inhibits DNA synthesis in sensitive cells while allowing
A. Santos et al./Fungal Genetics and Biology 50 (2013) 44–54
cells to progress through the cell cycle, asynchronous exponential-
phase cells exposed to toxin should accumulate with unreplicated
In order to map more precisely the point in the cell cycle at
which PMKT2 toxin exhibits its lethal effect, we used sensitive cells
that had been arrested at START in G1 by a-factor treatment or at
the transitional G2-phase by adding MBC. We added a-factor or
MBC for 3 h, and studied what had happened to the cells in the
presence or absence of PMKT2 simultaneously with these drugs
or after being released from arrest. As shown in Fig. 4c, cell division
was arrested after the addition of a-factor or MBC, with rapid inhi-
bition of cellular growth. In the absence of PMKT2, when a-factor
or MBC were removed after 3 h, cell growth restarted at the origi-
nal rate (Fig. 4c).
When lethal concentrations (2000 AU/ml) of PMKT2 were
added to G1- or G2-arrested cells in the presence of a-factor or
MBC, respectively, cells did not grow but remained fully viable,
indicating they were unaffected by PMKT2 in these stages of the
cell cycle (Fig. 4d). Interestingly, when arrested cells were washed
(to avoid the presence of a-factor or MBC) and then treated with
PMKT2 (Fig. 4e), viability dropped rapidly both in cells released
from a-factor and in unarrested cells (Fig. 1a). By contrast, cells ar-
rested with MBC started to grow again at the original rate, but only
for a short time, then viability dropped rapidly. These findings indi-
cated that cells released from G2-phase are initially able to divide,
but in the presence of high doses of PMKT2 they die after initiating
S-phase and adopt a terminal phenotype within that cycle. Due to
cell cycle inhibitors were not able to synergize PMKT2 activity con-
tributing with cell cycle arrest could indicate that the cell cycle
inhibition at S-phase, itself, is not the mechanism of action. An un-
known molecular event, directly associated with S-phase, should
be related with the mechanism of action, as it was described for
other toxins such as mycocine (Studte et al., 2008). Sensitive cells
need to pass through the S-phase of the cell cycle to initiate an un-
known molecular process which is interfered by PMKT2. At this
time we are initiating further investigations to determine. The rel-
atively rapid inhibition of DNA synthesis could reflect the inhibi-
tion of some separate event or of some unknown event common
to DNA replication and the completion of the bud cycle (Frohloff
et al., 2001).
3.4. Mechanism of action at low doses of PMKT2: Programmed cell
According to the results shown in Fig. 1, the presence of differ-
ent concentrations of PMKT2 generated different responses in the
sensitive cells, indicating that treatment with PMKT2 caused cell
death in S. cerevisiae in different ways depending on toxin concen-
tration. Once the mechanism of action of PMKT2 at high doses had
been studied, we sought to explain the results observed in Fig. 1b
at low doses. Initially, to gain a greater understanding of the tem-
poral progression of cell death in S. cerevisiae during PMKT2 expo-
sure at low doses, the cells were examined at a range of time points
for overall viability versus the ability to exclude propidium iodide
(Fig. 1b) and the generation of double-stranded DNA breaks (TUN-
EL assay) (Figs. 5a and 7a). Necrotic cells are usually defined by the
lack of integrity of the plasma membrane (usually detected by PI
exclusion), which can be measured by the flow cytometer or visu-
alized under an epifluorescence microscope. In contrast, the plas-
ma membrane of apoptotic cells is initially intact. Our studies
revealed that sensitive cells undergo a programmed cell-death re-
sponse when treated with low doses (200 AU/ml) of PMKT2,
whereas they did not with higher doses. Exposure to low doses
led to an increased proportion of apoptotic cells (TUNEL-positive)
ranging from 10% after the first hour of incubation with PMKT2
to 95% after 3 h of incubation (Fig. 5a). The presence of a basal level
(2–3%) of TUNEL-positive cells was determined for the highest con-
centrations (2000 AU/ml), similar to the level of TUNEL-positive
cells observed for control cells (not shown).
As stated above, cells treated for 180 min with low doses of
PMKT2 (200 AU/ml)were predominantly
(Fig. 5a). However, by the 180 min time point, only 2% of the cells
were necrotic (PI permeable, Fig. 1b). Additional incubation meant
that apoptotic cells became more permeable to DNA-binding dyes,
such as PI. The data in Fig. 1a show how apoptotic cells are first not
stained by PI and then, as the plasma membrane deteriorates, they
take up increasing amounts of PI. In conclusion, as a result of these
investigations, low doses of PMKT2 appeared to initiate a pro-
grammed cell death response, die in an apoptotic manner, and then
proceed to become necrotic as a secondary consequence.
Yeast apoptosis has previously been shown to include cleavage
of chromosomal DNA, which can be detected by a TUNEL assay
(Madeo et al., 1999). At a late stage in the apoptotic cascade, endo-
nucleases break the linkers between the nucleosomes – one of the
units of chromatin organization. Consequently, large numbers of
small fragments of DNA accumulate in the cell. If cells are fixed in
ethanol and subsequently rehydrated, some of the lower molecular
weight DNA leaches out, lowering the DNA content. These cells can
be observed as a hypodiploid or ‘sub-G1’ peak in a DNA histogram
(Gong et al., 1994). This method was used to estimate the percent-
age of apoptotic cells after PMKT2 treatment, showing a clear sub-
G1 peak (Fig. 5b) and confirming the presence of a programmed cell
death response in cells treated with 200 AU/ml of PMKT2. No such
sub-G1 peak was observed in control cells (Fig. 5c).
The results obtained by flow cytometry were compared to the
classic method of DNA ladder formation by gel electrophoresis. In
the DNA ladder formation assay, DNA fragmentation was observed
following 2 h incubation with PMKT2 (Fig. 6). It is evident from the
images showing DNA damage that although the chromosomes are
broken down to the point of being slightly visible, confirming
apoptosis, the resulting fragments are still several hundreds of
kilobases and do not seem to break down into smaller fragments,
as indicated by the sub-G1 peak observed by flow cytometry. These
results could be contradictory, even though both indicated the
presence of apoptosis, because of the different sizes of DNA frag-
ments shown by both techniques. We currently have no explana-
tion for the accounted divergence in these results.
Despite their apparent simplicity, the previous techniques
based on DNA fragmentation have their limitations in sensitivity
and, even more so, in specificity (Negoescu et al., 1998). Accord-
ingly, a further four different markers indicative of processes re-
lated to apoptosis were used: determination of increased ROS
production, phosphatidylserine externalization, presence of meta-
caspase activity and cytochrome c release from mitochondria
(Madeo et al., 1999).
S. cerevisiae has an asymmetric distribution of phospholipids
within the cytoplasmic membrane. An early morphological marker
of apoptosis is the exposure of phosphatidylserine at the outer leaf-
let of the cytoplasmic membrane, which is conserved from yeast to
mammalian cells (Cerbón and Calderón, 1991; Martin et al., 1995;
Madeo et al., 1999). In yeast, phosphatidylserine can be detected
by FITC-labeled annexin V staining upon cell wall digestion. During
apoptosis, phosphatidylserine (PS) residues, which are normally lo-
cated on the internal surface of the plasma membrane, relocate to
the external surface. PS binds the protein, annexin V, and the
change can be observed by incubating unfixed cells with labeled
annexin V (van Engeland et al., 1998). As shown in Fig. 7, cells ex-
posed to PMKT2 had strong fluorescence around the whole circum-
ference of the cell upon staining with annexin V, indicating
phosphatidylserine externalization. Our results show that more
than 90% of sensitive yeast cells were FITC-annexin V-positive
when treated with 200 AU/ml of PMKT for 1 h (Fig. 7c). Considering
A. Santos et al./Fungal Genetics and Biology 50 (2013) 44–54
that annexin V is an early apoptotic marker, these results therefore
suggest that PMKT2 induced early apoptosis in S. cerevisiae cells.
Green-stained cells were not detected in control assays performed
with heat-inactivated-PMKT2 (data not shown).
According to the above, metacaspase activation was detected in
cells treated with low doses of PMKT2. Metacaspases, which are
well-known apoptogenic factors of yeast, are typically activated
in the early stages of apoptosis and play a central role in the apop-
totic signaling network (Silva et al., 2005; Mazzoni and Falcone,
2008). To further characterize the apoptotic events induced by
PMKT2, we monitored metacaspase activity using FAM-FLICA™
probes. The result indicated that PMKT2- caused metacaspase acti-
vation, one of the important features of apoptosis, in S. cerevisiae.
Cells exposed to PMKT2 were significantly fluoresced green
(Fig. 7d), indicating metacaspase activation in the majority of the
cells. No fluorescence was observed in the control cells (data not
shown). Based on previous reports (Reiter et al., 2005; Silva
et al., 2005; Mazzoni and Falcone, 2008) it is likely that apoptosis
is dependent on metacaspases, but the causal relationship between
metacaspase activation by PMKT2 and apoptosis is not confirmed
by the data presented in this manuscript.
Furthermore, it is also known that in certain apoptotic settings,
cytochrome c (cyt c) relocates from inside the mitochondria to the
cytosol leading to the activation of a downstream caspase cascade.
In this study, we also studied the release of cyt c from mitochondria
as a central step in the initiation of caspase-dependent apoptosis.
Western blotting results indicate that the presence of cyt c in
cytosol was significantly increased in treated cells, showing that
low doses of PMKT2 also induced the release of cyt c from
mitochondria in S. cerevisiae (Fig. 8a). The release of cyt c from
mitochondria was detected 15 min after incubation with the toxin,
indicating that this is the first event related to apoptosis detected in
these investigations. By contrast, when high doses (2000 AU/ml)
were used, no cyt c was observed in the cytoplasm, indicating a
different mechanism of action to that observed at the high doses
causing cell cycle arrest (Fig. 8b).
tion with dihydroethidium, which is a red-fluorescent probe that
specifically detects superoxide anions (Fig. 7b). ROS, especially
superoxide anions, hydrogen peroxide and hydroxyl radicals, are
ions, which are relatively stable intermediates, are the precursors of
most ROS and mediate in oxidative chain reactions, so the produc-
tion of superoxide anions was determined in this study (Benaroudj
et al., 2001). A 90–95% of the cells grown for 2 h on buffered YMB
medium in the presence of PMKT2 (200 AU/ml) showed strong red
that the accumulated ROS, specifically the strong oxidant superox-
ide anion, may play a key role in PMKT2-induced apoptosis in
S. cerevisiae. Taken together, it seems likely that the accumulated
intracellular ROS at PMKT2-induced apoptosis, which are important
regulators of apoptosis, appear to contribute significantly to a mito-
chondria-mediated apoptotic pathway in S. cerevisiae.
The survival of yeast in the wild depends on its ability to cope
with dramatic physico-chemical changes, and the presence of tox-
ins, heavy metals or xenobiotics, etc. The important role of ROS in
the regulation of apoptosis may provide an explanation for the ori-
gin and primary function of the programmed suicide process in
killer toxin treated cells (Carmona-Gutierrez et al., 2010; Hwang
et al., 2011). As ROS are highly reactive and modify biomolecules,
ROS-induced cell damage is a frequent event. Cells have developed
mechanisms to repair oxidative damage. For a monoclonal
Fig. 7. Apoptotic markers observed in sensitive cells treated with PMKT2 (200 AU/ml) for 2 h. Micrographs show apoptotic cells by TUNEL-FITC (A), ROS production detected
by dihydroethidium (B), exposed phosphatidylserine detected by annexin V labeling (C) and metacaspase activation in S. cerevisiae cells detected using FAM-FLICA™ (D).
Control cells and cells treated with PMKT2 (2000 AU/ml) are not shown due to negative responses to these labeling techniques.
A. Santos et al./Fungal Genetics and Biology 50 (2013) 44–54
population of cells, it may be advantageous to reserve most of the
dwindling resources for a few healthy cells, rather than waste
these resources on potentially ROS-damaged cells or ones that
have a reduced chance of survival. Apoptosis may be an important
mechanism for yeast to adapt to these adverse environmental con-
ditions in a manner that ensures survival of the clone. Better
adapted cells are able to survive longer with substances released
by dying cells (Gourlay et al., 2006).
In previously published results (Santos and Marquina, 2011), it
was reported that PMKT poses a serious threat to cell survival by
inducing a programmed cell death in a manner akin to the stress
induced by certain oxidative agents. The results obtained from
DNA microarrays indicated that genes related with an oxidative
stress response were induced in response to proapoptotic concen-
trations of PMKT, showing that the coordinated transcriptional re-
sponse is not coincident with that obtained when ionophoric
concentrations of PMKT are used. As such, the PMKT-induced
stress response required the simultaneous expression of several
genes of the sensitive yeast. It remains to be understood why
PMKT2 treated cells, as well as PMKT, alter the oxidative homeo-
stasis of yeast cells, causing the programmed death of sensitive
cells. In sum, treating yeast with low doses of PMKT2 leads to
the typical membranous, cytoplasmic, mitochondrial and nuclear
markers of apoptosis.
Since the initial discovery of toxin-secreting killer strains in
Saccharomyces cerevisiae, ongoing research into this phenomenon
has substantially strengthened our knowledge in different areas of
biology, providing a deeperunderstandingofbasicaspectsofcellular
in particular the PMKT2 toxin. The alternative toxin’s strategy to
finally kill a sensitive cell depending on toxin dose is discussed.
The collective data presented in this study indicate that high
PMKT2 doses induce the cell cycle arrest of sensitive cells, whereas
no ionophoric effects were observed as indicated by different ap-
proaches, including Patch-Clamp techniques used on PMKT2-
containing liposomes. Furthermore, our investigations revealed a
mechanism related to cell cycle arrest at an early S-phase caused
by high PMKT2 doses. The molecular events underlying PMKT2-
cell cycle arrest are until now unknown and it must be considered
that the observed cell cycle arrest could be or not a direct effect of
PMKT2. By contrast, we conclude that induced mortality at low
PMKT2 concentrations is indeed of an apoptotic nature, as de-
scribed for PMKT. The mechanism by which programmed cell
death is induced in response to PMKT2, as well as other killer tox-
ins, is as yet unknown, but will prove to be an exciting area of
study in the future. Further studies are currently under way to ad-
vance knowledge on the properties of PMKT2 in the hope of con-
tributing to the understanding of a killer toxin with potential
biotechnological applications (Santos et al., 2009).
This study was supported by Grants from the Universidad Com-
plutense-Community of Madrid (CCG07-UCM/AGR-2612, GR58/08,
GR35/10-A). We are grateful to the Proteomic and Genomic Centre
of the Autonomous Community of Madrid.
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