Poster Category
4:
Fungal Physiology and Biochemistry
Paraskevi (Vivian) Georgakopoulos,
Robin A. Lockington, Joan M. Kelly
School of Molecular and Biomedical Sciences,
paraskevi.georgakopoulos@adelaide.edu.au
Acetate, in
A.
nidulans,
is a repressing carbon source that leads to similar levels of CreA mediated
repression as glucose. acdX
was identified in a mutation screen in
Aspergillus nidulans
to identify genes involved in acetate repression but not in glucose repression.
The conservation of the amino acid sequence of AcdX of
A.
nidulans
and Spt8 of
Saccharomyces cerevisiae
suggests that the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex may have a role
in acetate repression in
A.
nidulans,
since Spt8 is a component of the SAGA complex.
The SAGA complex is highly conserved from yeast to humans. In yeast it is
involved mostly in the regulation of highly regulated genes that respond to
environmental stresses, such as metabolic starvation, DNA damage and heat. SAGA
in yeast has been shown to have positive and negative functions on
transcription. Bioinformatic analysis indicates that the components of the
SAGA complex are also present in
A.
nidulans.
We report results of experiments undertaken to confirm the existence of the SAGA
complex in
A.
nidulans
and to determine whether AcdX is a component of the complex. The
A. nidulans
homologue
of the
S. cerevisiae
SAGA complex Spt3, designated SptC, was N terminally tagged with the TAP tag to
allow the purification of the SAGA complex. From these results it is
evident that AcdX is a component of a multiprotein complex and that it
co-immunoprecipitates with SptC, providing further evidence that the SAGA
complex exists in
A.
nidulans,
and includes AcdX.
Rosie Bradshaw[1]
Zhilun Feng[1] Arne Schwelm[1] Yongzhi Yang[2]
Shuguang Zhang[1]
Dothistromin is a non-host selective toxin that is toxic to most types of cells.
It is a close chemical relative of the aflatoxins and has similarities in both
genetics and biochemistry to these compounds.
Dothistroma septosporum,
a serious pine needle pathogen, is a prolific producer of dothistromin.
In
planta,
dothistromin that accumulates in necrotic disease lesions is visible due to its
red colouration and gives rise to the common name of ‘red-band needle blight’.
Studies with dothistromin-deficient mutants of
D.
septosporum
revealed that dothistromin is not required for pathogenicity to the susceptible
host
Pinus
radiata.
The current hypothesis is that dothistromin has a role in competition against
other microorganisms that are known to reside in pine needles, and
in vitro
studies with pine needle endophytes support this hypothesis. The purpose
of the current work was to determine how
D.
septosporum
is able to protect itself against its own potent toxin. A gene (dotC)
adjacent to known dothistromin biosynthetic genes is predicted to encode a Major
Facilitator Superfamily (MFS) transporter. To determine whether the DotC protein
is a membrane-bound transporter that can pump dothistromin out of the cell,
dotC-deficient
mutants, complemented mutants and strains containing DotC-GFP fusions were
studied. As predicted,
dotC
mutants secreted less dothistromin than wild type cells and the DotC-GFP fusions
indicated a membrane location. However the results also suggested that
DotC has an important role in regulating dothistromin biosynthesis and that
compartmentalization within the cell may be an important mechanism for
self-protection against dothistromin.
PR4.3
Michael Blatzer,
Schrettl Markus, Hubertus Haas
Virtually all organisms require iron as indispensable cofactor for various
metabolic processes. The opportunistic fungal pathogen
Aspergillus fumigatus
produces two major siderophores (low molecular-mass ferric iron chelators): it
excretes triacetylfusarinine C for iron uptake and accumulates ferricrocin for
intracellular iron storage. Biosynthesis of both triacetylfusarinine C and
ferricrocin has previously been shown to be crucial for virulence of
A.
fumigatus.
Here, we report the functional characterization of a new component of the fungal
siderophore biosynthetic machinery Afu1g04450, termed SidL. SidL is conserved in
siderophore-producing but not non-siderophore producing ascomycetes. The
C-terminal half of SidL shows similarity to acetylases involved in bacterial
siderophore biosynthesis, e.g.
Escherichia coli
IucB (a hydroxylysine acetylase required for aerobactin biosynthesis) and PvdY
(a hydroxyornithine acetylase required for pyoverdin biosynthesis), and the
hydroxyornithine:anhydromevalonyl coenzyme A-transacylase SidF that is essential
for triacetylfusarine C biosynthesis. Deletion of
sidL
in
A.
fumigatus
reduced ferricrocin biosynthesis during iron starvation and blocked ferricirocin
biosynthesis during iron-replete growth. Furthermore,
sidL-deficiency
blocked conidial ferricrocin accumulation under strict iron-replete conditions
but not when mycelia were transferred from iron-depleted to iron-replete
conditions before sporulation. In contrast, SidL-deficiency had no effect on
triacetylfusarinine C production. The expression of
sidL
was affected neither by iron availability nor the iron regulator SreA.
Taken together, these data show that SidL is a constitutively expressed
hydroxyornithine acetylase involved in ferricrocin biosynthesis. Moreover, the
data indicate the existence of a second hydroxyornithine acetylase, the activity
of which is induced by iron starvation. This study identified a novel component
of the fungal siderophore biosynthetic machinery and revealed unexpected
complexity.
Markus Schrettl[2]
Stephen Carberry[1] Kevin Kavanagh[1] Hubertus Haas[2]
Aine Nolan[1] Sean Doyle[1]
1Department
of Biology and National Institute for Cellular Biotechnology, National
University of Ireland Maynooth, Co. Kildare, Ireland, 2Biocenter,
Division of Molecular Biology, Innsbruck Medical University, Austria
markus.schrettl@i-med.ac.at
Gliotoxin, an epipolythiodioxopiperazine (ETP)-type toxin (326 Da) containing an
essential disulphide bridge, plays a major role in mediating the virulence of
the human pathogen,
Aspergillus fumigatus.
Gliotoxin toxicity in mammalian cells is generally enabled by direct
inactivation of essential protein thiols as well as redox cycling, leading to
hydrogen peroxide formation. In
A.
fumigatus,
enzymes involved in gliotoxin biosynthesis are located within a coordinately
expressed, multi-gene cluster. Here we report the functional characterisation of
a putative thioredoxin reductase encoded by
gliT
within this gene cluster. Expression of
gliT
is subject to regulation by the transcriptional activator GliZ and gliotoxin.
Deletion of
gliT
is detrimental for growth only in the presence of exogenously added gliotoxin,
which can be cured by supplementation with reduced glutathione. GliT is
localised in the cytoplasm and in the nucleus. GliT is not essential for
virulence of
A.
fumigatus
in larvae of the greater wax-moth
Galleria
mellonella.
The potential autoprotective role of GliT was investigated further by
heterologous expression of
gliT
in
Aspergillus nidulans.
And indeed, GliT conferred resistance to gliotoxin, making it a valuable tool
for transformation of fungi lacking an ortholog of
gliT.
Roberto do Nascimento Silva[1]
Andrei Stecca Steindorff[1] Cirano José Ulhoa[1] Carlos
Roberto Félix[2]
Trichoderma reesei (Hypocrea jecorina)
is widely used in industry and its potential for use in agriculture as a
biocontrol agent against phytophatogenic fungi has just started. We have
investigated the involvement of G proteins during mycoparasitism against plant
pathogens. Here we described the role of GNA1, a G-alpha protein which belongs
to alfai group in Cell Wall Degrading Enzymes (CWDEs) production by
T. reesei
during antagonism against
Pythium
ultimum.
For that, two mutants were used: Δgna1
and
gna1QL
(constitutively activated version of GNA1). The
gna1QL
mutant,
like
the parental TU-6, inhibited the growth of
P.
ultimum
in plate confrontation assay and grew faster than the parental TU-6 while the Δgna1
did not grow over
P.
ultimum.
Scanning electron microscopy showed that the
gna1QL
mutant promoted more morphological alterations of
P.
ultimum
cell wall than the parental TU-6 while the Δgna1
caused no effects.
The mutant Δgna1
produced less CWDEs than
gna1QL
and TU-6. The
gna1QL
mutant showed a better performance in production of CWDEs such as endochitinase,
N-Acetyl-β-D-glucosaminidase (NAGase), β-1,3-glucanase, protease, lipase and
acid phosphatase, after 72 hours of incubation. However, the parental TU-6
showed higher cellulase activity than gna1QL and Δgna1.
The intracellular content of cAMP in the strains after 72 hours of incubation
was:
gna1QL
(79.85 ± 12), Δgna1
(268.65
± 8.5) and TU-6 (109.70 ± 9.2) pmol/mg protein. We therefore suggest that the
production of some CWDEs during mycoparasitism by
T. reesei
against
P.
ultimum
can be mediated by GNA1 activity or cAMP levels.
Lisha Zhang,
Jan van Kan
D-galacturonic acid (GalA) is the major component of pectin, which can be
degraded by plant pathogens; GalA potentially is an important carbon source for
microorganisms living on decaying plant material. For bacteria, a catabolic
pathway of GalA has been described, which consists of five enzymes converting
GalA to pyruvate and glyceraldehyde-3-phosphate. A different catabolic pathway
is proposed in filamentous fungi. In
Hypocrea
jecorina,
GalA is converted to pyruvate and glycerol via D-galacturonate reductase,
L-galactonate dehydratase, 2-keto-3-deoxy-L-galactonate aldolase, and glycerol
dehydrogenase.
The
Botrytis
cinerea
genome contains a D-galacturonate reductase gene (BcgaaA),
a L-galactonate dehydratase gene (BcgaaB),
and a 2-keto-3-deoxy-L-galactonate aldolase gene (BcgaaC).
The three genes were cloned into a protein expression vector and the enzymatic
activity determined for each gene separately. The heterologous simultaneous
expression of BcgaaA, BcgaaB, and BcgaaC in an
E. coli
ΔuxaC
mutant which cannot grow on GalA is performed to determine whether the catabolic
pathway from
B.
cinerea
can restore the growth deficiency in
E.coli.
Targeted gene replacement of BcgaaC or both BcgaaA and BcgaaC resulted in
ΔgaaC
mutants and
ΔgaaAC
double knock-out mutants that displayed significantly reduced growth when
D-galacturonic acid was used as the sole carbon source. The mutants showed
similar virulence as the wild-type stain B05.10 on tomato leaves, indicating
that GalA is not the main carbon source for
B.
cinerea
growth during infection on tomato leaves. The virulence will be tested on other
pectin-rich plants and tissues.
PR4.7
Birgit S. Gruben1,
Ulla Christensen2, Igor Nikolaev2, Ronald P. de Vries1,3
1Microbiology,
Utrecht University, Utrecht, The Netherlands; 2 Danisco-Genencor,
Leiden, The Netherlands; 3CBS-KNAW Fungal Biodiversity Centre,
Utrecht, The Netherlands
b.s.gruben@uu.nl
D-Galactose is present in hemicelluloses and pectin which are constituents of
the plant cell wall. In pectin, β-1,4-linked D-galactose residues are present in
galactan or in arabinogalactan side chains. In hemicelluloses, D-galactose
residues are present in the form of side residues which are β-linked in to
xyloglucan and xylan, but α-linked in to galactoglucomannan.
For the degradation of these structures by filamentous fungi, several enzyme
classes are required depending on the linkage. These classes are α- and
β-galactosidases and endo- and exogalactanases.
Aspergillus nidulans,
a saprobic filamentous fungus, is able to use D-galactose efficiently as a
carbon source.
A.
nidulans
can convert D-galactose through two pathways: the common Leloir pathway as well
as the recently described alternative D-galactose utilization pathway.
Recently two regulators, GalR and GalX were identified that control D-galactose
metabolism in
A.
nidulans.
The interaction of these regulators, their control of the various genes of the
two D-galactose utilization pathways as well as genes encoding extracellular
galactose releasing enzymes will be discussed.
Nancy Alexander[1]
Susan McCormick[1] Cees Waalwijk[2] Robert Proctor[1]
1
National Center for Agricultural Utilization Research,
nancy.alexander@ars.usda.gov
In some regions of the world, most strains of the wheat head blight pathogen
Fusarium
graminearum
have one of two trichothecene mycotoxin production profiles (chemotypes), which
are designated as 3-ADON and 15-ADON. In a defined medium, strains with
the 3-ADON chemotype produce a trichothecene (3-acetyldeoxynivalenol) with an
acetate at carbon atom 3 (C3) but not at C15, whereas 15-ADON strains produce a
trichothecene (15-acetyldeoxynivalenol) with an acetate at C15 but not at C3.
Despite this, strains with both chemotypes possess enzymatic activities
necessary for production of trichothecenes with acetates at both C3 and C15,
e.g. 3,15-diacetyldeoxynivalenol (3,15-diADON), suggesting the chemical
modification responsible for the two chemotypes occurs near the end of the
trichothecene biosynthetic pathway. Polymorphisms in the
trichothecene biosynthetic genes
TRI3,
which encodes a C15 acetyltransferase,
and
TRI12,
which encodes a transport protein, are used as genetic markers to distinguish
between strains with 3-ADON and 15-ADON chemotypes. However, a causal
relationship between
TRI3/TRI12
and the chemotypes has not been demonstrated. Sequence analysis has
revealed marked differences in the coding sequence of the esterase gene
TRI8
in 3-ADON versus 15-ADON strains. To determine whether these differences
can affect trichothecene chemotype, we examined the activity of
TRI8
as well as
TRI3.
The data indicate that differences in activity of the
TRI8
esterase, rather than the
TRI3
acetyltransferase, are the basis of 3-ADON and 15-ADON chemotypes in
F.
graminearum.
PR4.9
Martha B. Arnaud[1]
Jonathan Binkley[1] Marcus C. Chibucos[2] Maria C.
Costanzo[1] Jonathan Crabtree[2] Diane O. Inglis[1]
Joshua Orvis[2] Prachi Shah[1] Marek S. Skrzypek[1]
Gail Binkley[1] Stuart R. Miyasato[1] Jennifer R. Wortman[2]
Gavin Sherlock[1]
1Department
of Genetics, Stanford University School of Medicine, Stanford, CA, 2Institute
for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD
arnaudm@genome.stanford.edu
The
Aspergillus
Genome Database (AspGD) is an online genomic resource designed to facilitate
research on Aspergilli and on other medically and economically important fungal
pathogens. We provide an online reference for
Aspergillus
genomics and molecular biology, with up-to-date, high-quality information
curated from the scientific literature, as well as web-based research tools for
exploration and analysis of these data. The Sybil Comparative Genomics
tool at AspGD displays alignments of the genomic regions encoding clusters of
homologous proteins from ten
Aspergillus
genomes (A.
nidulans,
A.
fumigatus,
A. flavus,
A. oryzae,
A. niger,
A.
clavatus,
A.
terreus,
and
Neosartorya fischeri),
as well as various displays for exploration of syntenic regions among these
organisms. The GBrowse Genome Browser supports navigation and searching of
genes and chromosomal regions of the
A.
nidulans
FGSC A4 and
A.
fumigatus
Af293 genomes, and will be extended to other Aspergilli in the future.
Additional tools are available for search and retrieval of
A.
nidulans
sequence and gene and protein information that has been curated from the
scientific literature. The suite of sequence analysis tools includes BLAST,
pattern matching, restriction mapping, and primer design. In addition,
AspGD offers keyword-based and gene-property-based searches, Gene Ontology
(GO)-based analysis of gene lists by function and localization, bulk data query
and retrieval, and downloadable files. While AspGD curation has initially
focused on
A.
nidulans,
we will begin curation of the scientific literature on
A.
fumigatus
and other
Aspergillus
species in 2010 and will provide the full suite of AspGD tools for each of these
species in the future. We also provide tools for community interaction,
including a colleague registry by which
Aspergillus
research community members may share contact information and research interests
to facilitate collaboration, and a list of
Aspergillus
research laboratories. Our mission is to be responsive to the needs of the
research community, and we welcome your feedback and suggestions, at
aspergillus-curator@genome.stanford.edu. All of the data in AspGD are
freely available to the public from http://www.aspgd.org/. AspGD is
supported by grant RO1 AI077599 from the NIAID at the NIH.
PR4.10
Jean-Paul Ouedraogo[1],
Silke Hagen[1], Anja Spielvogel[1], Ulf Stahl[1],
Vera Meyer[1,2]
2Leiden University,
jpaul_oued@yahoo.fr
The antifungal protein AFP, secreted by
Aspergillus giganteus
is able to inhibit the growth of a variety of filamentous fungi (e.g.
A.
fumigatus,
A.
In order to understand the resistance mechanism of yeast strains, we have
screened
S.
cerevisiae
mutants deleted for the components of the CWIP and chitin synthesis. Most of the
~ 70 screened strains remained AFP-resistant, except the knock out mutants
Δwsc1,
Δtor1,
Δvps34
and
Δchs1,
which became moderate-sensitive towards AFP. The plasma membranes of these
mutants became readily permeabilized by AFP. Interestingly, the presence of AFP
provoked increased chitin synthesis in these strains, an observation which we
also made for the AFP-resistant filamentous fungus
Penicillium chrysogenum
and the moderate-sensitive mutant of
F.
oxysporum
ΔchsV.
The obtained results stipulate the hypothesis that moderate-sensitive and
resistant filamentous fungi counteract AFP inhibitory effects by strongly
increasing their chitin levels, thereby making the cell walls presumably more
rigid. However, this response does not occur in AFP-sensitive fungi. Apparently,
the classical CWIP is not sufficient to counteract AFP inhibitory effects and
seems not to be involved in increased chitin synthesis. Our findings give rise
to the assumption that fungal strains which only use the classical CWIP are
AFP-sensitive.
Maria Olmedo,
D. Lenssen, M. Merrow
Department of Chronobiology,
m.olmedo@rug.nl
The circadian clock creates a temporal structure within cells in animals,
plants, fungi and cyanobacteria. One aspect of the clock is a self-sustained
oscillation with a period of ~24h in constant conditions. However, circadian
clocks in nature are rarely subjected to constant conditions. They are normally
exposed to a rhythmic environment, where signals (zeitgebers) such as light and
temperature entrain the oscillations to the 24 h day. Blue-light responses in
Neurospora
require the
wc-1
and
wc-2
genes and these proteins also regulate the circadian clock. However, the
Neurospora
genome contains additional genes coding for putative photoreceptors, including a
cryptochrome, an opsin, and two phytochrome genes. We obtained knockout mutants
for the genes coding for putative photoreceptors from the
Neurospora
Functional Genomics Project and screened them in a variety of protocols for
circadian rhythm and entrainment. When assayed under constant conditions
Δcry,
Δphy-1,
Δphy-2,
Δnop-1
and Δorp-1
strains have the same free running period relative to the wild-type control. In
cycling conditions, however, all of the knockouts show differences in phase of
entrainment compared to the wild-type strain. The most extreme phenotype in our
study is the Δcry,
Δphy-2,
matA
strain, which is arrhythmic under all conditions tested. In summary, our results
show that additional photoreceptors play a role in entraining the
Neurospora
circadian clock. We conclude that
Neurospora
indeed models circadian clocks of in other organisms with respect to light
input, because they also show striking contributions of input pathway components
on circadian clock behaviour.
Jolanda van Munster[3]
1Institute
of Biology Leiden, Leiden University, Molecular Microbiology and Biotechnology,
Kluyver Centre for Genomics of Industrial Fermentation, Sylviusweg 72, 2333 BE
Leiden, The Netherlands
2TNO Quality of Life, Dep. Food and Biotechnology Ingredients,
Utrechtseweg 48, 3704 HE Zeist, The Netherlands
3Microbial Physiology Research Group, Groningen Biomolecular Sciences
and Biotechnology Institute (GBB), Groningen University, Kerklaan 30, 9751 NN
Haren, The Netherlands.
J.M.van.Munster@rug.nl
The filamentous fungus
Aspergillus
The mycelium of this fungus is highly differentiated. After stationary growth
phase, part of the mycelium is degraded in a process called autolysis. Autolysis
is characterized by hyphal fragmentation, loss of biomass, ammonia release and
the production of enzymes such as proteases and glycoside hydrolases. These
glycoside hydrolases could function in degradation of cell wall polymers such as
chitin. However, knowledge about the exact mechanism of autolysis is currently
limited.
During industrial fermentation processes, autolysis can cause the productive
biomass to decrease, causing reduced product yield. A better understanding of
autolysis can contribute to the formation of strategies to increase efficiency
of fermentations.
In order to increase understanding of the dynamics of the fungal mycelium, a
consortium of academic and industrial partners investigates autolysis and
differentiation in
Aspergillus
By using microarrays to monitor transcription levels during growth, we have
identified genes that are upregulated during the autolytic phase compared to
exponential growth phase. Four of these genes belong to glycoside hydrolyse
family 18, which consists mainly of (putative) chitinases. In order to
investigate the properties of these enzymes we performed heterologous gene
expression in
E. coli
with subsequent purification using affinity tags. The activity of purified
proteins is investigated.
Diane Inglis[1]
Martha B. Arnaud[1] Jon Binkley[1] Maria C. Costanzo[1]
Marcus C. Chibucos[2] Jonathan Crabtree[2] Joshua Orvis[2]
Prachi Shah[1] Marek S. Skrzypek[1] Gail Binkley[1]
Stuart R. Miyasato[1] Jennifer Wortman[2] Gavin Sherlock[1]
1Department
of Genetics, Stanford University School of Medicine, Stanford, CA
2Institute for Genomic Sciences, University of Maryland School of
Medicine, Baltimore MD
dinglis@stanford.edu
The
Aspergillus
Genome Database (AspGD;
www.aspgd.org) is
a web-based genomics resource for researchers studying the genetics and
molecular biology of an important group of fungal microorganisms, the
aspergilli. AspGD provides high-quality manual curation of the experimental
scientific literature, including gene names, general descriptions, phenotype
data, and Gene Ontology (GO) annotations, as well as tools for exploring these
data. AspGD is based on the framework of the
Saccharomyces
Genome Database (SGD) and
Candida
Genome Database (CGD), two genomic resources with which many users within the
fungal research community are already familiar. The manual annotation of gene
information, phenotype data and GO annotations is the focus of this
presentation. Initially, we have focused on the manual curation of genomic
information for
Aspergillus nidulans,
the best-characterized species of the group. We will expand our efforts to
include curation of
A.
fumigatus, A. flavus, A. oryzae, A.
Tina Kogej,
Cene Gostincar, Nina Gunde-Cimerman
tina.kogej@bf.uni-lj.si
Purpose:
Hortaea
werneckii
is an ascomycetous halophilic black yeast naturally inhabiting hypersaline
waters of solar salterns. It is remarkable for its growth at a wide range of
NaCl concentrations (0 – 5.2 M) and is a novel eukaryotic organism for studying
cellular responses to extremely elevated environmental salinity. We have found
previously that
H.
werneckii
adapts to high NaCl concentration by accumulating glycerol and erythritol. The
purpose of this study was to search for and identify a glycerol transporter of
the halophilic black yeast
Hortaea
werneckii.
Methods: The partial gene sequence was obtained by suppression subtractive
hybridization and extended by genome walking. The complete cDNA sequence was
obtained by SMARTer rapid amplification of cDNA ends. The gene was cloned and
sequenced, and the putative protein was characterized
in silico.
Results: In the halophilic black yeast
Hortaea
werneckii,
we have identified a gene encoding a putative protein with a considerable degree
of similarity to a number of uncharacterized sugar transporters, and also to
Stl1p, a well-characterized member of the sugar transporter family, the
glycerol/H+ symporter of the plasma membrane in
Saccharomyces cerevisiae.
We have named it HwSTL1. HwSTL1 consists of 1631 bp, encodes a protein of 543 aa
with a calculated MW of 59417. Conclusions: We have identified, cloned and
characterized the gene HwSTL1 encoding a glycerol-transporter-like protein of
H.
werneckii.
In our further studies we will conclude whether the protein encoded by the
HwSTL1 indeed functions as a glycerol transporter protein in
H.
werneckii.
glycerol transport, Hortaea werneckii, MFS superfamily, osmoadaptation,
STL1-like transporter,
PR4.15
Hiroto Morita[1]
Ayako Okamoto[1] Youhei Yamagata[1] Ken-Ichi Kusumoto[2]
Yoshinao Koide[3] Hiroki Ishida[4] Michio Takeuchi[1]
1Tokyo
University of Agriculture and Technology, 2National Food Research
Institute, 3Amano enzyme Inc., 4Gekkeikan Sake Co. Ltd
morita25@cc.tuat.ac.jp
Serine-type carboxypeptidase (SCP) is an exopeptidase that has Ser, His, and Asp
residues as a catalytic triad construct and can sequentially release C-terminal
amino acid residues of peptides and proteins. In the genome of
Aspergillus oryzae
RIB40, 12 genes have been predicted to encode SCPs. However, the
carboxypeptidase activities of the gene products have not yet been confirmed
experimentally. Therefore, we have constructed those gene overexpressing strains
using
Aspergillus nidulans
and characterized their overproduced recombinant proteins. Here, we report
enzymatic character of six of the gene products. The recombinant proteins were
able to release amino acid residues from the C terminus of peptides, and the
activity of the enzymes was inhibited by phenylmethylsulfonyl fluoride,
indicating the enzymes to be SCPs. The enzymes were stable at lower than
40-55°C, at low and neutral pH. The optimum pHs of the enzymes except for AOCP16
were around pH 4. That of AOCP16 was pH5.5. The substrate specificities of each
enzyme for
N-acyl-peptides
were different. Result of transcriptional analysis of these genes suggested
differences in transcriptional regulation between these genes. The enzymatic
properties of AOCP6 and AOCP9 were different from those of any reported SCP.
AOCP4 and AOCP13 had similar enzymatic properties to carboxypeptidases O1 and O2
and carboxypeptidase O from
A. oryzae
IAM2640. Results of sequence analysis of DNA and N-terminal amino acid sequences
showed AOCP4 correspond to carboxypeptidases O1 and O2, and AOCP13 correspond to
carboxypeptidase O.
This study was supported by the Program for Promotion of Basic Research
Activities for Innovative Biosciences.
Youhei Yamagata[1]
Hiroshi Maeda[1] Ken-ichi Kusumoto[2] Yoshinao Koide[3]
Hiroki Ishida[4] Michio Takeuchi[1]
1Dep.
of Agriscience & Bioscience. Tokyo University of Agriculture & Technology,
Tokyo, Japan
2NFRI, Ibaraki, Japan, 3Amano Enzyme Inc., Gifu, Japan,
4Gekkeikan Sake Company Ltd., Kyoto, Japan
y-yama@cc.tuat.ac.jp
Aspergillus oryzae
is one of industrial microorganism as using for the Japanese traditional
fermented food. The genome project of
A. oryzae
clarified that there were 134 genes coding proteolytic enzymes. We have been
trying to characterize all proteolytic enzymes.
We found that
A. oryzae
had twelve genes for metallo-carboxypeptidases. Nine of them do not have the
signal peptide, and it is presumed that these enzymes would be localized
intracellularly. The enzymes were all classified in M20 super-family. As four of
the enzyme genes were translated under the liquid culture condition, the genes
were cloned and expressed in
E. coli.
The two purified enzymes (AOEXE305 and AOEXE306) showed maximum activity at
alkaline pH in spite of those are intracellular enzymes. It was shown that
acidic amino acid was favorable in P1’ site for AOEXE305. AOEXE306 showed wider
substrate specificity than AOEXE305. The enzyme could cleavage between even
Xaa-Pro bonds but did not favor acidic amino aicd in S1’ in substarates. The
results might indicate that fungal intracellular metallo-carboxypeptidases had
different roll in fungal cells.
This study was supported by the Program for Promotion of Basic Research
Activities for Innovative Biosciences (PROBRAIN).
Grainne O' Keeffe,
Christoph Jöchl, Sean Doyle
NUI Maynooth
grainneokeeffe@gmail.com
Aspergillus fumigatus
is an opportunistic pathogen predominantly affecting immunocompromised
individuals, resulting in pulmonary illnesses such as Invasive Aspergillosis.
Sequencing of the genome has led to an increased understanding of the organism;
however the functions of many genes remain unknown. A putative translation
elongation factor 1Bγ (EF1Bγ, termed
elfA;
750 bp) is expressed, and exhibits glutathione s-transferase activity, in
A.
fumigatus
[1]. Normally, EF1Bγ plays a key role in the elongation step of protein
synthesis. Our hypothesis is that
elfA
may also play a role in regulating the cellular redox state adjacent to the
ribosome during protein synthesis. Consequently,
elfA
was disrupted in
A.
fumigatus
ATCC46645 (wild-type) using a bipartite construct containing overlapping
fragments of a pyrithiamine resistance gene (ptrA).
The
elfA
mutant (ΔelfA)
was complemented using a hygromycin resistance marker
(hph).
Southern Blot analysis was used to confirm the generation of ΔelfA
and the complemented strain. RT-PCR confirmed the expression of
elfA
in wild-type and complemented strains, and absence of expression in ΔelfA.
The availability of the mutant has facilitated phenotypic analysis of
elfA
functionality.
A.
fumigatus
wild-type and ΔelfA
were grown on AMM plates with the oxidant H2O2 (1 - 5 mM),
voriconazole (0.25 - 1 µg/ml), and the thiol-reactive reagent,
4,4’-dithiodipyridine (3 - 7.5 µM). At 37°C, the
elfA
mutant was significantly more sensitive (p=0.0003) to H2O2
than wild-type. However, ΔelfA
was significantly less sensitive (p=0.0251) to voriconazole than wild-type. At
37°C, the ΔelfA
was significantly more sensitive (p=0.0056) to 4,4’-dithiodipyridine than
wild-type. These results implicate
elfA
in the oxidative stress response in
A.
fumigatus
and also strongly indicate that
elfA
may play a role in the sensitisation of
A.
fumigatus
to voriconazole. Global proteomic studies are currently underway using 2D-PAGE
and MALDI-MS to explore alterations in the proteome consequential to
elfA
disruption with a view to gaining further insight into the function of
elfA
in
A.
fumigatus.
1. Carberry, S, et al. (2006), Biochem Biophys Res Commun, 341, 1096-1104.
Géraldine MEY,
Heber Gamboa-Meléndez, Bénédicte Gayrin, Tim Rollenske, Marie-Josèphe Gagey,
Geneviève Billon-Grand, Michel Droux
Functional Genomics of Phytopathogenic Fungi, UMR 5240 CNRS-UCBL-INSA-Bayer
CropScience
geraldine.mey@univ-lyon1.fr
How phytopathogenic fungi fulfill their nutritional needs during the interaction
with their hosts remains poorly documented. They may first mobilize their
storage compounds during the early stages of the infection process. Then they
may use the simple compounds, released by the subsequent host constituent
enzymatic degradation, to complete their development
in planta.
The metabolism of amino acids may thus be redirected and adapted to the
different stages of the plant infection strategy. Our studies focus on the
amino-acid triggered regulation mechanisms required to successfully achieve
plant infections. The monomeric GTPase Rheb (Ras Homologue Enriched
in Brain) was known to activate the kinase TOR and to be involved in the
regulation of arginine and lysine uptake in yeasts.
Botrytis
cinerea
Rheb
orthologue was inactivated using different approaches (RNA interference,
promoter replacement). The assay of the amino acid content, using reversed
chromatography (HPLC), highlighted the implication of Rheb in the control of
amino acid metabolism. The putative involvement of Rheb in the amino acid uptake
control was suggested with complementation experiments of a
S.
cerevisiae
mutant strain. Rheb importance for
B. cinerea
development was analyzed using different model host systems and the microscopic
observations of the early differentiation stages. A comparative study of Rheb
functions in other phytopathogenic models is being performed by expressing Rheb
hyper- and hypo-active mutant forms in
Magnaporthe grisea.
The results obtained with both fungi will give insights on the molecular
mechanisms controlling amino acid uptake/metabolism and their requirement for
the parasitic development of fungal plant pathogens.
Moriyuki Kawauchi[1]
Mika Nishiura[1] Kazuhiro Iwashita[2]
Osamu yamada[2]
1Hiroshima
univ., 2NRIB
ysnbt575@ybb.ne.jp
Aspergillus oryzae
is ubiquitous filamentous fungi in nature and has been used in a number of
industries such as Japanese traditional fermented foods and pharmaceutical
products. In nature and industries,
A. oryzae
adapt to various environmental conditions by global change of transcriptional
regulation. In the previous study, we revealed that the expression of
histone acetylation related genes were affected by the growth phase and
alteration of growing environment, such as culture conditions and stress
exposing. In general, histone acetylation plays the fundamental roles for
the genes expression, and closely relates to growth, morphology, differentiation
and stress responses. Recently, several reports indicate that histone
acetylation also plays important roles in filamentous fungi. In this
context, we focused on histone acetylation related genes, particularly histone
deacetylases (HDACs). We attempted to disrupt 11 HDACs homologue of
A.
oryzae
and 10 of them were disrupted. However, in the deletion of RPD3 homologue,
only the heterokaryon transformants had been isolated. This result
suggests that RPD3 homologue is essential in
A. oryzae.
The phenotypes of 10 HDACs disruptants were observed in submerged and plate
culture with/without several stress conditions. As the results, four
disruptants showed considerable growth and developmental defects in these
conditions, especially, HOS2 homologue null mutant showed significant decline of
growth in submerged culture.
In microscopic analysis, two disruptants showed abnormal hyphal branches and
aberrant distribution of hyphae on the interface of medium and air. This
study has indicated that HDACs play important roles in the growth and stress
adaptation of
A.
oryzae.
Jerica Sabotic[2]
Sreedhar Kilaru[1] Jože Brzin[2] Andy Bailey[1]
Gary Foster[1] Janko Kos[2]
1School
of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG,
United Kingdom
2Department of Biotechnology, Jožef Stefan Institute, Jamova 39, 1000
Ljubljana, Slovenia
jerica.sabotic@ijs.si
Mycocypins, clitocypin and macrocypins, are cysteine protease inhibitors
isolated from basidiomycetes clouded agaric (Clitocybe
nebularis)
and parasol mushroom (Macrolepiota
procera).
Two new families of protease inhibitors in the MEROPS classification have been
established, I48 for clitocypin and I85 for macrocypins, based on their unique
primary sequences and biochemical characteristics. Mycocypins are exceptionally
stable proteins, exhibiting high thermal and broad pH stability. The
physiological function of the two mycocypin families is proposed to be defence
against pathogen infection and/or predation by insects or other pests,
analogously to the phytocystatins that are involved in plant defence by
inhibiting exogenous cysteine proteases during herbivory or infection. Sequence
diversity of clitocypin genes is limited to 18 discreet positions that have no
influence on its inhibitory activity. On the other hand, the sequence diversity
of macrocypin genes is higher and includes amino acid sites of positive
selection. The variations in inhibitory profile between different members of the
macrocypin family reveal different specificities and strengths of inhibition of
cysteine proteases of different evolutionary families, and even a serine
protease. These findings together suggest an adaptation process and the
selection of appropriate inhibitor isoforms providing effective defence.
Analysis of expression regulation of mycocypins using different mycocypin
promoters and green fluorescent protein as the reporter gene showed different
patterns of expression during fruiting body development for clitocypin and
macrocypins. In view of their proposed defensive roles different expression
profiles suggest different target organisms as both families of mycocypins are
present in each mushroom.
Violeta Díaz Sánchez[1]
Alejandro F. Estrada[1] Salim Al-Babili[2] Javier Avalos[1]
1Universidad
de Sevilla, 2University of
violetads@us.es
The gibberellin producing fungus
Fusarium
fujikuroi,
is also used as a model for genetic and biochemical analysis of carotenoid
biosynthesis. Its major carotenoid product is neurosporaxanthin (NX), an acidic
apocarotenoid formerly discovered in
N. crassa.
The NX precursor beta‑apo‑4’‑carotenal is produced by
F.
fujikuroi
through the activity of the enzymes encoded by genes
carRA
(cyclase and phytoene synthase),
carB
(phytoene desaturase), and
carT
(torulene cleaving oxygenase). The enzyme responsible for the oxidation of the
aldehyde group of beta‑apo‑4’‑carotenal to yield NX, has not been described in
this fungus. Based on our former results with
ylo-1
in
N.
crassa,
we have cloned the
F.
fujikuroi
gene
carD,
coding for an aldehyde dehydrogenase putatively responsible of this enzymatic
reaction. Crude protein extracts from an
E. coli
strain expressing a
carD
cDNA version were able to convert beta‑apo‑4’‑carotenal into the corresponding
apocarotenoic acid, confirming the expected enzymatic activity. CarD was also
active on shorter carotenoids, including acyclic ones, such as 8’-lycopenal,
indicating the irrelevance of the cycled end of the molecule for substrate
recognition. Also, we expressed the enzyme in a beta-apo-4’-carotenal producing
E. coli
strain and we got NX production
in vivo.
In contrast to other
car
genes, real-time RT-PCR analyses of
carD
mRNA levels showed a light-independent expression. However, the mRNA levels were
increased in a carotenoid overproducing mutant, indicating common regulatory
mechanisms for all the
car
genes in this fungus. Phenotypic effect of targeted
carD
disruption, currently in progress, will be reported.
Ellen L. Lagendijk[1]
Benjamin M. Nitsche[1] Vera Meyer[2] Cees A.M.J.J. van den
Hondel[1] Arthur F.J. Ram[1]
1Leiden
University, Institute of Biology Leiden, Section of Molecular Microbiology &
Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands
2Berlin University of Technology, Institute of Biotechnology,
Department Microbiology and Genetics, Gustav-Meyer-Allee 25, D-13355 Berlin,
Germany
e.l.lagendijk@biology.leidenuniv.nl
Increased resistance to currently used antifungal compounds and the fact that
these agents are often harmful to man and environment have resulted in a growing
demand for new antifungals, which selectively act on cellular processes that are
unique to fungi. To meet this demand, we have established a luciferin/luciferase
based reporter system for high-throughput screening of extracts obtained from
natural sources. This system allows us to identify compounds that specifically
target fungal cell wall biosynthesis. It has been well established in the yeast
Saccharomyces cerevisiae
that cell wall synthesis is a highly dynamic process, which is orchestrated by
the cell wall integrity (CWI) pathway. Such CWI pathway is also present in the
filamentous fungus
Aspergillus
Angelique Franken[2]
B. Christien Lokman[1] Arthur F.J. Ram[2] Cees A.M.J.J.
van den Hondel[1,2] Sandra de Weert[2]
1HAN
Biocentre, Laan van Scheut 2, 6525 EM Nijmegen, The Netherlands
2Institute of Biology Leiden, Leiden University, Molecular
Microbiology & Biotechnology, Kluyver Centre for Genomics of Industrial
Fermentation, Sylviusweg 72, 2333 BE Leiden, The Netherlands
a.c.w.franken@biology.leidenuniv.nl
The incorporation of heme as a cofactor is a putative limiting factor in the
overproduction of heme-containing fungal peroxidases in
Aspergillus
species. Addition of hemin to growth medium has been reported to improve the
production of peroxidase. However, hemin uptake and the effect of hemin addition
on the transcriptional regulation of the heme biosynthesis pathway genes have
hardly been studied in
Aspergillus.
To gain more insight into the heme biosynthesis pathway, the genes encoding the
eight different enzymes in the pathway were identified in the
A.
Carol Davis[1]
Stephen Carberry[1] Markus Schrettl[1] Dermot Brougham[2]
Kevin Kavanagh[1] John Stephens[1] Sean Doyle[1]
1NUI
Maynooth,
carol.davis@nuim.ie
Biosynthesis of gliotoxin is directed by the multi-gene (gli)
cluster in the opportunistic fungal pathogen,
Aspergillus fumigatus.
Minimal functional cluster annotation is available. The gene
gliG,
located in the
gli
cluster, is classified as a glutathione
s-transferase
by
in silico
analysis and recombinant GliG exhibits GST and glutathione reductase activity.
Two overlapping constructs, each containing part of a marker gene (ptrA)
and with homology to
gliG
flanking regions, were used to disrupt
gliG
in
A.
fumigatus
(Daku80
and Af293 strains). The generation of a
gliG
mutant was confirmed using Southern Blot analysis using a digoxigenin-labelled
probe specific for an
XbaI
digested fragment size of 2124 bp in the wild-type and 1668 bp in the
gliG
mutant. Absence of
gliG
expression in the
mutant was confirmed by Northern analysis. RP-HPLC-DAD and LC-MS analysis of
extracts from
A.
fumigatus
wild-type and ΔgliG
revealed that gliotoxin (Rt= 14.4 min) was absent from the mutant strains,
strongly indicating that
gliG
is involved in gliotoxin biosynthesis. Interestingly, an additional metabolite
(Rt = 12.3 min) was present in mutant culture supernatants which may represent a
precursor of gliotoxin (GTP). LC-ToF analysis determined that the
metabolic intermediate had a mass of 263 Da and targeted alkylation demonstrated
the lack both free thiol residues and an intact disulphide bridge.
Reconstitution of
gliG
into
A.
fumigatus
DgliG
restored gliotoxin biosynthesis. Unlike another component of the
gli
cluster,
gliA,
it appears that
gliG
is not involved in the auto-protection of
A.
fumigatus
against exogenous gliotoxin. In conclusion, we confirm a key role for the
glutathione
s-transferase,
GliG, in the biosynthesis of, and not auto-protection against, gliotoxin- which,
to our knowledge, is the first time this enzyme has been shown to play a pivotal
function in ETP biosynthesis.
CHIARA NOBILI[4]
A. Ricelli[1] M. Reverberi[2] S. Gatta[2] V.
Scala[3] G. Aureli[3] A.A. Fabbri[2] C. Fanelli[2]
1ICB-CNR,
2Dipartimento di Biologia Vegetale, Università “Sapienza”, Largo
Cristina di Svezia 24, 00165, Roma, Italy, 3Unità di Ricerca della
Valorizzazione Qualitativa dei Cereali, CRA-QCE, Via Cassia 166, 00191, Roma,
Italy, 4ENEA
chiara.nobili@enea.it
Under suitable conditions, some fungal species, such as
Fusarium,
growing on many food commodities, can produce secondary metabolites dangerous
for humans and animals.
It has been assessed that one of the main virulence factor involved in the
Fusarium
head blight (FHB) disease of wheat (leading to a severe reduction of grain yield
and quality) is the
Fusarium
graminearum
production of toxins, predominantly deoxynivalenol (DON) that delays germination
and growth of wheat plants, inducing hydrogen peroxide (H2O2)
production, inhibiting protein synthesis and stimulating cell death in
planta.
Mycotoxigenic fungi contamination is a real issue, especially for cereal
industry. Therefore, in order to reduce the diffusion of plant disease and
health risks, there is a real need to develop analytical methods able to
identify DON-producing fungal variety and to quantify mycotoxins.
In this work, the interaction between two
Triticum
aestivum
varieties, BLASCO (tolerant) and SAGITTARIO (susceptible), inoculated with two
F.
graminearum
strains (Fg126 and Fg8308), was studied.
Recent advances in DNA-based techniques confer to Real Time-PCR (RT-PCR) assays
an important role because of the accelerated diagnostic outcome, so that, this
method is providing new tools for fungal detection and quantification in complex
matrix. Thus, two primer pairs, designed by other authors, on the gene sequences
belonging to the thricothecene gene cluster were used to identify high DON
producing
Fusarium
strains through PCR method.
Furthermore, a SYBR green Real Time-PCR assay was developed to quantify
F. graminearum
strains in artificially contaminated soft wheat. These results were correlated
with the quantification of ergosterol by HPLC. Moreover, the expression of
different genes activated in the interaction environment, was analysed by a
relative RT-PCR approach. In the pathogen these genes encode for Fgap1
that is a transcription factor active in the cell defence against oxidative
stress,
ePG
a poligalatturonase involved in cell degradation and
tri6,
one of the thricotecenes biosynthesis regulator. In
T. aestivum,
the expression analysis of one glucosyl transferases (gt),
one of biochemical mechanisms of resistance to DON is the plant ability to
convert DON in a less toxic glucosylated form, and of the pathogenesis-related
protein PR1 (PR1)
were carried out.
It is known that, among the broad range of defence responses, occurring in
planta
when
Fusarium
invasion occurs, the generation of reactive oxygen species (ROS), such as
hydrogen peroxide (H2O2), is one of the earliest events.
The activities of three antioxidant enzymes (catalase, superoxide dismutase and
glutathione peroxidase) correlated to ROS and of one more enzyme related to the
defensive response (lipoxygenase), were monitored.
Finally, the application of HPLC method for the quantitative detection of DON
and 15-acetyl DON produced from
Fusarium
species present on samples, confirmed, also, through ELISA analysis, was
described.
In conclusion, as far as fungal diseases are wide diffused, the control of
contaminated matrices it’s a priority. Thus, it’s very important to deepen
plant-pathogen interaction study, in order to develop control strategies (i.e.
quantitative, specie-specific methods) to be applied in diagnostics (i.e.
advanced analytical method for mycotoxin detection).
PR4.26
Patrizia De Rossi[4]
Ricelli, Alessandra[1] Reverberi Massimo[2] Nicoletti
Isabella[3]
Antonella del Fiore[4]
Bello Cristiano[3] De Rossi Antonella[3] Corradini Danilo[3]
Fabbri Anna Adele[2] Fanelli Corrado[2]
1CRB-CNR,
P.le Aldo Moro 5, 00185, Roma, Italy
2Università degli Studi “Sapienza”, Largo Cristina di Svezia 24 00165
Roma, Italy
3Istituto di Metodologie Chimiche, CNR Area della Ricerca di Roma,
C.P. 1000016, MonteRotondo Stazione, Italy
4ENEA
C.R.-CASACCIA, Via Anguillarese 301 00123 Roma, Italy
The objective of this research was to investigate whether
A. carbonarius
contamination induces resveratrol production in grape berry. A possible
correlation between OTA production and resveratrol biosynthesis has also been
considered.
Aspergillus carbonarius
is an important ochratoxin A (OTA) producing fungus which is responsible for
toxin contamination of grapes and wine. OTA is a secondary metabolite which has
been shown to be nephrotoxic, nephrocarcinogenic, teratogenic and
immunosuppressive. Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a natural
polyphenolic antioxidant found in red wine and grapes has been described as
stress metabolite produced by
Vitis vinifera
in response to biotic and abiotic stress as well as to fungal infection and it
has been demonstrated to have a particularly wide spectrum of mycotoxin control.
In this study,
Vitis vinifera
berries were infected, during ripening, by a conidial suspension of
A. carbonarius
and incubated for 6, 12, 24, 48, 120 hours at 30°C. After incubation, each berry
was analyzed, at each time interval considered, for quantifying
A. carbonarius,
OTA and resveratrol in grapes. Real Time PCR method with specie-specific primers
(Acpks),
designed on the basis of the OTA-related polyketide synthase sequences, was
carried out quantifying the fungal development in grapes. Our results show a
correlation between the growth of the fungus and biosynthesis of OTA and
resveratrol content into grape berries, leading to hypothesize that some
grapevine cultivars are more capable of self-protection against fungal
contamination.
Hiroki Sato,
Takahiro Shintani, Katsuya Gomi
hiro._.s@biochem.tohoku.ac.jp
We have observed that Taka-amylase (TAA) activity disappeared in submerged
culture of
Aspergillus oryzae
at the later-stage of cultivation. This disappearance was revealed to be caused
by adsorption of TAA on fungal mycelia, but not by degradation by own
extracellular proteolytic enzymes. We have also showed that cell wall of
A. oryzae
prepared from mycelia at the later-stage of cultivation has an adsorption
ability for TAA. This suggested that a certain cell wall factor(s) can adsorb
TAA, resulting in the disappearance of TAA in liquid medium during cultivation.
To identify the adsorption factor(s) in fungal cell wall, we carried out
stepwise fractionation of cell wall prepared from mycelia at the later
cultivation stage by alkali extraction and cell wall lytic enzymes. The
alkali-insoluble fraction of cell wall, CW4, showed high adsorption ability for
TAA, but digestion of CW4 with chitinase resulted in a significant decrease in
the adsorption ability. These results indicated that the adsorption factor for
TAA is chitin, which is one of major polysaccharides in fungal cell wall.
However, the cell wall prepared from mycelia at the earlier cultivation stage
barely adsorbed TAA, although it contained equivalent amount of chitin to that
of later-stage mycelia. Taken together, it is suggested that there exists
unidentified factor(s) that could prevent from adsorption of TAA onto the cell
wall at the earlier-stage of cultivation and the factor(s) would be removed from
or decreased in the cell wall with longer cultivation periods.
PR4.28
Karen O'Hanlon[1]
D. Stack[1] M. Schrettl[2] T. Larsen[3] K.
Kavanagh[1] S. Doyle[1]
1National
Institute for Cellular Biotechnology, Department of Biology, National University
of Ireland, Maynooth, Co. Kildare, Ireland, 2Division of Molecular
Biology, Innsbruck Medical University, A-6020 Innsbruck, Austria
3Center for Microbial Biotechnology, Department of Systems Biology,
Technical University of Denmark, Søltofts Plads 221, 2800 Kgs, Lyngby, Denmark
karen.a.ohanlon@nuim.ie
Aspergillus fumigatus
is a ubiquitous filamentous fungus, and a serious opportunistic human pathogen.
Availability of the complete genome sequence for
A. fumigatus
has revealed that there are at least eighteen genes coding for non-ribosomal
peptide synthetases (NRPS). NRPS’s are usually large, multi-modular enzymes,
comprised of discrete domains, which synthesise bioactive peptides via a
thiotemplate mechanism. To date, a wide range of virulence factors have been
reported for
A. fumigatus,
including adhesions, conidial pigments and proteases. Some of the best
documented virulence factors for
A. fumigatus
include Gliotoxin and the iron-chelating Siderophores, which are of NRPS origin.
Despite these important findings, there have been few studies relating the
majority of
A. fumigatus
NRPS encoding genes to specific peptide products. This work aims to elucidate
the peptide product encoded by a mono-modular NRPS,
pesL
(Afu6g12050/NRPS11), and to determine a possible role in virulence. A
pesL
deletion strain was generated, termed
∆pesL.
∆pesL
displays severely reduced virulence in the
Galleria mellonella
model (p < 0.0001). Phenotypic analysis has confirmed increased sensitivity of
∆pesL
to H2O2 (> 1 mM) compared to the wild-type (p = 0.05), and
severely increased susceptibility towards the antifungal voriconazole (> 0.25
µg/ml) compared to wild-type (p < 0.01). These results indicate a role for
pesL
in protection against oxidative and antifungal stress within
A. fumigatus.
Comparative RP-HPLC analysis identified conidial specific material (Rt = 15.9
min; λmax at 220 nm) synthesised by
A. fumigatus
wild-type. This metabolite was absent from
∆pesL
conidia. Increased production of this metabolite was observed in conidial
extracts cultured in 2 mM H2O2, indicating up-regulation
in response to oxidative stress. This material is currently undergoing further
analysis. Furthermore, a recombinant PesL enzyme has been purified for use in an
assay to determine the specific PesL amino acid substrate. This will contribute
to the currently limited information on fungal NRPS substrate selectivity.
Interestingly, another NRPS mutant generated previously, termed
∆pes3
(∆Afu5g12030/∆NRPS8)
displays increased virulence in the
Galleria mellonella
model (p < 0.0001). Furthermore,
∆pes3
exhibited severely increased susceptibility towards the antifungal voriconazole
(> 0.5 µg/ml) compared to wild-type (p < 0.001). RP-HPLC has not yet revealed a
candidate
pes3
peptide. However, the search is on-going. This data further highlights the
importance that NRPS plays in this serious human pathogen, and may reveal novel
drug targets in the future.
PR4.29
Alessandra Salvioli[1]
Marco Chiapello[1] Joel Fontaine[2] Anne
Grandmougin-Ferjani[2] Luisa Lanfranco[1] Paola Bonfante[1]
1Department
of Plant Biology and IPP-CNR, University of Torino, Torino, Italy
2Laboratoire Mycologie/Phytopathologie/Environnement, Université du
Littoral, Côte d’Opale, BP 699, 62228 Calais cedex,
alessandra.salvioli@unito.it
AM fungi are obligate biotrophs of a large spectrum of plants with which they
establish a mutualistic symbiosis. The presence of endobacteria living inside
the cytoplasm of some AM fungi has long been documented, but the impact of these
prokaryotes on fungal biology is still unknown.
The aim of this work was to understand whether the endobacterium
Candidatus
Glomeribacter gigasporarum has an impact on the biology of its fungal host
Gigaspora margarita
through the study of the modifications induced on the fungal proteome and lipid
profile. The availability of
G. margarita
cured spores (i.e. spores that do not contain bacteria), represented a crucial
tool to enable the comparison between two fungal homogeneous populations in the
presence and the absence of the bacterial component. A differential protein
expression was detected between wild type and cured spores under different
physiological conditions (quiescent, germinating and strigolactone-elicited
spores). The results obtained indicate that the fungal primary metabolism does
not seem to be affected by the absence of the endosymbiont. By contrast, heat
shock proteins are unambiguously upregulated, suggesting that the fungus has to
face a stress situation when endobacteria are lacking. Furthermore, the fungal
fatty acid profile resulted to be modified both quantitatively and qualitatively
in the absence of endobacteria, being fatty acids more abundant in the presence
of the endobacterium.
The results not only revealed that endobacteria have important impacts on the
host fungal biology, but also offered one of the first contributions to the
knowledge of the metabolic features of
G. margarita.
Janneke van Gent, Franziska Wanka, Mark Arentshorst, Cees A.M.J.J van den
Hondel, Arthur F.J. Ram, Vera Meyer
Leiden University, Institute of Biology Leiden, Department Molecular
Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands &
Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA
Delft, The Netherland
v.meyer@biology.leidenuniv.nl
The function of genes is usually inferred from mutants in which the desired gene
has been deleted or strongly overexpressed. However, studies at these extreme
discrete points give only limited information about the gene functions.
Moreover, many overexpression studies make use of metabolism-dependent promoters
which often cause pleiotropic effects and thus impose further limitations on
their use and significance.
Here we report a promoter system for
Aspergilus
PR4.31
Purification and cloning of trans-3- and trans-4-proline hydroxylase from the
fungus Glarea lozoyensis
Loubna Youssar;
Wolfgang
Hüttel and Michael Müller
Institut für Pharmazeutische Wissenschaften
Lehrstuhl für Pharmazeutische und Medizinische Chemie.
Freiburg;
Glarea lozoyensis
is an anamorph fungus, which was initially assigned
Zalerion arboricola based on
morphological traits. G. lozoyensis
is of pharmaceutical interest since it is producing the antifungal secondary
metabolite pneumocandin B0. This cyclic lipopeptide is chemically converted into
a water-soluble derivative (caspofungin acetate) that is used against clinically
relevant fungi pathogens.
In Pneumocandin B0 trans-4- and trans-3-hydroxyprolines are incorporated which
are derived from hydroxylation of L-Proline by proline trans-3-hydroxylase (P3H)
and proline trans-4-hydroxylase (P4H),respectively. The P3H activity discovered
in G. lozoyensis is unique and specific for pneumocandin B0 biosynthesis.
We are interested in this new selectivity for biocatalysis, but also on the
molecular and genetic level. To understand the physiological parameters that
influence pneumocandin B0 and C0 production better, we proceed by purification
of potential P3H and P4H proteins using gel filtration. The activity is checked
in each step by HPLC and LC-MS. In parallel, we are trying to clone these genes
by use of some conserved domains known from bacterial proline hydroxylases for
designing degenerative primers. In future experiments, we want to express the
genes heterologously in E. coli,
knock them out in the native strain and study their expression which will allow
a deeper understanding of the mechanism of pneumocandin biosynthesis.
Transcriptomic insights into the physiology of
Aspergillus
Thomas R. Jørgensen,1 2 Benjamin M. Nitsche,1 Gerda E.
Lamers,1 Mark Arentshorst,1 2 Cees A. van den Hondel1
2 and Arthur F. Ram1 2
1. Sylvius Laboratory,
2. Kluyver Centre for Genomics of
Industrial Fermentation,
The physiology of filamentous fungi at growth rates approaching zero has been
subject to limited study and exploitation. With the aim of uncoupling product
formation from growth, we have revisited and improved the retentostat
cultivation method for Aspergillus
PR4.33
The main cell cycle genes in the pathogenic yeast
Cryptococcus neoformans
Susumu Kawamoto1,
Eric V. Virtudazo1,
Misako Ohkusu1,
Tomoko Sonoda2,
Satoshi Miura2,
Kanji Takeo1
1Medical
Mycology Research Center,
Chiba University, Chiba,
Japan
2
We
have been involved in
studies towards
molecular understanding of cell cycle
regulation
in the pathogenic yeast
Cryptococcus
neoformans.
Our group
has reported the unique cell cycle pattern of
C. neoformans, different from that of
the model yeast Saccharomyces cerevisiae.
In contrast to S. cerevisiae, very
little is known about the molecular regulation of
C. neoformans cell cycle.
To clarify
cell
regulation
at the molecular level,
cell cycle control genes in C. neoformans
were cloned
and analyzed, and further studies are
currently
being done to confirm their function in
C. neoformans cell cycle.
The
homologues
of CDC28/Cdc2
(CnCdk1),
the main cell cycle gene which regulates the major processes in eukaryotic cell
cycle,
and its cyclin counterparts,
known to interact with CDC28/Cdc2 and activate it to carry out specific controls
throughout different stages of the cell cycle,
were
isolated
and identified
from C. neoformans.
In addition to CnCdk1, at
least three cell-cycle related cyclin homologues
were identified in C. neoformans.
Analysis of putative amino acid sequences
of these cyclin homologues
showed that
one
is a G1 cyclin homologue,
named CnCln1. The molecular characterization of the two main cell cycle genes,
CnCdk1
and
CnCln1, in the pathogenic yeast C.
neoformans, will be reported and discussed.