Molecular adaptations to hypersaline environments in Hortaea werneckii and

some other selected fungi – I.Nina Gunde-CimermanAna Plemenitaš

(Hyper)saline environment

Salts dissolved in water Lowered water potential (Ψ) – less water is available to organisms

Cl-

Hypertonic environmentNa+

Loss of H2ONaCl

Ψs = - CiRT

[NaCl] Water potential

Isolation and detection of fungi

Samples: brine, immersed wood, surface of halophites, soil, microbial mats

Classic isolation: filtering, direct inoculaton, very selective media (17-32% NaCl), aerobic, microaerophilic, anaerobic incubation

Molecular methods:DNA isolation from

natural samples,PCR, Temporal Temperature

Gradient gel electrophoresis (TGGE),

Clone Sequencing Libraries

Salt as source of contamination of food with fungi

Fungi are inhabitants of solar salterns and salty lakes around the

world

Population dynamics of fungal communities in hypersaline environments (Statistical canonical correspondence analysis)– temporal factor, – water activity,– nutrients (P, N)

Numbers: hypersaline water up to 40.000 CFU/l

For comparison: ocean water 10 CFU/l, polluted sea water 5.000 CFU/l

Emericella FusariumAcremonium,Aspergillus, Penicillium, yeasts

EurotiumAternaria Cladosporium

Black yeasts

Wallemia

3% 17% 35%

Halotolerant and halophilic black yeasts

• Growth within a wide range of NaCl (0 - 32%)

• Constant presence in salterns/ rare occurrence elsewhere

• Extremophilic ecotype: slow, often meristematic growth, proliferation with endoconidiation, thick, melanized cell walls

Understanding life at low water activity –important for agronomy and biotechnology

Almost 400 Mha of land is salinized(Nachtergaele and Young, 2000)

20% of irrigated soil is salinized (Yeo, 1999)

On a global scale we lose every minute 10 hectares of arable land, 3 due to salinization (Kovda, 1983)

Model organisms for adaptations to hypersaline conditions

Saccharomyces cerevisiae

• Aspergillus nidulans• Candida sorbitophila• Candida versatilis• Debaryomyces hansenii• Pichia guilliermondii• Rhodotorula mucilaginosa • Zygosaccharomyces rouxii

New model organisms needed!!!

Halotolerant model -Aureobasidium pullulans

var. pullulans: plant, Salterns, osmotic subst.

var. melanogenum: glaciers, water, deep sea

var. subglaciale:glaciers

var. namibiae: desert

Freezing – dehydration of cells due to iceformation and deminished water absorption

High salinity - dehydration of cells due to osmotic disbalances

10th European Conference on Fungal Genetics: Dothideomycetes Comparative Genomics Workshop, March 29, 2010

US Department of Energy Joint Genomes Institute (DOE JGI), Walnut Creek, Ca, USA.

Genomes of 4 A. pullulans varieties?

Hortaea werneckii – extremely halotolerant model organism

Hortaea werneckii

0

10

20

30

40

0 5 10 15 20 25 30

NaCl (%)

max

pre

mer

kol

onije

(mm

)

H. werneckii

Ecological niches

Genom of H. werneckii already in progress...

Wallemia ichthyophaga – obligate halophile

Wallemia sebi (up to 2005)

0

1

2

3

4

5

6

7

8

9

10

0,999 0,984 0,976 0,959 0,957 0,88 0,876 0,852 0,832 0,805 0,771

water activity (aw)

aver

age

colo

ny d

iam

eter

(mm

)

Wallemia sebi Wallemia muriae Wallemia ichthyophaga

Groth of 3 Wallemia species on media with different water activity

AFTOL project:

Hibbett et al.: A higher-level phylogenetic classification of the Fungi. Mycological

research 111 (2007): 509-547.

BASIDIOMYCOTA

10

Trichosporon faecale AB001728 TSTrichosporon japonicum AB001749 TSTrichosporon aquatile AB001730 TS

Cryptococcus longus AB035586 TSCryptococcus fragicola AB035588 TSBullera unica D78330 TLBulleromyces albus X60179 TLSterigmatosporidium polymorphum D64120 TLCryptococcus neoformans var. gattii AJ560318 TLFilobasidiella neoformans D12804 TLFilobasidiella depauper AJ568017 TL

Filobasidium elegans AB075545 FBFilobasidium globisporum AB075546 FBFilobasidium capsuligenum AB075544 FBMrakia frigida D12802 CFBMrakia psychrophilia AJ223490 CFBCryptococcus aquaticus AB032621 CFB

Udeniomyces megalosporus D31657 CFBUdeniomyces puniceus D31658 CFB

Udeniomyces piricola D31659 CFBCystofilobasidium infirmo-miniatum AB072226 CFB

Dacrymyces chrysospermus L22257 DMRhodotorula bacarum AB038129 MS

Rhodotorula phylloplana AJ496258 MSSympodiomycopsis paphiopedili D14006 MS

Malassezia furfur AY083223 MZTilletia caries U00972 TT

Tilletiopsis lilacina AB023414 ETTilletiopsis cremea AB023413 ET

Tilletiopsis derxii AB045704 ETTilletiopsis fulvescens D83189 GF

Tilletiaria anomala D83193 GFTilletiopsis minor D83190 GF

Exobasidium vaccinii AJ271380 EBExobasidium rhododendri AJ271381 EBGraphiola cylindrica D63929 EB

W. sebi AF548107W. sebi CBS 196.56W. sebi EXF-757W. sebi AF548108

W. ichthyophaga EXF-994Taphrina nana AB000955 Taphrina ulmi AB000959

Taphrina deformans U00971 Taphrina sadebeckii AJ495820 Taphrina epiphylla AJ495823 Taphrina robinsoniana AJ495830 Protomyces macrosporus D85143

Pneumocystis carinii S83267Dipodascopsis uninucleata U00969

Dipodascus aggregatus AB000645

TS TrichosporonalesTL TremellalesFB FilobasidialesCFB CystofilobasidialesDM DacrymycetalesMS MicrostromatalesMZ MalassezialesTT TilletialesET EntylomatalesGF GeorgefischerialesEB Exobasidiales

92

100

64

10099

99

99

56

76

64

100

99

60

Wal

lem

ia

Wal

lem

iace

ae

Wal

lem

inal

esW

alle

mio

myc

etes

Het

erob

asid

iom

ycet

esU

stila

gino

myc

etes

Taph

rino-

myc

etes

Basidiomycota

PneumocystidomycetesSaccharomycetesSaccharomycetes

100

77

100

100

85

100

65

92

99

100

97 100

93

100

99

61

55

98

98

100

97

10

Trichosporon faecale AB001728 TSTrichosporon japonicum AB001749 TSTrichosporon aquatile AB001730 TS

Cryptococcus longus AB035586 TSCryptococcus fragicola AB035588 TSBullera unica D78330 TLBulleromyces albus X60179 TLSterigmatosporidium polymorphum D64120 TLCryptococcus neoformans var. gattii AJ560318 TLFilobasidiella neoformans D12804 TLFilobasidiella depauper AJ568017 TL

Filobasidium elegans AB075545 FBFilobasidium globisporum AB075546 FBFilobasidium capsuligenum AB075544 FBMrakia frigida D12802 CFBMrakia psychrophilia AJ223490 CFBCryptococcus aquaticus AB032621 CFB

Udeniomyces megalosporus D31657 CFBUdeniomyces puniceus D31658 CFB

Udeniomyces piricola D31659 CFBCystofilobasidium infirmo-miniatum AB072226 CFB

Dacrymyces chrysospermus L22257 DMRhodotorula bacarum AB038129 MS

Rhodotorula phylloplana AJ496258 MSSympodiomycopsis paphiopedili D14006 MS

Malassezia furfur AY083223 MZTilletia caries U00972 TT

Tilletiopsis lilacina AB023414 ETTilletiopsis cremea AB023413 ET

Tilletiopsis derxii AB045704 ETTilletiopsis fulvescens D83189 GF

Tilletiaria anomala D83193 GFTilletiopsis minor D83190 GF

Exobasidium vaccinii AJ271380 EBExobasidium rhododendri AJ271381 EBGraphiola cylindrica D63929 EB

W. sebi AF548107W. sebi CBS 196.56W. sebi EXF-757W. sebi AF548108

W. ichthyophaga EXF-994Taphrina nana AB000955 Taphrina ulmi AB000959

Taphrina deformans U00971 Taphrina sadebeckii AJ495820 Taphrina epiphylla AJ495823 Taphrina robinsoniana AJ495830 Protomyces macrosporus D85143

Pneumocystis carinii S83267Dipodascopsis uninucleata U00969

Dipodascus aggregatus AB000645

TS TrichosporonalesTL TremellalesFB FilobasidialesCFB CystofilobasidialesDM DacrymycetalesMS MicrostromatalesMZ MalassezialesTT TilletialesET EntylomatalesGF GeorgefischerialesEB Exobasidiales

92

100

64

10099

99

99

56

76

64

100

99

60

Wal

lem

ia

Wal

lem

iace

ae

Wal

lem

inal

esW

alle

mio

myc

etes

Het

erob

asid

iom

ycet

esU

stila

gino

myc

etes

Taph

rino-

myc

etes

Basidiomycota

PneumocystidomycetesSaccharomycetesSaccharomycetes

100

77

100

100

85

100

65

92

99

100

97 100

93

100

99

61

55

98

98

100

97

BGI Genomics Institute Shenzhen; China

H. werneckii and other fungi adapt to high concentrations of NaCl on many levels

Kogej, T., Wheeler, M.H., Lanišnik Rižner, T. and Gunde-Cimerman, N. 2004. Evidence for 1,8-dihydroxynaphthalene melanin in three halophilicblack yeasts grown under saline and non-saline conditions. FEMS Microbiol. Lett. 232, 203-209.

Melanisation of cell walls is salt dependent (distribution of granules, expression of genes)

Turk M., Mejanelle L., Šentjurc M., Grimalt J. O., Gunde-Cimerman N., Plemenitaš A. 2004. Salt-induced changes in lipid composition andmembrane fluidity of halophilic yeast-like melanized fungi. Extremophiles, 8, 1: 53-61.

Membrane fluidity changes

Kogej T., Stein M., Volkmann M., Gorbushina A. A., Galinski E. A., Gunde-Cimerman N. 2007. Osmotic adaptation of the halophilic fungusHortaea werneckii: role of osmolytes and melanization. Microbiology, 153, Pt 12: 4261-4273.

High levels of compatible solutes (glycerol, erythritol, arabitol, manitol) are synthesized

Vaupotič T, Plemenitaš A. 2007. Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerantblack yeast Hortaea werneckii, BMC Genomics 8:280-295.

Differential expression of many genes