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Lichen symbioses as a bioresource for natural products
Martin Grube1
1Karl-Franzens-University, Graz, Austria
in collaboration within collaboration with
Joel Boustie2, Delphine Parrot3, Sophie Tomasi2
2Université de Rennes 1, France3INRIA, Lyon, France
King George Island, Antarctica
More than 10% of terrestrial surface are dominated by lichensof terrestrial surface are dominated by lichens
What are lichens?
Not all fungal–algal combinations will form lichens !
Specific fungal–algal combinations for mutual support !
lichens:up to 86°S latitudeup to 7400 m altitudephotosynthesis at < 0°C
c. 2300 m alt.
Bacteria develop biofilm-like structures on lichensBacteria develop biofilm like structures on lichens
Cl d i b l L l tU bili i li d iCladonia arbuscula Lecanora polytropaUmbilicaria cylindrica
Grube et al. (2009) ISME J
Erlacher et al. Front Microbiol 2015
Bacteria on lower side seem to form different communities
Image: Ines Aschenbrenner
Reproduction modes of lichens
sexual asexual
Mitotic propagules (soredia)Apothecia (meiotic Ascospores)
Aschenbrenner et al. 2014 Environ Microbiol
Bacterial colonization on vegetative propagules
Volume rendering
Green: Algae
Microbial Cargo: Do bacteria on symbiotic propagules reinforce the microbiome of lichens? (2014) Ines Aline Aschenbrenner1,2, Massimiliano Cardinale1,2, Gabriele Berg1 and Martin Grube2
DOI: 10.1111/1462-2920.12658
3D re-construction
Yellow: Alphaproteobacteria
Red: other eubacteria
Putative new bacterial lineages are present in lichens
Bacteria have a high survival rate in storedmaterial (Cernava et al accepted) !material (Cernava et al. accepted) !
Frondihabitans cladoniiphilum sp novFrondihabitans cladoniiphilum sp. nov., new in ActinobacteriaCells are non-motile, non-spore-forming, aerobic, irregular, short rods (1.2–2.0 μm) that stain Gram-positive Growth occurs at temperature between 1 andpositive. Growth occurs at temperature between 1 and 30°C (optimum temperature, 22−26 °C) and at pH 3–8 (optimum, pH 3.7−5.4). Catalase-positive and oxidase-negative. The type B2β peptidoglycan contains the amino acids ornithine, alanine, glycine, homoserine and glutamic acid in an approximate molar ratio of 1.0 : 0.5 :
Selbmann et al. (2009) Polar BiolCardinale et al. (2011) IJSEM
1.0 : 0.5 : 1.0, respectively. Ornithine is the diagnostic diamino acid. Hydroxyglutamic etc.etc.
From diversity to function: a metagenomic approach
• Metagenomic DNA was extracted for Illumina gnext generation sequencing
DNADNA Extraction
170 mio paired-end reads were obtained pwith an average read length of 100 bp
Analysis and public repository
Tera-BLASTTM (blastN and blastX)
15Metagenomic studies – functional profile
Distribution of particular functions in the Lobaria metagenome and the most abundant taxa therein. Grube et al. (2015) ISME J
16 Metaproteomes – Taxonomic (A) and functional (B) assignment
Grube et al. (2015) ISME J(Voronoi treemaps)
From diversity to function: environmental proteomics
Schneider et al. (2011) Proteomics
A refined model for the lichen symbiosis
Vitamins, Cofactors,growth-promoting hormones Provision of carbohydratesgrowth promoting hormonesDegradation and recycling,Resistance against stress factorsNitrogen fixation,
Provision of carbohydratesProtection from stress
System regulationg ,
phosphate solubilization CO2 - fixation
Lichen secondary metabolitesLichen secondary metabolites
• Compounds from lichen fungi: 854 glichen substances (Elix 2016)
• Compounds from photobionts
C f• Compounds from associated fungi
C d f• Compounds from associated bacteria
Typical compounds from acetate polymalonate pathwayTypical compounds from acetate-polymalonate pathway
orcinols and orsellinic acid are often the building blocks for more complex compounds
Boustie & Grube 2005 Pl Gen Res
optional reducing domains
KS KRMT ACPERDH TEAT ACPPTSAT
Extension ProcessingLoading
type I PKS generalstructure
optional reducing domains
• Evolution of type I polyketide synthases in fungi includes mutation, domain acquisition,fungi includes mutation, domain acquisition, gene duplication, and horizontal transfer frombacteria
• Typically, many more genes are encoded inTypically, many more genes are encoded in fungal genomes than compounds expressed
Kroken et al. 2003 PNAS
Overview of the biosynthesis of dibenzofuran metabolites
Millot et al. 2016 Nat Prod Rev
Usnic acid
OH OO AntioxidantAntiinflamatory O
H3C
HO O
CH3H3C
Antioxidant
Gastroprotective
Cardioprotective
Antiinflamatory
Immunoestimulatory
OCH3
Cardioprotective
Cytoprotective
Antimicrobial
AnticarcinogenicPro-oxidant
• Antimicrobial effects of usnic acid, especially against gram positive bacteria, are widely reported in literature while mechanisms of action remain poorly studiedp y
• the medicinal use of usnic acid remains limited due to its hepatotoxicity
Millot et al. 2016 Nat Prod Rev
Usnic acid actsagainstbiofilm formation ofbiofilm formation ofStaphylocooccusaureus
Confocal images showing S. aureus biofilm on the surfaces of the control polyurethane (A) and (� )-usnicacid-loaded polyurethane (B) disks 3 days postinoculation. The grey arrow indicates a cluster of cocci, and the white arrow indicates the biofilm slime matrix. Bar, 55 � m. S. aureus adhered to the usnic acid-loaded polyurethane disk after 30 min (C) and 24 h (D) stained with the Live/Dead BacLight viability kit. Bar,
Francolini et al. 2004 Appl Env Microbiol
125 � m. The predominance of red cells after 24 h indicated a progressive loss of viability.
Xanthone dimers from lichens
Effective against Staphylococcus aureus and Bacillus subtilis
Wezeman et al. (2015) Nat Prod Rev
Eumitrins A1 and A2 could be used as inhibitors for nitric oxide formation by macrophages and thus be useful for a wide range of illnesses
Lichen compounds: effects on bacteria
Protolichesterinsäure
Active against:Interferes with the QS based communication systems ofgEscherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Proteusvulgaris, Streptococcus faecalis, Bacillus cereus, Bacillussubtilis, Pseudomonas aeruginosa, Listeria monocytogenes, Helicobacter pylori,
Interferes with the QS-based communication systems of Pseudomonas aeruginosa and Burkholderia cenocepacia and represses protease production in both organisms without inhibiting growthMycobacterium aurum, M. tuberculosis
Chromobacter violaceum J26 assay
Vulpinic acidEffects on bacterial gene expression g p
Alterations in secondary metabolism of aposymbiotically grown mycobionts of
voucher specimen aposymbiotically grown mycobionts of
Xanthoria elegans and cultured resynthesis stages
l dcultured mycobiont
resynthesis
Brunauer et al 2007 Pl Phys Biochem
Unusual compounds in lichens
Boustie & Grube 2005 Pl Gen Res
Mycosporines as light protectantsMycosporines as light-protectants
Roullier et al. 2011 Phytochemistry
Nosperin expression in natural thalli of the lichen Peltigera membranacea: the first cyanobacterial trans-AT polyketide biosynthetic pathway in the Nostocy p y y p ysymbiont discovered by metagenomics
Kampa et al. 2013 PNAS
Bioactive compounds of lichen-associated bacteria: an untapped treasure chest
Bioactive compounds of lichen-associated bacteria: an untapped treasure chest
So far only few actinobacterial isolates were analysed for their biosynthetic potential
uncialamycin dinemicin angucylin butenolide JBIR98
cladoniiamides A-Gcladoniiamides A-G
Parrot et al. 2016, Suzuki et al. 2016Coumabiocins A-F, novobiocin, isonovobiocin
Bacterial bioconversion of lichen metabolites
erythtrennes #67-314 RT: 2,24-9,60 AV: 133 SB: 80 26,29-31,07 NL: 5,11E6T: c ESI Full ms [ 50 00 2000 00]
T: - c ESI Full ms [ 50,00-2000,00]167,15T: - c ESI Full ms [ 50,00-2000,00]
3000000
4000000
5000000
ty
271,08
167,15 60000
sity
,
intermediated 0 d + 15
1000000
2000000
3000000
Inte
nsit
421,11149,14 306,98123,15 542,96357,08 422,18175,03 596,06 693 03
20000
40000
Inte
ns
123,12 168,13 359,10312 93 496 21100 200 300 400 500 600 700
0175,03 596,06 693,03
100 200 300 400 500 6000
,312,93 496,21382,60 6107,13 517,63
Esterase Esterase
Orsellinic acid/
MontagnetolErythrin/ [ ] m/z [M-H]- 167m/z [M-H]- 271m/z [M-H]- 421
Parrot et al. ms.
Compounds of lichen-associated bacteria: Marine lichens as a potential source
cyaneodimycin
Streptomyces cyaneofuscatus
usnic acidPhenoxazine Diketopiperazines
cyaneodimycin
Parrot et al. EurJOC accepted
37 Imaging chemistry – LDI-MSg g y
Le Pogam et al. 2015 Anal Chem
38 Lichen symbiosis – chemical variationLichen symbiosis – chemical variation
inherited variation, abiotic and biotic induction of variation...
Islandoquinone
Coculturing will be a fruitful approach in the future to detect the influenceof symbiotic partners
39 Coculturing of fungal, algal and bacterial partners
Muggia et al. submitted
40
Conclusions
Lichens harbor a highly diverse well structured bacterial Lichens harbor a highly diverse, well-structured bacterial microbiome – the 3rd component of the symbiosis
Lichen fungi photobionts associated fungi and bacteriaLichen fungi, photobionts, associated fungi and bacteria produce secondary metabolites of considerable interest
Metagenomic approaches reveal biosynthetic pathways inMetagenomic approaches reveal biosynthetic pathways in various members of the lichen symbioses
Experimental work requires coculturing of symbiotic partnersp q g y pto assess and utilize the extended biosynthetic potential
Acknowledgments
Austrian lichen team:
Gabriele Berg (TU Graz)Tomislav Cernava (TU Graz) ( )Ines Aschenbrenner (KFU Graz)Lucia Muggia (KFU Graz)
French lichen team:
Marcelino Suzuki (CNRS Banyuls)Marylène Chollet-Krugler (Univ. Rennes)Pierre Le Pogam (Univ. Rennes)
42 ConclusionsConclusions Lichens harbor a highly diverse well Lichens harbor a highly diverse, well-
structured bacterial microbiome – the 3rd t f th bi i ?component of the symbiosis?
Specific taxa are well-adapted and Welcome to Graz!p ppredominantly present on various lichens.
The lichen microbiome fulfills essential
Welcome to Graz!
The lichen microbiome fulfills essential functions within the symbiosis.
A combination of –omics and conventional techniques was useful to obtain a holistictechniques was useful to obtain a holistic view on microbial contributions.