Modèles de biofilms sociaux issus de la diversité marine
Régis GRIMAUD UMR5254, CNRS/ UNIV PAU & PAYS ADOUR, INSTITUT DES SCIENCES ANALYTIQUES ET DE PHYSICO - CHIMIE POUR L'ENVIRONNEMENT ET LES MATERIAUX.
Mardi 28 février 2017
Didier STIEN LABORATOIRE DE BIODIVERSITÉ ET BIOTECHNOLOGIES MICROBIENNES LBBM - USR3579 – CNRS- OBSERVATOIRE DE BANYULS - UNIVERSITÉ PIERRE ET MARIE CURIE
Biofilms are bacterial communities living at interfaces
Planktonic
Biofilms mono-species multi-species
1 Modèles de biofilms sociaux issus de la diversité marine
Neu, T.R. and Lawrence, J.R. (2015)
Trends in Microbiology 23: 233–242
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Biofilms are ubiquitous in ecosystems
Natural ecosystems Biofilms drive biogeochemical cycles C, N, S, P, Fe…
Artificial ecosystems Biofilms are responsible for major damages Bio-corrosion bio-fouling … Contamination of medical implants
Host associated ecosystems Biofilms are responsible for difficult to treat infections Biofilms are also essential for hosts: root or gut microbiota
Modèles de biofilms sociaux issus de la diversité marine
From: Flemming, H.-C., et al (2016) Nature Reviews Microbiology 14: 563–575.
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Biofilms are complex organized and diverse structures Heterogeneity in biofilms
Transport of solutes is controlled by diffusion
Immobilized cells High cell density Chemical micro-gradients Micro-habitat Physiological/taxonomic Diversity
Modèles de biofilms sociaux issus de la diversité marine
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Biofilms are complex organized and divers structures Social interactions in biofilms Communication Cooperation Competition
Modèles de biofilms sociaux issus de la diversité marine
Flemming, H.-C., et al (2016) Nature Reviews Microbiology 14: 563–575.
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Structure and properties of biofilms results from chemical heterogeneity and social interaction
Modèles de biofilms sociaux issus de la diversité marine
Structure function
Knowledge on biofilms at the molecular level comes from a few model bacteria, most often pathogenic
Estimated diversity of bacteria: ~30 000 described species
6 Modèles de biofilms sociaux issus de la diversité marine
Bacteria are obligate osmotrophs
Assimilation of substrates is limited by size and solubility
Dissolved carbon
< 1000 Da
Benner, Ronald, and Rainer M. W. Amon. “The Size-Reactivity Continuum of Major
Bioelements in the Ocean.” 2015 In Annual Review of Marine Science, Vol 7, edited
by C. A. Carlson and S. J. Giovannoni, 7:185–205.
Particles, Gels, colloids,
macromolecules
Adsorbed or dissolved in
non-aqueous phase
Particulate carbon
In ocean ~ 25% of organic carbon is not dissolved
~76% dissolved organic carbon: water-soluble low molecular weight compounds
small metabolites, organic acids and monomers or
small oligomers of biopolymers and their diagenetic
alteration products.
~22% Colloidal organic carbon: nm µm
~2 % Particulate organic carbon µm mm, cm
sinking = marine snow
80% proteins, polysaccharides, lipids
Reactive
Total Organic Carbon
TOC
Recalcitrant
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Biofilms enable solubilization of Particulate Organic Carbon Neige marine « hot spot » de l’activité bactérienne A marine snow aggregate 37 µm x 27 µm colonized by bacteria (~109 bacteria per ml) stained blue with DAPI
Modèles de biofilms sociaux issus de la diversité marine
Azam, Farooq, and Richard A. Long. “Sea Snow Microcosms.” Nature 414, no. 6863 (November 29, 2001): 495–98.
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Objective Establish new models for the study of interspecific social behaviors taking place within biofilms from Marine lytic biofilms
Modèles de biofilms sociaux issus de la diversité marine
Social behaviors: -cooperation: metabolites exchange, intercellular communications -competition: public goods exploitation, toxins production
Intercellular interactions are crucial for accessing to substrates not directly available.
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Marinobacter hydrocarbonoclasticus SP17 forms biofilms on lipids and hydrocrabons
Modèles de biofilms sociaux issus de la diversité marine
Biofilm on an alkane droplet
Biofilm on solid alkane
Triglycerides
Wax esters
Alkanes
Fatty acids
Fatty acids
Fatty alcohol
Biofilm on triglyceride
10 Modèles de biofilms sociaux issus de la diversité marine
M. hydrocarbonoclasticus biofilm on n-hexadecane produces a proteinaceous matrix
SEM image of M. hydrocarbonoclasticus biofilm grown on n-hexadecane
The matrix extract contained per gram (dried weight) of biofilm: 71 ± 7 mg of EPS 20.0 ±1.1 mg of proteins 3.5 ±0.1 mg of carbohydrates 0.5 ±0.1 mg of nucleic acids
11 Modèles de biofilms sociaux issus de la diversité marine
12 Modèles de biofilms sociaux issus de la diversité marine
The matrix comprises both cytoplasmic and extra-cytoplasmic proteins
Two –dimensional gel electrophoresis of matrix proteins extracts of M.
hydroarbonoclastcus SP17 biofilm grown on n-hexadecane.
.
Identified matrix proteins classified according to
COG categories.
Cellular localization of matrix proteins
predicted with PSORTb 3.0.2 and lipoP 1.0
13 Modèles de biofilms sociaux issus de la diversité marine
MARHY2041
35% 38% 44% 67% 42% 34% 65% 60% 28% 29% 37%
ΔCTGCTGGG
Pos.233 1KB miniTn5
Organization of T2SS genes in M. hydrocarbonoclasticus SP17
Pseudomonas aeruginosa PAO1
M. hydrocarbonoclasticus SP17
0.00
1.00
2.00
3.00
4.00
Extracellular Lipase activity p-NP_octanoate
hydrolysis (nmol.min-1)
Growth substrates
wild-type
gspG::miniTn5
The gspG::miniTn5 mutant is defective in lipase secretion
wt
gspG + lipase
gspG
Biofilm on trioleine
gspG + esterase
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Looking for oleolytic biofilms
Modèles de biofilms sociaux issus de la diversité marine
Microscope slide
Substrate coating
Biofilm
Mineral medium
Screening of collections (MOLA Banyuls) Crystal violet assay
Biofilms isolation from seawater: North Sea Bizerte Bay (Tunisia)
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Marine strains forming oleolytic biofilms
Modèles de biofilms sociaux issus de la diversité marine
Gamma Proteobacteria
Alpha Proteobacteria
Bacteriodetes
Actinobacteria
Paraffin (alkanes)
Triglyceride
Wax ester
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Marine strains forming oleolytic biofilms
Modèles de biofilms sociaux issus de la diversité marine
Class 1
Class 2
Class 3
Class 4
Class 5
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Revealing social interactions in oleolytic strains. Interstitial biofilm
Mixed species biofilm
Modèles de biofilms sociaux issus de la diversité marine
Identification of extracellular metabolites
Development of
genetic tools
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Des difficultés liées au milieu de culture (énormément de lipides) ? Il faut rechercher des conditions
adéquates pour améliorer la proportion de métabolites bactériens
-4 -2 0 2 4 6 8
Log2 Fold Change
0
1
2
3
4
5
-Lo
g1
0 P
-va
lue
Volcano plot généré par Compound Discoverer pour la recherche de biomarqueurs
Groupe des acyl-homosérine lactones de Maribius sp. déterminé par le GNPS. En bleu : AHLs standards
Notez que la majorité des masses dans le cluster
sont paires.
Remerciements
Claudie Barnier Laurent Urios Pierre Sivadon Florence Hakil Fanny Soula
Didier STIEN Raphaël LAMI
Laurent INTERGLIA Philippe LEBARON
PEPS ExoMOD