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

2

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.

3

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.

5

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