Oleolytic biofilm biodiversity and functionality in marine environments

En milieu marin le carbone organique particulaire (POC) représente 25 % du carbone organique total. Sa dégradation est réalisée par des microorganismes hétérotrophes ayant mis en place diverses stratégies pour parvenir à le dissoudre et l’assimiler. Peu d’études se sont intéressées à la dégradation des composés polymériques et/ou hydrophobes, quasiment insolubles dans l’eau constituant le POC. Parmi ces composés, on retrouve les lipides et les hydrocarbures regroupés sous le terme de COH (composés organiques hydrophobes). La dégradation des COH est réalisée par des bactéries dîtes oléolytiques ayant entre autre pour stratégie la formation de biofilms également qualifiés d’oléolytique. Nos connaissances sur la diversité et la fonctionnalité des biofilms oléolytiques se limitent actuellement aux bactéries spécifiquement étudiées pour leur capacité à dégrader les HC. Ainsi la dégradation des lipides est souvent négligée alors que cette famille de molécules représente une part significative du POC.La diversité taxonomique des bactéries formant des biofilms oléolytiques a été déterminée par un criblage de 199 souches marines sur 4 substrats : un alcane (paraffine), un triglycéride (tristéarine), un acide gras (acide palmitique) et une cire (l’hexadécyl palmitate). Cette étude a révélé que les bactéries oléolytiques (formant un biofilm sur au moins 1 des substrats) sont relativement répandues parmi les bactéries marines puisque qu’elles représentent 18.7 % des souches testée. Cette étude montre également que les bactéries capables d’assimiler les alcanes sont également capables d’assimiler au moins un lipide. Les bactéries hydrocarbonoclastes, jusqu’alors décrites comme spécialisées, voir restreintes à l’assimilation des hydrocarbures, présentent donc une gamme de substrats s’étendant aux lipides. La corrélation positive entre la capacité d’assimilation des alcanes et l’assimilation des lipides suggère un lien physiologique entre l’assimilation de ces deux familles de COH. L’activité lipase qui est essentielle à l’assimilation des triglycérides mais pas à l’assimilation des alcanes, a été mesurée dans des cultures de souches oléolytiques poussant sur acétate, triglycéride ou hexadécane. Comme attendu, les cultures sur triglycérides montrent toutes une surexpression de l’activité lipases par rapport aux cultures sur acétate. Les cultures sur hexadécane montraient aussi une surexpression de l’activité lipase renforçant l’idée d’un lien physiologique entre dégradation des alcanes et dégradation des lipides. De plus les souches oléolytiques n’ont pas montré de capacité à former un biofilm sur une surface inerte hydrophobe telle que le polystyrène ou sur une surface hydrophile telle que le verre à la hauteur de celles constaté sur COH. Une étude quantitative de l’adhésion sur COH et substrats inertes réalisée par microscopie montre que l’adhésion (dans les conditions testées) n’est pas un facteur déterminant de la formation de biofilm sur ces mêmes substrats. Cela suggère que la spécificité de formation de biofilm sur les substrats COH, ne réside pas dans l’adhésion mais vraisemblablement dans les étapes de développement du biofilm plus tardives.Enfin, les biofilms oléolytiques mettant en jeux des produits extracellulaires (enzymes et facteurs de solubilisation) qui constituent des biens communs, sont propices à l’établissement de comportements sociaux. Nous avons mis en évidence des comportements synergiques (5/8 des comportements observés) ou compétitifs au sein de biofilm oléolytiques (3/8 des comportements observés).Particulate organic carbon (POC), in marine environment, accounts for 25% of total organic carbon. POC degradation is carried out by heterotrophic microorganisms which have developed strategies to dissolve and assimilate it. Few studies have investigated the degradation of the polymeric and / or hydrophobic components of POC, which are almost insoluble in the water. Among these compounds, there are lipids and hydrocarbons (HC) grouped under the term of HOCs (hydrophobic organic compounds). The degradation of the HOCs is carried out by oleolytic bacteria which form biofilms at the HOC– water interface. Our knowledge of the diversity and functionality of oleolytic biofilms is mostly limited to HC degrading bacteria, while the degradation of lipids is often neglected although this family of molecules represents a significant part of the POC. A screening of 199 marine strains on 4 substrates: an alkane (paraffin), a triglyceride (tristearin), a fatty acid (palmitic acid) and a wax ester (hexadecyl palmitate) was performed to determine the taxonomic diversity of bacteria able to form oleolytic biofilms. This study revealed that oleolytic bacteria (forming a biofilm on at least 1 substrate) were relatively widespread among marine bacteria since they represented 18.7% of tested strains. This study also showed that bacteria able to assimilate alkanes were also able to assimilate at least one lipid. Hydrocarbonoclastic bacteria, previously described as specialized, or restricted to the assimilation of hydrocarbons, have actually a substrate range spanning from HC to lipids. The positive correlation between the ability to form a biofilm on alkanes and on lipids suggested a physiological link between the assimilation of these two HOC families. The lipase activity, which is essential for triglycerdides assimilation but not for the alkanes assimilation, was measured in oleolytic strains cultures growing on acetate, triglyceride or hexadecane. As expected, overexpression of lipase activity was observed in cultures on triglycerides compared to cultures on acetate. Moreover, overexpression of lipase activity was also observed in cultures on hexadecane reinforcing the idea of a physiological link between alkanes and lipids degradation.Oleolytic strains exhibited a very weak ability to form a biofilm on the inert surfaces (non-nutritive) polystyrene or glass compared to the HOC nutritive surface indicating that oleolytic strains have a specificity for HOC to form a biofilm. A quantitative study of adhesion on HOC and inert substratums carried out by microscopy shows that adhesion (in the tested conditions) is not a determining factor of the biofilm formation on these same substrates. This suggests that the specificity of biofilm formation on HOC substrates does not reside in adhesion but presumably in later biofilm development stages.Lastly, oleolytic biofilms, involving extracellular products (enzymes and solubilization factors) that constitute public goods, are favorable to the establishment of social behaviors. We have demonstrated synergistic behaviors (5/8 of observed behaviors) or competitive behaviors (3/8 of observed behaviors) in oleolytic biofilms

Oleolytic biofilm biodiversity and functionality in marine environments

En milieu marin le carbone organique particulaire (POC) représente 25 % du carbone organique total. Sa dégradation est réalisée par des microorganismes hétérotrophes ayant mis en place diverses stratégies pour parvenir à le dissoudre et l’assimiler. Peu d’études se sont intéressées à la dégradation des composés polymériques et/ou hydrophobes, quasiment insolubles dans l’eau constituant le POC. Parmi ces composés, on retrouve les lipides et les hydrocarbures regroupés sous le terme de COH (composés organiques hydrophobes). La dégradation des COH est réalisée par des bactéries dîtes oléolytiques ayant entre autre pour stratégie la formation de biofilms également qualifiés d’oléolytique. Nos connaissances sur la diversité et la fonctionnalité des biofilms oléolytiques se limitent actuellement aux bactéries spécifiquement étudiées pour leur capacité à dégrader les HC. Ainsi la dégradation des lipides est souvent négligée alors que cette famille de molécules représente une part significative du POC.La diversité taxonomique des bactéries formant des biofilms oléolytiques a été déterminée par un criblage de 199 souches marines sur 4 substrats : un alcane (paraffine), un triglycéride (tristéarine), un acide gras (acide palmitique) et une cire (l’hexadécyl palmitate). Cette étude a révélé que les bactéries oléolytiques (formant un biofilm sur au moins 1 des substrats) sont relativement répandues parmi les bactéries marines puisque qu’elles représentent 18.7 % des souches testée. Cette étude montre également que les bactéries capables d’assimiler les alcanes sont également capables d’assimiler au moins un lipide. Les bactéries hydrocarbonoclastes, jusqu’alors décrites comme spécialisées, voir restreintes à l’assimilation des hydrocarbures, présentent donc une gamme de substrats s’étendant aux lipides. La corrélation positive entre la capacité d’assimilation des alcanes et l’assimilation des lipides suggère un lien physiologique entre l’assimilation de ces deux familles de COH. L’activité lipase qui est essentielle à l’assimilation des triglycérides mais pas à l’assimilation des alcanes, a été mesurée dans des cultures de souches oléolytiques poussant sur acétate, triglycéride ou hexadécane. Comme attendu, les cultures sur triglycérides montrent toutes une surexpression de l’activité lipases par rapport aux cultures sur acétate. Les cultures sur hexadécane montraient aussi une surexpression de l’activité lipase renforçant l’idée d’un lien physiologique entre dégradation des alcanes et dégradation des lipides. De plus les souches oléolytiques n’ont pas montré de capacité à former un biofilm sur une surface inerte hydrophobe telle que le polystyrène ou sur une surface hydrophile telle que le verre à la hauteur de celles constaté sur COH. Une étude quantitative de l’adhésion sur COH et substrats inertes réalisée par microscopie montre que l’adhésion (dans les conditions testées) n’est pas un facteur déterminant de la formation de biofilm sur ces mêmes substrats. Cela suggère que la spécificité de formation de biofilm sur les substrats COH, ne réside pas dans l’adhésion mais vraisemblablement dans les étapes de développement du biofilm plus tardives.Enfin, les biofilms oléolytiques mettant en jeux des produits extracellulaires (enzymes et facteurs de solubilisation) qui constituent des biens communs, sont propices à l’établissement de comportements sociaux. Nous avons mis en évidence des comportements synergiques (5/8 des comportements observés) ou compétitifs au sein de biofilm oléolytiques (3/8 des comportements observés).Particulate organic carbon (POC), in marine environment, accounts for 25% of total organic carbon. POC degradation is carried out by heterotrophic microorganisms which have developed strategies to dissolve and assimilate it. Few studies have investigated the degradation of the polymeric and / or hydrophobic components of POC, which are almost insoluble in the water. Among these compounds, there are lipids and hydrocarbons (HC) grouped under the term of HOCs (hydrophobic organic compounds). The degradation of the HOCs is carried out by oleolytic bacteria which form biofilms at the HOC– water interface. Our knowledge of the diversity and functionality of oleolytic biofilms is mostly limited to HC degrading bacteria, while the degradation of lipids is often neglected although this family of molecules represents a significant part of the POC. A screening of 199 marine strains on 4 substrates: an alkane (paraffin), a triglyceride (tristearin), a fatty acid (palmitic acid) and a wax ester (hexadecyl palmitate) was performed to determine the taxonomic diversity of bacteria able to form oleolytic biofilms. This study revealed that oleolytic bacteria (forming a biofilm on at least 1 substrate) were relatively widespread among marine bacteria since they represented 18.7% of tested strains. This study also showed that bacteria able to assimilate alkanes were also able to assimilate at least one lipid. Hydrocarbonoclastic bacteria, previously described as specialized, or restricted to the assimilation of hydrocarbons, have actually a substrate range spanning from HC to lipids. The positive correlation between the ability to form a biofilm on alkanes and on lipids suggested a physiological link between the assimilation of these two HOC families. The lipase activity, which is essential for triglycerdides assimilation but not for the alkanes assimilation, was measured in oleolytic strains cultures growing on acetate, triglyceride or hexadecane. As expected, overexpression of lipase activity was observed in cultures on triglycerides compared to cultures on acetate. Moreover, overexpression of lipase activity was also observed in cultures on hexadecane reinforcing the idea of a physiological link between alkanes and lipids degradation.Oleolytic strains exhibited a very weak ability to form a biofilm on the inert surfaces (non-nutritive) polystyrene or glass compared to the HOC nutritive surface indicating that oleolytic strains have a specificity for HOC to form a biofilm. A quantitative study of adhesion on HOC and inert substratums carried out by microscopy shows that adhesion (in the tested conditions) is not a determining factor of the biofilm formation on these same substrates. This suggests that the specificity of biofilm formation on HOC substrates does not reside in adhesion but presumably in later biofilm development stages.Lastly, oleolytic biofilms, involving extracellular products (enzymes and solubilization factors) that constitute public goods, are favorable to the establishment of social behaviors. We have demonstrated synergistic behaviors (5/8 of observed behaviors) or competitive behaviors (3/8 of observed behaviors) in oleolytic biofilms

Quorum-quenching limits quorum-sensing exploitation by signal-negative invaders

Some bacteria produce and perceive quorum-sensing (QS) signals that coordinate several behaviours, including the costly processes that are exoenzyme production and plasmid transfer. In the case of plasmid transfer, the emergence of QS signal-altered invaders and their policing are poorly documented. In Agrobacterium tumefaciens, the virulence Ti-plasmid encodes both synthesis and sensing of QS-signals, which promote its transfer from a donor to a recipient cell. Here, we reported that QS-altered A. tumefaciens mutants arose during experimental evolution. All showed improved growth compared to their ancestor. Genome sequencing revealed that, though some had lost the Ti-plasmid, most were defective for QS-signal synthesis and Ti-plasmid conjugation (traR mutations) and one exhibited a QS-signal exploitation behaviour, using signal produced by other cells to enhance its own Ti-plasmid transfer. We explored mechanisms that can limit this QS-hijacking. We showed that the A. tumefaciens capacity to inactivate QS-signals by expressing QS-degrading enzyme could attenuate dissemination of the QS signal-negative Ti-plasmids. This work shows that enzymatic QSdisruption whether encoded by the QS-producing Ti-plasmid itself, by a companion plasmid in the same donor cells, or by one in the recipient cells, in all cases can serve as a mechanism for controlling QS exploitation by QS signal-negative mutants

Quorum-quenching limits quorum-sensing exploitation by signal-negative invaders

Some bacteria produce and perceive quorum-sensing (QS) signals that coordinate several behaviours, including the costly processes that are exoenzyme production and plasmid transfer. In the case of plasmid transfer, the emergence of QS signal-altered invaders and their policing are poorly documented. In Agrobacterium tumefaciens, the virulence Ti-plasmid encodes both synthesis and sensing of QS-signals, which promote its transfer from a donor to a recipient cell. Here, we reported that QS-altered A. tumefaciens mutants arose during experimental evolution. All showed improved growth compared to their ancestor. Genome sequencing revealed that, though some had lost the Ti-plasmid, most were defective for QS-signal synthesis and Ti-plasmid conjugation (traR mutations) and one exhibited a QS-signal exploitation behaviour, using signal produced by other cells to enhance its own Ti-plasmid transfer. We explored mechanisms that can limit this QS-hijacking. We showed that the A. tumefaciens capacity to inactivate QS-signals by expressing QS-degrading enzyme could attenuate dissemination of the QS signal-negative Ti-plasmids. This work shows that enzymatic QSdisruption whether encoded by the QS-producing Ti-plasmid itself, by a companion plasmid in the same donor cells, or by one in the recipient cells, in all cases can serve as a mechanism for controlling QS exploitation by QS signal-negative mutants

Atomic Force Microscopy Stiffness Mapping in Human Aortic Smooth Muscle Cells

International audienceAortic smooth muscle cells (SMCs) play a vital role in maintaining mechanical homeostasis in the aorta. We recently found that SMCs of aneurysmal aortas apply larger traction forces than SMCs of healthy aortas. This result was explained by the significant increase of hypertrophic SMCs abundance in aneurysms. In this study, we investigate whether the cytoskeleton stiffness of SMCs may also be altered in aneurysmal aortas. For that, we use atomic force microscopy (AFM) nano-indentation with a specific mode that allows subcellular-resolution mapping of the local stiffness across a specified region of interest of the cell. Aortic SMCs from a commercial human lineage (AoSMCs, Lonza) and primary aneurysmal SMCs (AnevSMCs) are cultured in conditions promoting the development of their contractile apparatus, and seeded on hydrogels with stiffness properties of 12 kPa and 25 kPa. Results show that all SMCs exhibit globally a lognormal stiffness distribution, with medians in the range 10–30 kPa. The mean of stiffness distributions is 16 kPa in aneurysmal SMCs and 12 kPa in healthy cells, but the differences are not statistically significant due to the large dispersion of AFM nano-indentation stiffness. We conclude that the possible alterations previously found in aneurysmal SMCs do not affect significantly the AFM nano-indentation stiffness of their cytoskeleton

Description of Palleronia rufa sp. nov., a biofilm-forming and AHL-producing Rhodobacteraceae, reclassification of Hwanghaeicola aestuarii as Palleronia aestuarii comb. nov., Maribius pontilimi as Palleronia pontilimi comb. nov., Maribius salinus as Palleronia salina comb. nov., Maribius pelagius as Palleronia pelagia comb. nov. and emended description of the genus Palleronia

International audienceStrain MOLA 401T was isolated from marine waters in the southwest lagoon of New Caledonia and was shown previously to produce an unusual diversity of quorum sensing signaling molecules. This strain was Gram-negative, formed non-motile cocci and colonies were caramel. Optimum growth conditions were 30 °C, pH 8 and 3% NaCl (w/v). Based on 16S rRNA gene sequence analysis, this strain was found to be closely related to Pseudomaribius aestuariivivens NBRC 113039T (96.9% of similarity), Maribius pontilimi DSM 104950T (96.4% of similarity) and Palleronia marisminoris LMG 22959T (96.3% of similarity), belonging to the Roseobacter group within the family Rhodobacteraceae. As its closest relatives, strain MOLA 401T is able to form a biofilm on polystyrene, supporting the view of Roseobacter group strains as prolific surface colonizers. An in-depth genomic study allowed us to affiliate strain MOLA 401T as a new species of genus Palleronia and to reaffiliate some of its closest relatives in this genus. Consequently, we describe strain MOLA 401T (DSM 106827T = CIP 111607T = BBCC 401T) for which we propose the name Palleronia rufa sp. nov. We also propose to emend the description of the genus Palleronia and to reclassify Maribius and Hwanghaeicola species as Palleronia specie