Effects of Osmotic Stress on Rhamnolipid Synthesis and Time-Course Production of Cell-To-Cell Signal Molecules by Pseudomonas aeruginosa

Biosynthesis of biosurfactant rhamnolipids by Pseudomonas aeruginosa depends on two hierarchical quorum sensing systems, LasRI and RhlRI, which synthesize and sense the signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and N-butyryl-L-homoserine lactone (C4-HSL), respectively. The Pseudomonas Quinolone Signal (PQS) is a third cell-to-cell signal molecule connecting these two systems, and its precursor, 2-heptyl-4-quinolone (HHQ), also constitutes a signal. The chronology of the production of signal molecules and rhamnolipids was determined during growth in PPGAS medium. Hyperosmotic condition (0.5 M NaCl) moderately affected growth, and led to intra-cellular accumulation of compatible solutes. Production of signal molecules was delayed and their highest concentrations were 2.5 to 5 fold lower than in NaCl-free PPGAS, except for HHQ, the highest concentration of which was increased. The presence of NaCl prevented rhamnolipid synthesis. When the osmoprotectant glycine betaine was added to PPGAS/NaCl medium, it was imported by the cells without being metabolized. This did not improve growth, but reestablished the time-courses of HSL and HHQ accumulation and fully or partially restored the HSL and PQS levels. It also partially restored rhamnolipid production. Quantification of mRNAs encoding enzymes involved in HSL, PQS, and rhamnolipid biosyntheses confirmed the effect of hyperosmotic stress and glycine betaine at the gene expression level

The Temperature-Regulation of Pseudomonas aeruginosa cmaX-cfrX-cmpX Operon Reveals an Intriguing Molecular Network Involving the Sigma Factors AlgU and SigX

Pseudomonas aeruginosa is a highly adaptable Gram-negative opportunistic pathogen, notably due to its large number of transcription regulators. The extracytoplasmic sigma factor (ECFσ) AlgU, responsible for alginate biosynthesis, is also involved in responses to cell wall stress and heat shock via the RpoH alternative σ factor. The SigX ECFσ emerged as a major regulator involved in the envelope stress response via membrane remodeling, virulence and biofilm formation. However, their functional interactions to coordinate the envelope homeostasis in response to environmental variations remain to be determined. The regulation of the putative cmaX-cfrX-cmpX operon located directly upstream sigX was investigated by applying sudden temperature shifts from 37°C. We identified a SigX- and an AlgU- dependent promoter region upstream of cfrX and cmaX, respectively. We show that cmaX expression is increased upon heat shock through an AlgU-dependent but RpoH independent mechanism. In addition, the ECFσ SigX is activated in response to valinomycin, an agent altering the membrane structure, and up-regulates cfrX-cmpX transcription in response to cold shock. Altogether, these data provide new insights into the regulation exerted by SigX and networks that are involved in maintaining envelope homeostasis

Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition

A unique collection of oceanic samples was gathered by the Tara Oceans expeditions (2009-2013), targeting plankton organisms ranging from viruses to metazoans, and providing rich environmental context measurements. Thanks to recent advances in the field of genomics, extensive sequencing has been performed for a deep genomic analysis of this huge collection of samples. A strategy based on different approaches, such as metabarcoding, metagenomics, single-cell genomics and metatranscriptomics, has been chosen for analysis of size-fractionated plankton communities. Here, we provide detailed procedures applied for genomic data generation, from nucleic acids extraction to sequence production, and we describe registries of genomics datasets available at the European Nucleotide Archive (ENA, www.ebi.ac.uk/ena). The association of these metadata to the experimental procedures applied for their generation will help the scientific community to access these data and facilitate their analysis. This paper complements other efforts to provide a full description of experiments and open science resources generated from the Tara Oceans project, further extending their value for the study of the world's planktonic ecosystems

Influence de l'environnement et des communications inter-bactériennes sur la production de biosurfactants par Pseudomonas Aeruginosa

La bioremèdiation, qui utilise la capacité naturelle des micro-organismes à disperser et dégrader les hydrocarbures, est l'une des perspectives des plus attractives pour la décontamination des zones souillées par les marées noires. Pseudomonas aeruginosa produit des biosurfactants (dispersants) de type glycolipides nommés rhamnolipides, dont la régulation dépend d'un système relatif à la densité cellulaire, le quorum sensing (QS). Lors de cette étude, la hiérarchie du QS fut pour la première fois mise en évidence par des cinétiques de production des deux molécules signal : la N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) et la N-butyryl-L-homoserine lactone (C4-HSL) (HSL), les rhamnolipides étant produits ultérieurement. Nous avons montré que le facteur sigma RpoN était impliqué dans l'arrêt de synthèse des 3OC12-HSL et qu'il n'avait aucune influence sur la production des C4-HSL, alors qu'il inhibait la synthèse des rhamnolipides. En revanche, RpoS activait indirectement la production des rhamnolipides, via les HSL. Nous avons observé qu'une concentration en sel proche de celle de l'eau de mer inhibait la production des rhamnolipides, en diminuant l'expression des gènes impliqués dans la synthèse des biosurfactants (rhlAB et rhlC). L'expression de rhlI et la synthèse des HSL qui en dépend, étaient également diminuées. Le stress hyperosmotique agissait sur d'autres facteurs associés au QS comme la pyocyanine et la formation du biofilm. Un apport en osmoprotecteur (glycine bétaïne) a permis une restauration partielle des expressions génétiques et des phénotypes, démontrant l'intérêt de son utilisation pour la bioremèdiation maritime. Les facteurs environnementaux agissent également sur la production des rhamnolipides indépendamment du QS, puisqu'une carence en phosphate favorisait la production des biosurfactants alors qu'elle diminuait la concentration en HSL. Contrairement aux autres gènes impliqués dans la production des rhamnolipides, rhlG était induit par le stress salin. Nos résultats indiquent que RhlR (régulateur de transcription fixant C4-HSL) est le principal inhibiteur de rhlG et que l'inhibition est augmentée par une concentration élevée en C4-HSL. Les facteurs sigma RpoN et 70 paraissent impliqués à parts égales dans la transcription de rhlG, et le complexe C4-HSL/RhlR inhiberait la transcription dépendante de RpoN. Nous n'avons en revanche pas pu confirmer que rhlG soit indispensable à la production des rhamnolipides. rhlG et rcsF qui sont organisés en opéron, seraient toutefois impliqués dans la croissance de P. aeruginosa en milieu limité en oxygène.The ability of micro-organisms to disperse and degrade hydrocarbons is used in bioremediation, which is one of the most attractive methods for the removal of crude oil in marine areas. Pseudomonas aeruginosa produces glycolipidic biosurfactants named rhamnolipids, the synthesis of which is regulated by a system depending of bacterial density: the quorum sensing (QS). In this study, the QS hierarchy was for the first time clearly shown by a time-course analysis of production of the two cell-to-cell signal molecules: N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and N-butyryl-L-homoserine lactone (C4-HSL), the rhamnolipids being subsequently produced. We showed here that the sigma factor RpoN stopped the production of 3OC12-HSL but had no effect on the C4-HSL one, despite its inhibitory effect on rhamnolipid production. RpoS was able to induce rhamnolipid production, probably by activating HSL production. We observed that a sea-water saline concentration inhibited rhamnolipid production, by reducing the expression level of the rhlAB, C and I genes, which are responsible for C4-HSL and rhamnolipid syntheses. Hyperosmotic stress also altered other QS-depending phenotypes such as pyocyanin production and biofilm formation. The osmoprotectant glycine betaine was able to partially restore genetic and phenotypic expressions, suggesting that its use is of interest for bioremediation procedures. Environmental factor could act on rhamnolipid production independently from the QS, since phosphate starvation increased the biosurfactant production although it decreased HSL concentration. Contrary to the other genes involved in rhamnolipid production, rhlG was induced by hyperosmotic stress. Our results indicate that RhlR (the transcription regulator which binds C4-HSL) is the main inhibitor of rhlG expression and that the inhibition is increased by high C4-HSL concentrations. RpoN and 70 sigma factors could be equally involved in rhlG transcription, and the C4-HSL/RhlR complex could inhibit the RpoN-dependent transcription. Surprisingly, the requirement of rhlG for rhamnolipid production was not confirmed in our conditions. The co-transcribed rhlG and rcsF genes seemed to be involved in P. aeruginosa growth in a poorly oxygenated medium.LORIENT-BU (561212106) / SudocSudocFranceF

Multispecies Biofilm Development of Marine Bacteria Implies Complex Relationships Through Competition and Synergy and Modification of Matrix Components

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Red and brown seaweeds differently inhibit colonization by epiphytic bacteria

International audienceThe inhibition of bacterial colonization by seaweeds belonging to the Florideophyceae and Phaeophyceae classes, collected along the Brittany coast (France), was evaluated using two epiphytic bacteria. Organic and aqueous extracts were screened for their potential to prevent colonization by the bacterium Zobellia galactanivorans and the commensal Pseudoalteromonas sp. 3J6. Among the 14 species evaluated in a colorimetric 96-well bioassay, the methanolic extracts of 5 Florideophyceae showed the highest activities on Z. galactanivorans but revealed no activity on Pseudoalteromonas 3J6. These results suggest that unknown polar compounds may control adhesion and/or biofilm development of epiphytic bacteria on seaweed surfaces

Anti-vibrio strategy: Antibiofilm activity of a marine bacterium against Vibrio tapetis CECT4600 biofilm, the causative agent of Brown Ring Disease.

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Antibiofilm Activity of the Marine Bacterium Pseudoalteromonas sp. 3J6 Against Vibrio tapetis, the Causative Agent of Brown Ring Disease.

International audienceVibrio tapetis CECT4600 is a pathogenic Gram-negative bacterium causing the brown ring disease in the Manila clam Ruditapes philippinarum. This vibriosis is induced by bacterial attachment on the periostracal lamina, yielding a decalcification of the bivalve shell. As in many bacterial species, pathogenesis is likely related to biofilm formation. The proteinaceous exoproducts of the marine bacterium Pseudoalteromonas sp. 3J6 inhibit the formation of biofilm by most of the tested marine bacteria without affecting their planktonic growth. In the present work, we examined the sensitivity of V. tapetis to Pseudoalteromonas sp. 3J6 and its exoproducts. In V. tapetis CECT4600-GFP-Pseudoalteromonas sp. 3J6 co-cultures, the latter outcompeted V. tapetis whatever the growth mode (planktonic or biofilm), which could result from a slower growth of V. tapetis. Biofilms containing only V. tapetis were grown in vitro on a glass substratum under dynamic conditions. When the glass was coated with a culture supernatant of Pseudoalteromonas sp. 3J6 (SN3J6) prior to inoculating V. tapetis CECT4600-GFP, the bacterial attachment was about fivefold lower than in control experiment without SN3J6 and the biofilm formation was delayed by about 24 h: A full biofilm was obtained at 48 versus 24 h for the control. Moreover, a preformed V. tapetis biofilm (grown on SN3J6-free glass substratum) could be disrupted by incubating it with SN3J6. This data suggest that Pseudoalteromonas sp. 3J6 is a good candidate to set up an anti-V. tapetis strategy usable in aquaculture to grow V. tapetis-free Manila clam spats

Complete genome sequence of the halophilic PHA-producing bacterium Halomonas sp. SF2003: insights into its biotechnological potential

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