100 research outputs found
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 (ECFÏ) AlgU, responsible for alginate biosynthesis, is also involved in responses to cell wall stress and heat shock via the RpoH alternative Ï factor. The SigX ECFÏ 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 ECFÏ 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
Marine bacteria from the French Atlantic coast displaying high forming-biofilm abilities and different biofilm 3D architectures
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
The Pseudomonas aeruginosa rhlG and rhlAB genes are inversely regulated and RhlG is not required for rhamnolipid synthesis.
International audienceBACKGROUND: Pseudomonas aeruginosa produces rhamnolipid biosurfactants involved in numerous phenomena including virulence. The transcriptional study of the rhlAB operon encoding two key enzymes for rhamnolipid synthesis led to the discovery of the quorum sensing system RhlRI. The latter positively controls the transcription of rhlAB, as well as of rhlC, which is required for di-rhamnolipid synthesis. The rhlG gene encodes an NADPH-dependent beta-ketoacyl reductase. Although it was reported to be required for the biosynthesis of the fatty acid part of rhamnolipids, its function in rhamnolipid synthesis was later questioned. The rhlG transcription and its role in rhamnolipid production were investigated here. RESULTS: Using 5[prime]-RACE PCR, a luxCDABE-based transcriptional fusion, and quantitative reverse transcription-PCR, we confirmed two previously identified sigma70- and sigma54-dependent promoters and we identified a third promoter recognized by the extra-cytoplasmic function sigma factor AlgU. rhlG was inversely regulated compared to rhlAB and rhlC: the rhlG transcription was down-regulated in response to N-butyryl-l-homoserine lactone, the communication molecule of the RhlRI system, and was induced by hyperosmotic stress in an AlgU-dependent manner. Consistently with this transcriptional pattern, the single or double deletions of rhlG and PA3388, which forms an operon with rhlG, did not dramatically impair rhamnolipid synthesis. CONCLUSION: This first detailed study of rhlG transcription reveals a complex regulation involving three sigma factors and N-butyryl-l-homoserine lactone. We furthermore present evidences that RhlG does not play a key role in rhamnolipid synthesis
Influence de l'environnement et des communications inter-bactériennes sur la production de biosurfactants par Pseudomonas Aeruginosa
La bioremĂšdiation, qui utilise la capacitĂ© naturelle des micro-organismes Ă disperser et dĂ©grader les hydrocarbures, est l'une des perspectives des plus attractives pour la dĂ©contamination des zones souillĂ©es par les marĂ©es noires. Pseudomonas aeruginosa produit des biosurfactants (dispersants) de type glycolipides nommĂ©s rhamnolipides, dont la rĂ©gulation dĂ©pend d'un systĂšme relatif Ă la densitĂ© cellulaire, le quorum sensing (QS). Lors de cette Ă©tude, la hiĂ©rarchie du QS fut pour la premiĂšre fois mise en Ă©vidence par des cinĂ©tiques de production des deux molĂ©cules signal : la N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) et la N-butyryl-L-homoserine lactone (C4-HSL) (HSL), les rhamnolipides Ă©tant produits ultĂ©rieurement. Nous avons montrĂ© que le facteur sigma RpoN Ă©tait impliquĂ© dans l'arrĂȘt de synthĂšse des 3OC12-HSL et qu'il n'avait aucune influence sur la production des C4-HSL, alors qu'il inhibait la synthĂšse des rhamnolipides. En revanche, RpoS activait indirectement la production des rhamnolipides, via les HSL. Nous avons observĂ© qu'une concentration en sel proche de celle de l'eau de mer inhibait la production des rhamnolipides, en diminuant l'expression des gĂšnes impliquĂ©s dans la synthĂšse des biosurfactants (rhlAB et rhlC). L'expression de rhlI et la synthĂšse des HSL qui en dĂ©pend, Ă©taient Ă©galement diminuĂ©es. Le stress hyperosmotique agissait sur d'autres facteurs associĂ©s au QS comme la pyocyanine et la formation du biofilm. Un apport en osmoprotecteur (glycine bĂ©taĂŻne) a permis une restauration partielle des expressions gĂ©nĂ©tiques et des phĂ©notypes, dĂ©montrant l'intĂ©rĂȘt de son utilisation pour la bioremĂšdiation maritime. Les facteurs environnementaux agissent Ă©galement sur la production des rhamnolipides indĂ©pendamment du QS, puisqu'une carence en phosphate favorisait la production des biosurfactants alors qu'elle diminuait la concentration en HSL. Contrairement aux autres gĂšnes impliquĂ©s dans la production des rhamnolipides, rhlG Ă©tait induit par le stress salin. Nos rĂ©sultats indiquent que RhlR (rĂ©gulateur de transcription fixant C4-HSL) est le principal inhibiteur de rhlG et que l'inhibition est augmentĂ©e par une concentration Ă©levĂ©e en C4-HSL. Les facteurs sigma RpoN et 70 paraissent impliquĂ©s Ă parts Ă©gales dans la transcription de rhlG, et le complexe C4-HSL/RhlR inhiberait la transcription dĂ©pendante de RpoN. Nous n'avons en revanche pas pu confirmer que rhlG soit indispensable Ă la production des rhamnolipides. rhlG et rcsF qui sont organisĂ©s en opĂ©ron, seraient toutefois impliquĂ©s dans la croissance de P. aeruginosa en milieu limitĂ© en oxygĂšne.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 24Â h: A full biofilm was obtained at 48 versus 24Â 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
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