Multicolour whole-cell bacterial sensors for in vivo identification of bio-physicochemical interactions in mineral-bacteria systems

Le monitoring des écosystèmes terrestres nécessite une connaissance approfondie des interactions entre microorganismes, minéraux et métaux dans les sols. Afin d'évaluer in vivo la disponibilité de métaux tel que le fer dans des systèmes bactéries-minéraux, une approche basée sur l’utilisation de biosenseurs bactériens fluorescents et d’une analyse spectroscopique non-invasive a été explorée. Ce travail a notamment conduit à la construction chez Pseudomonas aeruginosa de fusions génétiques couplant des promoteurs régulés par le fer à des rapporteurs fluorescents multicolores. Les souches obtenues ont été utilisées comme senseur de la disponibilité du fer constitutif de différents minéraux (Nontronites). La réponse de ces biosenseurs bactériens a été étudiée en couplant la spectroscopie de fluorescence à balayage synchrone (SFS) à la décomposition canonique polyadique Candecomp / Parafac (CP). Avec des plans d’expérience privilégiant la diversité des réponses, le couplage SFS-CP garantit une estimation conjointe et rapide de l’expression de plusieurs promoteurs d’intérêts, y compris dans des milieux auto-fluorescents. Cette méthode originale permet, entre autres, de s’affranchir des problèmes liés aux recouvrements spectraux des protéines fluorescentes et fournit une estimation robuste et précise de la réponse des biosenseurs. Appliquée à d’autres plans d’expériences, elle démontre également que la bio-dissolution des nontronites par P. aeruginosa est assurée par la production de sidérophores et contrôlée par la cristallochimie des feuillets des smectites, notamment par la distribution des atomes de fer(III) entre les tétraèdres et les octaèdresMonitoring terrestrial ecosystems requires a better understanding of the interactions between microorganisms, minerals and metals in the environment. To assess in vivo availability of metals such as iron in bacteria-mineral system, an approach based on whole-cell fluorescent biosensors and non-invasive spectroscopy was explored. This work led to the construction in Pseudomonas aeruginosa of a set of gene fusions coupling iron-regulated promoters to multicolour fluorescent reporters. The recombinant strains were used as sensors of structural iron availability in nontronites NAu-1 and NAu-2. The response of these biosensors was studied by coupling synchronous fluorescence spectroscopy (SFS) with canonical polyadic Candecomp/Parafac (CP) decomposition. On the basis of experimental designs favouring response diversity, the coupled SFS-CP method guarantees a joint estimate of gene expression from multiple promoters, even in highly fluorescent media. This novel method can solve the issue of spectral bleed-through of fluorescent proteins and provides a means to integrate multiple signals from combinations of whole-cell fluorescent bioreporters. In addition, we could show using SFS-CP that P. aeruginosa indirectly mobilize Fe(III) from nontronites primarily through the production of pyoverdine siderophore. The structural Fe(III) present on the edges of NAu-2 rather than NAu-1 particles appears to be more bioaccessible, suggesting that the distribution of Fe, in the tetrahedron and/or in the octahedron sites, governs the solubilization proces

Conception de biosenseurs fluorescents multicolores pour l'identification in vivo des interactions bio-physicochimiques dans les systèmes minéral-bactérie

Monitoring terrestrial ecosystems requires a better understanding of the interactions between microorganisms, minerals and metals in the environment. To assess in vivo availability of metals such as iron in bacteria-mineral system, an approach based on whole-cell fluorescent biosensors and non-invasive spectroscopy was explored. This work led to the construction in Pseudomonas aeruginosa of a set of gene fusions coupling iron-regulated promoters to multicolour fluorescent reporters. The recombinant strains were used as sensors of structural iron availability in nontronites NAu-1 and NAu-2. The response of these biosensors was studied by coupling synchronous fluorescence spectroscopy (SFS) with canonical polyadic Candecomp/Parafac (CP) decomposition. On the basis of experimental designs favouring response diversity, the coupled SFS-CP method guarantees a joint estimate of gene expression from multiple promoters, even in highly fluorescent media. This novel method can solve the issue of spectral bleed-through of fluorescent proteins and provides a means to integrate multiple signals from combinations of whole-cell fluorescent bioreporters. In addition, we could show using SFS-CP that P. aeruginosa indirectly mobilize Fe(III) from nontronites primarily through the production of pyoverdine siderophore. The structural Fe(III) present on the edges of NAu-2 rather than NAu-1 particles appears to be more bioaccessible, suggesting that the distribution of Fe, in the tetrahedron and/or in the octahedron sites, governs the solubilization processLe monitoring des écosystèmes terrestres nécessite une connaissance approfondie des interactions entre microorganismes, minéraux et métaux dans les sols. Afin d'évaluer in vivo la disponibilité de métaux tel que le fer dans des systèmes bactéries-minéraux, une approche basée sur l’utilisation de biosenseurs bactériens fluorescents et d’une analyse spectroscopique non-invasive a été explorée. Ce travail a notamment conduit à la construction chez Pseudomonas aeruginosa de fusions génétiques couplant des promoteurs régulés par le fer à des rapporteurs fluorescents multicolores. Les souches obtenues ont été utilisées comme senseur de la disponibilité du fer constitutif de différents minéraux (Nontronites). La réponse de ces biosenseurs bactériens a été étudiée en couplant la spectroscopie de fluorescence à balayage synchrone (SFS) à la décomposition canonique polyadique Candecomp / Parafac (CP). Avec des plans d’expérience privilégiant la diversité des réponses, le couplage SFS-CP garantit une estimation conjointe et rapide de l’expression de plusieurs promoteurs d’intérêts, y compris dans des milieux auto-fluorescents. Cette méthode originale permet, entre autres, de s’affranchir des problèmes liés aux recouvrements spectraux des protéines fluorescentes et fournit une estimation robuste et précise de la réponse des biosenseurs. Appliquée à d’autres plans d’expériences, elle démontre également que la bio-dissolution des nontronites par P. aeruginosa est assurée par la production de sidérophores et contrôlée par la cristallochimie des feuillets des smectites, notamment par la distribution des atomes de fer(III) entre les tétraèdres et les octaèdre

Multicolor whole-cell bacterial sensing using a synchronous fluorescence spectroscopy-based approach

International audienceThe wide collection of currently available fluorescent proteins (FPs) offers new possibilities for multicolor reporter gene-based studies of bacterial functions. However, the simultaneous use of multiple FPs is often limited by the bleed-through of their emission spectra. Here we introduce an original approach for detection and separation of multiple overlapping fluorescent signals from mixtures of bioreporters strains. The proposed method relies on the coupling of synchronous fluorescent spectroscopy (SFS) with blind spectral decomposition achieved by the Canonical Polyadic (CP) decomposition (also known as Candecomp/Parafac) of three-dimensional data arrays. Due to the substantial narrowing of FP emission spectra and sensitive detection of multiple FPs in a one-step scan, SFS reduced spectral overlap and improved the selectivity of the CP unmixing procedure. When tested on mixtures of labeled E. coli strains, the SFS/CP approach could easily extract the contribution of at least four overlapping FPs. Furthermore, it allowed to simultaneously monitor the expression of three iron responsive genes and pyoverdine production in P. aeruginosa. Implemented in a convenient microplate format, this multiplex fluorescent reporter method provides a useful tool to study complex processes with different variables in bacterial systems

Multi-way signal processing for multicolor fluorescent biosensing

Evry Spring School on advances in Systems and Synthetic BiologyIn this paper, we investigate the possibility of modelling the concomitant response of multiple bacterial genes to environmental factors by using fluores-cent whole cell biosensors. An experimental procedure is proposed to obtain three-way data sets by synchronous fluorescence spectroscopy (SFS). A Candecomp/Parafac algorithm (CP) is then proposed to separate simultaneously all the fluorescence signals of reporter proteins and to interpret the response of the corresponding promoters. The joined estimation of several gene responses is the main original point of this blind identification procedure. The method proves to be more powerful than the traditional principal components analysis (PCA) or the singular value decomposition (SVD) and provides a promising multi-way strategy for in vivo monitoring of gene expression in system biology studies

Spectral decomposition of fluorescence from mixtures of three <i>P</i>. <i>aeruginosa</i> PAO1 iron bioreporter strains harboring <i>pvdS-gfp</i>, <i>pvdA</i>-<i>dsred-express2</i> and <i>bfrB-e2-orange</i> fusions.

<p>(A) Raw synchronous spectra of strain mixtures incubated with 0.3, 1 and 3 μM FeCl₃ showing the relative intensity of fluorescent signals. (B) Spectra of the four fluorescent sources identified from CP analysis performed independently on 400–470 nm and 470–600 nm wavelength ranges. (C) Profile of fluorescence sources as a function of iron concentration. (D) Profile of fluorescence sources as a function of the ratio of <i>bfrB-e2-orange</i> reporter strain in the mixture. The mixture pattern for this experiment is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122848#pone.0122848.s003" target="_blank">S3 Fig</a>. PVD: pyoverdine; af-PVD and bf-PVD: “acid” and “basic” forms of pyoverdine.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Fluorescence spectra of GFP, E2-Orange and mCherry.

<p>Shown are normalized excitation and emission (A) and synchronous (B) fluorescence spectra of <i>E</i>. <i>coli</i> cultures expressing GFP, E2-Orange and mCherry.</p

Spectral decomposition of fluorescence from mixtures of two <i>P</i>. <i>aeruginosa</i> PAO1 iron bioreporter strains harboring <i>pvdA</i>-<i>dsred-express2</i> and <i>bfrB-e2-orange</i> fusions.

<p>Shown are (A) the spectra of the four fluorescent sources identified from CP analysis and their profile as a function of (B) iron concentration and (C) the ratio of bioreporter strains in the mixture. The mixture pattern for this experiment is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122848#pone.0122848.s002" target="_blank">S2 Fig</a>. (D) Synchronous spectra of purified pyoverdine (solid line) and cultures of PAO1 wild type (WT, dashed line) and PAO1 ∆<i>pvdA</i> (large dashed lines) cells grown in low-iron DCAA medium. The later spectrum was normalized with respect to that obtained for PAO1 wild type. (E) Synchronous spectra of WT PAO1 culture supernatant as a function of pH. The DCAA growth medium supernatant was diluted tenfold in different buffers with pH adjusted to 5.2 and 6.2 (40 mM MES), 7.4 (40 mM MOPS) and 8, 9 and 10 (40 mM Tris-HCl).</p

Interactions of three soil bacteria species with phyllosilicate surfaces in hybrid silica gels

International audienceTo simulate iron consumption in soils, iron leaching from silicate minerals due to three heterotrophic bacterial strains and a chemical treatment was studied using hybrid silica gel (HSG) doped with two phyllosilicates, nontronite (NAu-2) or low-iron-content montmorillonite (SWy-2). HSG methodology, a novel way of separating bacteria cells from a colloidal mineral source, consisted in embedding colloidal mineral particles into an amorphous porous silica matrix using a classical sol-gel procedure. Pantoae agglomerans PA1 and Rahnella aquatilis RA1 were isolated from silicate-rich soils, that is, beech and wheat rhizospheres (Vosges, France); Burkholderia sp. G5 was selected from acidic and nutrient-poor podzol soils (Vosges, France). Fe release from clay minerals and production of bacterial metabolites, that is, low molecular weight organic acids (LMWOA) and siderophores, were monitored. Two LMWOA profiles were observed with major gluconate production (>9000M) for Burkholderia sp. G5 and moderate production of lactate, acetate, propionate, formate, oxalate, citrate, and succinate (<300M) for R.aquatilis RA1 and P.agglomerans PA1. HSG demonstrated its usefulness in revealing clay mineral-microorganisms interactions. The effect of bacterial exsudates was clearly separated from physical contact effect