Taxonomical composition and functional analysis of biofilms sampled from a nuclear storage pool

Sampling small amounts of biofilm from harsh environments such as the biofilm present on the walls of a radioactive material storage pool offers few analytical options if taxonomic characterization and estimation of the different biomass contributions are the objectives. Although 16S/18S rRNA amplification on extracted DNA and sequencing is the most widely applied method, its reliability in terms of quantitation has been questioned as yields can be species-dependent. Here, we propose a tandem-mass spectrometry proteotyping approach consisting of acquiring peptide data and interpreting then against a generalist database without any a priori. The peptide sequence information is transformed into useful taxonomical information that allows to obtain the different biomass contributions at different taxonomical ranks. This new methodology is applied for the first time to analyze the composition of biofilms from minute quantities of material collected from a pool used to store radioactive sources in a nuclear facility. For these biofilms, we report the identification of three genera, namely Sphingomonas, Caulobacter, and Acidovorax, and their functional characterization by metaproteomics which shows that these organisms are metabolic active. Differential expression of Gene Ontology GOslim terms between the two main microorganisms highlights their metabolic specialization

A simple and efficient method for the long-term preservation of plant cell suspension cultures

<p>Abstract</p> <p>Background</p> <p>The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority.</p> <p>Results</p> <p>Sycamore (<it>Acer pseudoplatanus</it>) and Arabidopsis cell were preserved over six months as suspensions cultures in a phosphate-free nutrient medium at 5°C. The cell recovery monitored via gas exchange measurements and metabolic profiling using <it>in vitro </it>and <it>in vivo </it>¹³C- and ³¹P-NMR took a couple of hours, and cell growth restarted without appreciable delay. No measurable cell death was observed.</p> <p>Conclusion</p> <p>We provide a simple method to preserve physiologically homogenous plant cell cultures without subculture over several months. The protocol based on the blockage of cell growth and low culture temperature is robust for heterotrophic and semi-autotrophic cells and should be adjustable to cell lines other than those utilised in this study. It requires no specialized equipment and is suitable for routine laboratory use.</p

An extremely radioresistant green eukaryote for radionuclide bio-decontamination in the nuclear industry

International audienceNuclear activities generate radioactive elements which require processes for their decontamination. Although biological remediation has proved efficient in industrial applications, no biotechnology solution is currently operational for highly radioactive media. Such a solution requires organisms that accumulate radionuclides while withstanding radioactivity. This paper describes the potentialities of an extremophile autotrophic eukaryote, Coccomyxa actinabiotis nov. sp., that we isolated from a nuclear facility and which withstands huge ionizing radiation doses, up to 20,000 Gy. Half the population survives 10,000 Gy, which is comparable to the hyper-radioresistant wellknown prokaryote Deinococcus radiodurans. Cell metabolic profile investigated by nuclear magnetic resonance was hardly affected by radiation doses of up to 10,000 Gy. Cellular functioning completely recovered within a few days. This outstanding microalga also strongly accumulates radionuclides, including 238U, 137Cs, 110mAg, 60Co, 54Mn, 65Zn, and 14C (decontamination above 85% in 24 h, concentration factor, 1,000-450,000 mL g-1 fresh weight). In 1 h, the microalga revealed as effective as the conventional physico-chemical ion-exchangers to purify nuclear effluents. Using this organism, an efficient real-scale radionuclide bio-decontamination process was performed in a nuclear fuel storage pool with an important reduction of waste volume compared to the usual physico-chemical process. The feasibility of new decontamination solutions for the nuclear industry and for environmental clean-up operations is demonstrated

The Phosphate Fast-Responsive Genes <i>PECP1</i> and <i>PPsPase1</i> Affect Phosphocholine and Phosphoethanolamine Content

International audiencePhosphate starvation-mediated induction of the HAD-type phosphatases PPsPase1 (AT1G73010) and PECP1 (AT1G17710) has been reported in Arabidopsis (Arabidopsis thaliana). However, little is known about their in vivo function or impact on plant responses to nutrient deficiency. The preferences of PPsPase1 and PECP1 for different substrates have been studied in vitro but require confirmation in planta. Here, we examined the in vivo function of both enzymes using a reverse genetics approach. We demonstrated that PPsPase1 and PECP1 affect plant phosphocholine and phosphoethanolamine content, but not the pyrophosphate-related phenotypes. These observations suggest that the enzymes play a similar role in planta related to the recycling of polar heads from membrane lipids that is triggered during phosphate starvation. Altering the expression of the genes encoding these enzymes had no effect on lipid composition, possibly due to compensation by other lipid recycling pathways triggered during phosphate starvation. Furthermore, our results indicated that PPsPase1 and PECP1 do not influence phosphate homeostasis, since the inactivation of these genes had no effect on phosphate content or on the induction of molecular markers related to phosphate starvation. A combination of transcriptomics and imaging analyses revealed that PPsPase1 and PECP1 display a highly dynamic expression pattern that closely mirrors the phosphate status. This temporal dynamism, combined with the wide range of induction levels, broad expression, and lack of a direct effect on Pi content and regulation, makes PPsPase1 and PECP1 useful molecular markers of the phosphate starvation response

Metaproteomics for deciphering biomass contributions and functions of complex aquatic microbiota

International audienceMetaproteomics aims at comprehensively identifying the preeminent metabolic pathways for gaining insight into the functions of microbiota. This large scale molecular phenotypic analysis is highly complementary to 16S rRNA metabarcoding and metagenomics. We have developed a new method for exploiting metaproteomic datasets and defining the biomass contribution of the organisms present in the sample. The phylopeptidomics concept is based on a signature describing the number of peptide sequences shared with all other organisms calculated by mathematical modeling and phylogenetic relationships. Its efficiency was exemplified with artificial mixtures, as well as with more complex microbiota models. This methodology was applied on aquatic microbiota such as those discovered within the water used to cool a nuclear reactor core. We highlighted in a nuclear reactor at shutdown the predominance of two phyla: Proteobacteria with the genus Methylobacterium (50% of the signal) and Actinobacteria with two genera Asanoa (25%) and Streptomyces (25%). This approach proved to be highly complementary to the metabarcoding analysis of the same samples. We also successfully applied the method to biofilms sampled from the walls of a pool used to store radioactive sources. These examples applied to aquatic microbial communities pave the way to new discoveries regarding novel microorganisms of biotechnological interest

Resistance to irradiation of micro-algae growing in the storage pools of a nuclear reactor investigated by NMR and neutron spectroscopies

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First Isolation and Characterization of Bacteria from the Core’s Cooling Pool of an Operating Nuclear Reactor

International audienceMicrobial life can thrive in the most inhospitable places, such as nuclear facilities with high levels of ionizing radiation. Using direct meta-analyses, we have previously highlighted the presence of bacteria belonging to twenty-five different genera in the highly radioactive water of the cooling pool of an operating nuclear reactor core. In the present study, we further characterize this specific environment by isolating and identifying some of these microorganisms and assessing their radiotolerance and their ability to decontaminate uranium. This metal is one of the major radioactive contaminants of anthropogenic origin in the environment due to the nuclear and mining industries and agricultural practices. The microorganisms isolated when sampling was performed during the reactor operation consisted mainly of Actinobacteria and Firmicutes, whereas Proteobacteria were dominant when sampling was performed during the reactor shutdown. We investigated their tolerance to gamma radiation under different conditions. Most of the bacterial strains studied were able to survive 200 Gy irradiation. Some were even able to withstand 1 kGy, with four of them showing more than 10% survival at this dose. We also assessed their uranium uptake capacity. Seven strains were able to remove almost all the uranium from a 5 µM solution. Four strains displayed high efficiency in decontaminating a 50 µM uranium solution, demonstrating promising potential for use in bioremediation processes in environments contaminated by radionuclides