Development of a digital droplet PCR approach for the quantification of soil micro-organisms involved in atmospheric CO2 fixation.

Carbon-fixing micro-organisms (CFMs) play a pivotal role in soil carbon cycling, contributing to carbon uptake and sequestration through various metabolic pathways. Despite their importance, accurately quantifying the absolute abundance of these micro-organisms in soils has been challenging. This study used a digital droplet polymerase chain reaction (ddPCR) approach to measure the abundance of key and emerging CFMs pathways in fen and bog soils at different depths, ranging from 0 to 15 cm. We targeted total prokaryotes, oxygenic phototrophs, aerobic anoxygenic phototrophic bacteria and chemoautotrophs, optimizing the conditions to achieve absolute quantification of these genes. Our results revealed that oxygenic phototrophs were the most abundant CFMs, making up 15% of the total prokaryotic abundance. They were followed by chemoautotrophs at 10% and aerobic anoxygenic phototrophic bacteria at 9%. We observed higher gene concentrations in fen than in bog. There were also variations in depth, which differed between fen and bog for all genes. Our findings underscore the abundance of oxygenic phototrophs and chemoautotrophs in peatlands, challenging previous estimates that relied solely on oxygenic phototrophs for microbial carbon dioxide fixation assessments. Incorporating absolute gene quantification is essential for a comprehensive understanding of microbial contributions to soil processes. This approach sheds light on the complex mechanisms of soil functioning in peatlands.

Quantitative Determination of Iron-Siderophore Complexes in Peat by Isotope-Exchange Size-Exclusion UPLC-Electrospray Ionization High-Resolution Accurate Mass (HRAM) Mass Spectrometry.

A method was developed for the quantification of iron-siderophore complexes by electrospray high-resolution accurate mass (HRAM) mass spectrometry (MS) without the need for authentic standards. The bulk of iron-siderophore complexes was purified by solid-phase extraction (SPE) and concentrated by evaporation. The individual complexes were identified by fast size-exclusion chromatography (FastSEC)-Orbitrap MSn on the basis of the exact molecular mass (±1 ppm) and MS2 or MS3 fragmentation. Their capability to readily exchange the natural 56Fe for the added 58Fe was demonstrated by SEC with ICP MS and ESI MS detection. The method was applied to the analysis of peat sampled in the eastern part of the French Pyrenean mountains. Nineteen siderophores belonging to four different classes were identified and quantified. The results were validated using ICP MS detection of iron by matching the sum of iron complexes determined by isotope exchange-ESI MS within each peak observed by FastSEC-ICP MS.

Unveiling the Pool of Metallophores in Native Environments and Correlation with Their Potential Producers.

For many organisms, metallophores are essential biogenic ligands that ensure metal scavenging and acquisition from their environment. Their identification is challenging in highly organic matter rich environments like peatlands due to low solubilization and metal scarcity and high matrix complexity. In contrast to common approaches based on sample modification by spiking of metal isotope tags, we have developed a two-dimensional (2D) Solid-phase extraction-Liquid chromatography-mass spectrometry (SPE-LC-MS) approach for the highly sensitive (LOD 40 fmol per g of soil), high-resolution direct detection and identification of metallophores in both their noncomplexed (apo) and metal-complexed forms in native environments. The characterization of peat collected in the Bernadouze (France) peatland resulted in the identification of 53 metallophores by a database mass-based search, 36 among which are bacterial. Furthermore, the detection of the characteristic (natural) metal isotope patterns in MS resulted in the detection of both Fe and Cu potential complexes. A taxonomic-based inference method was implemented based on literature and public database (antiSMASH database version 3.0) searches, enabling to associate over 40% of the identified bacterial metallophores with potential producers. In some cases, low completeness with the MIBiG reference BCG might be indicative of alternative producers in the ecosystem. Thus, coupling of metallophore detection and producers' inference could pave a new way to investigate poorly documented environment searching for new metallophores and their producers yet unknown.

Use of Endophytic and Rhizosphere Bacteria To Improve Phytoremediation of Arsenic-Contaminated Industrial Soils by Autochthonous Betula celtiberica.

The aim of this study was to investigate the potential of indigenous arsenic-tolerant bacteria to enhance arsenic phytoremediation by the autochthonous pseudometallophyte Betula celtiberica The first goal was to perform an initial analysis of the entire rhizosphere and endophytic bacterial communities of the above-named accumulator plant, including the cultivable bacterial species. B. celtiberica's microbiome was dominated by taxa related to Flavobacteriales, Burkholderiales, and Pseudomonadales, especially the Pseudomonas and Flavobacterium genera. A total of 54 cultivable rhizobacteria and 41 root endophytes, mainly affiliated with the phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria, were isolated and characterized with respect to several potentially useful features for metal plant accumulation, such as the ability to promote plant growth, metal chelation, and/or mitigation of heavy-metal stress. Seven bacterial isolates were further selected and tested for in vitro accumulation of arsenic in plants; four of them were finally assayed in field-scale bioaugmentation experiments. The exposure to arsenic in vitro caused an increase in the total nonprotein thiol compound content in roots, suggesting a detoxification mechanism through phytochelatin complexation. In the contaminated field, the siderophore and indole-3-acetic acid producers of the endophytic bacterial consortium enhanced arsenic accumulation in the leaves and roots of Betula celtiberica, whereas the rhizosphere isolate Ensifer adhaerens strain 91R mainly promoted plant growth. Field experimentation showed that additional factors, such as soil arsenic content and pH, influenced arsenic uptake in the plant, attesting to the relevance of field conditions in the success of phytoextraction strategies.IMPORTANCE Microorganisms and plants have developed several ways of dealing with arsenic, allowing them to resist and metabolize this metalloid. These properties form the basis of phytoremediation treatments and the understanding that the interactions of plants with soil bacteria are crucial for the optimization of arsenic uptake. To address this in our work, we initially performed a microbiome analysis of the autochthonous Betula celtiberica plants growing in arsenic-contaminated soils, including endosphere and rhizosphere bacterial communities. We then proceeded to isolate and characterize the cultivable bacteria that were potentially better suited to enhance phytoextraction efficiency. Eventually, we went to the field application stage. Our results corroborated the idea that recovery of pseudometallophyte-associated bacteria adapted to a large historically contaminated site and their use in bioaugmentation technologies are affordable experimental approaches and potentially very useful for implementing effective phytoremediation strategies with plants and their indigenous bacteria.

Isolation, purification and chemical characterization of a new angucyclinone compound produced by a new halotolerant Nocardiopsis sp. HR-4 strain.

A halotolerant Actinobacteria strain HR-4 was isolated from a salt lake soil sample in Algerian Sahara. Analysis of 16S rDNA gene sequence showed that strain HR-4 belonged to the genus Nocardiopsis. The similarity level ranges between 97.45 and 99.20% with Nocardiopsis species and Nocardiopsis rosea being the most closely related one. Morphological, physiological and phylogenetic characteristics comparisons showed significant differences with the nearest species. These data strongly suggest that strain HR-4 represents novel species. The antimicrobial activity of strain HR-4 showed an antibacterial activity against Gram-positive bacteria as well as an antifungal one. Two major natural products including a new one were isolated from the culture broth using various separation and purification procedures. The chemical structure established on the basis of spectroscopic studies NMR and by comparing with spectroscopic data from the literature of the two compounds affirm that they are classified in the group of Angucyclinones. This is the first report of a production of this type of molecules by the genus Nocardiopsis. The new natural compound was established as (-)-7-deoxy-8-O-methyltetrangomycin with a new configuration.

Postprandial kinetics of gene expression of proteins involved in the digestive process in rainbow trout (O. mykiss) and impact of diet composition.

The impact of increased incorporation of plant ingredients on diets for rainbow trout was evaluated in terms of gene expression of gastric (gastric lipase, pepsinogen) and intestinal (prolidase, maltase, phospholipase A2) digestive enzymes and nutrient transporters (peptide and glucose transporters), as well as of postprandial levels of plasma glucose, triglycerides and total free amino acids. For that purpose, trout alevins were fed from the start of exogenous feeding one of three different experimental diets: a diet rich in fish meal and fish oil (FM-FO), a plant-based diet (noFM-noFO) totally free from fish meal and fish oil, but containing plant ingredients and a Mixed diet (Mixed) intermediate between the FM-FO and noFM-noFO diets. After 16 months of rearing, all fish were left unfed for 72 h and then given a single meal to satiation. Blood, stomach and anterior intestine were sampled before the meal and at 2, 6 and 12 h after this meal. The postprandial kinetics of gene expression of gastric and intestinal digestive enzymes and nutrient transporters were then followed in trout fed the FM-FO diet. The postprandial profiles showed that the expression of almost all genes studied was stimulated by the presence of nutrients in the digestive tract of trout, but the timing (appearance of peaks) varied between genes. Based on these data, we have focused on the molecular response to dietary factors in the stomach and the intestine at 6 and 12 h after feeding, respectively. The reduction in FM and FO levels of dietary incorporation induced a significant decrease in the gene expression of gastric lipase, GLUT2 and PEPT1. The plasma glucose and triglycerides levels were also reduced in trout fed the noFM-noFO diet. Consequently, the present study suggests a decrease in digestive capacities in trout fed a diet rich in plant ingredients.

16S rRNA and As-Related Functional Diversity: Contrasting Fingerprints in Arsenic-Rich Sediments from an Acid Mine Drainage.

To gain an in-depth insight into the diversity and the distribution of genes under the particular evolutionary pressure of an arsenic-rich acid mine drainage (AMD), the genes involved in bacterial arsenic detoxification (arsB, ACR3) and arsenite oxidation (aioA) were investigated in sediment from Carnoulès (France), in parallel to the diversity and global distribution of the metabolically active bacteria. The metabolically active bacteria were affiliated mainly to AMD specialists, i.e., organisms detected in or isolated from AMDs throughout the world. They included mainly Acidobacteria and the non-affiliated "Candidatus Fodinabacter communificans," as well as Thiomonas and Acidithiobacillus spp., Actinobacteria, and unclassified Gammaproteobacteria. The distribution range of these organisms suggested that they show niche conservatism. Sixteen types of deduced protein sequences of arsenite transporters (5 ArsB and 11 Acr3p) were detected, whereas a single type of arsenite oxidase (AioA) was found. Our data suggested that at Carnoulès, the aioA gene was more recent than those encoding arsenite transporters and subjected to a different molecular evolution. In contrast to the 16S ribosomal RNA (16S rRNA) genes associated with AMD environments worldwide, the functional genes aioA, ACR3, and to a lesser extent arsB, were either novel or specific to Carnoulès, raising the question as to whether these functional genes are specific to high concentrations of arsenic, AMD-specific, or site-specific.

Environmental integrons: the dark side of the integron world.

Integrons are bacterial genetic elements notorious for their role in spreading antibiotic resistance in clinical settings. In the natural environment, integrons present a wide and hidden diversity, raising questions as to their broader role in bacterial adaptation. From the One Health perspective, they must be considered a threatening pool of resistance determinants.

Culturing the desert microbiota.

Over the last 30 years, the description of microbial diversity has been mainly based on culture-independent approaches (metabarcoding and metagenomics) allowing an in-depth analysis of microbial diversity that no other approach allows. Bearing in mind that culture-dependent approaches cannot replace culture-independent approaches, we have improved an original method for isolating strains consisting of "culturing" grains of sand directly on Petri dishes (grain-by-grain method). This method allowed to cultivate up to 10% of the bacteria counted on the surface of grains of the three sites studied in the Great Western Erg in Algeria (Timoudi, Béni Abbès, and Taghit), knowing that on average about 10 bacterial cells colonize each grain. The diversity of culturable bacteria (collection of 290 strains) predicted by 16S rRNA gene sequencing revealed that Arthrobacter subterraneus, Arthrobacter tecti, Pseudarthrobacter phenanthrenivorans, Pseudarthrobacter psychrotolerans, and Massilia agri are the dominant species. The comparison of the culture-dependent and -independent (16S rRNA gene metabarcoding) approaches at the Timoudi site revealed 18 bacterial genera common to both approaches with a relative overestimation of the genera Arthrobacter/Pseudarthrobacter and Kocuria, and a relative underestimation of the genera Blastococcus and Domibacillus by the bacterial culturing approach. The bacterial isolates will allow further study on the mechanisms of tolerance to desiccation, especially in Pseudomonadota (Proteobacteria).

CRISPR-Cas9 enrichment, a new strategy in microbial metagenomics to investigate complex genomic regions: The case of an environmental integron.

Environmental integrons are ubiquitous in natural microbial communities, but they are mostly uncharacterized and their role remains elusive. Thus far, research has been hindered by methodological limitations. Here, we successfully used an innovative approach combining CRISPR-Cas9 enrichment with long-read nanopore sequencing to target, in a complex microbial community, a putative adaptive environmental integron, InOPS, and to unravel its complete structure and genetic context. A contig of 20 kb was recovered containing the complete integron from the microbial metagenome of oil-contaminated coastal sediments. InOPS exhibited typical integron features. The integrase, closely related to integrases of marine Desulfobacterota, possessed all the elements of a functional integron integrase. The gene cassettes harboured mostly unknown functions hampering inferences about their ecological importance. Moreover, the putative InOPS host, likely a hydrocarbonoclastic marine bacteria, raises questions as to the adaptive potential of InOPS in response to oil contamination. Finally, several mobile genetic elements were intertwined with InOPS highlighting likely genomic plasticity, and providing a source of genetic novelty. This case study showed the power of CRISPR-Cas9 enrichment to elucidate the structure and context of specific DNA regions for which only a short sequence is known. This method is a new tool for environmental microbiologists working with complex microbial communities to target low abundant, large or repetitive genetic structures that are difficult to obtain by classical metagenomics. More precisely, here, it offers new perspectives to comprehensively assess the eco-evolutionary significance of environmental integrons.

Les intégrons environnementaux sont omniprésents dans les communautés microbiennes naturelles, mais la plupart ne sont pas caractérisés et leur rôle reste obscur. A ce jour, les limitations méthodologiques ont restreint leur étude. Ici, nous avons utilisé avec succès une approche innovante, combinant l'enrichissement par CRISPR-Cas9 et le séquençage nanopore longs-fragments, pour cibler, dans une communauté microbienne complexe, un intégron environnemental potentiellement adaptatif, InOPS, et pour révéler sa structure complète et son contexte génétique. Un contig de 20 kb contenant l'intégron complet a été obtenu à partir du métagénome microbien de sédiments côtiers contaminés par du pétrole. InOPS présente les caractéristiques typiques d'un intégron. Son intégrase, proche des intégrases des Desulfobacterota marines, possède tous les éléments d'une intégrase d'intégron fonctionnelle. Les cassettes de gène ont des fonctions pour la plupart inconnues, ce qui empêche d'inférer leur importance écologique. De plus, l'hôte présumé d'InOPS, probablement une bactérie marine hydrocarbonoclaste, interroge sur le potentiel adaptatif d'InOPS en réponse à la contamination par le pétrole. En outre, la présence de plusieurs éléments génétiques mobiles dans le contig met en évidence une probable plasticité génomique qui pourrait être source de remaniements génétiques. Cette étude de cas a montré la puissance de l'enrichissement par CRISPR-Cas9 pour élucider la structure et le contexte de régions d'ADN spécifiques pour lesquelles seule une courte séquence est connue. Cette méthode fournit un nouvel outil aux microbiologistes environnementaux travaillant avec des communautés microbiennes complexes pour cibler des structures génétiques peu abondantes, larges ou répétées, qui sont difficiles à obtenir par métagénomique classique. Plus précisément, elle offre ici de nouvelles perspectives pour évaluer de manière exhaustive l'importance éco-évolutive des intégrons environnementaux.