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.

Integron diversity in marine environments.

Integrons are bacterial genetic elements known to be active vectors of antibiotic resistance among clinical bacteria. They are also found in bacterial communities from natural environments. Although integrons have become especially efficient for bacterial adaptation in the particular context of antibiotic usage, their role in natural environments in other contexts is still unknown. Indeed, most studies have focused on integrons and the spread of antibiotic resistance in freshwater or soil impacted by anthropogenic activities, with only few on marine environments. Notably, integrons show a wider diversity of both gene cassettes and integrase gene in natural environments than in clinical environments, suggesting a general role of integrons in bacterial adaptation. This article reviews the current knowledge on integrons in marine environments. We also present conclusions of our studies on polluted and nonpolluted backgrounds.

First gene cassettes of integrons as targets in finding adaptive genes in metagenomes.

The first gene cassettes of integrons are involved in the last adaptation response to changing conditions and are also the most expressed. We propose a rapid method for the selection of clones carrying an integron first gene cassette that is useful for finding adaptive genes in environmental metagenomic libraries.

FurA modulates gene expression of alr3808, a DpsA homologue in Nostoc (Anabaena) sp. PCC7120.

The DNA-binding protein from stationary phase (Dps) protein family plays an important role in protecting microorganisms from oxidative and nutritional stresses. In silico analysis of the promoter region of alr3808, a dpsA homologue from the cyanobacterium Nostoc sp. PCC7120 shows putative iron-boxes with high homology with those recognized by FurA (ferric uptake regulator). Evidence for the modulation of dpsA by FurA was obtained using in vitro and in vivo approaches. SELEX linked to PCR was used to identify P(dpsA) as a FurA target. Concurrently, EMSA assays showed high affinity of FurA for the dpsA promoter region. DpsA expression analysis in an insertional mutant of the alr1690-alphafurA message (that exhibited an increased expression of FurA) showed a reduced synthesis of DpsA. These studies suggest that FurA plays a significant role in the regulation of the DpsA.