Mining of unexplored habitats for novel chitinases—chiA as a helper gene proxy in metagenomics

The main objective of this study was to assess the abundance and diversity of chitin-degrading microbial communities in ten terrestrial and aquatic habitats in order to provide guidance to the subsequent exploration of such environments for novel chitinolytic enzymes. A combined protocol which encompassed (1) classical overall enzymatic assays, (2) chiA gene abundance measurement by qPCR, (3) chiA gene pyrosequencing, and (4) chiA gene-based PCR-DGGE was used. The chiA gene pyrosequencing is unprecedented, as it is the first massive parallel sequencing of this gene. The data obtained showed the existence across habitats of core bacterial communities responsible for chitin assimilation irrespective of ecosystem origin. Conversely, there were habitat-specific differences. In addition, a suite of sequences were obtained that are as yet unregistered in the chitinase database. In terms of chiA gene abundance and diversity, typical low-abundance/diversity versus high-abundance/diversity habitats was distinguished. From the combined data, we selected chitin-amended agricultural soil, the rhizosphere of the Arctic plant Oxyria digyna and the freshwater sponge Ephydatia fluviatilis as the most promising habitats for subsequent bioexploration. Thus, the screening strategy used is proposed as a guide for further metagenomics-based exploration of the selected habitats

Resupply of mesopelagic dissolved iron controlled by particulate iron composition

The dissolved iron supply controls half of the oceans’ primary productivity. Resupply by the remineralization of sinking particles, and subsequent vertical mixing, largely sustains this productivity. However, our understanding of the drivers of dissolved iron resupply, and their influence on its vertical distribution across the oceans, is still limited due to sparse observations. There is a lack of empirical evidence as to what controls the subsurface iron remineralization due to difficulties in studying mesopelagic biogeochemistry. Here we present estimates of particulate transformations to dissolved iron, concurrent oxygen consumption and iron-binding ligand replenishment based on in situ mesopelagic experiments. Dissolved iron regeneration efficiencies (that is, replenishment over oxygen consumption) were 10- to 100-fold higher in low-dust subantarctic waters relative to higher-dust Mediterranean sites. Regeneration efficiencies are heavily influenced by particle composition. Their make-up dictates ligand release, controls scavenging, modulates ballasting and may lead to the differential remineralization of biogenic versus lithogenic iron. At high-dust sites, these processes together increase the iron remineralization length scale. Modelling reveals that in oceanic regions near deserts, enhanced lithogenic fluxes deepen the ferricline, which alter the vertical patterns of dissolved iron replenishment, and set its redistribution at the global scale. Such wide-ranging regeneration efficiencies drive different vertical patterns in dissolved iron replenishment across oceanic provinces

The great screen anomaly—a new frontier in product discovery through functional metagenomics

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product

Achievements and new knowledge unraveled by metagenomic approaches

Metagenomics has paved the way for cultivation-independent assessment and exploitation of microbial communities present in complex ecosystems. In recent years, significant progress has been made in this research area. A major breakthrough was the improvement and development of high-throughput next-generation sequencing technologies. The application of these technologies resulted in the generation of large datasets derived from various environments such as soil and ocean water. The analyses of these datasets opened a window into the enormous phylogenetic and metabolic diversity of microbial communities living in a variety of ecosystems. In this way, structure, functions, and interactions of microbial communities were elucidated. Metagenomics has proven to be a powerful tool for the recovery of novel biomolecules. In most cases, functional metagenomics comprising construction and screening of complex metagenomic DNA libraries has been applied to isolate new enzymes and drugs of industrial importance. For this purpose, several novel and improved screening strategies that allow efficient screening of large collections of clones harboring metagenomes have been introduced

Metagenomics: DNA sequencing of environmental samples

While genomics has classically focused on pure, easy-to-obtain samples, such as microbes that grow readily in culture or large animals and plants, these organisms represent but a fraction of the living or once living organisms of interest. Many species are difficult to study in isolation, because they fail to grow in laboratory culture, depend on other organisms for critical processes, or have become extinct. DNA sequence-based methods circumvent these obstacles, as DNA can be directly isolated from live or dead cells in a variety of contexts, and have led to the emergence of a new field referred to as metagenomics

Analysis of bacterial and archaeal diversity in coastal microbial mats using massive parallel 16S rRNA gene tag sequencing

Coastal microbial mats are small-scale and largely closed ecosystems in which a plethora of different functional groups of microorganisms are responsible for the biogeochemical cycling of the elements. Coastal microbial mats play an important role in coastal protection and morphodynamics through stabilization of the sediments and by initiating the development of salt-marshes. Little is known about the bacterial and especially archaeal diversity and how it contributes to the ecological functioning of coastal microbial mats. Here, we analyzed three different types of coastal microbial mats that are located along a tidal gradient and can be characterized as marine (ST2), brackish (ST3) and freshwater (ST3) systems. The mats were sampled during three different seasons and subjected to massive parallel tag sequencing of the V6 region of the 16S rRNA genes of Bacteria and Archaea. Sequence analysis revealed that the mats are among the most diverse marine ecosystems studied so far and consist of several novel taxonomic levels ranging from classes to species. The diversity between the different mat types was far more pronounced than the changes between the different seasons at one location. The archaeal community for these mats have not been studied before and revealed a strong reaction on a short period of draught during summer resulting in a massive increase in halobacterial sequences, whereas the bacterial community was barely affected. We concluded that the community composition and the microbial diversity were intrinsic of the mat type and depend on the location along the tidal gradient indicating a relation with salinity.