Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph

A variety of culture-independent techniques have been developed that can be used in conjunction with culture-dependent physiological and metabolic studies of key microbial organisms, in order to better understand how the activity of natural populations influences and regulates all major biogeochemical cycles. In this study, we combined DNA-stable isotope probing with metagenomics and metaproteomics to characterize an as yet uncultivated marine methylotroph that actively incorporated carbon from 13C-labeled methanol into biomass. By metagenomic sequencing of the heavy DNA, we retrieved virtually the whole genome of this bacterium and determined its metabolic potential. Through protein-stable isotope probing, the RuMP cycle was established as the main carbon assimilation pathway, and the classical methanol dehydrogenase-encoding gene mxaF, as well as three out of four identified xoxF homologues were found to be expressed. This proof-of-concept study is the first in which theculture-independent techniques of DNA- and protein-stable isotope probing have been used to characterize the metabolism of a naturally-ocurring Methylophaga-like bacterium in the marine environment (i.e. M. thiooxydans L4) and thus provides a powerful approach to access the genome and proteome of uncultivated microbes involved in key processes in the environment

Genome- and transcriptome-assisted development of nuclear insertion/deletion markers for Calanus species (Copepoda: Calanoida) identification

Copepods of the genus Calanus are key zooplankton species in temperate to arctic marine ecosystems. Despite their ecological importance, species identification remains challenging. Furthermore, the recent report of hybrids among Calanus species highlights the need for diagnostic nuclear markers to efficiently identify parental species and hybrids. Using next-generation sequencing analysis of both the genome and transcriptome from two sibling species, Calanus finmarchicus and Calanus glacialis, we developed a panel of 12 nuclear insertion/deletion markers. All the markers showed species-specific amplicon length. Furthermore, most of the markers were successfully amplified in other Calanus species, allowing the molecular identification of Calanus helgolandicus, Calanus hyperboreus and Calanus marshallae.

Next Generation Sequencing Reveals the Hidden Diversity of Zooplankton Assemblages

Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel next generation sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify richness and diversity of a mixed zooplankton assemblage from a productive time series site in the Western English Channel. Methodology/Principle Findings Plankton net hauls (200 µm) were taken at the Western Channel Observatory station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,041 sequences were obtained for all samples. The sequences clustered into 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 135 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 58 taxonomic groups. Conclusions Metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and hard-to-identify meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for elucidating the true diversity and species richness of zooplankton communities. While this approach allows for broad diversity assessments of plankton it may become increasingly attractive in future if sequence reference libraries of accurately identified individuals are better populated.