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

Implications of streamlining theory for microbial ecology

Whether a small cell, a small genome or a minimal set of chemical reactions with self-replicating properties, simplicity is beguiling. As Leonardo da Vinci reportedly said, 'simplicity is the ultimate sophistication'. Two diverging views of simplicity have emerged in accounts of symbiotic and commensal bacteria and cosmopolitan free-living bacteria with small genomes. The small genomes of obligate insect endosymbionts have been attributed to genetic drift caused by small effective population sizes (Ne). In contrast, streamlining theory attributes small cells and genomes to selection for efficient use of nutrients in populations where Ne is large and nutrients limit growth. Regardless of the cause of genome reduction, lost coding potential eventually dictates loss of function. Consequences of reductive evolution in streamlined organisms include atypical patterns of prototrophy and the absence of common regulatory systems, which have been linked to difficulty in culturing these cells. Recent evidence from metagenomics suggests that streamlining is commonplace, may broadly explain the phenomenon of the uncultured microbial majority, and might also explain the highly interdependent (connected) behavior of many microbial ecosystems. Streamlining theory is belied by the observation that many successful bacteria are large cells with complex genomes. To fully appreciate streamlining, we must look to the life histories and adaptive strategies of cells, which impose minimum requirements for complexity that vary with niche.