title: Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time.
abstract: The identification of predominant microbial taxa with specific metabolic capabilities remains one the biggest challenges in environmental microbiology, because of the limits of current metagenomic and cell culturing methods. We report results from the direct analysis of multiple genes in individual marine bacteria cells, demonstrating the potential for high-throughput metabolic assignment of yet-uncultured taxa. The protocol uses high-speed fluorescence-activated cell sorting, whole-genome multiple displacement amplification (MDA), and subsequent PCR screening. A pilot library of 11 single amplified genomes (SAGs) was constructed from Gulf of Maine bacterioplankton as proof of concept. The library consisted of five flavobacteria, one sphingobacterium, four alphaproteobacteria, and one gammaproteobacterium. Most of the SAGs, apart from alphaproteobacteria, were phylogenetically distant from existing isolates, with 88-97% identity in the 16S rRNA gene sequence. Thus, single-cell MDA provided access to the genomic material of numerically dominant but yet-uncultured taxonomic groups. Two of five flavobacteria in the SAG library contained proteorhodopsin genes, suggesting that flavobacteria are among the major carriers of this photometabolic system. The pufM and nasA genes were detected in some 100-cell MDA products but not in SAGs, demonstrating that organisms containing bacteriochlorophyll and assimilative nitrate reductase constituted <1% of the sampled bacterioplankton. Compared with metagenomics, the power of our approach lies in the ability to detect metabolic genes in uncultured microorganisms directly, even when the metabolic and phylogenetic markers are located far apart on the chromosome.
authors.affiliation: Bigelow Laboratory for Ocean Sciences, P.O. Box 475, West Boothbay Harbor, ME 04575-0475, USA. rstepanauskas@bigelow.org
authors.forename: Ramunas
authors.initials: R
authors.lastname: Stepanauskas
authors.forename: Michael E
authors.initials: ME
authors.lastname: Sieracki
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D005784:Gene Amplification; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics; D008386:Marine Biology; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics; D010802:Phylogeny; D012335:RNA, Ribosomal / Q000235:genetics; D025604:Rhodopsins, Microbial / Q000235:genetics; D012623:Seawater / Q000382:microbiology*; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; C098543:PufM protein, Bacteria; D012335:RNA, Ribosomal; D025604:Rhodopsins, Microbial