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Thursday, November 12

Redefining Microbial Genomics:
Sequencing Individual Cells

Ramunas Stepanauskas, Bigelow Laboratory for Ocean Sciences,
West Boothbay Harbor, Maine
Biological and Environmental Sciences Directorate Seminar
10:00 AM, Building 1505, Ocoee Room (189)
Contact: Martin Keller (kellerm@ornl.gov), 865.574.4333

Abstract

The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomics. Single cell sequencing offers a powerful, novel approach to recover discrete genomes and complete metabolic pathways of the microbial "uncultured majority". We developed a high-throughput pipeline for such analyses, which employs fluorescence-activated cell sorting and multiple displacement amplification of individual microbial cells to obtain sufficient amount of genomic DNA for downstream PCR of multiple loci and for whole genome shotgun sequencing. We employed this pipeline to analyze multiple genes in uncultured microorganisms from diverse aquatic environments, including surface and deep ocean, freshwater lakes, hypersaline ponds, etc. Shotgun sequencing of several coastal Flavobacteria representatives yielded up to 90% genome recoveries, with insignificant DNA contamination. In contrast to all cultured strains of marine flavobacteria, the single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches and oligotrophic lifestyle. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation.