Assembling the marine metagenome, one cell at a time

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 metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two 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. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex microbial community of marine bacterioplankton. A combination of single cell genomics and metagenomics enabled us to analyze the genome content, metabolic adaptations, and biogeography of these taxa.

Figure 1. Global Ocean Sampling metagenome fragment recruitment reveals SAGs MS024-2A and MS024-3C as strong recruiters.

The available marine flavobacteria isolate genomes, the non-marine Flavobacterium johnsoniae, and the three best GOS fragment recruiters Pelagibacter, Prochlorococcus and Synechococcus served as reference genomes. Fragment recruitment was performed with MUMMER and only ≥400 bp alignments were counted. Psychroflexus torquis ATCC 700755 was excluded from the analysis due to its poor genome assembly quality. The following marine flavobacteria genomes had fewer than 10 recruits and are not shown: Croceibacter atlanticus HTCC2559, Robiginitalea biformata HTCC2501, Gramella forsetii KT0803, Kordia algicida OT-1, isolate ALC-1, isolate HTCC2170, and isolate BBFL7. Croceibacter atlanticus HTCC2559 and Robiginitalea biformata HTCC2501 were originally collected at or near GOS sampling stations.

Figure 2. Biogeography of microorganisms closely related to MS024-2A and MS024-3C.

A. Geographic distribution of the Global Ocean Sampling (GOS) metagenome fragments with >95% identity to MS024-2A and MS024-3C DNA. Numerals on the map indicate GOS station numbers. B. Sea surface temperature in December 2003, which demonstrates hydrological separation of GOS aquatic samples collected north and south of Cape Hatteras (near GOS station 13). Provided is a composite Aqua-MODIS image for December 2003 (http://oceancolor.gsfc.nasa.gov). The GOS stations were numbered in the order of their sampling, and stations 12, 13 and 14 were sampled on December 18, 19 and 20, 2003.

Figure 3. Genome streamlining in MS024-2A and MS024-3C.

Genome streamlining was evidenced by small genome sizes, low fraction of genes in paralog families, and low fraction of non-coding bases. Included are all available genomes of the Bacteroidetes/Chlorobi group. The number of genes in paralog families was estimated using the BLASTCLUST tool from the NCBI BLAST software (>30% sequence similarity, across >50% of their length and E<10⁻⁶).