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.

Fig. 1.

Maximum-likelihood phylogenetic trees of bacterial SSU rRNA genes and proteorhodopsins. The SSU rRNA tree includes SAGs from this study and all Flavobacteriaceae isolates with completed or currently undergoing whole-genome sequencing. The proteorhodopsin tree includes SAGs from this study and the most closely related sequences in GenBank, based on a BLASTP search. Colors indicate SAGs (yellow background), isolates (blue text), and environmental clones (black text). GenBank accession nos. are provided for each clone. Nodes marked with circles have >70% neighbor-joining bootstrap support.

Fig. 2.

Maximum-likelihood phylogenetic trees of the PufM and NasA. Included are protein sequences obtained from 100-cell MDA reactions and most closely related sequences in GenBank, based on BLASTP searches. Colors indicate sequences from this study (yellow background), isolates of Alphaproteobacteria (purple text), isolates of Betaproteobacteria (green text), isolates of Gammaproteobacteria (blue text), and environmental clones (black text). GenBank accession nos. are provided for each clone. Nodes with yellow circles have >70% neighbor-joining bootstrap support.