Matching Phylogeny and Metabolism in the Uncultured Marine Bacteria, One Cell at a Time

Bigelow Laboratory for Ocean Sciences, P.O. Box 475, West Boothbay Harbor, ME 04575-0475, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2007; 104(21):9052-7. DOI: 10.1073/pnas.0700496104
Source: PubMed


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.

Download full-text


Available from: Michael Sieracki, Oct 08, 2015
41 Reads
  • Source
    • "In recent years, investigators have tried different approaches to ask targeted ecological questions at the resolution of single cells. The most common approach to connect phylogeny with function combines single-cell FACS sorting with whole-genome amplification and PCR screening for target genes (Stepanauskas and Sieracki, 2007; Siegl and Hentschel, 2010; Martinez-Garcia et al., 2012; Bayer et al., 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells.
    The ISME Journal 09/2015; DOI:10.1038/ismej.2015.124 · 9.30 Impact Factor
  • Source
    • "Single cell sorting, whole-genome amplification, PCRamplification and sequencing of the small subunit ribosomal RNA (SSU rRNA) genes, as well as the shotgun sequencing and de novo assembly of the selected SAGs were performed at the Bigelow Laboratory Single Cell Genomics Center (, as described previously (Stepanauskas and Sieracki, 2007; Swan et al., 2011; Martinez-Garcia et al., 2012; Field et al., 2015). PCR-amplified SSU rRNA gene sequences (∼800–900 bp) of SAGs were edited using Sequencher v4.7 (Gene Codes) and compared with previously deposited sequences using the RDP v10 Classifier (SSU rRNA) (Wang et al., 2007) and National Center for Biotechnology Information (NCBI) BLAST (Altschul et al., 1990) nucleotide database (nt). "
    [Show abstract] [Hide abstract]
    ABSTRACT: A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT) and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a 3 km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32% of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.
    Frontiers in Microbiology 04/2015; 6:349. DOI:10.3389/fmicb.2015.00349 · 3.99 Impact Factor
  • Source
    • "The method performs well on complex sediment microbial population spiked with low amounts of E. coli XL1, despite the rate of false positive events that increases with dilution. Until now, such target-gene screening of genomes has been done by testing single amplified genomes on 96-well plates by PCR (Stepanauskas and Sieracki, 2007), an approach that would require screening an average of 10 plates to find one target genome with a target-gene frequency of 0.1%. Clearly, a high-throughput screening procedure such as that described here would be invaluable for studying microbes that are not predominant community members. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Recent progress in environmental microbiology has revealed vast populations of microbes in any given habitat that cannot be detected by conventional culturing strategies. The use of sensitive genetic detection methods such as CARD-FISH and in situ PCR have been limited by the cell wall permeabilization requirement that cannot be performed similarly on all cell types without lysing some and leaving some nonpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. We describe an emulsion-based procedure to trap individual microbial cells into picoliter- volume polyacrylamide droplets that provide a rigid support for genetic material and therefore allow complete degradation of cellular material to expose the individual genomes. The polyacrylamide droplets are subsequently converted into picoliter-scale reactors for genome amplification. The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry. Using the Escherichia coli strains XL1 and MC1061, which differ with respect to the presence (XL1), or absence (MC1061) of a single copy of a tetracycline resistance gene per genome, we demonstrate that XL1 genomes present at 0.1% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to highly complex environmental microbial communities as a target gene-based screen for individual microbes. The method provides a novel tool for enumerating functional cell populations in complex microbial communities. We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.
    Frontiers in Microbiology 03/2015; 6(195):1-10. · 3.99 Impact Factor
Show more