In this study, using a laser-assisted microfluidic device, a single cell belonging to candidate division OP11 was isolated, and its genomic DNA was partially amplified and sequenced, yielding a 270-kb partial genome. In-depth analysis revealed a heterotrophic, fermentative lifestyle with the capacity for cellulose, starch, and potentially lignin metabolism. In addition, evidence for a cell wall of Gram-negative bacteria, multiple secretion systems, antibiotic and bactericidal peptidase production, antibiotic resistance, and stress response mechanisms was identified. Within Zodletone Spring source sediments from which the OP11 cell has been sorted, phototrophic and chemolithoautotrophic CO2 fixation coupled to sulfide and sulfur oxidation results in an extremely eutrophic habitat with an extremely biomass-rich prokaryotic community. Therefore, the reported heterotrophic, polymer-degrading capabilities of OP11 suggest a possible role for members of this candidate phylum, or at least the OP11 cell from which ZG1 assembly is obtained, in carbon cycling. Specifically, OP11 could be involved in the breakdown of polymers derived from microbial biomass and subsequent degradation of the fermentable products obtained to fermentation end products (e.g., fatty acids and CO2) that could be utilized by other metabolic guilds within the community (e.g., sulfate and sulfur-reducing and photoautotrophic and chemolithautotrophic bacteria), respectively.
Members of OP11 have been encountered in a wide variety of habitats. In general, ecosystems where OP11 has been identified are eutrophic, with high prokaryotic biomass and high phylogenetic diversity (e.g., hydrocarbon-impacted habitats and hydrothermal vents [16
]). In addition, OP11 members were also identified in various soils (20
), ecosystems characterized by constant fluctuations in environmental conditions and high competition for nutritional resources by soil prokaryotes and soil fungi. Bacterial survival in such ecosystems requires constant adaptation to environmental fluctuations and possession of multiple survival weapons to enhance competitiveness. The ZG1 partial genome appears to have a moderately high ratio of transcription-associated genes (3.55%), indicating a high capability to respond to environmental fluctuations (11
). In addition, ZG1 genomes have multiple antibiotic production and resistance mechanisms, which enhance its survivability in highly diverse and competitive ecosystems. The possession and retention of a large array of regulatory and other nonhousekeeping genes are in stark contrast to the characteristics of streamlined genomes of marine picoplankton, e.g., Pelagibacter ubique
) or genomes of prokaryotic parasites (e.g., Mycoplasma
spp.), where the lack of environmental fluctuations and relaxation of positive selection for genes used in biosynthesis or regulation favor genome reduction as an energy-saving strategy.
This study yielded interesting metabolic insights (e.g., cellulose- and starch-degrading capability, resistance to β-lactam and aminoglycoside antibiotics, and potential capability to metabolize galactose) that could help in designing enrichment strategies for some members of OP11 in Zodletone Spring source sediments. However, the capability of a single genome assembly, much like a single bacterial isolate from a high-order bacterial lineage (e.g., phylum or class), is not an adequate representation of the panmetabolic capability of an entire lineage. Therefore, we strongly caution against regarding these findings as a general indication of the overall metabolic capabilities of all members of OP11. This is partially due to the small size of the ZG1 genome assembly but, more importantly, to the ubiquity and expected high level of genomic diversity within various members and lineages within candidate division OP11. As such, multiple diverse capabilities in different OP11 lineages or even within the same lineage are entirely possible. Indeed, a previous study enriching for anaerobes from an uranium-contaminated environment in field research center (FRC) sediments (Oak Ridge, TN) enriched for members of OP11 (GenBank accession numbers EF508021
) during syntrophic growth on ethanol (46
The sequencing of nucleic acids derived from a phenotypically identified single cell in a chemically fixed sample constitutes a powerful new tool available to microbiologists. This tool is capable of providing great insights both in cases where complete or nearly complete genome sequences can be recovered (6
) and when partial genomes are recovered, as in the study described in this report. The analysis of a partial single-cell genome is reminiscent of the analysis of environmental fosmid sequences (8
) or fragments assembled from metagenomic data (57
). One difference is that single-cell data sets can span much greater genomic distances than fosmids or conventional metagenomic assemblies. The key to confident interpretation of single-cell data is an assurance that only one cell is, in fact, being analyzed and that the sample is not contaminated, as the potential for contamination of material amplified from a single cell is extraordinarily high.
Metagenomic mining using a stringent genome recruitment approach was utilized to identify hitherto unclassified metagenomic fragments belonging to OP11. Our goal was to demonstrate the utility of these OP11 gene markers in improving phylogenetic binning in metagenomics and to determine whether additional characteristics of OP11 in other environments could be gleaned from such efforts. Unfortunately, only an additional 18 kb of total fragments was identified from three different metagenomic data sets. Multiple reasons could possibly explain the limited success in such effort: (i) the fact that members of OP11 are almost always encountered in low numbers in highly diverse ecosystems (16
), which renders obtaining large (if any) OP11 fragments unlikely, except in extremely ambitious metagenomic surveys (e.g., GOS); (ii) the stringent genome recruitment criteria applied to positively identify OP11 metagenomic fragments coupled to the potentially high level of intralineage diversity within members of OP11; and (iii) the rapid increase in dependence on short-read technology (pyrosequencing/Illumina) in recent metagenomic studies and the subsequent difficulty and uncertainty associated with assembly under such conditions due to potential chimeras (39
). It is telling that all the metagenomic OP11 fragments obtained were from Sanger sequencing-based metagenomic studies. Nonetheless, there is undoubtedly a tremendous potential for synergy between single-cell genomic data and metagenomic data (6
). Our limited success with recruitment of database sequences highlights the novelty of the sequence that we report here, the extreme diversity of environmental microbial communities, and the importance of pairing analyses of single-cell data sets and metagenomic data sets derived from the same sample. Additional OP11 single-cell data sets, long-read sequencing technologies (17
) with sufficient throughput to deeply sample complex communities, and improvements in assembly and binning strategies could each lead to much better identification of OP11 fragments from current and future data sets.