Partial genome assembly for a candidate division OP11 single cell from an anoxic spring (Zodletone Spring, Oklahoma)

Abstract

Members of candidate division OP11 are widely distributed in terrestrial and marine ecosystems, yet little information regarding their metabolic capabilities and ecological role within such habitats is currently available. Here, we report on the microfluidic isolation, multiple-displacement-amplification, pyrosequencing, and genomic analysis of a single cell (ZG1) belonging to candidate division OP11. Genome analysis of the ∼270-kb partial genome assembly obtained showed that it had no particular similarity to a specific phylum. Four hundred twenty-three open reading frames were identified, 46% of which had no function prediction. In-depth analysis revealed a heterotrophic lifestyle, with genes encoding endoglucanase, amylopullulanase, and laccase enzymes, suggesting a capacity for utilization of cellulose, starch, and, potentially, lignin, respectively. Genes encoding several glycolysis enzymes as well as formate utilization were identified, but no evidence for an electron transport chain was found. The presence of genes encoding various components of lipopolysaccharide biosynthesis indicates a Gram-negative bacterial cell wall. The partial genome also provides evidence for antibiotic resistance (β-lactamase, aminoglycoside phosphotransferase), as well as antibiotic production (bacteriocin) and extracellular bactericidal peptidases. Multiple mechanisms for stress response were identified, as were elements of type I and type IV secretion systems. Finally, housekeeping genes identified within the partial genome were used to demonstrate the OP11 affiliation of multiple hitherto unclassified genomic fragments from multiple database-deposited metagenomic data sets. These results provide the first glimpse into the lifestyle of a member of a ubiquitous, yet poorly understood bacterial candidate division.

Fig. 1.

Optofluidic apparatus for isolating individual OP11 cells and amplifying their genomic contents. (A) Computer-controlled microscope fitted for fluorescence imaging and laser trapping. (B) Plan view of the two-layer 32-channel microfluidic chip used in this study. Control lines (25-μm depth, square profile, bottom layer) are shown in red, flow lines (10-μm depth, rounded profile, top layer) are shown in blue, and large channels/chambers (60-μm depth, rounded profile, top layer) are shown in green. (C) Photograph of the microfluidic chip with tubing to power control lines attached. (D) Plan view zoom of the box in panel B showing a single sorting/amplification channel. Cell suspension flows vertically in the blue channel at the left. Reagents are supplied to the indicated T junction from a supply line dedicated to 1 of the 32 reaction channels. Each reagent solution is flushed to the left from the T junction to backwash the blue channel, before being applied for the single-cell reaction by redirection to the right of the T junction. (E) Plan view micrograph of the chip region shown in panel D. (F) Elevation view (cross-sectional) schematic indicating components visible in panel E and layup of the microfluidic device. PDMS, polydimethylsiloxane. (G) Plan view zoom of the box in panel D showing the path by which cells are sorted using the optical trap. Cells traverse about 1.5 mm of channel containing clean buffer across two valves, which are opened sequentially to allow cells to pass. (H to N) Micrographs depicting device and MDA reaction setup. (H) Bare device with air-filled channels; (I) device with control lines filled with water (low-contrast channels) and pressurized (valves closed, visible where control channels cross air-filled flow lines); (J) device with reagent and sample lines prefilled with buffer (high-contrast channels, air; low-contrast channels, buffer); sorting takes place with this device configuration; (K) lysis chamber 1 (3.5-nl capacity) after reagent flush and dead-end fill; (L) lysis chamber 2 (3.5-nl capacity) after reagent flush and dead-end fill; (M) reaction chamber (60-nl capacity) initial filling by dead-end method after reagent flush; (N) reaction chamber with nearly complete dead-end fill.

Fig. 2.

(A) Phase-contrast micrograph of candidate division OP11 ZG1 cell. (B) 16S rRNA gene tree highlighting the phylogenetic affiliation of the ZG1 cell. Bootstrap values (expressed as percentages) are based on 1,000 replicates and are shown for branches with more than 50% support. The evolutionary distance-based tree was constructed with the neighbor-joining algorithm with the Jukes-Cantor corrections. TCE, trichloroethylene.

Fig. 3.

Pentanucleotide content of ZG1 versus that of sequenced bacteria and archaea. (A) PCA of pentanucleotide content of ZG1, P. cryohalolentis K5, E. coli O157:H7, and M. acetivorans. (B to E) Distance histograms of sequences from each organism present in panel A to sequences from the other three organisms. Units are normalized variance and can be compared within and across panels B to E.

Fig. 4.

Phylogenetic affiliation of ZG1 first hits for all genes identified within the partial ZG1 genome.