Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage - PubMed (original) (raw)

Paul C Blainey, Senthil K Murugapiran, Wesley D Swingley, Christian A Ross, Susannah G Tringe, Patrick S G Chain, Matthew B Scholz, Chien-Chi Lo, Jason Raymond, Stephen R Quake, Brian P Hedlund

Affiliations

Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage

Jeremy A Dodsworth et al. Nat Commun. 2013.

Abstract

OP9 is a yet-uncultivated bacterial lineage found in geothermal systems, petroleum reservoirs, anaerobic digesters and wastewater treatment facilities. Here we use single-cell and metagenome sequencing to obtain two distinct, nearly complete OP9 genomes, one constructed from single cells sorted from hot spring sediments and the other derived from binned metagenomic contigs from an in situ-enriched cellulolytic, thermophilic community. Phylogenomic analyses support the designation of OP9 as a candidate phylum for which we propose the name 'Atribacteria'. Although a plurality of predicted proteins is most similar to those from Firmicutes, the presence of key genes suggests a diderm cell envelope. Metabolic reconstruction from the core genome suggests an anaerobic lifestyle based on sugar fermentation by Embden-Meyerhof glycolysis with production of hydrogen, acetate and ethanol. Putative glycohydrolases and an endoglucanase may enable catabolism of (hemi)cellulose in thermal environments. This study lays a foundation for understanding the physiology and ecological role of the 'Atribacteria'.

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Figures

Figure 1

Figure 1

Chimera-filtering increases contig N50 in the cSCG assembly. Stepwise assembly of composite OP9 SCG before (grey points) or after (black points) filtering out potentially chimeric reads. The increase in (A) total assembly size, (B) number of contigs, and (C) contig N50 are shown as a function of the number of reads (sampling with replacement) used.

Figure 2

Figure 2

Identification of OP9-like contigs from the 77CS metagenome. (A) Principal component analysis (PCA) of tetranucleotide frequency of contigs in the 77CS metagenome and ten individual OP9 SCG assemblies. 77CS contigs in dark blue were binned as OP9-like, as defined by their placement within an ellipsoid with a centroid and semi-axis lengths equal to the mean and 2.5 times the standard deviation, respectively, of the first three principle components (PC1-PC3, with the percent variation explained by each in parentheses) of the OP9 SCG contigs. Only contigs greater than 2 kb in length are shown. Contigs <2kb inside this ellipsoid were also included in the OP9 bin if they had significant nucleotide identity (>85% identity over >100 nt) to contigs in the OP9-cSCG by BLASTn. (B) Total length of OP9 SCG and 77CS contigs >2kb contained within ellipsoids with different semi-axis lengths defined by multiplication of the standard deviation of PC1-PC3 of the OP9 SCG contigs by increasing scalar quantities (x-axis). Black points on the curves indicate the multiplier (2.5) used to define the OP9 bin, chosen at an approximate inflection point of the CS77 contig length curve to maximize inclusion of metagenome contigs in the cluster overlapping the OP9 SCGs but minimize inclusion of contigs in adjacent clusters. (C) Plot of coverage depth vs. contig length for the CS77 metagenome assembly, highlighting contigs in the OP9 bin with (dark blue circles) and without (open circles) >85% nucleotide identity over >100 bp to the OP9-cSCG assembly.

Figure 3

Figure 3

Relationship of OP9-SCG and OP9-CS77 to other bacterial groups. (A) Neighbor-joining tree based on a distance matrix of 1349 aligned positions of 16S rRNA genes from selected bacterial phyla with cultured representatives and the candidate phyla JS1 and OP9, including those in the OP9-SCG and OP9-CS77 assemblies. Black (100%), grey (>80%), and white (>50%) circles indicate bootstrap support (100 pseudoreplicates) for selected nodes in phylogenies inferred using neighbor-joining (left half of circle) and maximum-likelihood (right half) methods. (B) Maximum-likelihood phylogeny inferred from concatenated alignments of predicted amino acid sequences of 31 housekeeping genes identified by AMPHROA in genomes representing a variety of bacterial phyla and the OP9-SCG and OP9-CS77 assemblies. The number of genomes represented in each wedge is indicated in parentheses, and black circles indicate bootstrap support of >80% for 100 pseudoreplicates. (C) Binning of CDSs in the OP9 assemblies based on the phylogenetic affiliation of their top BLASTP hit to a database of sequenced bacterial and archaeal genomes. Phylogenetic groups with fewer than 1% of top hits were aggregated (‘other groups’). (D) Phylogenetic binning of CDSs on contigs or scaffolds containing markers diagnostic for a diderm cell envelope structure, emphasizing that top BLASTP hits were distributed among both monoderm and diderm Bacteria. Diderm Firmicutes includes members of the Negativicutes and Halanaerobiales.

Figure 4

Figure 4

Overview of features and potential metabolic capabilities of the OP9 lineage represented by the OP9-SCG and OP9-CS77 genomes as discussed in the text. CDSs in the OP9-cSCG and OP9-77CS associated with predicted functions are listed in Supplementary Table S5. Proteins involved in specific processes are identified by color: secretion and transporters (purple); saccharide catabolism and fermentation (red); energy conservation (orange); flagellar motility and chemotaxis (green); and nitrogen transport and assimilation (blue). Substrates and products are not necessarily balanced in the reactions depicted. Abbreviations not indicated in the text: ABC, ATP-binding cassette; Fdox, oxidized ferredoxin; AK, acetate kinase; PTA, phosphotransacetylase; AlDH, aldehyde dehydrogenase; ADH, alcohol dehydrogenase; PPi, pyrophosphate; Pi, inorganic phosphate.

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