What's New Is Old: Resolving the Identity of Leptothrix ochracea Using Single Cell Genomics, Pyrosequencing and FISH

Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms

Capturing Single Cell Genomes of Active Polysaccharide Degraders: An Unexpected Contribution of Verrucomicrobia

Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation

Depth distribution of single amplified genome (SAG)-related thaumarchaea determined by metagenomic fragment recruitment.

<p>Thaumarchaea cultures and SAGs are listed along the y-axis and metagenomes are listed along the x-axis. SAGs are colored according to source; red, South Pacific; blue, North Pacific. The scale bar indicates the percentage of aligned metagenome sequences that had ≥95% nucleotide sequence identity and an alignment length ≥200 base pairs for the BLASTN-based recruitment, normalized by the length of each genome. <i>C. symbiosum</i>, <i>Cenarchaeum symbiosum</i>; <i>N. maritimus</i>, <i>Nitrosopumilus maritimus</i>; HOT, Hawaii Ocean Time Series station ALOHA; NESAP, North Eastern Subarctic Pacific; GB, Guaymas Basin hydrothermal vent plume; ETSP, Eastern Tropical South Pacific; SA, Subtropical South Atlantic; KM3, Ionian Sea Station KM3.</p

Homology and characterization of proteins from MGI single amplified genomes (SAGs) and thaumarchaea marine cultures.

<p>BLAST Score Ratio (BSR) analysis of the non-redundant protein set from 37 MGI SAGs (n = 2,988) (<b>A</b>), and characterization of selected homolog protein groups using Clusters of Orthologous Groups (COG) categories (<b>B</b>). BSR scores >0.4 (∼30% protein identity) are considered homologous. Proteins are color coded by homology pattern: red, shared among all genomes; blue, shared among SAGs and <i>N. maritimus</i>; green, shared among SAGs and <i>C. symbiosum</i>; yellow, not homologous to either culture. Proteins identified as a genomic island are represented by black squares. Arrows indicate enriched genomic island COG categories. COG categories: B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control, mitosis, and meiosis; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation; K, transcription; L, replication, recombination, and repair; M, cell wall/membrane biogenesis; N, cell motility; O, posttranslational modification, protein turnover, and chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolite biosynthesis, transport, and catabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking and secretion; V, defense mechanisms; Mixed (multiple categories); None (no COG category). <i>C. symbiosum</i>, <i>Cenarchaeum symbiosum</i>; <i>N. maritimus</i>, <i>Nitrosopumilus maritimus</i>.</p

Syntenic and phylogenetic analysis of genes involved in urea hydrolysis.

<p>Arrangement and similarity of genes involved in the urea hydrolysis pathway <i>C. symbiosum</i> and SAGs (<b>A</b>), and an inferred phylogenetic tree of α-subunit of urease (<i>ureC</i>) gene sequences from SAGs and selected cultures and environmental samples (<b>B</b>). SAGs are colored according to source; red, South Pacific; blue, North Pacific. The scale bar indicates tblastx similarity values between genes. The tree was inferred using maximum likelihood in RAxML and bootstrap (1000 replicates) values ≥50% are indicated at nodes. <i>C. symbiosum</i>, <i>Cenarchaeum symbiosum</i>.</p

Phylogenetic analysis of archaeal single amplified genomes (SAGs) from South Atlantic and North Pacific gyres.

<p>The phylogenetic composition of archaeal SAG libraries (<b>A</b>) and an inferred phylogenetic tree of partial SSU rRNA sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095380#pone.0095380.s005" target="_blank">Table S2</a>) amplified from Marine Group I (MGI) Thaumarchaeota SAGs (<b>B</b>). Numbers in parentheses represent the number of SAGs in each archaeal group. The tree was inferred using maximum likelihood in RAxML and bootstrap (1000 replicates) values ≥50% are indicated at nodes. Sequences from South Atlantic SAGs are colored red, and North Pacific (HOT station ALOHA) SAG sequences colored blue. MGI Thaumarchaeota sequences with ≥99% similarity were grouped into phylotypes (bold), representative sequence(s) are in colored parentheses, and total number of sequences in each phylotypes is in parentheses (bold).</p

Evidence for the laminarinase gene in the single amplified genome AAA168-F10.

<p>(A) Active site, including the catalytic residues responsible for laminarin hydrolysis, derived from Conserved Domain Protein, SWISS-MODEL, and PROSITE databases. (B) Neighbor-joining phylogenetic tree of amino acid sequences, applying the Kimura evolutionary model and indicating bootstrap values above 50.</p

Comparative analysis of genes encoding hydrolytic enzymes in prokaryote genomes.

<p>The bar chart indicates the genome-wide frequency of glycoside hydrolase genes in various microbial groups, average ± standard deviation. The number of publicly available genomes found in the IMG database (as of February 2012) for each taxonomic group is provided in parentheses. The average enrichment of glycoside hydrolases was also estimated for the <i>Bacteria</i> domain. The small pie chart shows the number and composition of genes involved in polysaccharide hydrolysis in the <i>Verrucomicrobia</i> SAG AAA168-F10. The large pie chart shows CAZy families of glycoside hydrolase genes detected in SAG AAA168-F10. Each glycoside hydrolase family is indicated as GH-xxx, according to CAZy database nomenclature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035314#pone.0035314-Weiss1" target="_blank">[25]</a>.</p

Flow-cytometric sort gates (A) and taxonomic composition (B) of single amplified genomes (SAGs) generated from coastal bacterioplankton using various fluorescent probes.

<p>Bacterioplankton were probed with (from top to bottom): 1) nucleic acid stain SYTO-9, targeting high- and low-nucleic acid content cells (HNA and LNA cells) representing a random subset of the entire microbial assemblage; 2) fluorescently-labeled laminarin; 3) fluorescently-labeled xylan; 4) 5-cyano-2,3-ditolyltetrazolium chloride (ETS-active cells) and 5) carboxyfluoresceindiacetate (esterase-active cells). Gates used for cell sorting are indicated in blue.</p

Optimization of cell probing conditions with fluorescently labeled laminarin.

<p>Flow cytometric dot plots of heat-killed and live freshwater samples incubated for various lengths of time with 4 or 40 µM fluorescently labeled laminarin. Red polygons indicate gates used to count putative laminarin-positive cells.</p