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

Draft genome sequence of Thauera sp. strain SWB20, isolated from a Singapore wastewater treatment facility using gel microdroplets

We report here the genome sequence of Thauera sp. strain SWB20, isolated from a Singaporean wastewater treatment facility using gel microdroplets (GMDs) and single-cell genomics (SCG). This approach provided a single clonal microcolony that was sufficient to obtain a 4.9-Mbp genome assembly of an ecologically relevant Thauera species.Published versio

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

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

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

Phylogenetic composition of <i>Verrucomicrobia</i> SAGs.

<p>(A) Maximum likelihood phylogenetic analysis of the SSU rRNA gene sequences. Bootstrap (1000 replicates) values ≥50 are displayed. Each phylotype, indicated in red (coastal) or blue (freshwater) is formed by SAGs with ≥99% SSU rRNA gene sequence similarity. Five SAGs from the most abundant putative polysaccharide degrader phylotype in the coastal sample were selected for whole genome sequencing (red star). (B) Phylotype relative abundances in SAG libraries generated using various fluorescent probes. (nd) = not detected in a SAG library.</p

Genomic Comparison of <em>Escherichia coli</em> O104:H4 Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity, Including Shiga Toxin Encoding Phage stx2

<div><p>In May of 2011, an enteroaggregative <em>Escherichia coli</em> O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical <em>E. coli</em> O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the <em>mer/tet</em> antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of <em>Yersinia pestis</em>. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing <em>E. coli</em> O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.</p> </div