Census of the Viral Metagenome within an Activated Sludge Microbial Assemblage

Abstract

The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.Bacteriophages play an active role in the ecology of natural environments, influencing prokaryotic population dynamics (5, 15) and mediating lateral gene transfer between diverse bacterial species, for example. Activated sludge (AS) microbial assemblages in wastewater treatment plants have been shown to harbor great numbers of viruses with a wide range of genome sizes (7,9,10, 16). Historically, the focus of wastewater viral studies has been on specific host-virus interactions, the application of phages as tools in microbial source tracking, or the use of phages to improve the efficiency of the wastewater treatment process (e.g., foam and pathogen control) (2,4,8,12,17). Despite the interest in the wastewater viral community, a census of the activated sludge total viral community has not, to our knowledge, been investigated using both culture-based and metagenomic approaches.Taxonomic assignment of viral metagenomic clone sequences. Samples were collected from the AS aeration basin at the H. C. Morgan Water Pollution Control Facility in Auburn, AL, treated with 10% beef extract buffer (to separate viruses from aggregates), precipitated with polyethylene glycol, and subjected to cesium chloride gradient centrifugation. Viral DNA was then extracted by benzonase, proteinase K, and sodium dodecyl sulfate treatments and purified by phenolchloroform extraction and isopropanol precipitation. An AS viral metagenomic library was constructed at the Lucigen Corporation (Middleton, WI) using a linker-amplified shotgun library approach (3, 18), and 1,161 cloned insert DNA sequences were determined using a single-vector primer. Trimmed viral insert sequences were classified as known (E value Ͻ 0.001; n ϭ 694), unknown (n ϭ 97), or novel (n ϭ 370) as described previously (18). The known sequences were assigned to their taxonomic affiliations by BLASTx comparisons to the GenBank nonredundant nucleotide (nr/nt) database and selection of the top BLASTx hits for phylogenetic affiliation (Fig. 1).The dominance of viral metagenomic sequences with significant homology to bacterial DNA (nearly 60% of the known sequences) is likely the consequence of the high frequency of prophage sequences within bacterial genomes deposited in GenBank databases (1). Considerable care was taken to prevent inclusion of prokaryotic (or eukaryotic) chromosomal DNA within the viral metagenomic library (i.e., CsCl purification and nuclease treatment were performed), and the high percentage of viral metagenomic sequences with homology to bacterial genomes has been observed in other environments (3). Of the 191 known sequences with homology to viral genomes...

High Variety of Known and New RNA and DNA Viruses of Diverse Origins in Untreated Sewage

“…A metagenomics analysis of reclaimed water (for nonpotable public uses) using pyrosequencing of viral particle-associated DNA indicated 98% prokaryotic versus 2% eukaryotic viral sequences, while the viral RNA population showed a more equally mixed distribution of viruses from a variety of inferred hosts more akin to that reported here (82). A metagenomics analysis of DNA viruses purified from activated sludge (a product of wastewater treatment) also showed a predominance (95%) of prokaryotic DNA viruses (76). A pyrosequencing analysis of the DNA viruses in the influent, activated sludge, effluent, and anaerobic digester of a wastewater treatment plant showed Ͼ90% of viral reads to be of likely bacterial origin (89).…”

“…Recent studies have shown that besides human viruses, untreated sewage also contains a wide diversity of other animal, plant, insect, and bacterial viruses (11,76,82,89). We performed a metagenomic deep-sequencing analysis of viral particles in sewage from four countries on three continents.…”

Metagenomic Analysis of Kimchi, a Traditional Korean Fermented Food

“…Bacteriophages are ubiquitous and abundant components of microbial communities and have significant influence upon bacterial diversity, horizontal gene transfer, and genetic diversity in environmental habitats (43). By counting the number of reads mapped to each phage-related contig and normalizing by the total read count, it was possible to estimate the relative abundance of phages.…”

“…Low-complexity sequence regions (simple sequence repeats) were identified and excluded from consideration during initial pair-wise comparison but were included during final alignment and consensus building. Assembled contigs longer than 10 kb were assigned to their taxonomic affiliations by BLASTN comparisons to the GenBank nonredundant nucleotide (nr/nt) database and selections of the top BLASTN hits for phylogenetic affiliation as described previously (43,44). Clustered regularly interspaced short palindromic repeats (CRISPR) elements were searched from assembled contigs longer than 10 kb by using the Web-based tool CRISPRFinder (18).…”

Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis

“…2). It has previously been shown that environmental phages in wastewater, sludge and soil contain 16S rRNA genes of bacterial origin (Sander & Schmieger, 2001, Del Casale et al, 2011aParsley et al, 2010;Ghosh et al, 2008).…”