scholarly journals Viral recombination blurs taxonomic lines: examination of single-stranded DNA viruses in a wastewater treatment plant

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2585 ◽  
Author(s):  
Victoria M. Pearson ◽  
S. Brian Caudle ◽  
Darin R. Rokyta
Keyword(s):  
Wastewater Treatment ◽  
Microbial Communities ◽  
Rolling Circle Amplification ◽  
Treatment Plant ◽  
Microbial Pathogens ◽  
Model Organisms ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Depth Analysis ◽  
Ssdna Viruses

Understanding the structure and dynamics of microbial communities, especially those of economic concern, is of paramount importance to maintaining healthy and efficient microbial communities at agricultural sites and large industrial cultures, including bioprocessors. Wastewater treatment plants are large bioprocessors which receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a broad range of hosts. To examine this complex collection of viruses, full-length genomes of circular ssDNA viruses were isolated from a wastewater treatment facility using a combination of sucrose-gradient size selection and rolling-circle amplification and sequenced on an Illumina MiSeq. Single-stranded DNA viruses are among the least understood groups of microbial pathogens due to genomic biases and culturing difficulties, particularly compared to the larger, more often studied dsDNA viruses. However, the group contains several notable well-studied examples, including agricultural pathogens which infect both livestock and crops (CircoviridaeandGeminiviridae), and model organisms for genetics and evolution studies (Microviridae). Examination of the collected viral DNA provided evidence for 83 unique genotypic groupings, which were genetically dissimilar to known viral types and exhibited broad diversity within the community. Furthermore, although these genomes express similarities to known viral families, such asCircoviridae,Geminiviridae, andMicroviridae, many are so divergent that they may represent new taxonomic groups. This study demonstrated the efficacy of the protocol for separating bacteria and large viruses from the sought after ssDNA viruses and the ability to use this protocol to obtain an in-depth analysis of the diversity within this group.

scholarly journals An improved experimental pipeline for preparing circular ssDNA viruses for next-generation sequencing

2020 ◽  
Author(s):  
Catherine D. Aimone ◽  
J. Steen Hoyer ◽  
Anna E. Dye ◽  
David O. Deppong ◽  
Siobain Duffy ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rolling Circle Amplification ◽  
Next Generation ◽  
Viral Dna ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Short Read ◽  
Rolling Circle ◽  
Optimized Protocol ◽  
Generation Sequencing

AbstractWe present an optimized protocol for enhanced amplification and enrichment of viral DNA for Next Generation Sequencing of begomovirus genomes. The rapid ability of these viruses to evolve threatens many crops and underscores the importance of using next generation sequencing efficiently to detect and understand the diversity of these viruses. We combined enhanced rolling circle amplification (RCA) with EquiPhi29 polymerase and size selection to generate a cost-effective, short-read sequencing method. This optimized protocol produced short-read sequencing with at least 50% of the reads mapping to the viral reference genome. We provide other insights into common misconceptions about RCA and lessons we have learned from sequencing single-stranded DNA viruses. Our protocol can be used to examine viral DNA as it moves through the entire pathosystem from host to vector, providing valuable information for viral DNA population studies, and would likely work well with other CRESS DNA viruses.HighlightsProtocol for short-read, high throughput sequencing of single-stranded DNA viruses using random primersComparison of the sequencing of total DNA versus size-selected DNAComparison of phi29 and Equiphi29 DNA polymerases for rolling circle amplification of viral single-stranded DNA genomes

scholarly journals Time-series genome-centric analysis unveils bacterial response to operational disturbance in activated sludge

2019 ◽  
Author(s):  
María Victoria Pérez ◽  
Leandro D. Guerrero ◽  
Esteban Orellana ◽  
Eva L. Figuerola ◽  
Leonardo Erijman
Keyword(s):  
Growth Rate ◽  
Time Series ◽  
Wastewater Treatment ◽  
Activated Sludge ◽  
Microbial Communities ◽  
Wastewater Treatment Plant ◽  
Treatment Plant ◽  
Operating Conditions ◽  
The Face

ABSTRACTUnderstanding ecosystem response to disturbances and identifying the most critical traits for the maintenance of ecosystem functioning are important goals for microbial community ecology. In this study, we used 16S rRNA amplicon sequencing and metagenomics to investigate the assembly of bacterial populations in a full-scale municipal activated sludge wastewater treatment plant over a period of three years, including a period of nine month of disturbance, characterized by short-term plant shutdowns. Following the reconstruction of 173 metagenome-assembled genomes, we assessed the functional potential, the number of rRNA gene operons and thein situgrowth rate of microorganisms present throughout the time series. Operational disturbances caused a significant decrease in bacteria with a single copy of the ribosomal RNA (rrn) operon. Despite only moderate differences in resource availability, replication rates were distributed uniformly throughout time, with no differences between disturbed and stable periods. We suggest that the length of the growth lag phase, rather than the growth rate, as the primary driver of selection under disturbed conditions. Thus, the system could maintain its function in the face of disturbance by recruiting bacteria with the capacity to rapidly resume growth under unsteady operating conditions.IMPORTANCEIn this work we investigated the response of microbial communities to disturbances in a full-scale activated sludge wastewater treatment plant over a time-scale that included periods of stability and disturbance. We performed a genome-wide analysis, which allowed us the direct estimation of specific cellular traits, including the rRNA operon copy number and the in situ growth rate of bacteria. This work builds upon recent efforts to incorporate growth efficiency for the understanding of the physiological and ecological processes shaping microbial communities in nature. We found evidence that would suggest that activated sludge could maintain its function in the face of disturbance by recruiting bacteria with the capacity to rapidly resume growth under unsteady operating conditions. This paper provides relevant insights into wastewater treatment process, and may also reveal a key role for growth traits in the adaptive response of bacteria to unsteady environmental conditions.

scholarly journals Single-Stranded DNA Viruses in Antarctic Cryoconite Holes

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1022 ◽  
Author(s):  
Pacifica Sommers ◽  
Rafaela S. Fontenele ◽  
Tayele Kringen ◽  
Simona Kraberger ◽  
Dorota L. Porazinska ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Open Reading Frames ◽  
Pairwise Identity ◽  
Capsid Protein Gene ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Viral Genomes ◽  
Freshwater Pond ◽  
Dry Valley ◽  
Population Structures

Antarctic cryoconite holes, or small melt-holes in the surfaces of glaciers, create habitable oases for isolated microbial communities with tightly linked microbial population structures. Viruses may influence the dynamics of polar microbial communities, but the viromes of the Antarctic cryoconite holes have yet to be characterized. We characterize single-stranded DNA (ssDNA) viruses from three cryoconite holes in the Taylor Valley, Antarctica, using metagenomics. Half of the assembled metagenomes cluster with those in the viral family Microviridae (n = 7), and the rest with unclassified circular replication associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses (n = 7). An additional 18 virus-like circular molecules encoding either a Rep, a capsid protein gene, or other unidentified but viral-like open reading frames were identified. The samples from which the genomes were identified show a strong gradient in microbial diversity and abundances, and the number of viral genomes detected in each sample mirror that gradient. Additionally, one of the CRESS genomes assembled here shares ~90% genome-wide pairwise identity with a virus identified from a freshwater pond on the McMurdo Ice Shelf (Antarctica). Otherwise, the similarity of these viruses to those previously identified is relatively low. Together, these patterns are consistent with the presence of a unique regional virome present in fresh water host populations of the McMurdo Dry Valley region.

scholarly journals Nitrifying and Denitrifying Microbial Communities in Centralized and Decentralized Biological Nitrogen Removing Wastewater Treatment Systems

Water ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1688 ◽  
Author(s):  
Sara K. Wigginton ◽  
Elizabeth Q. Brannon ◽  
Patrick J. Kearns ◽  
Brittany V. Lancellotti ◽  
Alissa Cox ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
Microbial Communities ◽  
High Throughput Sequencing ◽  
Treatment Plant ◽  
Onsite Wastewater Treatment Systems ◽  
N Removal ◽  
Composition And Structure ◽  
Management Approaches ◽  
Biological Nitrogen ◽  
Wastewater Treatment Systems

Biological nitrogen removal (BNR) in centralized and decentralized wastewater treatment systems is assumed to be driven by the same microbial processes and to have communities with a similar composition and structure. There is, however, little information to support these assumptions, which may impact the effectiveness of decentralized systems. We used high-throughput sequencing to compare the structure and composition of the nitrifying and denitrifying bacterial communities of nine onsite wastewater treatment systems (OWTS) and one wastewater treatment plant (WTP) by targeting the genes coding for ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ). The amoA diversity was similar between the WTP and OWTS, but nosZ diversity was generally higher for the WTP. Beta diversity analyses showed the WTP and OWTS promoted distinct amoA and nosZ communities, although there is a core group of N-transforming bacteria common across scales of BNR treatment. Our results suggest that advanced N-removal OWTS have microbial communities that are sufficiently distinct from those of WTP with BNR, which may warrant different management approaches.

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