Phage-host Evolution in a Model Ecosystem

Skennerton, Connor (2013). Phage-host Evolution in a Model Ecosystem PhD Thesis, School of Chemical Engineering, The University of Queensland.

       
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Author Skennerton, Connor
Thesis Title Phage-host Evolution in a Model Ecosystem
School, Centre or Institute School of Chemical Engineering
Institution The University of Queensland
Publication date 2013-01-01
Thesis type PhD Thesis
Supervisor Gene Tyson
Philip Hugenholtz
Total pages 203
Language eng
Subjects 060309 Phylogeny and Comparative Analysis
080301 Bioinformatics Software
060504 Microbial Ecology
Formatted abstract
Microorganisms are key drivers of global biogeochemical cycles and are being used increasingly in industrial processes. Enhanced biological phosphorus removal (EBPR) is a microbial-mediated processes commonly used in wastewater treatment for the removal of carbon and phosphorus. EBPR reactors harbour diverse microbial communities however phosphorous removal is only performed by some members of the community called polyphosphate- accumulating organisms (PAOs). The most widely studied PAO is Candidatus Accumulibacter phosphatis (Accumulibacter), because it the most commonly enriched PAO in lab-scale EBPR reactors. Despite decades of research aimed at cultivating Accumulibacter it has not yet been obtained in pure culture, therefore culture-independent techniques, such as metagenomics, have been used to study Accumulibacter and its interactions with other community members.

An interaction in microbial communities that is becoming increasingly recognised is the role phage (viruses that infect bacteria) play in shaping community composition. However, very little is known about phage in EBPR systems and whether their predation on Accumulibacter and other PAOs contributes to the periodic poor performance observed in EBPR reactors. In this thesis research, metagenomics has been used to investigate the role of phage in shaping EBPR communities, and detailed analysis of co-evolving phage and microbial genomes provides insights into evolutionary dynamics shaping these populations.

Chapter 2 of this thesis research involved the reanalysis of a phage metagenome from a lab-scale EBPR reactor generated by researchers at Joint Genome Institute. De novo assembly of this metagenome lead to the recovery of three complete and six partial phage genomes. One of complete phage, EPV1, contained a transcriptional repressor known as histone-like nucleoid structuring protein (H-NS) that appeared to be horizontally transferred from its host, Accumulibacter. Bioinformatic analysis suggested that many of the phage defence mechanisms in Accumulibacter were under the control of H-NS, which are likely suppressed by EPV1-coded H-NS during infection.

It has recently been discovered that some bacteria and most archaea possess an adaptive immune system called clustered regularly interspaced short palindromic repeats (CRISPR). The CRISPR and associated genes enable small fragments of phage DNA to be stored in the host genome (called spacers). These spacers are then used as a targeting mechanism to degrade phage genomes upon infection. CRISPRs are very dynamic in environmental communities however the bioinformatic tools used to analyse them are not suited to metagenomic analysis. Motivated by these limitations a new tool, called Crass, was developed specifically to interrogate CRISPRs from short-read metagenomic data (Chapter 3). Crass identified significantly more CRISPRs in a short-read EBPR derived dataset and allowed for complicated strain variation found in an Accumulibacter CRISPR to be visualised and assembled.

In order to gain a better understanding of the genomic diversity of Accumulibacter populations and to study phage-host interactions and evolution, deep metagenomics sequencing of two lab-scale EBPR reactors was performed during the course of one year of operation. Six Accumulibacter genomes were recovered including four from previously unsequenced clades. Metabolic reconstruction revealed differences in the nitrogen and carbon metabolism between these genomes suggesting a molecular basis for reactor operational differences and niche differentiation (Chapter 4).

From the same EBPR reactors, 21 phage metagenomes spanning the 12 months of reactor operation revealed that the phage community was highly dynamic with many phage being present in only a few of the sampling points (Chapter 5). There were 28 phages that became very abundant in the community (>5%) at one or more time points suggesting that they were the most active members of the phage community. CRISPRs from the microbial metagenomes allowed for ~300 phage to be paired with their host bacteria. The pattern of CRISPR targeting indicated that some of the phage contained a broad host range. Many of the Accumulibacter phages were targeted by multiple Accumulibacter types and one of these phage was also targeted by another member of the Rhodocyclaceae. One of the most abundant phage in the system was targeted by CRISPRs in genome bins representing members of two separate phyla, which contradicts the long-standing assumption that phage a highly specific predators (Chapter 5).

Surprisingly the CRISPR spacer complement in the Accumulibacter genomes did not evolve as rapidly as expected. Three out of four Accumulibacter CRISPRs did not change in over more than 100 days between the first and last sampling points. The changes in the fourth CRISPR was minor, with two new spacers being inserted. These results suggest that in a closed EBPR reactor the role that CRISPRs play in combating phage infection in Accumulibacter may be minor and that other phage defence mechanisms may be more important. Other meta’omic approaches, such as metatranscriptomics will be required to determine if CRISPR loci are expressed in Accumulibacter or if there are other mechanisms that are preventing their evolution.

The findings presented in this thesis represent the largest and most comprehensive microbial and phage metagenomic study of EBPR reactor undertaken to date. 
Keyword Accumulibacter
Metagenomics
Phage
Enhanced Biological Phosphorus Removal
Crispr
Phage-host interactions

 
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Created: Sat, 15 Mar 2014, 02:24:55 EST by Mr Connor Skennerton on behalf of Scholarly Communication and Digitisation Service